Indian Phytopathology 69 (4s) : 739-743 (2016) 739Indian Phytopath. 69 (4s) : 739-743 (2016)

RESEARCH ARTICLE

*Corresponding author: nogabashan@bashanfoundation.org

The Bashan Institute of Science (BIS) is an American

non-profit scientific research organization. Its mission

is creating novel cutting edge scientific advances by

continuously exploring the unknown, development of

new scientific ideas and concepts for the benefit and

advancement of mankind and enhancing quality of life.

The driving force behind its operation is the constant

needs of society for better living. This is done through

creating new knowledge and new, and continuously

changing, scientific solutions and new technologies,

mostly in biology.

The main research efforts of BIS are focused on

environmental science and biotechnology. The BIS is

committed to a long-term, continuous evolvement as a

scientific research organization. Its main goal is creating

useful, outstanding science and technologies. The BIS

is an organization for current scientific needs and for

the needs of future generations.

The BIS is a self-sustaining organization. Its surplus

funds are invested only in further development of the

facilities and research capacities of the organization.

Details of the initial research facility and BIS future

development plans

- Conference rooms and 3 research laboratories and

auxiliary spaces focused on Environmental

Sciences located on 25 hectare of forest.

- Few offices for the staff and graduate students

to allow operation of the entire small campus.

What’s more, once BIS is established and growing

it will continue to expand. So where does BIS go from

here?

- A pond for ecological research in the flooding zone

(in 2019).

- Conserve forever, as a forest, the entire flooding

zone for environmental research (immediately).

- Few dorms for graduate students studying on

campus (in 2020).

- Additional research buildings as needed to meet

future needs.

Background

BIS is the research facili ty of the international

database institute the Bashan Foundation;

www.bashanfoundation.org. The Bashan Foundation

was founded in 1999, and is registered with the state of

Oregon since 2003 as a non-profit organization. The

Bashan Foundation has 7 branches worldwide, over

90 research scientists located in over 65 research units

in 12 countries and 9 units in the USA all sharing

information with the public.The Bashan foundation

database is growing annually and is doing exceptionally

well with its main purpose: sharing scientific information

free-of-charge.

Inoculant formulations are essential for successfulinoculation with plant growth-promoting bacteria andbusiness opportunities

NOGA BASHANThe Bashan Institute of Science (BIS), Auburn, Alabama, USA

ABSTRACT: Inoculation of plants with plant growth-promoting bacteria (PGPB/PGPR) either rhizoplane, endophytesand plant symbionts to enhance performance of plants is centuries old. The majority of field inoculations done todayare on cereals and legumes. Yet, numerous other crops are inoculated mostly in developing countries. In general,shortly after suspensions of bacteria, the most primitive inoculant, are inoculated into the soil without a proper carrier,the bacterial population declines rapidly for most species of PGPB/PGPR. This phenomenon, combined with productionof bacterial biomass, the need to sustain activity in the rhizosphere, and the physiological state of the bacteria atapplication time, can prevent the buildup of a sufficiently large PGPB/PGPR population in the rhizosphere. Theseunprotected, inoculated bacteria must compete with the often better-adapted native microflora and withstand predationby soil microfauna. Consequently, a major role in the formulation of inoculants is to provide a more suitablemicroenvironment, combined with physical protection for prolonged periods to prevent a rapid decline of bacteriaintroduced into the soil. Inoculants for field-scale use have to be designed to provide a dependable source of bacteriathat survives in the soil and become available to the plant, when needed. The first goal when considering inoculationof cereals with PGPB/PGPR is to find the best strain of bacteria or a microbial consortium for the intended effect on thetarget crop. The next step is to design a specific inoculant formulation for specific target plants and a method ofpractical application, considering the limitations of the growers. Currently, many inoculants are in the market place,some that substantially improve yield. This inoculation strategy opens significant business opportunities in Asia ingeneral and India in particular.

740 Indian Phytopathology 69 (4s) : 739-743 (2016)

The Bashan Institute of Science is a new

development of Bashan Foundation and is registered,

independently, with the state of Alabama Since 2014

as a non-profit scientific research institute with

authorization to apply for federal USA grants.

Main goals of Bashan Institute of Science

- Create and operate a new high-education,

research-intensive academic facility concentrating

on environmental science.

- Build a research facility based on graduate

students, post-doctoral fellows and high skill

training.

- Create youth scientific programs for senior high

schools students in Alabama (BOUKS; Bring OUr

Kids to Science).

- Use the capacity of retired scientists of Bashan

Foundation to continue to create new knowledge

(RAS; Retired Active Scientists)

- Create an educational facility such as research

laboratories and preserved areas that will allow

this level of education and skills.

Formulations for inoculants of PGPR

One of the research goals of BIS is creating new

formulations for PGPB/PGPR. “Formulation” refers to

the laboratory or industrial process of unifying the carrier

with the microbial strain. “Inoculant” refers to the final

product of formulation containing a carrier and microbial

agent.

Inoculation of plants with microorganisms to

enhance crop yields or native plants has been practiced

for several decades. Two factors predominate in the

success of inoculation: effectiveness of the bacterial

isolate and the technology of application. Technologies

of microorganism encapsulation in these areas are

experimental, and no commercial encapsulation

products currently exist. It is well known that inoculant

formulations are essential for successful inoculation with

plant growth-promoting bacteria (Bashan et al. 2014,

2016; Calvo et al. 2014).

Formulation is an industrial/experimental mixture

consisting of bacteria or fungi with inert material. The

inert material can be of different substances as long as

the material is not alive. Industrial mixture of the inert

material with the microbial agent is the formulation.

Formulations is the crucial issue for successful

commercial inoculants. The bacterial strain is frequently

an IP, intellectual property, and the formulation is the

second IP and together it is a market product, an

inoculant.

Kinds of inoculants that exist in the current inoculant

market

PGPB/PGPR, that are beneficial bacteria in general,

are absolutely essential for most crops. Instead of using

extremely expensive fertilizer, PGPR and PGPB are

being used to raise the productivity of the plant and

reduce production costs of food.In particular, PGPR-

plant growth-promoting rhizobacteria. These bacteria,

mostly biological control agents, are part of the larger

definition PGPB. PGPB and PGPR- are the living part of

the inoculant.

First group of inoculants is organic inoculants .The

most common ingredient is peat. It is suitable for most

PGPB/PGPR. Yet, peat is rarely available and expensive

in most of Asia and Africa. Many other organic waste

materials and composts can be used instead of peat

(Bashan et al., 2014).

Second group is inorganic inoculants, for example:

clay, soil and minerals such as talc. Those materials are

also being used to make inoculants mainly in Asia.

Third group is liquid inoculants. It is culture medium

in water or oil plus additives useful for thickness,

stabilization, surfactant and dispersal. It is possible to

use the living bacteria or dry bacteria, mix them in liquid-

and this will form the inoculants. But what is the problem

with it? The problem is that those inoculants do not

protect the bacteria even if dispersal is easy.

Fourth group is polymeric inoculants, the most

common is alginate. It is a polymer of sugars that is

extracted from large cold sea algae, for example in

California, Chile, Norway and New Zealand. Alginate is

being used for stabilizing ice cream and dental work.

This purified alginate is relatively expensive.Industrial

level cheap alginate is being used for inoculants. The

cost is only 2$ per kilogram (in China). And from one

Kg it is possible to produce inoculants for large fields.

But there is more to it than the price, the material is

flexible and it is possible to manufacture from it macro-

beads- 2-4 mm, or micro-beads- 200-150 micron. The

formulations can be dry or wet. Wet formulation is

useful for spraying or irrigating. Dry formulations can

be spread or attached to seeds.

Optimal characteristics of a carrier for inoculants

Above all, the carrier should deliver the right number

of viable cells. In addition, it should have high water-

holding capacity (for wet carriers).

- What’s more, it should be nearly sterile or easily

sterilized to reduce the risk of contamination.

Fig. 1. Polymeric dry microbead inoculants of Azospirillumbrasilense made of alginate (Bashan et al., 2015)

Indian Phytopathology 69 (4s) : 739-743 (2016) 741

- It is also a matter of chemically and physically

uniform for industrial process.

- And finally, it should be non-toxic, leave no carbon

footprint, biodegradable, and non-polluting.

The main problem is that it is impossible to find a carrier

with all those characteristics at top quality. Obviously, it

is significantly important to find a carrier with major of

those characteristics. Not all, but most. Alginate is a

good candidate for such a carrier.

Advantages and disadvantages of alginate

First of all, alginate carrier controls slow release of

bacteria into the soil allowing inoculation of the plants

over a desired period of time. Therefore, the customer

can decide whether he/she wants slow release or faster

release of PGPB/PGPR according to their needs. For

example for improving the growth of trees, BIS will create

slow release inoculants and for fungal dumping-off soil-

borne diseases a fast release inoculant.

- Second, alginate is a polysaccharide, therefore, it

is degradable into water and sugars. After several

weeks nothing left from the inoculants and all

PGPG/PGPR are released.

- Third, it is extremely important to the industry that

the inoculants will be non-toxic. The US Food and

Drug Administration (FDA) requests testing costs

of ~10 million dollars for registration of pesticides.

The alginate passed all the tests long time ago and

has approval for human use by the FDA.

- Fourth, it is easy to use by the grower and produce

very little or nil additional work.

- Fifth, nowadays the alginate is cheap. When in the

past it was expensive and prohibitive to create from

it inoculants.

Comparison between encapsulation of bacteria for

use in industrial fermentation and as an inoculant for

agricultural/environmental use

The process starts the same in the fermentor to

grow the bacterial strain, then encapsulated

(immobilized) in polymers. Bacteria for industrial

production of metabolites are transferred to another

production fermentor and there is constant release of

useful compounds, such as vitamins or antibiotics. The

main issue is that there is no degradation of the bead

and no release of microorganisms to the surrounding

medium.

The inoculants for agriculture use are not transfer

to fermentor but to the seeds. Initially, the inoculant

needs to protect the bacteria from soil stressors and

micro predators. Once the seed germinates, there is no

need for encapsulation of the bacteria and there is a

need to protect the plant or to enhance its growth directly.

To this end there is gradual degradation over time of

the bead unti l complete disappearance of the

inoculant.

The major difference is that in the industry there is

no degradation of the beads. On the other hand for

environmental usage there must be degradation.

How does slow release of Azospirilum from dry

alginate inoculant work?

It starts by exposing alginate beads to soil environment.

Alginate is polysaccharide and it is available for bacteria

and fungi in the ground as carbon source to consume

it. And what are the results? The results aredegradation

of the alginate layers releasing in the process of

entrapped PGPB/PGPR. This is how slow release of

the PGPB Azospirilum from dry alginate inoculants

works. This is the first degradation phase of this product.

Now again, the bacteria and fungi in the ground

consume the next layer, and again a release of

Azospirilum from the new layer of the inoculants. The

degradation of the layers takes time and therefore the

release is slow.

Fig. 2. Wet macro-alginate inoculant, immediately after for-mation; First generation created in 1986. (Bashan, 1986)

Fig. 3. Surface of wet alginate inoculants containing largepopulation of the PGPB Azospirilum brasilense (Bashan, 1986)

742 Indian Phytopathology 69 (4s) : 739-743 (2016)

Disadvantages of macro-alginate beads

Macro-alginate beads as inoculants has two unsolved

disadvantages: (1) an additional treatment during

sowing is needed by the grower, even if the inoculant is

planted by seeding machines; (2) microorganisms

released from the inoculant need to migrate through

the soil to the plants. Under agricultural practices, when

beads are loosely mixed with seeds and sown together,

the beads might land up to several millimetres or even

few centimetres away from the plant. The bacteria

released from the beads must move through the soil,

facing competition with, and predation by the native

micro flora. Consequently, the future of macro-beads

inoculant in agriculture is uncertain.

Survival of PGPB in inoculants

A main bottleneck in the production of any inoculant for

agricultural and environmental improvements is shelf

l ife. Shelf-l ife should be increased rather than

maintaining high bacterial counts in the original

inoculant because the number of bacteria eventually

decreases during storage. From commercial and

agricultural standpoints, longer survival of bacteria in

polymeric preparations makes dry formulations

extremely attractive.

Studies shows that in peat, the common

inoculants,bacteria will survive about 1 year.Two years

is an advantage that not many inoculants can meet.

Survival in liquid inoculant can be measured by months.

In alginate, 1-3 years has been demonstrated in many

studies. The record of survival oftwo PGPB in alginate

is14 years (Bashan and Gonzalez, 1999). These

inoculants were forgotten after production in the

laboratory in Israel and after 14 years they were sent to

Mexico for evaluation. The amazing thing is that these

inoculants did not lose their promotional activity on

plants- none at all.

This shelf life of this dry alginate formulation is

superior, by far, to any existing commercial microbial

inoculant.

Micro-bead concept

As mentioned, macro-beads have deficiencies, so in

practice what we suggest is the new generation of

inoculants. The micro-bead concept (50-200 µm beads

or even smaller). It was developed to overcome the two

difficulties of macro-beads. It was developed long time

ago in fact (Bashan et al., 2002). However, as the

performance is excellent, almost no improvements

were required over the years.The concept says that:

If the beads are small enough,yet, still able to

encapsulate sufficient numbers of bacteria,then, it is

possible to produce powder-like formulations. This

“bead-dust” can be coated on seeds in the

factory.Therefore, grower purchases “improved seeds”.

And above all, no special application technology is

necessary.This creates new opportunities for the

industry.

Today, seeds coated with fertilizers, fungicides, or

hormones are commonplace and universally accepted

by most farmers. In developed countries with large-

scale agricultural practices, pre-coating seeds with

micro-bead inoculants would eliminate the need for an

additional expensive field treatment and provide the

ultimateconvenience and incentive to farmers.

Application of formulations in micro-alginate beads

to inoculate plants in the soil has been successful on

several occasions, mostly for environmental plant and

also for crop plants.

Fig. 4. Cross section of dry macro-bead alginate inoculantsformed in a shape of cabbage containing many layers andcavities (Bashan, 1986)

Fig. 5. An enlargement of one layer of alginate from aboveFig 4. Every bulge is a colony of dry microorganisms-thePGPB Azospirilum brasilense (Bashan, 1986)

Fig. 6. Alginate macro bead inoculant immediately after

sowing of wheat in the field,beads are white and seeds

are brown

Indian Phytopathology 69 (4s) : 739-743 (2016) 743

Detection and verification of purity of a strain in the

synthetic inoculants

There are several techniques that can check the purity

of the inoculant and can be used for quality control

purposes. Our technique is Fluorescence-in-situ-

hybridization (FISH).

The yellow color indicated 100% verification of the

correct bacteria in the inoculants

Slow release of Azospirillum from alginate micro-

spheres

Most important is that the release will be according to

the needs of the customer.

For creation of fast release inoculant- for example,

the alginate can contains protein which in the ground

is digested very fast. Therefore release of bacteria will

be rapid in the first week.

And lastly, creation of harden inoculants will enable

very slow release over time.

The main conclusion is that it is possible to control

the release of bacteria over time according to the

definition of the customer.

Fig. 7.Enlargement of the micro-bead. The size of thesemicro-beads is ~100 micro-meter. In one mm there are 10beads (Bashan et al., 2002)

Fig. 8. Detections of Azospirillum using FISH

Fig. 9. In black (artificially colored)are alginate micro-beadsattached to wheat seeds

Alginate micro-beads attached to wheat seeds

Conclusion and future potentials for developments of

inoculants at BIS

What action does BIS propose to take in the nearby

future?

- Immobilization of vaccine iRNA for Aquaculture.

- Extending shelf life of Azospirillum inoculant for

corn.

- Spray foliar applications of PGPB for plants

- Inoculant from endophytes.

- Inoculants for phosphate solubilizing.

- Inoculants for wastewater treatment.

- Inoculant for wetlands/wastewater treatment.

- Inoculant for mangrove restoration.

- Inoculant for domestication of endangered

ironwood trees.

REFERENCES

Bashan, Y.(1986). Alginate beads as synthetic inoculantcarriers for the slow release of bacteria that affect plantgrowth.Applied and Environmental Microbiology51: 1089-1098.

Bashan, Y. and de-Bashan, L.E. (2015). InoculantPreparation and Formulations for Azospirillum spp. In:Handbook for Azospirillum (pp. 469-485). SpringerInternational Publishing.

Bashan, Y., de-Bashan, L.E., Prabhu, S.R. andHernandez, J.P. (2014). Advances in plant growth-promoting bacterial inoculant technology: formulationsand practical perspectives (1998-2013). Plant and Soil378: 1-33.

Bashan, Y. and Gonzalez, L.E. (1999). Long-term survivalof the plant-growth-promoting bacteria Azospirillumbrasilense and Pseudomonas fluorescens in dry alginateinoculant.Applied Microbiology and Biotechnology 51:262-266.

Bashan, Y., Hernandez, J.P., Leyva, L.A. and Bacilio, M.(2002). Alginate microbeads as inoculant carrier for plantgrowth-promoting bacteria. Biology and Fertility of Soils35: 359-368.

Bashan, Y., Kloepper, J.W., de-Bashan, L.E. andNannipieri, P. (2016). A need for disclosure of theidentity of microorganisms, constituents, and applicationmethods when reporting tests with microbe-based orpesticide-based products. Biology and Fertility of Soils52: 283-284.

Calvo, P., Nelson, L. and Kloepper, J.W. (2014). Agriculturaluses of plant biostimulants. Plant and Soil 383: 3-41.