EXERCISE 18
TO VERIFY THE NITROGENFIXING POTENTIAL OF GLASSHOUSE SELECTED SOYBEAN RHIZOBIA IN THE FIELD ENVIRONMENT
Strains of rhizobia, previously selected in potted field soil, are evaluated in the field environment so as to further identify the most effective strains for inoculant production. The effectiveness of a multistrain inoculant is compared with singlestrain inoculants.
Key steps/objectives
1)Select rhizobial strains and prepare the inoculants
2)Prepare the field and apply fertilizers
3)Inoculate the seeds and plant
4)Determine the number of rhizobia on the inoculated seeds
5)Inspect the field and weed as necessary
6)Harvest at 50% flowering (early harvest)
7)Harvest for grain yield (final harvest)
8)Analyze the data
(a)Setting up the experiment
Set up the experiment as a randomized complete block with four replications (Fig. 18.1). Set up eight treatments; six inoculated (five singlestrain and one multistrain); a plusnitrogen; and noninoculated control without nitrogen.
Field Dimensions:A field area of 360 m2 (24 m x 15 m) is required. Make rows 7.5 m in length and 0.5 m apart. Each treatment plot is flanked by an uninoculated guard (border) row along each side, with two center harvest rows (see Figure 18.2). The area of each plot is 11.25 m2 (0.001125 ha). The area harvested for grain yield is 3.75 m2.
Figure 18.1. Field layout and dimensions.
Choice of rhizobial strains:Use five of the best strains, according to their order of ranking in Exercise 17. From this group select three serologically distinct strains for the preparation of the multistrain inoculant for use in this exercise and later in Exercise 19.
Figure 18.2. Field layout and dimensions.
(b)Selecting rhizobia for the experiment
(Key step 1)
Serologically distinct and/or antibiotic resistant labeled rhizobia may be selected using methods described in Section B.
If selection of serologically distinct strains is not possible (because of crossreactions amongst the strains chosen), a multistrain inoculant can still be prepared but may not be suitable for studying aspects of strain ecology (competition, persistence, etc.) in the soil by serological methods. Antibiotic labeling offers an alternative to the use of serologically distinct strains. However, the antibiotic labeling method is suggested as more reliable only when each of the component strains in the multistrain inoculant has double antibiotic resistance labels. Singlelabel strains may also be used but with caution.
Since three strains are used in the multistrain inoculant, the process of labeling (multilabeling) and identification of the strains may become too involved, especially when more antibiotics are needed in the development of the resistant strains. Moreover, the labeled strains need to be confirmed for retention of symbiotic effectiveness (Leonard jar screening, Exercise 16) when compared with the parent strains prior to their use in the inoculants.
(c)Preparing inoculants
(Key step 1)
In this experiment, inoculate the seeds (except controls) with peat cultures.
Prepare the peat inoculants of the five strains following procedures described in Exercise 21 using gammairradiated or autoclaved peat. In preparing the multi-strain inoculant, grow the 3 chosen strains of rhizobia separately. Aseptically mix equal volumes of each strain in a sterile Erlenmeyer flask. Use this mixture to inoculate the peat.
Prepare the inoculants in advance of the experiment and allow them to mature for at least 2 weeks at 2530C. Determine and record the quality of each inoculant (number of viable rhizobia per g peat) by plate counts (Chapter 5 or 6). After the 2 weeks of curing, the inoculants may be stored up to 4 weeks in a refrigerator (4C).
(d)Preparing seeds for inoculation and planting
(Key steps 3 and 4)
A planting distance of 3.7 cm between seeds is optimal for good soybean yields. Based on this planting distance, approximately 203 seeds are needed per 7.5 m row. Since there are two inoculated rows per plot and four replications, a total of 1624 seeds will be needed for each inoculated treatment. Count or weigh 2000 seeds for each treatment to make allowances for losses and for samples to be taken for determining the number of rhizobia per seed at planting. The seed numbers should be converted to weight measures for convenience. Weigh out the seeds for each treatment in clean plastic bags and label accordingly.
For soybean, 10 g of peatbased inoculant and 3.0 ml of gum arabic for 100 g of seed are recommended for experiments. Inoculate the seeds as described in Exercise 23.
Inoculate the seeds just before planting. Keep the seeds in their plastic bags and in a cool place away from direct sunlight.
Set aside 20 seeds of each inoculated treatment and with minimum delay determine the number of rhizobia per seed (inoculation rate) as described in Exercise 23.
(e)Preparing the field
(Key step 2)
Conduct the experiment in the field site from where soil was previously collected for Exercise 17. Drive posts into the soil at the four corners of the field to indicate the boundary of the experimental site. Clear and remove all surface vegetation and treat the field with herbicide. Plow the field after sufficient time has been given for the herbicide to take effect in killing the weeds. Remove large rocks, plant roots, and other forms of debris. Till the soil to break up lumps and prepare a smooth, firm seed bed. Alternatively, the sowing may be done without plowing. This will minimize disturbance to the soil and release of soil nitrogen.
Mark the plots and designate treatments for the different plots (Treatment should be randomized in advance of planting and recorded.)
(f)Controlling crosscontamination by modifying irrigation methods
(Key step 2)
Rhizobia are soil bacteria and are easily spread when soil borne or in soil suspension. Surface overflow resulting from heavy rains and the floodirrigation method may cause serious crosscontamination. In this particular exercise, where several different strains of rhizobia are tested, the methods of irrigating the field may be modified to control heavy crosscontamination.
Surface overflow resulting from heavy rains and the flood-irrigation method may cause serious cross-contamination.
Crosscontamination from rainwash may be controlled by the preparation of elevated seed beds (bunds). This method will result in the creation of shallow ditches between the seed beds (rows). Alternately, an elevated plot with a surrounding ditch would be suitable for areas of heavy rains. Elevated plots may be preferred over elevated rows, as the latter are more susceptible to erosion. Rain water can be efficiently drained away during heavy rains if the rows are prepared so as to follow the general inclination of the slope if the slope is not too great.
In locations of very low rainfall where irrigation water is obtained from canals or rivers, flood irrigation is frequently practiced (Egypt, Sudan, etc.). In this situation, ditches between rows are preferred as they deliver water more efficiently to the roots of plants growing on elevated rows than to rows of plants on an elevated plot. However, if plots are not elevated, irrigation by flooding the entire surface of the plot may be done. This would require the construction of an elevated bund around each treatment plot to prevent water flow from one plot to neighboring plots. Water must be controlled to flow only from the main stream into the plot. Backflow into the mainstream must be prevented. Successive irrigation by channeling water from one plot to neighboring plots must be prevented.
Sprinkler and dripirrigation methods may be used if these are available.
(g)Applying fertilizer
(Key step 2)
Fertilize the field soil to optimize conditions for growth. Follow levels of fertility as recommended for the potted soils (Exercise 17).
Lime the soil to pH 6.06.5. The quantity of lime may vary from 50010,000 kg ha1 (depending on the soil and its initial pH) to bring about appreciable changes in the soil pH. Apply the lime 2 weeks prior to the application of the other fertilizers. Use the lime requirement data from Appendix 16.
To facilitate the application of the fertilizers, each of the four blocks is fertilized individually by broadcasting. The rates per block (90 m2) are as follows:triple superphosphate, 4.5 kg; potassium chloride, 3.44 kg; zinc sulphate, 0.42 kg; ammonium molybdate, 0.016 kg; magnesium sulfate 0.45 kg.
Weigh out the fertilizer quantities in containers (plastic bags or buckets) of adequate size and apply by broadcasting. The smaller quantities, for example, zinc sulphate, ammonium molybdate and magnesium sulfate, may be mixed with an inert carrier (e.g., sand) and broadcasted or sprayed on. Do not apply the urea with the other fertilizers as this is applied at planting only to the plusN controls. Till in the fertilizers soon after application.
The field is ready for planting one day after the application of the fertilizers.
(h)Planting the experiment
(Key step 3)
Make furrows 7.5 m long, 0.5 m apart, and four per plot and 33.5 cm in depth. Make furrows for only a few plots at a time so that open furrows are not subjected to drying out from prolonged exposure in the sun.
Irrigate the experimental site just enough to moisten the soil the evening prior to the day of sowing, if the soil is dry. Irrigate again immediately after sowing the trial.
A straight 2 m long wooden stick with 3.7 cm graduations, placed alongside the furrow, is a useful guide for even placement of seeds.
Plant the controls and guard rows first and cover the seeds on completion of each row.
Prevent contamination of the seeds by sterilizing your hands when handling each batch of seeds inoculated with a different strain. Hands are easily sterilized by thorough washing with soap and water followed by swabbing with alcohol after the hands are dry.
Apply urea, only to the plusnitrogen controls, at the rate of 0.23 kg urea per plot with 25% (58 g per plot) at planting and the remaining 75% (174 g per plot) at 4 weeks. Weigh out 58 g each of urea in four bags, one for each of the four replicates.
Make a furrow 45 cm deep, parallel to and 45 cm away from the planted row. Evenly distribute the urea with your hands. Cover the furrows immediately after application. Exposure of the urea will result in hydrolysis and loss of N (as ammonia) to the atmosphere.
(i)Monitoring the trial and harvest
(Key steps 5, 6, and 7)
Inspect the field frequently for plant damage by disease and insect pests. Take appropriate measures to control these pests. Weed the plots whenever necessary.
Make frequent observations of plant growth and color. Note treatments with early signs of N fixation.
Record the time taken for 50% of plant population to initiate flowering. Make an early harvest at this time.
The area of the plots for early harvest and harvest for grain yield are indicated in Figure 18.2. Harvest plants for dry matter yield. Observe nodule size, color, and distribution on the root. Obtain the fresh and dry weight of nodules.
If facilities are available, perform the acetylene reduction assay to determine nitrogenase activity as described in Appendix 15.
Record time for the plants to reach maturity. Process the plants for determining grain yield (dried to 56% storage moisture). Express grain yield on a kg ha1 basis.
(j)Analyzing the data
(Key step 8)
Analyze the data from the early harvest for correlation (Appendix 18) tops vs. nodule weight; tops vs. nodule numbers; tops vs. nitrogenase activity (if available); nodule weight vs. nitrogenase activity. In addition, perform a correlation analysis to correlate total nitrogen with all the parameters measured.
Rank the strains according to nitrogen fixing potential, and compare your data with that from the Leonard jars and pottedsoil experiments.
Compare the performance of the multistrain inoculant with singlestrain inoculants. What could be the advantage of a multistrain inoculant? Rank the data obtained for grain yield. Does ranking of strains according to dry matter production at early harvest and at grain yield agree?
Requirements
(a)Setting up the experiment
Measuring tape (50 m)
Field site (24 m x 15 m)
Five best rhizobial strains from Exercise 17
(b)Selecting strains for the experiment
Serologically distinct or antibiotic labeled strains of rhizobia
(c)Preparing inoculants
Agar slant cultures from (a)
Five Erlenmeyer flasks (250 ml) each containing 100 ml YM broth
Six sterile plastic syringes (50 ml); six sterile needles (3/4 in, 18 gauge)
Six bags of peat (50 g per bag) autoclaved or irradiated
Sterile 10 ml pipettes
Incubator
Quality check of inoculants (materials as in Exercise 21)
(d)Inoculating the seeds
Soybean seeds, balance, plastic bags
Peat inoculants, gum arabic solution, 10 ml pipette with widebore tip (for pipetting gum arabic solution)
Samples of inoculated seed
(e)Preparing the field
Field area
Four wooden posts for marking field perimeter
Herbicide(s) and spraying equipment
Plowing and tilling machinery
Other field preparation accessories
(f)Controlling crosscontamination by modifying irrigation methods
Suitable field design to control crosscontamination
(g)Applying fertilizer
Magnesium sulfate 0.45 kg x 4 blocks = 1.8 kg
Triple superphosphate 4.5 kg x 4 blocks = 18 kg
Potassium chloride 3.44 kg x 4 blocks = 13.76 kg
Zinc sulfate 0.42 kg x 4 blocks = 1.68 kg
Ammonium molybdate 0.016 kg x 4 blocks = 0.064 kg
Balance, plastic bags or plastic buckets
Tiller or hoes
(h)Planting the experiment
Inoculated and noninoculated soybean seeds from (d)
Irrigation water
Metric tape, hoes or suitable equipment for making furrows
Planting guide for even placement of seeds
Soap, water, clean rags, alcohol in spray bottle
Urea for Ncontrols
Covered container to keep seeds
(i)Monitoring the trial and harvest
Insecticides and spraying equipment
Weeding tools, hoes
Scissors/snips, paper bags, aluminum weighing boats
Coarse sieve
Drying oven (70C)
Balance
(j)Analyzing the data
Calculators and statistical tables
Statistical assistance