RHIZO MIC RANGE OF PRODUCTS A practical guide

for the inoculation of legume crops.

Legume Crops

1. Introduction 3

2. What is Rhizobium? 4

3. What is the right Bacteria? 4

4. Why is Rhizobium so specific? 4

5. Isn’t there enough Rhizobium already in the soil? 6

6. How many Rhizobia do you need for effective nodulation? 6

7. What is needed for Rhizobium to survive? 7

8. Factors which influence the survival of Rhizobium. 7

9. What is inoculation? 7

10. How does nodulation work? 7

11. When must I inoculate? 8

12. What happens if I use to much Rhizobium or overdose? 8

13. How should I handle inoculants? 9

14. Powder inoculants 9

15. Applying of inoculant as a seed treatment 10

16. Liquid inoculants: Application in-furrow 11

17. Are there compatibility issues between seed applied inoculantsand fertilizers, including chemical remedies? 11

18. Addition of molybdenum during inoculation 13

19. Was inoculation successful? 13

20. What went wrong if no or poor nodulation occurred? 13

Table of Contents

Introduction

Inoculation of legume crops with Rhizobia is one of agriculture’s greatest sucess stories. It is also one of the most cost effective practises in agriculture. Years before the scientific basis of binding of nitrogen by legume crops was understood, farmers used rudementary methods of inoculation. For example they would take soil from fields with inoculated crops and move it to fields which did not inoculate.

Inoculation of legume seeds with pure Rhizobium-cultures was made possible by the pioneering work of German and Holland microbiologists during the last two decades of the 19th century. Within a couple of years after the invention European farmers

had access to cultures of Rhizobium strains for the inoculation of a broad spectrum of legume crops.

Nitrogen is required by all living organisms for the synthesis of proteins, nucleic acids and other nitrogen-containing compounds. The earth’s atmosphere contains almost 80% nitrogen gas. It cannot be used in its current form by most living organisms. However, nitrogen can be converted into a useable form by legumes when it grows in the presence of a specific Rhizobium bacterium. These bacteria can infect the roots of leguminous plants, leading to the formation of nodules where the nitrogen fixation takes place. The host plant and the

bacteria have a symbiotic relationship. The bacterium’s enzyme system supplies a constant source of reduced nitrogen to the host plant and the plant furnished nutrients and energy for the activity of the bacterium. After harvest legume roots left in the soil decay, releasing organic nitrogen compounds for uptake by the next generation of plants. Where legumes are planted for the production of hay, a large volume of the nitrogen will be removed from the soil by the hay.

1

Rhizobium is specialized soil bacteria. Due to their unique biological characteristics that enables them to bind nitrogen from the atmosphere there are many established mutually beneficial interactions with the roots of legumes. This symbiosis leads to the formation of specialized structures on legume roots, known as nodules. Inside these root nodules rhizobia absorbs carbohydrates from the plant and in return it binds nitrogen from the atmosphere for use by the plant. Legumes are unable to bind nitrogen from the atmosphere by itself, although it is able to absorb nitrogen from minerals in the soil. Rhizobium only binds nitrogen if it is present inside the root nodules. Rhizobia are microscopic singlecelled organisms. They are small organisms and can only been seen under a microscope. Thousands of cells of rhizobia fit onto a needle tip. Registered commercial inoculant contains single strains of specific rhizobia that provide optimal nitrogen fixation within the specific legume crop.

The relationship between certain rhizobia and a specific legume is very specific. Different legumes are inoculated by clearly different rhizobia. Specific Rhizobium will only nodulate and fix nitrogen with a specific legume. Ex, lupines are inoculated by slow growing acid-tolerant Rhizobium bacteria species while medics are inoculated by the rapidly growing, acid-sensitive Sinorhizobium species.

In different soils inoculant bacteria must compete with indigenous soil organisms or other strains of bacteria, so either the one outcompetes the other or after a period of time (some years) both will be in balance. This is one of the main reasons why soybeans need to be inoculated each year or season.

What is Rhizobium?

What is the right Bacteria?

Why is Rhizobium so specific?

3

It is a specifically selected rhizobia bacterium specific to each crop with the following characteristics:

Competitiveness Most soil contains Rhizobium,

and many of them outcompete other rhizobia during infection. Therefore, a strong competitive strain is essential.

Infectious The Rhizobium must be able to

effectively penetrate the root hair and form a nodule.

Effectiveness The Rhizobium must effectively

fix nitrogen in the nodule. An effective nodule is large and pink inside. Ineffective nodules are small and white. An effective strain meets all the above requirements.

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Legume Crop Inoculant1 pack inoculant(200 g or 250 g) treat how many seed?

Cowpea: Cajanus cajan (Pigeon pea), Crotalaria-spp., Cyamopsis tetragonolobus (Guar), Indigofera-spp., Lablab purpureus (Dolichos), Microptilium artopurpureum (Siratro), M. lathyroides (Velvet bean), vigna radiata (Mung bean), V. unguiculata (Cowpea), V.subterranea (Juga bean or bambara)

Groundnut and cowpea group inoculant Registration No. L 5798 Act 36 of 1947 50 kg

Cowpea: Neonotonia Wightii, Pueraria thunbergiana (Kudzu), Stylosanthes spp, except S. Guianesis cv. Oxley

Groundnut and cowpea group inoculant Registration No. L 5798 Act 36 of 1947 12,5 kg

Lupins: Lupinus albus (Whitelupin), L. angustifolius (Narrow leafed lupin), L. perennis (Perrennial lupin), L. luteus (yellow lupin)

Lupins and Serradella inoculant Registration No. L 1729 Act 36 of 1947 50 kg

Serradella: Ornithopus sativus (Blue leaf serradella), O. compressus (Yellow leaf serradella)

Lupins and Serradella inoculant Registration No. L 1729 Act 36 of 1947 25 kg

Clover: Trifolium repens (White clover), T. subterraneum (Subterranean clover), T. pratense (Red clover)

Clover Inoculant Registration No. L 1728 Act 36 of 1947 12,5 kg

Trifolium spp.: Trifolium subterraneum (woodgenellup clover), T. fragiferum (strawberry clover) and T. visculosum (arrow leaf clover) Not suitable for Indigenous clover

Woogenellup clover inoculant Registration No. L 1728 Act 36 of 1947 12,5 kg

Birdsfoot: Lotus corniculatus Lotus corniculatus Inoculant Registration No. L 550 Act 36 of 1947 12,5 kg

Boyds trefoil: Lotus Pedunculatus- en L. Hispidus Lotus Pedunculatus- en L. Hispidus Inoculant Registration No. L 550 Act 36 of 1947 12,5 kg

Groundnut: Arachis Hypogaea Groundnut and cowpea group inoculant Registration No. L 5798 Act 36 of 1947 50 kg

Medicago: Medicago murex (cv. Zodiac), M. polymoprha (cv. circlevalley, serena and Santiago) Not suitable for Medicago sativa and other annual “Medics”

Polymorpha Inoculant Registration No. L 1734 Act 36 of 1947 12,5 kg

Lespedeza: Lespedeza cuneata- spp: L.Sericea and L. Striata Lespedeza Inoculant Registration No. L 4354 Act 36 of 1947 12,5 kg

Lucerne: Medicago sativa (lucerne), Annual medics and sweet clover. Not suitable for Medicago rugosa (paragosa medic)

Lucerne Inoculant Registration No. L 5879 Act 36 of 1947 12,5 kg

Peas: Pisum sativum (garden peas), field peas and other Pisum-species.

Peas and vetch inoculant Registration No. L 1794 Act 36 of 1947 25 kg

Vetches: Vica villosa (hairy vetches), vicia sativa and other vicia species

Peas and vetch inoculant Registration No. L 1794 Act 36 of 1947 25 kg

Broadbean: Vicia faba Peas and vetch inoculant Registration No. L 1794 Act 36 of 1947 25 kg

Dry and green beans: phaseolus vulgaris and P. Coccineus (kidney beans)

Bean inoculant Registration No. L 1795 Act 36 of 1947 50 kg

Soy bean: Glycine Max Rhizo-Mic SOYABEANS (powder) Registration No. L 5799 Act 36 of 1947 50 kg

Soy bean: Glycine Max Soy bean inoculant (liquid) Registration No. L 9012 Act 36 of 1947 50 kg

Desmodium: Desmodium uncinatum (Silver leaf), D.intortum (Green leaf desmodium)

Desmodium Inoculant Registration No. L 1482 Act 36 of 1947 12,5 kg

Crown vetch: Coronilla varia Crown vetch inoculant Registration No. L 1489 Act 36 of 1947 12,5 kg

Sainfoin: Onobrychis viciifolia Sainfoin inoculant Registration No. L 1491 Act 36 of 1947 12,5 kg

Which crop can be inoculated?

Isn’t there enough Rhizobium already in the soil?

How many Rhizobia do you need for effective nodulation?

5 6

Some publications suggest that inoculation is not necessary if the legume host has been planted and inoculated in the previous four years on the specific land. The problem with this statement is that it does not take into account the level of the nodulation of the previous crop, and that the current population of rhizobia in the soil may be affected.

There are two factors which give rise to this:

1. Most soils are not beneficial to the survival of large numbers of rhizobia over a long period due to factors such as extremes in pH, low clay content, high temperatures and dry periods.

2. Colonies of rhizobia developed during the cultivation of legumes year after year is often less efficient binding nitrogen with the passage of time. So every time preferably inoculate plants when it is planted. So plants preferably needs to be inoculated every time when it is planted.

The number of Rhizobium that is needed in the soil for quick nodulation is somewhere between 100 and 1000 Rhizobia per gram of soil.

There is mainly two reasons for this specific number:

• When commercial inoculants with Rhizobia are applied according to the recommended dosage, the rate of rhizobia that is applied is equal to 100 rhizobia per gram of soil for a dept of 10 centimeter. This may lead to fast nodulation.

• Results from many field and glasshouse experiments shows that poor nodulation happens when the rhizobia counts drops below 100 rhizobia per gram of soil. With a rhizobia count of more than 100, plants tend to develop a large number of nodules (>10) on the primary root system, especially near the crown of the root system. Lower numbers of Rhizobia in the soil may lead to slower nodulation en less and smaller nodules per plant.

What is needed for Rhizobium tosurvive?

Factors which

influence the survival of

Rhizobium.

What is inoculation?

How does nodulation

work?

7

8

9

10

There is various factors which influeneces the survival of Rhizobia in the soil. These factors can lead to poor nodulation or even the death of the Rhizobium.

1. Influence of the host legume crop The right Rhizobium must be used in combination with the correct host

legume crop, otherwise the Rhizobium will be ineffective and nodulation will not take place. For example soybean inoculant will not nodulate effectively on a groundnut plant.

2. Influence by the type of soil A soils chemical and physical caractaristics will influence the survival of the

rhizobia. Especially pH, texture (clay-content) and organic matter. (See also point : What rhizobia needs to survive)

3. Herbicide The large scale use of herbicides is know to have a negative influence on the

simbiotic relationship between rhizobia and the legume plant. The influence seems to be more on the host plant than on the survival of the rhizobia. Even where high numbers of rhizobia are present the damage caused by herbicide to the rootsystem may lead to poor or no nodulation.

Inoculation is the process when Rhizobia (soilbacteria) are applied to seed or the zone in the soil where the legume crop is going to be planted in. This is done to facilitate root nodulation (forming of nodules on the roots). Improving the rate of nodules forming on a legume crop, may lead to better symbiotic nitrogen binding and increase of biomass, yield and quality. It will also increase the organic nitrogen levels in the soil were the legume crops are grown, which in return are available to the next crop grown on the same field.

Nodulation starts with the colonization of the legume roots by the Rhizobium. The sooner the colonization takes place, the sooner the development of root nodules and the sooner the Rhizobium will start nitrogen fixation. In optimal conditions, there is a series of events that has to happen in a specific order for nodulation to take place. Rhizobium infects the legume through the root hairs on new young roots. This can happen approximately 4-12 days after germination. The initial infection develops very quickly in visible nodules, more or less 3-5 weeks after the plant has germinated, depending on the plant species and the growing conditions.

Rhizobium only exists as living vegetative cell and is therefore very sensitive to any changes in their environment. If exposed to stress factors like heat, extreme pH and toxic chemicals, the Rhizobium will struggle to survive. Like all other bacteria, Rhizobium will survive and grow at its best in optimum conditions when they are provided with nutritional elements and water at a suitable pH. Rhizobium is also aerobic bacteria and needs oxygen to survive.

Requirements DescriptionFood and energy Normally carbohydrates (sugars, example glucose)

Nutrients All macro and micro nutrients

Water Rhizobium needs moisture to survive

Temperature Optimum between15 and 30°C

pH Optimum between the pH of 5 and 7,5

Air Rhizobium is an aerobic organism, it needs air for respiration

When must I inoculate?

What happens if I use to much Rhizobium or overdose?

11

12

Anytime when legumes are planted, inoculation can be considered because of the potential nitrogen fixation and potential increase in yield.

Overdosing of inoculants on legume plants/seeds is not harmful. One cannot overdose with inoculants. The cfu (colony forming units) will just be very high. Less problems will occur when overdosing that applying less product than the recommended rate. Excessive application of inoculants is a small price to pay, compared to poor nodulation and nitrogen deficiency on crop yield.

Important points to remember when inoculation is considered:

• The specific legume crop has never been planted in the specific field before.

• To increase the number of Rhizobium which are already in the soil

• The soil is acid or very alkaline which could negatively affect the Rhizobium count in the soil

• The soil is exposed to high temperatures and dry summers which could reduce the number of Rhizobium from previous seasons or inoculation

• The crop is Rhizobium specific so each time a different crop is planted the right Rhizobium needs to be used.

• Always follow the manufacturer’s label / instructions

• Store inoculants in a cool, dry place (not necessary for refrigeration) and keep below 25 °C

• Do not leave the inoculant in direct sunlight

• Do not use inoculants past the expiry date.

Powder inoculants14Powder inoculants is cost effective and reliable. It is the most common method of product formulation. There is also a liquid formula available but mostly only for soybean inoculants.

Powder inoculants consists of a carrier (the powder) with the different strains of Rhizobium single or in mixtures together. The Rhizobium are produced in fermentors were the inoculants are grown in high concentrate together with a feeding culture.

The Rhizobium grows and multiply further in the sterile bags with the carrier. Powder based inoculants are mostly applied as a slurry directly with the seed. The powder inoculant can also be applied as a in-furrow application mixed with water of between 30-150L per hectar.

Mixing of powder inoculants

• Always use clean equipment for mixing the inoculants. (Do not use for example old herbicide drums for mixing inoculants, there may be residue left in the drums)

• Mix the inoculant with clean water, add the sticker if using a powder with the inoculants and water. The sticker is used to ensure better adhesion between the inoculant and the seed.

• The optimum pH of the water needs to be between 5 and 7. The inoculnat can be killed if the pH is to high or to low.

• A preserver can also be added to extend the life of the Rhizobium to prevent the Rhizobium from dying. This may extend the life of the Rhizobium on treated seed for a couple of days. (Anything from 3 to 30 days)

• Some inoculants contain a sticker already inside the pack, with some inoculants the sticker has to be bought seperately.

• Make sure the sticker is cool before the inoculant is added. If it is to hot it can be detremental for the inoculant.

• Seed must be planted as soon as possible after inoculation. This will ensure the maximum number of colony forming units (cfu) per seed.

• Inoculants are highly effective when planted in moist soil. Rhizobia needs moisture to live.

• The size of the inoculants packaging differs between different suppliers. Smaller packs are in the rangeof between (100- 250 gr/ ml) and bigger packs (1- 3 kg/ L).

• Powder inoculants which are applied over the seed in the seed hopper is not recommended as not every seed is coated properly.

13 How should I handle inoculants?

Applying of inoculant as a seed treatment15

Concrete Mixer

With the use of a concrete mixer a certain amount of seed is weighed out. (Example 50kg of seeds)The seed together with the product mix (slurry of water and inoculant) are then added together and mixed. Usually a higher slurry volume are used with this method vs the other two methods which are more suited for treating seed. The mix of seed with a concrete mixer works good, but may be slower than with the other methods. Care must be taken not to wet the seed to much. Some farmers do not use a slurry mix but apply the water and the sticker together and then apply the powder inoculant over the seed as a dry powder.

Rotating Drum Mixer (Batch Treater)

With this method a certain amount of seed are also measured beforehand and the “batch” of seed added to the rotating drum. The slurry are applied using a rotating disc which spreads the product/ inoculant over the seed evenly. The seed is further mixed with the slurry/ inoculant while the seeds rotates inside the drum. This method is especially good if a very low slurry volume needs to be applied. This method happens at a very high speed and is the best if a very good coverage of seed to seed is needed.

Auger treaters

This method is the best if high volumes of seeds needs to be treated in a very short period. Anything from 3 to 6 tons of seeds per hour. With this method continous flowing seed is used, the seed is not weighed in batches, only the seed quanity is measured per hour. As the seed flows throught the treater it is treated with the product which was calibrated using the seed quantity. After the initial treatment the seed is further mixed in a drum to get better coverage of the seed.

The slurry are applied onto the seed by using a concrete mixer, rotating batch treater or a auger type treater. (See pictures)

The three different treatments mentioned above ensures propper application onto the seed at high speeds vs other methods. For example mixing of seed and inoculants on a tarp with a shovel.

Are there compatibility

issues between seed applied

inoculantsand fertilizers,

includingchemical

remedies?

Inoculants should only be applied were the seedbed is moist when in-furrow application is used. The practice of applying inoculants in-furrow is on the increase in South-Africa, because of the ease of application. No need to mix seed with the inoculant anymore. The inoculant is added to a tank which is mounted on top of the planter and applied in-furrow using a pipe which coats the seed in the soil.

Rhizobia is living organisms and it is important that inoculants are cept away from toxic products which can influence the viability.

• Inoculated seed must not come into contact with certain fertilizer mixtures. This can cause the mortatlity of Rhizobia because of drying out en exposure to acid and salts. A trial needs to be done before Rhizobia and fertilizers are put into contact with each other to determine the effect the specific fertilizer has on the inoculants.

• Certain herbicides can also have a impact on the nodulation of the Rhizobia.

It is very important to ensure that the tanks used to apply the inoculants are clean and free of residue of pesticides. If not clean it can cause a certain amount of mortality of the inoculants. If a powder inoculant is used it must be pre-mixed with water to ensure that there is no lumps in the mixture which might block nozzles.(If a powder inoculant is used) The mixture can be apllies to the mixing tank by throwing the mixture through a seeve to ensure that any lumps stay behind.

There is three important factors which needs to be considered when inoculants and fertilizers are brought into contact with each other:

• Is the chemical in the mixture acidic? Most Rhizobia is sensitive for solutions with a pH lower than 5 or higher than 7,5.

• Does the solution which are going to be applied to the seed contain toxic chemicals? Metals like copper are toxic to inoculants. Negative side effects of active ingredients could be difficult to determine and needs to be tested beforehand to determine the effect it will have on inoculants.

• Is the contact period between the inoculant and toxic materials short or long periods? If the period is short the negative effect can be reduced.

Liquid inoculants: Application in-furrow

16

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Superphosphate and relevant products are acidic and tocix towards inoculants if it comes into direct contact. Contact between seed and fertilizers should be avoided.

Fungicides which are applied onto the seed for disease control, can cause mortality of the Rhizobia. All fungicides needs to be tested with the inoculants before it is applied in combination to the seed.

• A common rule of thumb is that if the seed is treated with a fungicide, the seed needs to be treated with double the rate of inoculant. This will ensure that enough live rhizobia are still left if the fungicide causes a certain rate of mortality.

• Another practice is to not treat the seed too long before planting, this will reduce the period within

which the inoculant and fungicide are in contact with each other.

• When treating the seed the fungicide can be applied, then a couple of seconds later when the fungicide has dried on the seed the inoculant can be applied over the fungicide. (Like a second coating). This will reduce the impact the fungicide has on the inoculant.

See the compatibility list in the manual for products already tested.

Inoculant

B-Ru

s

Pseu

dom

onas

Azom

onas

Soyb

ean

Luce

rne

Grou

ndnu

ts

Clov

er

Lupi

ns

Peas

Cow

Pea

s &Dr

y Be

ans

HerbicidesBastaBuctrilChlorimuronCossackHussarMetalachlorMetribuzinGlyphosate

FungicidesApron XLBayleton WPCaptanCelest XLFolicurShavitTeldor 500 SCTwist 500 SCVitavaxSeed PlusDividend

InsecticidesBulldock 050CruiserCuraterrCypermetrinDecisDimetoaatGauchoEndosulfanTamaronThioflo

Compatibility

Product is compatible with inoculant

Product not yet tested

Product is not safe to use with inoculant

Low levels of molybdenum (Mo) in the soil (especially in soils with a pH below 6) may lead to poor nodulation, as well as poor nitrogen fixation in legume crops. The addidtion of molybdenum onto the seed is cost-effective and ensures even distribution of molybdenum throughout the field. This is especially true if molybdenum is needed for instance close to the seed. The risk is that molybdenum is detrimental for Rhizobium if it comes in direct contact with it. Before molybdenum is applied together with Rhizobium onto seed, it must be tested to determine if the Molybdenum is affecting the Rhizobium negatively.

The effectiveness of inoculation can be evaluated by looking at the amount, placement and colour of the nodules.

• Amount and size of nodules: It isdetermined by the size of the plantand the nitrogen requirements of thehost at the time.

• Placement of the nodules:The placement of the nodules onthe roots indicates when nodulationtook place. A few large pink noduleshigh on the stem indicate early andeffective nodulation. The lower thenodules are on the root system, thelater nodulation occurred and thishappens typically when a high level ofnitrogen is present in the soil.

• Colour: The colour of a nodule that iscut open also indicates effectivenessand activity. Pink nodules indicates an infective and effective strain of rhizobia that nodulated the plant. Later in theseason the nodule turns green/blackthat indicates nitrogen fixation hadstopped. A lot of small, white nodulesover the whole root system indicatean ineffective strain was used toinoculate the plant or that residentrhizobia have nodulated the plant and no nitrogen fixation has taken place.

Dr Peoples of Australia worked with the PPRI to investigate poor results obtained in the RSA. They found that:

• RSA inoculants are of a highquality.

• The problem lies mainly withincorrect application of theinoculant. It is very importantto spend some time on studyingthe instructions carefully beforeseed treatment.

1. Make sure that the rightinoculant is used. The bacteriaare very specific and inoculantsfor beans, lucerne or groundnuts cannot be used for soybeans,and vice versa.

2. The application must be correct, i.e. at least one packet ofinoculant per 50 kg soybeanseed under normal conditions.A sticker should be used toattach the inoculant to the seed. Stimulym can be used at 1.25 gfor 50 kg soybeen seed.

Was inoculation successful?

19

Addition of molybdenum during inoculation

What went wrong if no or poor nodulation occurred?

18 20

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