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Methods for Testing Legume Inoculant
and
Pre-Inoculated Seed Products

Fertilizers Act, Section 23, Regulations

PLANT PRODUCTION DIVISION
Canadian Food Inspection Agency
Ottawa, Ontario, K1A 0Y9, Canada

Approved by the Director, Plant Production Division

_____________________
(Signature and date)

Latest Revision: May, 2005

Method I: Most-Probable-Number Plant Grow Out Test

General

This testing method is based on the following major assumptions:

1. If a viable rhizobium cell is inoculated onto its specific host in a nitrogen-free medium, nodules will develop on the roots of the inoculated legume plant.
2. Nodulation of the roots of the inoculated plant is proof of the presence of infective rhizobia.
3. Test cleanliness is demonstrated by the absence of nodules on plants grown from uninoculated seeds.
4. Absence of nodules is proof of the absence of infective rhizobia.
5. The MPN assessment of rhizobia on pre-inoculated legume seed is based on the ability of the inoculant to induce nodules on an aseptic test legume seedling.

Inoculant and pre-inoculated seed products sampled by inspectors of the Canadian Food Inspection Agency are protected from adverse environmental conditions which might affect the viability of the bacterial cultures. Insulated bags or containers are used to protect the samples from exposure to extreme temperature variations or fluctuations during shipping of the samples. They are accompanied by a control sample of an inoculant containing a known number of viable cells of a specific rhizobial species. The samples are shipped by the fastest means of transportation, usually air express. Samples are put under refrigerated conditions (4°C-10°C) immediately upon arrival at the laboratory.

The product samples and the control sample accompanying the product samples are tested by the analysts according to the procedures specified in this testing method. Results for product samples received with a control sample that is tested and found to be unsatisfactory are nullified.

The analyst issues a certificate of analysis stating the result of the analysis for each sealed sample submitted by an inspector. These certificates are then sent to the appropriate inspector for follow up and a copy is forwarded to the Fertilizer Section, Ottawa.

MPN Testing

Disposable growth pouches (for use with all seeds except chickpea)

The pouch (16 x 20 cm) is made of a strong and transparent polyester film capable of withstanding steam sterilization at 100 kPa (15 lb/inch2) for up to 20 minutes. Inserted in the pouch is a sleeve-like paper germination towel that is folded along the top edge into a trough and perforated to permit roots to escape from the seeding area. Pouches may be split by hermetically sealing the pouch itself, and splitting the paper germination towel. Prepared pouches are then steam sterilized.

Thirty (30) ml of sterile, nitrogen-free nutrient solution are added to the sterilized pouches (15 ml in each compartment for double compartment pouches) which are placed singly in appropriate containers so as to provide support during seeding, inoculation and growth. Each growth unit is aseptically seeded with 20, 15 and 5 seeds of the small, intermediate, and large size legume seeds, respectively. Surface disinfected seeds are placed directly in the sterile growth pouch and incubated in darkness at 20°C (for about four days) until the radicles have elongated to 0.5-1.0 cm. Seeds of large-seeded legumes (e.g., peas, soybean) may be pre-germinated before placement in the pouch. In this case the seeds are placed on 3 layers of Kimpak germination paper. The rolls are placed in a humid chamber with the seed radicles pointing downward. They are incubated at 20°C (for about four days) until the radicles have elongated to 0.5-1.0 cm. Seeds may be also pre-germinated in sterile humid vermiculite. (NOTE: pre-germinated seeds can be stored for up to two weeks at 4°C).

Growth cups (for chickpea)

Clear, autoclavable, 250-ml polypropylene specimen collection cups (e.g., Fisher Scientific) are filled with very fine vermiculite. The vermiculite is moistened with distilled water, the cups are covered with squares of heavy-duty aluminum foil, and the assembly is autoclaved at 121°C and 100 kPa for 20 minutes. Two sterilized seeds are transferred aseptically to the sterile growth medium in the cups and allowed to germinate in darkness at 20°C. Chickpea seeds may also be pre-germinated as for large seeds above.

Seed preparation

Undamaged seeds are surface disinfected by immersion in 95% ethanol for 30 seconds followed by, either: (1) 10 minutes in 3-5% hydrogen peroxide (H₂0₂) solution, or (2) 10 minutes in 5% sodium hypochlorite (NaClO) solution. The seeds are then washed or rinsed thoroughly with at least 5 changes of sterile, distilled water.

Plant nutrient solution

For plant culture, the following sterilized nitrogen-free mineral salts solution is used:

Preparation of dilutions of peat-based inoculants

A sterile phosphate-peptone buffer solution of the following composition is used for all dilutions, including the initial suspension: peptone, 1.0 g; KH₂PO₄, 0.34 g; K₂HPO₄, 1.21 g; distilled water, 1,000 ml; pH = 7.0 + 0.1.

A 10-fold dilution (w/v) using a minimum of 10 grams of inoculant is made in the sterile phosphate-peptone buffer diluent in a Waring Blender and dispersed for 2 minutes at 12,600 rpm, or in a Stomacher bag and dispersed for 1 minute. A 10-fold dilution (v/v) using a minimum 1 ml aliquot of this buffer-inoculant suspension is made using buffer diluent, and the container is shaken for 5 minutes. Ten-fold serial dilutions (v/v) using a minimum 1 ml buffer-inoculant suspension in buffer diluent, are made as required. Five-fold dilution series are prepared by mixing 1.0 ml of the final 10-fold bacterial suspension dilution and 4.0 ml of sterile phosphate-peptone buffer. Six consecutive 5-fold dilutions are made, and all but the last dilution are used to inoculate four growth units (pouches or cups) with 1.0 ml of the suspension at the appropriate 5-fold dilution level (see Figure 1. MPN Dilutions). The last dilution is used to inoculate five growth units (pouches). The inoculation is carried out as described below in the section entitled “Inoculation”. Each series includes an uninoculated control pouch/cup between each four pouch/cup dilution levels.

Figure 1. MPN Dilutions

Preparation of dilutions of liquid inoculants

An initial 10-fold dilution using a minimum 10 ml of inoculant is made with the sterile phosphate-peptone buffer and shaken for 5 minutes. Subsequent 10 and 5-fold dilutions are prepared as required according to the section “Preparation of dilutions of peat-based inoculants”.

Preparation of dilutions of clay-based inoculants

1. Weigh out 24 g clay inoculant into a small beaker.
2. Slowly add 8 ml of sterile water drop-wise to the sample and mix with a spatula.
3. Allow the sample to rehydrate for 10 min.
4. Weigh out 20 g of this sample.
5. Measure 130 ml of sterile water, pour into a small Waring blender cup (Eberbach 8580), place the cup on the blender, and turn the blender on LO.
6. Slowly sprinkle the 20 g sample into the blender and mix for 90 seconds.
7. Remove the desired volume for serial dilution and enumeration.

The initial dilution is 10^-1 expressed as rhizobia per g of original dry sample. Ten and 5-fold dilutions are prepared as required, as described above under "Preparation of dilutions of peat-based inoculants".

Preparation of dilutions of clay and peat-based granular inoculants

The granular inoculant is rehydrated slowly by placing a minimum of 10 g of inoculant into an empty dilution bottle and equilibrating for 1 h in a controlled environment of at least 80% relative humidity at room temperature. A 10-fold dilution is made by adding sterilized buffer diluent into the dilution bottle, and the bottle is shaken for 1 h. Dilutions are then made as for peat-based inoculants, as described above under "Preparation of dilutions of peat-based inoculants".

Preparation of dilutions of pre-inoculated seed (coated or uncoated)

1. For medium- or large-seeded legumes, two hundred (200) pre-inoculated seeds are placed in a sterilized container.

2. For small-seeded legume seeds, 10 g of pre-inoculated seed are added to an empty dilution bottle. For mixed small seed samples, the percentage of the legume in the mix is used to calculate the sample size required to provide 10 g of legume seed.

   EXAMPLE: For a mixture of 90% alfalfa and 10% timothy, the sample size is calculated according to the following formula:

   Alternatively, the pre-inoculated legume seed is physically separated from the mixture based on morphological examination of the seed (200 seeds are counted).

3. For coated seed samples, the weight of the seed used is adjusted based on the % of coating material.

4. All types of seed samples are slowly rehydrated by equilibrating the container or bottle containing the seeds for 1 h in a controlled environment of at least 80% relative humidity at room temperature.

5. Following equilibration, for medium- or large-seeded legumes, 200 ml of sterile 0.1% peptone-phosphate buffer is added to the container containing 200 seeds. The initial suspension of 200 seeds in 200 ml buffer corresponds to one seed per ml. The seed/buffer solution is then shaken for 1 hr.

6. For small-seeded legumes, 100 ml of sterile peptone-phosphate buffer is added to the dilution bottle. The initial seed/buffer suspension is then shaken for 1 hr. Before serial dilutions are made the concentration of the initial seed-buffer suspension is adjusted to one seed per ml. The volume of the initial seed/buffer suspension required to make the final suspension equivalent to the number of rhizobia obtained from one seed per ml is calculated according to the following formula:

   

   where
   V_i = aliquot of initial suspension to be made up to final volume V_f(ml)
   V_t = total volume (ml) of initial suspension
   S = number of seeds/kg (from Table A)
   W_s = weight (g) of seed in V_t

   EXAMPLE: If the initial suspension was made with 10 g alfalfa seed in 100 ml buffer, and the final suspension is to be diluted to 100 ml, the volume of the aliquot required is calculated according to the following formula:

7. Five-fold dilutions for MPN testing are made from the final suspension (at a concentration of one seed per ml) after the adjustments for seed mixtures and/or coating materials are calculated and made as described above. When using large pre-inoculated seeds or when the number of rhizobia is expected to exceed the MPN value, it may be necessary to make additional 10-fold dilutions before the 5-fold dilutions. These should be made with a minimum initial transfer of 1 ml.

8. From the suspension, or from a 10-fold dilution, subsequent 5-fold dilution series (1 ml into 4 ml diluent) are prepared for 6 dilution steps. At the 5-fold dilution level, each of the four growth units (pouches or cups) is inoculated as described below with 1.0 ml of the suspension. At each dilution level one uninoculated control pouch/cup is included in addition to the four inoculated pouches/cups.

Inoculation

The growth units containing the germinated seeds are inoculated with the appropriate dilutions (dilutions for each product type are prepared as described above). The diluted inoculant is applied to the root zone of the germinated seeds and the entire unit is then transferred to a growth chamber (phytotron).

Growth conditions

General
After inoculation, the growth pouches are placed in a growth chamber providing approximately 16,000 lux. Temperature is maintained at 22°C during the 16-hour light period, and at 18°C during the dark period. Humidity is held constant at 65-70%.

Specifically for chickpea
After inoculation, the cups are re-covered with their original foil tops. As the seedlings emerge, the foil covers on the MPN cups are replaced with clear plastic drinking cups which have had the bottoms cut out, and which have been sterilized with ethanol. Plants are given water as required.

Calculation of Most Probable Number (MPN) results

The plants within any given pouch or cup are considered to be a growth unit. Growth units are kept for three weeks in the phytotron before examination for the presence of nodules is made; species of legumes that show slow development (e.g. chickpeas, soybeans) are examined after four weeks of test duration. Nodulation is recorded as "+" for a nodulated growth unit or as "-" for a growth unit showing absence of with no nodules. A single nodule on a single plant is sufficient for the growth unit to be recorded as "+". The number of positive growth units (i.e. with nodules) at each dilution level is recorded. Using the series of numbers for the 6 dilution steps from the least to the most dilute, the most probable number of rhizobia in the sample and the 95% confidence limits are determined using the Most-Probable-Number Enumeration System (MPNES) computer software program of Bennett, Woomer, and Yost; NifTAL Project and the University of Hawaii. The final MPN value corresponds to the number of rhizobia per g or per ml of product for solid or liquid inoculants respectively, and to the number of rhizobia per seed for pre-inoculated seed products.

If additional 10-fold dilutions are required the MPN number is multiplied by the 10-fold dilution level to obtain the number of rhizobia contained in the sample. Appearance of bona fide nodules on plants in uninoculated controls invalidates the results of the test.

Method II: Plate Count Enumeration

Plate counting media

For plate count procedures, the following sterilized yeast extract - mannitol agar media is used:

Congo red and cycloheximide are not required for plating of inoculants prepared using sterile carrier.

Monitor the pH of autoclaved media and adjust, if necessary, to pH = 6.8 - 7.2

Plate Count Procedures

Samples for plate count are prepared and diluted as per MPN procedures. One hundred microliters (100 µl) of dilutions are pipetted in triplicate onto petri plate media surfaces and spread with sterile glass "hockey sticks". Plates are incubated inverted at 30°C. Incubation periods are normally 2-3 days for fast growing rhizobia and 5-6 days for slow-growing rhizobia. Counts of the plates at a dilution level showing 20 to 300 colonies are averaged.

Compliance of Legume Inoculant and Pre-inoculated Seed Products

The standards for inoculants and pre-inoculated seed products are prescribed by the Fertilizers Regulations. According to these standards, an inoculant must contain, per gram of product, sufficient viable cells of the nodule- inducing species to provide, when used according to directions, a minimum number of viable cells per seed (N). The specific standard number of viable rhizobia per seed (N) varies with seed size for a given legume species:

In addition to these standards, the Fertilizers Regulations state that every package containing a supplement shall have a label that, among other information, guarantees a minimum number of active viable cells per gram of product (G) and none of the information on the label is incorrect or misleading. The label must always guarantee a sufficient number of viable cells per gram so that when the product is applied to seed it meets the specific standard number of rhizobia per seed. This is verified by the Fertilizer Section at the time of product registration.

Rating of Legume Inoculants and Pre-inoculated Seed Products

Inoculant samples tested by MPN are rated satisfactory if the upper 95% confidence limit for the number of viable rhizobia per gram of inoculant is equal to or larger than the G value (minimum guaranteed analysis on the label). For preinoculated seed samples to be rated satisfactory the upper 95% confidence limit for the number of viable rhizobia per seed must be equal to or larger than N. The product sample is deemed unsatisfactory if the upper 95% confidence limit for the number of viable rhizobia per gram fresh inoculant or per seed is less than the G or N values, respectively and the control accompanying the product sample is rated satisfactory. If this occurs, another MPN test on the same sample is done immediately. In order for a sample to be rated unsatisfactory, it must prove unsatisfactory in the two consecutive MPN tests. The product sample is not rated if the control sample accompanying the product is rated unsatisfactory or if the product sample was not accompanied by a control sample.

MPN 95% confidence limits are calculated by the MPNES computer program at the time that the MPN value is determined.

Control Samples

Control samples are prepared in sterile carrier and are tested by plate count enumeration. The known number of viable rhizobia per gram fresh inoculant control is established (by plate count) prior to distribution of the control samples to the inspectors of the Canadian Food Inspection Agency. A returned control sample is rated satisfactory if its plate count value is at least 500.0 x 10⁶ rhizobia/g.

NOTE: Official samples are NOT valid if the control fails to meet the standard defined here. These results will not be used as an indication of inoculant or pre-inoculated seed product compliance and no regulatory action shall be undertaken.

TABLE A. Important legumes cultivated in Canada: scientific name arranged alphabetically, common name, number of seeds per kilogram and nodule bacteria for inoculation