inoculant formulations are essential for successful
TRANSCRIPT
Indian Phytopathology 69 (4s) : 739-743 (2016) 739Indian Phytopath. 69 (4s) : 739-743 (2016)
RESEARCH ARTICLE
*Corresponding author: [email protected]
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.