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Tests for the Ability of Rhizobium Isolates to Nodulate |
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Tests for the Ability of Rhizobium Isolates to Nodulate
Apart from the existence of Rhizobium isolates with a range of effectiveness, occurrence of non-nodulating and also pathogenic strains among the isolates obtained by plating extracts of nodules cannot be ruled out. Therefore, testing strains for their ability to nodulate and for their effectiveness under sterile conditions is of prime importance.
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(a) Test-tube method for small seeded legumes: Agar-agar, vermiculite or sand can be used as the substrate for growing plants. Any nitrogen-free plant nutrient (e.g., Jensen's nutrient for seedlings which has the following composition: CaHPO4, 1.0 g; K2HPO4, 0.2 g; MgSO4 . 7H20, 0.2 g; NaCI, 0.2 g; FeCI3, 0.1 g; agar-agar, 8.0 g; and distilled water, 1000 ml; pH-6.8) is used to make agar slants in 2.5 x 15 cm or 4 x 20 cm test tubes, depending on the size of the seed. Seeds are surface sterilized with suitable agents and sown on the substrate in tubes. On germination, they are inoculated with a suspension of the bacterial isolate under test prepared in a small aliquot of the nutrient solution. At regular intervals, the moisture content of the tube is checked and the nutrient solution replenished when necessary. During the experiment which may run for several weeks, the plants are examined and nodulation data obtained. A general criterion for distinguishing the effectiveness is to note the relative dry weight of nodulated plants, provided the replication in the experiment is large enough to accommodate variations.
Testing Nodulating Ability of Rhizobium Isolates
| (1) Jensen's closed tube showing |
(2) Gibson's tube showing |
(3) The plastic cup method showing |
(4) A Leonard jar |
(5) A simple technique to follow infection of legume roots by Rhizobium which can be continuoulsy used for observation at different days of incubation |
(6) Technique for culture of excised roots |
| a. seedling |
a. cotton plug |
a. glass Petri dish lid |
a. glass Petri dish lid |
a. cotton plug |
a. holder of stainless steel wire |
| b. agar base |
b. aluminium foil cap |
b. metal cap |
b. brown paper wrapping the assembly and held on with rubber bands |
b. boiling tube |
b. organic nutrients in agar |
| c. 1/4 strength nitrogen-free nutrient liquid |
c. 1/4 strength nutrient solution solidified with agar |
c. open ended glass tubing for watering |
c. inverted bottomless wine or beer bottle |
c. small seed plant |
c. excised root |
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d. 1/4 strength nutrient solution |
d. sand |
d. snad |
d. microscope slide |
d. coarse sand with inorganic nutrients and Rhizobium inoculum |
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e. rimless tube |
e. plastic cup |
e. jar |
e. cover slip to hold the plant intact; the four corners of the ocver slip are attached to the slide by means of araldite |
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f. nutrient solution |
f. nitrogen-free nutrient medium |
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g. foam wick or cotton wick |
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The above method restricts the growth of seedlings in the closed test tube. Hence, the tube method was modified to allow the growth of seedlings in the air by covering the rimless test tube prepared as mentioned earlier with an aluminium foil cap with a hole covered with a cotton plug. When the seedling reaches the aluminium cap, the leaves are gently placed above the tube rim by adjusting the cap to allow the plant to grow freely. (2). Watering can be done by removing the cotton plug periodically under aseptic conditions.
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(b) Plastic cup method: By using a plastic cup and an open ended glass tube for watering, seedlings can be grown on sand and watered aseptically. The plastic cup is initially covered with a Petriplate and later when the seedlings become big, sand mixed with paraffin wax can be poured to keep the assembly in aseptic conditions.
(c) Testing for large seeded legumes: Bacterial isolates are tested for efficiency by growing the inoculated legume in sterilized sand with nitrogenfree plant nutrient solution. At the end of the desired period, data on the appearance of plants in terms of colour and vigour, the number of nodules formed on the root system, dry weight and nitrogen content of the plants are collected. During the experiment, it is necessary that temperature and light conditions under which the plants grow should be kept at optimum levels conducive for maximum performance of legume-Rhizobium symbiosis.
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A standard assembly known as Leonard Jar, consisting of a round bottomless screw-capped bottle and a glass jar designed to permit automatic and continuous water flow to the growing root system can be used for the routine testing of isolates of Rhizobium for efficiency in symbiosis. Alternatively, sterilized sand or soil in earthenware or porcelain pots can also be used to grow large seeded legumes.
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(d) Infection test: Seedlings may have to be examined for the density of infection with a view to improve the strains for virulence and early nodulation. This is done by growing plants on agar slants containing nitrogen-free medium and inoculating them with desired strains. At regular intervals, seedlings are fixed in 4% formalin or acrolein and examined later under a microscope for infection threads' in root hairs and emergence of nodule primordia on roots. Depending on the extent of infection, isolates may be rated for virulence. A simple device for continuous observation of the progress of infection has been designed which has proved useful in many laboratories .
(e) Nodulation in excised roots: In excised root cultures, the growing root
of a 2-4 day old legume seedling (grown aseptically) is cut with a sharp razor blade and the blunt end implanted in a small vial containing organic constituents (glycine, B-vitamins and sucrose) in solidified agar. The tip of the excised root is suspended in nitrate-free mineral salt medium in a Petri dish or in a test-tube into which a suspension of Rhizobium is added. In this way, the organic constituents are provided to the growing root through the cut end and the growing end of the root receives mineral constituents along with Rhizobium culture. Nodules develop after 2-3 weeks when lateral roots are fairly well developed.
(f) Tissue and cell cultures: The use of tissue cultures in plant morphogenetic studies is now well known. Explants of roots, stem and apical meristem can be grown into an undifferentiated callus mass on a suitable basal medium. New plantlets could be raised from the callus mass in test-tubes. When such plantlets are transplanted to soil, they grow into mature plants bearing flowers and fruits. Tissue culture techniques have had extensive use in plant disease control, especially in the control of seed-borne viruses.
In recent years, root calli have been used to study the interactions of plant tissues with Rhizobium or Azotobacter. In this method, a mass of undifferentiated callus or a suspension of isolated cells or protoplasts is inoculated with nitrogen-fixing bacteria such as Rhizobium or Azotobacter and incubated under controlled environmental conditions. Samples are analysed periodically for nitrogenase activity by the acetylene reduction technique. Samples of calli or cells could also be cut into thin sections and observed under light or electron microscope.
Such studies have revealed the following facts and possibilities: (1) Infection thread-like structures and bacteria or bacteroid containing cells could be seen in callus tissues or isolated cells inoculated with Rhizobium; (2) Definite nitrogenase activity could be detected in inoculated calli or cells indicating the establishment of symbiosis in such plant cell-bacteria associations; (3) when cells of Azotobacter and carrot were grown on nitrate;free media, not only the bacterial cells entered isolated cells of Carrot but such cells showed nitrogenase activity; and (4) If such studies are extended further, the day may not be far off when 'nitrogen-fixing' plants are raised. as clones through pIantlets regenerated from bacteria incorporated cells or tissues.
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