A work Outline of Maize for Resistance test

2. DEPARTMENT OF ENTOMOLOGY
DR. RAJENDRAPRASADCENTRALAGRICULTURAL UNIVERSITYPUSA, SAMASTIPUR
BIHAR– 848125
ProposedTopic
Studies on Various Factors of Resistance in
Maize Plants Affecting Spotted Stem Borer, Chilo
partellus (Swinhoe) Incidence.
Speaker
Reg. No.
Major advisor
:Manish Kumar Yadav
:Ph.D. (Ag.) Entomology
:D/Ento./107/2016-17
:Dr. A.K. Rai, Associate
Professor
Dept of Entomology

3. INTRODUCTION
 Maize (Zea mays L.) is a third most important
cereal after rice and wheat.
 Maize serves as staple food, raw material for
processed food industries and animal feed and
fodder for many countries throughout the
world
 The average maize productivity in India is 2.5
t/ha which is less as compared to U.S. (9.6 t/ha)
due to less mechanization and loss of yield in
fields by insect-pests and pathogens.

4. CONT…
 Maize is subjected to attack by a number of
insect-pests in which spotted stem borer is
major cause of damage during kharif season.

5. CONT.…
 Spotted stem borer Chilo partellus (Swinhoe)
is a major Lepidopteran pest of maize belong
to family Crambidae.
 Spotted stem borer lay eggs on leaves in a
batch of 20-50 eggs, generally close to the
midrib.
 It cause serious damage in maize crop during
Kharif season by making dead hearts and
Tunnels.
 Yield loss due to this pest ranges from 10 to 20
per cent and may go up to 80 per cent
(Rahman, 1994).

6. WHY WE CHOSES THIS CROP
 Maize is an important cereal grown
throughout the year.
 It is a major crop of Bihar, grown in large
areas.
 Maize has ability to produce yield in all types
of soil and environment.
 It is grown for many purposes and provide
raw materials to many industries.

7. Objectives of investigation
Physical, biochemicals and genotypic factors
are largely responsible for imparting resistance
into the maize plant against insects including
Chilo partellus (Swinhoe). The biochemical like
chlorophyll and proteins are present in maize
plant with significant role in insect-pest
resistance. Therefore, the study of the relation
of physical, biochemical and genotypic
resistance factors of maize against maize
spotted stem borer, Chilo partellus (Swinhoe) is
proposed with the following objectives.

8. 1. To assess the molecular diversity in maize
cultivars and establish correlation with different
degree of infestation of spotted stem borer, C.
partellus (Swinhoe),
2. quantitative estimation of total nitrogen,
potassium and other plant biochemical like total
chlorophyll and protein in maize cultivars and to
find out their correlation with C. partellus
(Swinhoe) infestation levels and
3. to study the physical resistance factors of
maize plant against spotted stem borer, C.
partellus (Swinhoe)

9. TECHNICAL PROGRAMME OF THE WORK
All cultivars, will be sown during Kharif in the
month of August (first week) at research farm,
DRPCAU Pusa. Test insect i. e. Chilo partellus
will be reared in laboratory and neonate larvae
will be used for artificial infestation in the field.

10. Crop : Maize
Genotypes : DRPCAU Cultivars: RHM-1, RHM-2, RHM-
3,SM-1, SM-2, SM-3, SM-4, SM-5, Dewaki,
Lakshmi, Suwan, Hemant and Deep Jwala
Monsanto: 900M Gold, 9144 SC, 7074 SC
and Double- DC
Pioneer: P-3322, P-3335 and P-3535
Pipeline Cross of DRPCAU: 12 New Cross,
13 New Cross, 14 New Cross and 15 New
Cross
Total 26 genotypes
Location : Research Farm, DRPCAU Pusa
Row length : 4 m
Row to Row Space : 60 cm
Plant to Plant Space : 20 cm
Total rows for one
genotype
: 2
Layout of the experiment

11. 1. To assess the molecular diversity in maize cultivars and
establish correlation with different degree of infestation of
spotted stem borer, C. partellus (Swinhoe).
 Assessment of molecular diversity of maize cultivars will be
done by SSR marker at 35 DAS. For this experiment, DNA
will be extracted from plant samples by CTAB method
described by Doyle and Doyle, 1987. After DNA extraction,
PCR reactions will be carried out in a 20ml volume
containing 50 ng genomic DNA×PCR buffer and primers.
The amplification consist denaturing step of 2 min at 94oC.
Amplification products will be separated by electrophoresis
in horizontal gel system at 110V for 2.0 hour in 3% agarose
gel. Finally, the gels will be photographed under UV light
attached to a gel documentation system with Biocapture
software and the bands will be binary coded for each
genotype.

12.  The following data will be recorded at 35 DAS.
 Different injury symptoms on plants created by C.
partellus will be recorded by screening (Guthrie et.
al., 1960).
 Average score of results (infestation) will be
compared with molecular diversity which will show
the different degree of susceptibility of different
genotypes against spotted stem borer, C. partellus.

13. 2. Quantitative Estimation of total nitrogen,
potassium and other plant biochemicals like total
chlorophyll and protein in maize plants and to
find out their correlation with C. partellus
(Swinhoe) infestation levels.
 Quantification of above components will be
done at 35 DAS and just before start of
tasseling. The correlation between infestation
and plant nutrients will be established.
Sampling of leaves will be done in all plants of
each genotypes to form a single composite
sample. The following observations will be
recorded:

14. Nitrogen estimation: By Kjeldahl method, The
leaf sample will be heated with sulphuric acid,
which decomposes the organic substance by
oxidation. In this step, potassium sulphate will
be added to increase the boiling point of the
medium. Then the solution will be distilled with
a small quantity of sodium hydroxide, which
converts the ammonium salt to ammonia. The
amount of nitrogen present in the sample, is
determined by titration. (AOAC, 1970)

15.  Potassium estimation: For calculation of
potassium, 5gm plant material will be taken
and placed in a 100 ml flask with 10 ml
digestion acid in the ratio of 9:4
(HNO3:HClO4). Then the flask will be placed
on low heat hot plate in digestion chamber
and heated upto a higher temperature to
reduce total volume (3-5 ml) by evaporation.
The composition of digestion will be
confirmed when the liquid will become
colourless and then the samples will be read in
flame photometer. (Amjad et al., 2015)

16. Estimation of total chlorophyll: 0.5g of fresh
plant leaf sample will be taken, and
homogenized in tissue homogenizer with 10 ml of
extractant solvent like 80% acetone or 95%
ethanol. Then the sample mixture will be
centrifuged at 4000 rpm for 20 min at 40⁰C.
Then the solution mixture will be analyzed for
total chlorophyll content in spectrophotometer.
The readings will be taken at 645 and 663 nm.

17. Protein estimation: Total protein content in
each genotype will be calculated by the given
formula (Winkleman et al., (1986).
Protein per cent = Nitrogen per cent×6.25

18. 3. To study the physical resistance factors of maize
plant against spotted stem borer, C. partellus
(Swinhoe)
 Physical attributes of plant like plant architect,
stem diameter, plant height, internode length and
number of nodes will be recorded just before start
of tasseling in all grown genotypes.
 Study of Trichome density on leaf surface will be
done by selecting and cutting an area of 2.5 cm
squire at 3 different points of a leaf at just before
tasseling stage. All the Leaves from middle point to
whorl will be selected. The counting of leaf
trichomes density will be done with the help of
ocular.

19.  The associated effect of leaf trichomes i. e.,
viral and bacterial infection on larvae due to
trichome injury, will be assessed by walking the
neonate larvae from mid-point of the leaf to
stem-leaf junction on the upper surface of
matured leaf. This experiment will be
conducted just before tasseling. Observations
(Larval mortality and Tunnel length) will be
recorded by cutting and splitting stem of 10
plants from each genotypes on each time (10,
15, 20 and 25 days after release of the larvae).

20. Crop : Maize
Genotypes : All 26 previously Selected genotypes
Sowing Time : August (First week)
Location : Research Farm, DRPCAU Pusa
Row length : 4 m
Row to Row Space : 60 cm
Plant to Plant Space : 20 cm
Total rows for one cultivar : 2
Area : 153.00 2
Experimental layout for Trichome injury and its associatedeffect study:

21.  Stem diameter at three height points of a
plant, plant height and number of nodes and
length will be recorded by counting and with
the help of measuring tape just before start of
tasseling (Amjad et al., 2015)
 Plant architect study will be done by
measuring leaves angle in respect of stem. It
will be done with the help of protactor.