Identification and Preliminary Screening of

Nitrogen-Fixing and Phosphate Solubilizing

Activities of Coix lacryma-Jobi L. (Adlai)Associated Bacteria
Kathleen DC. Vasquez1, Nathaniel R. Alcantara2, and Emmanuel L.
Bernardo3
1,2Microbiology

Division, Institute of Biological Sciences, University of the Philippines

Los Banos, College, Laguna 4031 Philippines
3Crop Science Cluster, College of Agriculture, University of the Philippines Los Banos,
College, Laguna 4031 Philippines

Coix lacryma-Jobi L.(Adlai)

erect and highly tillering monoecious grass
3 meters tall
stem diameter: 0.5 to 1 inch

leaves are large and sheathed

propagated by seeds, followed by subsequent
ratooning two to three times after the main crop

Adlai as an alternative to rice

cooked like rice and corn grits
made into flour for baking purposes
can tolerate low pH, poor soil quality,
waterlogging and is resistant to pests

development, promotion and utilization of Adlai

enhancement and sustainability as an alternative food source

Other Uses of Adlai

As feed source
As an ornament
As traditional medicine
Potential for fuel
Nutritive Value

White Corn
Grits*

Adlai**

Brown Rice***

White Rice***

Energy (kcal)

356

135

129

110

Carbohydrates (g)

73.9

24.6

27.9

22.9

Protein (g)

12.8

2.6

2.7

2.6

Fat (g)

1.0

0.7

0.3

0.9

Dietary Fiber (g)

0.3

0.7

0.4

1.8

* Nutrition

facts of White Corn Grits (IPB Var 6) released by Crop Science Cluster-Institute of Plant Breeding, UPLB
** Nutrition facts of Adlai Grits released by Food and Nutrition Research Institute (FNRI) chemical analysis, 2011
***Nutrition facts of White and Brown Rice released by FatSecret All Things Food and Diet (www.fatsecret.com)

Plant Tissue Culture Experiment

Presence of exudates

Longer coleoptiles
More profuse root system

Hypothesis:
The contaminant may be a plant-associated bacterium
which may produce substances with growth-promoting
properties

Challenge:

Isolate and identify the contaminant and screen for its

growth promoting ability to be able to determine promising
inoculants which may increase yield of Adlai

Objectives of the Study

1. To isolate the Adlai-associated organism after enrichment
and purification by subsequent streaking on basal medium.
2. To do morphocultural characterization of the pure
culture(s).
3. To do physiological/biochemical characterization by
qualitative determination of nitrogen-fixing and phosphate
solubilizing activities of the isolates(s). and;
4. To do molecular characterization of the isolate(s) for
identification.

Methodology
I.

Enrichment/Isolation/Purification of
Isolates
II. Morpho-cultural Characterization
a. Gram Stain Reaction
b. Cell Morphology
IV. Molecular Characterization
c. Cultural Characteristics
a. Genomic DNA Isolation
III. Physiological/Biochemical
b. 16s rDNA amplification
Characterization
c. Sequencing/Identification
a. Oxygen Requirement
b. Nitrogen Fixation
c. Phosphate Solubilization

Results and DiscussionEnrichment/Isolation/Purification of Isolates

Enrichment set-ups
a)

Static(S) grown in N broth with

1000 ppm Nystatin, 24h, RT
- 1.96 x 109 CFU/mL
- 12 isolated colonies

b) Rotating (R)-grown in N broth

with 1000 ppm Nystatin, 24h,
RT, 2000 rpm
- 3.05 x 109 CFU/mL
- 12 isolated colonies

Results and Discussion

Table 1 Morpho-cultural characterization of static (S)

isolates.
Isolate Code

Form

Margin

Color

Other distinguishing
Characteristics

S1

circular

entire

cream

shiny

S2

circular

entire

cream

shiny

S3

circular

entire

cream

shiny

S4

circular

entire

cream

shiny

S5

circular

entire

cream

shiny

S6

circular

entire

cream

shiny

S7

circular

entire

cream

shiny

S8

circular

entire

cream

shiny

S9

circular

entire

cream

shiny

S10

circular

entire

cream

shiny

S11

circular

entire

cream

shiny

S12

circular

entire

cream

shiny

Results and Discussion

Table 2. Morpho-cultural characterization of aerated (R)

isolates.
Isolate Code

Form

Margin

Color

Other distinguishing
Characteristics

R1

circular

entire

cream

shiny

R2

circular

entire

cream

shiny

R3

circular

entire

cream

shiny

R4

circular

entire

cream

shiny

R5

circular

entire

cream

shiny

R6

circular

entire

cream

shiny

R7

circular

entire

cream

shiny

R8

circular

entire

cream

shiny

R9

circular

entire

cream

shiny

R10

circular

entire

cream

shiny

R11

circular

entire

cream

shiny

R12

circular

entire

cream

shiny

Results and Discussion

.

Figure 2. Growth of R1 and S1 isolates on Nutrient agar

agar incubated at 37C.

Results and Discussion

.
Morpho-cultural
Characterization

Gram Reaction
-All S and R isolates were pleomorphic, occur singly and Gram-negative

Figure 3. Gram staining reaction, shape and arrangement of S and R isolates

Results and Discussion

Physiological/Biochemical
Characterization
.
A. Oxygen Requirement
S1 and R1 isolates were both obligate aerobes

- Growth halfway through FTM, more concentrated on top

- Most of the studied genera of plant growth promoting bacteria are those
belonging to the aerobic endospore-forming bacteria (de Araujo et al.,
2014)

Results and Discussion

.
Physiological/Biochemical
Characterization
B. Nitrogen Fixation and Phosphate Solubilization

Figure 5. Nitrogen fixation activities of S1 and R1 isolates as indicated by

change of color of Dobereiners medium from green to blue.

Results and Discussion

.
Physiological/Biochemical
Characterization
B. Nitrogen Fixation and Phosphate Solubilization

A.

B.

A.

B.

Figure 4. Phosphate solubilization and nitrogen fixation activities of S1

and R1 isolates after inoculating onto Phosphate Solubilization Medium
(A) and Dobereiners Medium (B)

Results and Discussion

.

Physiological/Biochemical Characterization
B. Nitrogen Fixation and Phosphate Solubilization

Phosphate solubilization is one trait being exploited in selecting

microorganisms as inoculants

while only 10-20% of fertilizer P can be utilized by plants, the major part
is deposited in soil and as such P becomes limiting (Hoflich et al., 1995)

Microorganisms solubilize inorganic P compounds by phosphatases,

making P compounds utilizable for plants (Lifshitz et al., 1987)

Results and Discussion

.

Physiological/Biochemical Characterization
B. Nitrogen Fixation and Phosphate Solubilization
ability of a plant to supply all or part of its requirements from biological
nitrogen fixation confers a great competitive advantage over non-nitrogenfixing plants
Nitrogen fixation activity by plant associative bacteria has been reported as
a mechanism by which beneficial bacteria stimulate plant growth
Significant nitrogen fixation has been demonstrated in Brazilian sugar cane
(Saccharum officinarum) cultivars known to form associations with a
number of diazotrophs, including Acetobacter diazotrophicus (James et al.,
1994)

Results and Discussion

16S rRNA Amplification
genomic DNA was isolated from pure cultures S1 and R1 using CTAB method

The~1.5 kb rDNA fragment was amplified through high-fidelity PCR polymerase

by using consensus primers 27F and 1492R (Lane, 1991)

Send for Sequencing ( Macrogen)

BLAST

Results and Discussion

1

Figure 6. Agarose gel electrophoresis (1.5% w/v) of genomic DNA prepared

from S1 and R1 isolates. Lane 1: Universal DNA ladder; Lane 2: S1; Lane 3: R1
isolate.

Results and Discussion

1 2 3 4 5

6 7

1600 bp
900 bp

Figure 7. PCR products obtained from amplification of 16s rDNA of S1 (Lane 2),
R1(Lane 3), S2(Lane 4), R2(Lane 5), S3(Lane 6) and R3(Lane 7) isolates.

Summary and Conclusion

These findings indicate that S1 and R1 isolates are promising plant growthpromoting bacteria for Adlai
No report yet of a plant growth-promoting species being applied as
microbial inoculant specific for Adlai and Adlai-associated bacteria being
incorporated into Adlai through tissue culture in the Philippines
The information obtained from the study will establish baseline
information on a plant associative bacteria capable of enhancing Adlai
growth and yield

The Adllai-associated bacteria can be used in developing/formulating

microbial inoculants for Adlai

Recommendations
Studies on quantitative determination of nitrogen fixation and
phosphate solubilization activities may be done via gas
chromatography and spectophotometric assay, respectively
In-vivo assays such as determination of effect of inoculation
of the adlai-associated bacteria on root elongation,
germination rate, plant height and biomass may be further
explored
Future studies involving the determination of mechanism for
colonization, survival and persistence of the Adlai-associated
bacteria in the host plant tissues/roots may be conducted

THANK YOU for

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