|III. RHIZOBIUM STRAIN SELECTION
A. Strain Variation and Host Specificity
One of the first and most important steps in legume inoculant production is the selection of Rhizobium strains to be used in inoculants for the various leguminous crops. In some countries, government agencies specify and provide the approved Rhizobium strains for use by inoculant manufacturers. In other countries such as the United States, strain selection is the responsibility of each manufacturer. In any case, the prospective inoculant manufacturer should obtain all available Rhizobium cultures which have been tested and judged effective on the legumes for which he will prepare inoculants. Such cultures can serve as standards for evaluating new strains or isolates.
The criteria used in selecting strains of rhizobia for use in inoculant production are:
1. Ability to form effective N-fixing nodules on the legume plants for which the inoculum is recommended.
2. Ability to grow well in media, in the carrier and in the soil.
3. The ability to induce nodulation and enhance yield of leguminous crops under a wide range of soil and climatic conditions.
4. Good persistence in the soil.
The manual focuses on inoculant production and assumes that strains of Rhizobium have been selected by a thorough procedure, such as described by Halliday (1984) or Date (1976). The professional inoculant production microbiologist should be trained in methods of testing Rhizobium strains and should realize the importance of using only the most effective strains of rhizobia. Some strains of rhizobia may prove effective on many leguminous species; these broad-spectrum strains are highly desirable.
B. Effectiveness Groupings of Leguminous Plants
In the early development of the legume inoculant industry, it was common practice to produce a single inoculant for all leguminous species nodulated by a common kind or species of Rhizobium. One inoculant was produced for each cross inoculation group of plants. Since the cross inoculation grouping of plants was based entirely on nodulation with no relation to nitrogen fixation or growth enhancement (symbiotic effectiveness), these inoculants were worthless on many leguminous species.
As pointed out earlier, in some cases one strain of rhizobia may be an effective nitrogen fixer on several species and genera of leguminous plants. Further, it has been noted that certain leguminous genera and species tend to give effective response to the same strains of rhizobia. Leguminous species may thus be grouped for inoculation with a common inoculant (Table 1).
This grouping of leguminous species is very useful because it facilitates production of effective multiple host inocula when production of individual inocula for each genus or species of legume is not practical. This grouping is particularly useful as a planning aid in searching and screening Rhizobium strains for legumes which have not been studied extensively. This grouping should not be used as a substitute for screening on strains of each legume on which the inoculant is to be used. Confirming tests with individual legumes should always be made.
GROUPING LEGUMINOUS PLANTS
1. Medicago falcata, M. minima, M. sativa, M. tribuloides, Melilotus alba, M. denticulata, M. indica, M. officinalis
2. Medicago arabica, M. hispida, M. lupulina, M. orbicularis, M. polymorpha, M. praecox, M. rigidula, M. rotata, M. scutellata, M. truncatula, Trigonella foenum-graecum
3. Medicago laciniata
4. Medicago rugosa
Rhizobium leguminosarum, biovar trifolii
5. Trifolium alexandrinum, T. angustifolium, T. arvense, T. hirtum, T. incarnatum, T. subterraneum
6. Trifolium fragiferum, T. glomeratum, T. hybridum, T. nigrescens, T. pratense, T. procumbens, T. repens
7. Trifolium berytheum, T. bocconi, T. boissiere, T. compactum, T. vesiculosum
8. Trifolium africanum, T. burchellianum var. burchellianum. burchellianum var. johnstonii, T. pseudostriatum, T. rueppelianum, T. steudneri, T. tembense, T. usambarense
9. Trifolium cheranganiense, T. masaiense, T. rueppellianum var. lanceolatum, T. semipilosum var. kilimanjaricum
10. Trifolium alpestre, T. medium, T. sarosience
11. Trifolium ambiguum
12. Trifolium heldreichianum
13. Trifolium rubens
14. Trifolium reflexum
15. Trifolium rubens
16. Trifolium semipilosum
GROUPING LEGUMINOUS PLANTS
Rhizobium leguminosarum, biovar viceae
17. Lathyrus aphaca, L. cicera, L. hirsutus, L. odoratus, L. sylvestris, Lens esculenta, Pisum sativum, Vicia hirsuta, V. tenuifolia, V. tetrasperma, V. villosa
18. Lathyrus ochrus, L. szenitzii, L. tuberosus
19. Lathyrus clymenum, L. sativus, L. tingitanus
20. Vicia faba, V. narbonensis
21. Vicia amphicarpa, V. sativa
Rhizobium leguminosarum, biovar phaseoli
22. Phaseolus angustifolia, P. coccineus, P. vulgaris
23. Lotus americanus, L. pedunculatus, L. strictus, L. strigosus, L. uliginosus, Lupinus albicaulis, L. albifrons, L. albus, L. angustifolius, L. arboreus, L. argenteus, L. benthami, L. formosus, L. luteus, L. micranthus, L. perennis, L. sericeus
24. Lupinus densiflourus, L. vallicola
25. Lupinus nanus
26. Lupinus polyphyllus
27. Lupinus subcarnosus
28. Lupinus succulentus
29. Anthyllis vulneraria, A. latoides, Dorycnium hirsutum, D. rectum, D. D. suffruiticosum, Lotus angustissimus, L. caucasicus, L. corniculatus, L. crassifolius, L. creticus, L. edulis, L. froindosus, L. subpinnatus, L. tenuis, L. tetragonologus, L. weilleri
GROUPING LEGUMINOUS PLANTS
30. Glycine soja
31. Glycine max
32. Alysicarpus vaginalis, Cajanus cajan, Crotalaria sp. Desmodium sp., Indigofera sp., Lespedeza stipulaceae, L. striata, Macroptilium lathyroides, M. atropurpureus, Psophocarpus sp., Vigna angularis, V. cylindrica, V. luteola, V. mungo, V. radiata, V. sesquipedalis, V. unguiculata
33. Canavalia ensiformis, C. lineata, Phaseolus aconitifolius, P. limensis, P. lunatus
34. Arachis glabrata, A. hypogaea, Cyamopsis tetragonoloba, Lespedeza bicolor, L. japonica, L. sericea
35. Centrosema pubescens, Galactia sp.
36. Lotononis bainesii
37. Lotononis angolensis
38. Coronilla varia, Leucaena leucocephala, L. retusa, Onobrychis viciaefolia, Petalostemum candidum, P. microphyllum, P. multiflorum, P. purpureum, P. villosum
39. Dalea alopecuroides
40. Strophostyles helvola
41. Robinia hispida, R. pseudoacacia
42. Amorpha canescens
43. Caragana arborescens, C. frutesceus
44. Oxytropis sericea
45. Astragalus canadensis, A. cicer, A. falcatus, A. mexicanus, A. orbiculatus
Competitiveness is a term used to describe a desirable quality in rhizobia. In essence, a Rhizobium strain with the ability to infect and dominate in the formation of nodules on a particular host when large numbers of other highly infective strains are in the rhizosphere is considered highly competitive. The factors contributing to this property are not understood. This quality is not related to the strain's ability to enhance growth of its host. It may be related in some way to the growth and survival of the rhizobia on the seed and in the rhizosphere, or to speed of infection. There is evidence that in some cases the host plant influences which strain infects its root hairs.
High competitiveness combined with good nitrogen fixing ability are essential for yield enhancement in soils which harbor infective rhizobia. Serology is one method used in screening strains for competitiveness but it can be laborious and time consuming. Further, interpretation of results be confounded by the lack of correlation between nodule numbers and nitrogen fixation. Field testing of individual high nitrogen fixing strains as seed inoculants is perhaps the most useful nitrogen fixation. Field testing is the best integration of the Rhizobium strain's overall performance, including competitive ability, soil stress tolerance and persistence.
D. Stock Culture Maintenance
Rhizobium strain selection is a continual process. It is important to start with the most effective and competitive strains available and to strive continuously to find even better strains. Stocks of Rhizobium cultures important in the area should be maintained in the best way possible and checked frequently to make certain that they have not lost their desirable qualities. In order to avoid or reduce undesirable mutations, Rhizobium cultures should be maintained in the lyophilized or freeze dried state in sealed glass ampoules. When lyophilization equipment is not available, stock cultures may be preserved satisfactorily by desiccation on porcelain beads. This method is described in Appendix C.
When bead materials are not available, cultures may be kept on yeast extract mannitol agar slants. Screw capped tubes should be used to reduce the need for frequent subculturing. The cultures should be allowed to grow before tightening the caps, then stored at 4C.