Rhizobia nodule bacteria




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3. RHIZOBIA   NODULE BACTERIA

Rhizobia are soil bacteria characterized by their unique ability to infect root hairs of legumes and induce effective nitrogen fixing nodules to form on the roots. Rhizobia commonly occur in soils but often fail to produce effective nodulation, either because too few are present or because those present cannot work effectively with the particular legume.

Rhizobia are rod shaped living plants which exist only in the vegetative state. Unlike many other soil microorganisms, rhizobia produce no spores. They are aerobic and motile. They multiply by simple cell division. The generation time ranges from two to four hours for so called "fast growers", which generally form relatively large (2  to 4 mm diameter) colonies in three to five days, to six to eight hours for the "slow growers", which yield colonies 1 mm in diameter in seven to ten days. The optimum growth temperature is 28 to 30 °C.

Rhizobia are not particularly fastidious in their nutritional requirements. They can use sugars, alcohols and some acids as sources of energy. Yeast extract provides growth factors and vitamins and usually enhances growth, but some species can produce their own growth factors.



Nodulation
Infection of root hairs
Steps in the infection and development of a nodule are illustrated in Figure 4. The mechanism of recognition between rhizobia and the leguminous seedling is not fully understood, but when an infective Rhizobium cell comes in contact with the root of a susceptible legume seedling, the Rhizobium cell increases in number and root hair colonization occurs. The root hair curls; a Rhizobium enters, mul-

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Figure 4. Infection process and nodule development on a soybean (Glycine max). root (see figure below)
(a) infection thread cells in this zone multiply actively and are contaminated with Rhizobium, but fixation does not take place

(b) meristem made up of small cells that are not contaminated by Rhizobium japonicum and that provide for the growth of the nodule


(c) fixation zone, made up of bacteroids, plant cells filled with rhizobia that have taken on a swollen or pleomorphic shape. These rhizobia contain an enzyme, nitrogenase, that enables them to fix atmospheric nitrogen. Nitrogenase contains iron and molybdenum, metals necessary in the transport of electrons required in the reduction reaction. The cells at the fixation site are tinted red by leghaemoglobin, a pigment that transports oxygen to the bacteroid



(d) degeneration zone, green or brown. No fixation takes place in degenerating cells

(e) vascular system derived from vessels of the stele, it irrigates the nodule, bringing in necessary carbohydrates for the fixation reaction and transporting the nitrogen compounds that are formed to the leaves

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a

b

c

d

e


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tiplies, and an infection thread is formed. This thread penetrates the root cortex. Other root cells are infected and cell division increases. The embryonic nodule develops.

Nodules vary widely in shape, size, colour, texture and location. Shape and location of effective nodules are largely determined by the host plant. Nodules produced by a strain of Rhizobium on one legume host may have no resemblance to nodules produced by the same strain on another host in the same "cross inoculation" group.

The size, colour and distribution of nodules on the roots of leguminous plants reflect the status of the Rhizobium legume association and its nitrogen gathering efficiency. This varies widely with the strain of Rhizobium. Various types of effective nodules are shown in Figures 5 and 6.

Nodules that can be seen with the naked eye usually develop in 10 to 14 days on seedlings grown in a nitrogen free substrate in a greenhouse or growth chamber. The time required varies somewhat, depending on the legume and the seed size. Under field conditions, visible nodules should appear in 21 to 28 days. With high levels of nitrogen in the soil or application of large amounts of nitrogen fertilizer, nodule development will be delayed. In tropical climates, however, small applications of nitrogen fertilizer are often used to stimulate early growth of leguminous crops. The overall effect can be beneficial.




Kinds and distribution of nodules
Effective nodules are generally large and are clustered on the primary and upper lateral roots. Maximum development of nodules as determined by weight or volume normally occurs in the late flowering stage. In contrast, ineffective nodules are small, numerous, and usually distributed throughout the root system. Examples of effective and ineffective nodules on Trifolium sp. are shown in Figure 7.
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Nodule colour

The effectiveness of the Rhizobium:leguminous plant association can also be determined by slicing the nodules of the legume host during the early flowering period and noting the nodule colour. Effective nodules are large and have a deep reddish colour inside. The red pigment, leghaemoglobin, is associated with active nitrogen fixation in legume nodules. It is not a part of nitrogenase, the nitrogen fixing enzyme, but it controls the oxygen necessary to activate this

enzyme.

Effective nodules on the prairie bean (Strophostyles helvola), cowpea (Vigrea unguiculata) and the hyacinth bean (Dolichos lablab) are sometimes black on the inside. These nodules usually contain both leghaemoglobin and melanin, the latter causing the black colour.



The red haemoglobin in effective nodules breaks down to green leghaemoglobin as the nodules become older and senescent. A single effective nodule on certain legumes crown vetch (Corortilla varia), sainfoin (Onobrychis viciifolia) and milk vetch (Astragalus titer)   may show white, red and green regions simultaneously during any stage of growth. These indicate, respectively, areas of nodule growth, active nitrogen fixation and senescence (Fig. 7). Ineffective nodules are white to pale green on the inside. They do not change colour as they age.

When legumes are supplied with nitrogen fertilizer, the nodules produced by effective strains of rhizobia remain small and exhibit the same characteristics as those produced by ineffective rhizobia. After the soil nitrogen is exhausted, the nodules usually increase in size and function normally.

By contrast, nodules produced by effective rhizobia on molybdenum deficient plants tend to grow large and appear normal except for colour. Molybdenum deficient nodules are green on the inside and appear

senescent.

The pigmentation of nodule interiors is shown in Figure 8.

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Figure 5. Kinds of nodules on grain legumes:

(a) and (b) groundnut (Arachis hypogaea)

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(c) soybean

(Glycine max)



(d) chick pea

(Cicer arietinum)

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Figure 6. Kinds of nodules on forage legumes:

(a) birdsfoot



(Lotus corniculatus)

(b) white clover

(Trifolium repens)

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(c) sainfoin

(Onobrychis viciifolia)

(d) guar

(Cyamopsis tetragonoloba)

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Figure 7. Effective and ineffective nodules:

(a) nodules of Trifolium spp.. ineffective on bottom.

effective on top

(b) effective nodules of crown vetch (Coronilla varia)





Figure 8. Section of soybean (Glycine max) nodules. Top to bottom, effective to ineffective

Nodule examination
When nodulation of field plants can be examined only once, the best time is at the peak of flowering. If more frequent examination is possible, nodule development at four to five weeks should reveal adequacy of the inoculation treatment and relative infectiousness of the Rhizobium strains. Late examination during pod formation can be useful in selecting the more compatible Rhizobium strains during the critical period of seed development. Whenever possible, all three examinations are recommended.
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Species and strains of rhizobia: host specificity
Early in the study of the Rhizobium:legume association, it was noted that certain plants showed preferences for certain rhizobia and vice versa. Leguminous species mutually susceptible to nodulation by a particular kind of rhizobia constitute a "cross inoculation" group. Strains of rhizobia capable of nodulating the plants in one of these groups were considered a species regardless of whether nitrogen fixation occurred.

The six Rhizobium species established under this system and the corresponding genera of plants that each nodulates are given in Table 2, Part 1.

The "cross inoculation" concept for grouping legumes and establishing Rhizobium species was questioned after numerous inharmonious bacteria plant responses were discovered. The idea was almost abandoned. No new Rhizobium species have been introduced during the past half century. Nonetheless, this grouping of plants and designation of Rhizobium species on the basis of nodulation have been useful both to scientists and to manufacturers of legume inoculants. While nodulation does not guarantee any benefit in terms of yield, it results in a beneficial association when soil nitrogen is low.

A system of classifying Rhizobium species based on numerical taxonomy has been developed and appears in the ninth edition of Bergey's Manual of determinative bacteriology. This is included in Part 2 of Table 2 for clarification during the forthcoming "change over" period.

The "cross inoculation" grouping of leguminous plants and designation of Rhizobium species is based entirely on nodulation and does not relate to host benefit. Numerous data show that nodules produced by some strains of rhizobia are of no benefit to the growth of their host. These ineffective nodules could even be considered parasitic because the plant provides energy for their growth without receiving any nitrogen in exchange.

Many legumes are "host specific". While they may be



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Table 2. RHIZOBIUM SPECIES

Bacteria

Genera of host plants




Part 1

R. meliloti

Medicago, Mehlotus and Trigonella

R. trifolii

Trifolium spp.

R. leguminosarum

Pisum, Vicia, Lathyrus, Lens

R. phaseoli

Phaseolus vulgaris, P. multifloris

R. lupini

Lupinus and Ornithopus

R. japonicum

Glycine max

Cowpea (type)

Vigna, many other genera and species




Part 2

Fast growers




R. meliloti

Medicago, Mehlotus, and Trigonella

R. leguminosarum




biovar.-trifolii

Trifolium spp.

biovar.-phaseoli

Phaseolus vulgaris, P. multifloris

biovar.-viceae

Pisum, Lathyrus, Lens, Vicia

R. loti

Lupinus, Lotus, Anthyllis, Ornithopus

Slow growers




Bradyrhizobium japonicum

Glycine max

Bradyrhizobium spp.




Bradyrhizobium sp. (Vigna)

Vigna - numerous other genera and

species


Bradyrhizobium sp. (Lupinus) Lotus pedunculatus, Lupinus sp.




Source: Bergey, Manual of determinative bacteriology, 9th ed., 1983.

nodulated by diverse strains of rhizobia, growth is enhanced only when the nodules are produced by particular strains of rhizobia. It is thus extremely important to match symbionts prudently for maximum nitrogen fixation.


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Effectiveness grouping
As the study of the responses of numerous legume species to different strains of rhizobia has been expanded, it has been noted that certain plants tend to respond similarly to particular strains of rhizobia. In other words, a strain of rhizobia that nodulates and fixes a large amount of nitrogen in association with one species may also do the same in association with certain other species. This must be verified by testing. Leguminous plants that demonstrate this tendency to respond similarly to particular strains of rhizobia are considered an "effectiveness" group.

The effectiveness groupings of leguminous species are given in Table 3. One inoculant can be prepared for an entire effectiveness group of plants rather than for each individual plant species.



Table 3. EFFECTIVENESS GROUPINGS OF LEGUME CROPS

Legumes that tend to respond similarly when inoculated

with the same strain of rhizobia



Rhizobium Effectiveness Leguminous

species groupings species



Rhizobium meliloti

1. Medicago sativa, M. falcata, M. minima, M. tribu-

loides, Melilotus denticulata, M. alba, M. officinalis,

M. indica

2. Medicago arabica, M. hispida, M. lupulina, M. or-

bicularis, M. praecox, M. truncatula, M. scutellata,

M. polymorpha, M. rotata, M. rigidula, Trigonella

foenum-graecum

3. Medicago laciniata

4. Medicago rugosa




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Table 3. (cont'd)

Rhizobium Effectiveness Leguminous

species groupings species



Rhizobium trifolii

5.

Trifolium incarnatum, T. subterraneum, T. alexan-

drinum, T. hirtum, T. arvense, T. angustifolium

6.

Trifolium pratense, T. repens, T. hybridum T. fragi-

ferum, T. procumbens, T. nigrescens, T. glome-

ratum

7.

Trifolium vesiculosum, T. berytheum, T. bocconei,

T. boissieri, T. compactum, T. leucanthum, T. mut-

abile, T. vernum, T. physodes, T. dasyurum

8.

Trifolium ruepellianum, T. tembense, T. usam-

barense, T. steudneri, T. burchellianum var. bur-

chellianum, T. burchellianum var. johnstonii, T. afri-

canum, T. pseudostriatum

9.

Trifolium semipilosum var. kilimanjaricum, T. ma-

saiense T. cheranganiense, T. ruepellianum var.

lanceolatum

10.

T. medium, T. sarosience, T. alpestre

11.

T. ambiguum

12.

T. heldreichianum

13.

T. masaiense

14.

T. reflexum

15.

T. rubens

16.

T. semipilosum

Rhizobium leguminosarum

17.

Pisum sativum, Vicia villosa, V. hirsuta, V. faba,

V. tenuifolia, V. tetrasperma, Lens esculenta, La-

thyrus aphaca, L. cicera, L. hirsutus, L. odoratus,

L. sylvestris

18.

Lathyrus ochrus, L. tuberosus, L. szenitzii

19.

Lathyrus sativus, L. clymenum, L. tingitanus

20.

Vicia faba, V. narbonensis

21.

Vicia saliva, V. amphicarpa




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Table 3. (cont'd)

Rhizobium Effectiveness Leguminous

Species groupings species



Rhizobium phaseoli

22. Phaseolus vulgaris, P. coccineus, P. angustifolius

Rhizobium lupini

23. Lupinus albicaulis, L. albifrons, L. albus, L. angusti-



folius, L. arboreus, L. argenteus, L. benthamii, L.

formosus, L. luteus, L. micranthus, L. perennis,

L. sericeus, Lotus uliginosus, L. americanus, L.

pedunculatus, L. strictus, L. strigosus

24. Lupinus densiflorus, L. vallicola

25. L. nanus

26. L. polyphyllus

27. L. subcarnosus

28. L. succulentus

Rhizobium japonicum

29. Glycine max

Rhizobium spp. (cowpea type)

30. Vigna unguiculata, V. sesquipedalis, V. luteola, V.



cylindrica, V. angularis, V. radiata, V. mungo, Des-

modium sp., Alysicarpus vaginalis, Crotalaria sp.,

Macroptilium lathyroides, M. atropurpureum, Pso-

phocarpus sp., Lespedeza striata, L. stipulacea,

Indigofera sp., Cajanus cajan, Cicer arietinum

31. Phaseolus limensis, P. lunatus, P. aconitifolius, Ca-

navalia ensiformis, C. lineata

32. Arachis hypogaea, A. glabrata, Cyamopsis tetrago-

noloba, Lespedeza sericea, L. japonica, L. bicolor

33. Centrosema pubescens, Galactia sp.

34. Lotononis bainesii




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Table 3. (concluded)

Rhizobium Effectiveness Leguminous

species groupings species



35. Lotononis angolensis

Rhizobium spp. (lotus)

36. Lotus corniculatus, L. tenuis, L. angustissimus, L. te-

tragonolobus, L. caucasicus, L. crassifolius, L:

creticus, L. edulis, L. frondosus, L. subpinnatus,

L. weilleri, Dorycnium hirsutum, D. rectum, D. suf-

fruticosum, Anthyllis vulneraria, A. lotoides

37. Lotus uliginosus, L. americanus, L. scoparius, L.

angustissimus, L. pedunculatus, L. strictus, L. stri-

gosus, Ornithopus sativus, Lupinus angustifolius,

L. albus, L. luteus

Rhizobium spp. (Coronilla, Petalostemon-Onobrychis)

38. Coronilla varia, Onobrychis viciifolia, Petaloste-

mon purpureum, P. candidum, P. microphyllum,

P. multiflorus, P. villosum, Leucaena leucocephala,

L. retusa

Rhizobium spp. (various)

39. Dalea alopecuroides

40. Strophostyles helvola

41. Robinia pseudoacacia, R. hispida

42. Amorpha canescens

43. Caragana arborescens, C. frutescens

44. Oxytropis sericea

Rhizobium spp. Astragalus sp.

45. A. cicer, A. falcatus, A. canadensis, A. mexicanus,

A. orbiculatus




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