morphological, biochemical and anatomical basis for biotic stress resistance in cotton

111 Pages·2014·0.96 MB·English

MORPHOLOGICAL, BIOCHEMICAL AND ANATOMICAL
BASIS FOR BIOTIC STRESS RESISTANCE IN COTTON

Thesis submitted to the
University of Agricultural Sciences, Dharwad
in partial fulfillment of the requirements for the
Degree of

Master of Science (Agriculture)
In
Genetics and Plant Breeding

By
YALLAPPA S. HARIJAN

DEPARTMENT OF GENETICS AND PLANT BREEDING
COLLEGE OF AGRICULTURE, DHARWAD
UNIVERSITY OF AGRICULTURAL SCIENCES,
DHARWAD – 580 005

JULY, 2013
ADVISORY COMMITTEE

DHARWAD (B. M. KHAD)
JULY, 2013 MAJOR ADVISOR

Approved by :
Chairman : ____________________________
(B. M. KHAD)
Members : 1. __________________________
(I. S. KATAGERI)

2. __________________________
(B. R. PATIL)

3. __________________________
(H. M. VAMADEVAIAH)

4. __________________________
(B. N. ARAVINDKUMAR)
CONTENTS
Sl. No. Chapter Particulars
CERTIFICATE
LIST OF TABLES
LIST OF FIGURES
LIST OF PLATES
1. INTRODUCTION
2. REVIEW OF LITERATURE
2.1 Screening for disease resistance cotton genotypes
2.2 Screening of sucking pest resistant/ tolerant cotton genotypes
2.3 Role of biochemical components for sucking pest tolerance/
resistance
2.4 Role of anatomical characters in host-plant resistance
2.5 Genetic variability for yield and yield contributing traits
2.6 Creation of genetic variability for fibre quality traits
2.7 Molecular marker studies
3. METHODOLOGY
3.1 Experimental site location and weather condition
3.2 Description of the experimental material and methods
3.3 Anatomical studies
3.4 Observation on yield contributing and yield traits
3.5 Fibre quality characters
3.6 Molecular studies
3.7 Statistical analysis
3.8 Genetic diversity analysis
4 EXPERIMENTAL RESULTS
4.1 Screening of RILs for disease resistance in unprotected
condition
4.2 Evaluation of recombinant inbred lines for sucking pest
reaction
4.3 Estimates of biochemical components in un-protected
condition of recombinant inbred lines
4.4 Anatomical study
4.5 Evaluation of recombinant inbred lines for yield and yield
related traits
4.6 Mean performance of recombinant inbred lines for fibre traits
4.7 Variability studies in recombinant inbred lines
4.8 Genetic diversity studies in recombinant inbred lines for yield,
yield related traits
4.9 Genetic diversity studies in recombinant inbred lines for fibre
traits
4.10 Molecular characterisation for heterologous loci in cotton
5. DISCUSSION
5.1 Evaluation of RILs for disease reactions
5.2 Evaluation of recombinant inbred lines for sucking pest
reaction
5.3 Estimates of recombinant inbred lines for biochemical
components in un-protected condition
5.4 Anatomical studies
5.5 Variability study among RILs for yield, yield related traits
5.6 Genetic diversity studies
5.7 Detailed fibre properties of RILs
5.8 Genetic diversity studies of RILs for fibre properties
5.9 Molecular study
6. SUMMARY AND CONCLUSIONS
REFERENCES

LIST OF TABLES

Table
Title
No.
1. List of safflower specific RGA primers used in molecular
characterization
2. List of pulse specific RGA primers used in the study
3. Grouping of recombinant inbred lines based on reaction to Alternaria
blight (Alternaria macrospora)
4. Grouping of recombinant inbred lines based on reaction to bacterial
blight (Xanthononas axomopodis pv. malvacerum)
5. Grouping of recombinant inbred lines based on reaction to grey mildew
(Ramularia areola)
6. Seed cotton yield (kg/ha) of resistant and susceptible RILs under natural
and unprotected condition
7. Seed cotton yield of selected RILs in protected and unprotected
condition
8. Status of fibre traits of selected RILs
9 Classification of recombinant inbred lines for thrips and jassids reaction
10 Mean pest load per leaf for Thrips and Jassids
11 Seed cotton yield (kg/ha) of sucking pest resistant and susceptible RILs
12 Estimates of biochemical components in recombinant inbred lines
13 Anatomy of leaf lamina and leaf midrib of RILs
14 Performance of recombinant inbred lines for yield and related traits
under unprotected condition
15 Analyses of variance for yield and yield related traits in recombinant
inbred lines
16 Performance of different recombinant inbred lines for yield and related
traits
17 Performance of recombinant inbred lines for fibre traits
18 Measurement of variability parameters in recombinant inbred lines
19 Cluster-wise distribution of recombinant inbred lines
20. Contribution of different yield and yield related characters towards
divergence in recombinant inbred lines
21 The cluster-wise mean values of recombinant inbred lines for yield
and yield related traits
22 Cluster-wise distribution of recombinant inbred lines for fibre traits
23 Contribution of different fibre characters towards divergence in
recombinant inbred lines
24 The cluster-wise mean values of recombinant inbred lines for fibre
traits
25 List of monomorphic and polymorphic loci detected using heterologous
primers in cotton
LIST OF FIGURES

Figure
Title
No.
1 Frequency distribution of yield and yield related traits in RILs
2 Dendrogram depicting genetic diversity for RILs
3. Dendrogram depicting genetic diversity for fibre quality traits
4. Estimates of biochemical components in recombinant inbred lines

LIST OF PLATES
Plate
Title
No.
1. Alternaria disease susceptible RIL
2 Alternaria disease resistant RIL
3 Bacterial blight disease resistant RIL
4 Bacterial blight disease susceptible RIL
5 Grey mildew disease susceptible RIL
6 Grey mildew disease resistant RIL
7 Sucking pest resistant RIL
8 Sucking pest susceptible RIL
9 Anatomical differences for leaf lamina in resistant and susceptible RILs
10 Anatomical differences for leaf midrib in resistant and susceptible RILs
11 Parental polymorphism for heterologous RGA primers from safflower
12 Parental polymorphism for heterologous RGA primers from pulses
LIST OF ABBREVIATIONS

% : Percentage
µg/in : Microgram per inch
CIA : Chloroform and Isoamyl Alcohol
CIRCOT : Central Institute for Research on Cotton Technology
cm : Centimeter
CTAB : Cetyl Trimotyl Ammonium Bromide
CV : Coefficient of Variation
DAS : Days After Sowing
dATP : Deoxyribo Adenine Trisphosphate
dCTP : Deoxyribo Cytocine Triphosphate
DF : Degrees of Freedom
dGTP : Deoxyribo Guonosine Triphosphate
DNA : Deoxyribo Nucleic Acid
dNTP : Deoxyribo Nucleotide Triphosphate
DPX : Dibutyl Phathalate Xylene
dTTP : Deoxyribo Trymedine phosphate
DNS : 3,5 – Dinitrosalicylic Acid
EDTA : Ethylene Diamine Tetra Acetic Acid
g : Grams
GA : Genetic Advance
GAM : Genetic Advance as per cent Mean
GCV : Genotypic Coefficient of Variation
GOT : Ginning outturn
HCl : Hydrochloric Acid
HVI : High Volume Instrument
kg/ha : Kilograms per hectare
max : Maximum
mg : Milligram
min : Minimum
mm : Millimeter
MSL : Mean Sea Level
OD : Optical Density
PCR : Polymerase Chain Reaction
PCV : Phenotypic Coefficient of Variation
QTL : Quantitative Trait Locus
RGA : Resistance Gene Analogues
RILs : Recombinant Inbred Lines
RNA : Ribo Nucleic Acid
RNase : Ribonuclease
SCY : Seed Cotton Yield
SPSS : Statistical Package for the Social Sciences
SSR : Simple Sequence Repeats
TAE : Tris Acetic Acid
Taq DNA Polymerase :Thermus aquaticus DNA Polymerase
TE : Tris-EDTA
Vg : Genotypic variability
Vp : Phenotypic variability
INTRODUCTION
Cotton is an important fibre crop of global importance. Cotton known as the “king of fibre” and
in recent times called as “white gold”, is the most vital crop of commerce to many countries including
India. Cultivated cotton (Gossypium spp.) is the world’s leading natural fibre crop and it is the
cornerstone of textile industries worldwide. Inspite of several competitions from synthetic fibres, cotton
continues to enjoy a place of prime importance in textile industry. In India, cotton provides means of
livelihood to millions of farmers and workers and sustains cotton textile industry which annually
produces cloth of the value exceeding a thousand crore rupees. Cotton seed had also gained the
additional economic importance as a major contributor to edible oil, protein and other by products.
The valuable biomass from cotton stalks has become very useful raw material for manufacture of
particle boards, paper and other stationaries. In total, cotton has become a highly agro-industrial crop.
No agricultural commodities in the world have exercised such a profound influence on men
and matters as cotton has done from times immemorial. India has a pride place in the global cotton
scenario, it has the distinction of having the largest cotton area of 117.73 lakh hectares, production of
330.0 lakh bales (1 bale= 170 kg lint) and productivity of 496.39 kg per ha (Anon., 2012).
Taxonomically, cotton is recognized under Kingdom Plantae, Subkingdom Tracheobionta,
Superdivision Spermatophyta, Division Magnoliophyta, Class Magnoliopsida, Subclass Dilleniidae,
Order Malvales, Family Malvaceae, Genus Gossypium. According to Kohel et al. (1990) the genus
Gossypium includes 49 species, four are cultivated, 43 are wild diploids and two wild tetraploids. Of
the four cultivated species, G. hirsutum and G. barbadense are allotetraploids (2n=4x=52), commonly
called as new world cotton. G. hirsutum also known as upland cotton, long staple cotton or Mexican
cotton, produces 90 per cent of the world’s cotton; G. barbadense, also known as Sea Island cotton,
extra long staple cotton, American Pima or Egyptian cotton, contributes 8 per cent of the world’s
cotton; Whereas G. arboreum and G. herbaceum are diploids (2n=2x=26) and commonly called as
old world or Asiatic cotton. G. herbaceum, known as Levant cotton, and G. arboreum, known as Tree
cotton, together provide 2 per cent of the world’s cotton (Hong-Bin et al., 2008).
Cotton is commercial crop producing 90 per cent raw material to textile industry and contributes
60 per cent of oil requirements. Cotton production is hampered by different biotic stresses viz.,
diseases like bacterial blight is most wide spread and destructive disease reported to cause yield
losses of about 10-30 per cent (Kalpana et al., 2004), Leaf curl disease caused by gemini virus
(CLCuV) and transmitted by insect vector white fly (Bemisia tabaci) reducing production by 30 per
cent (Mansoor et al., 1993), Seed cotton yield losses ranging between 26-66 per cent have been
reported under grey mildew epiphytotic conditions (Sangitrao et al.,1993) and in intra- hirsutum hybrid
H4, the loss in yield to extent of 62-68 per cent (Shivankar and Wangikar,1992), Alternaria leaf spot
and rust etc cause an average of 10-12 per cent losses in India.
Cotton is infested by a large number of insect pests right from the sowing till harvest. In the
early stages, sucking pests like aphids, thrips, leaf hoppers and white flies cause serious problem and
resulting reduction in yield and quality of cotton. The sucking pests cause heavy reduction in cotton
yield (Saptute et al., 1990). Jassid causing 24.45 per cent (Bhat et al., 1986) and 18.78 per cent
(Javed et al., 1992) reduction in cotton yield.
Resistance in varieties offer an inexpensive preventive measure which is generally
compatible with other methods of pest control (Choudhary and Arshad, 1989). Defense mechanisms
in resistant varieties involve either morphological barriers or elaborative array of phytochemicals,
which act as repellents, phagodeterrents and oviposition deterrents, these exhibiting resistances.
Polyphenol and phenols are considered to play an important role in plant defense mechanisms. In
Cotton, phenols and tannins are also found negatively correlated with white fly population densities
(Butter et al., 1992).
Anatomical characters are also known to play a major role in the resistance mechanism
against insects. Characters like thickness of leaf, vein toughness, vein turgidity, compactness of
tissue in leaf, petiole and stem tip are found to be important against aphid resistance.
Thin leaf lamina could be one of the factor imparting resistance against sucking pests.
Thinner leaf comprising of higher density of lower epidermal, upper epidermal and mesophyll cells
serves the genotypes to have compact leaf lamina. Leaf toughness could limit population build up of
certain pests (Kadapa et al., 1988).
In midrib of resistant genotypes compactly arranged cortical cells have less intercellular space
for sucking pests (Kadapa et al., 1988). This could favour low piercing rate and also low rate of
injection of the phloem and there by imparting resistance to sucking pests.
The importance of molecular markers used in the development of disease resistant lines over a
conventional breeding is recognized, as, it saves the time from substitution of complex field trials (that
need to be conducted at a particular times of a year or at specific locations, or technically
complicated) with molecular tests and selection of genotypes at seedling stage, gene pyramiding or
combining multiple genes can be done simultaneously. Estimation of unreliable phenotypic
evaluations associated with field trials due to environmental effects. Avoid the transfer of undesirable
or deleterious genes (linkage drag) and selecting for trials with low heritability. Testing for specific
traits where phenotypic evaluation is not feasible and also assessment of cultivar, genetic diversity
and parental selection and also used to exploit heterosis.
In the light of the above facts, present study on “Morphological, anatomical and biochemical
basis for biotic stress resistance in cotton” was undertaken with the following objectives.
Objectives of investigation
1. Screening for biotic stress resistance in recombinant inbred lines of the cross, DS28 (G.
hirsutum) × SBYF 425 (G. barbadence).
2. Screening for disease resistance blocks using heterologous primers in these recombinant
inbred lines.
REVIEW OF LITERATURE
Success of any crop breeding programme is based on the knowledge and availability of
genetic variability for efficient selection. Genetic similarity or genetic diversity estimates among
genotypes are helpful in selecting parental combinations for creating segregating populations so as to
maintain genetic diversity in a breeding programme.
A critical estimate and study of genetic variability is pre-requisite for initiating appropriate
breeding procedure for effective selection of superior genotypes. The partitioning of total variability
into heritable and non-heritable components by using suitable design will enable the breeders to know
whether the superiority of selection is inherited by the progenies.
The literature relevant to objectives of the present study is reviewed under the following
subheadings.
2.1 Screening of disease tolerance/resistance genotypes
2.2 Screening of sucking pest tolerance/resistance genotypes
2.3 Role of biochemical components for sucking pest tolerance/resistance
2.4 Role of anatomical characters in host-plant resistance
2.5 Genetic variability for yield, yield contributing traits
2.6 Creation of genetic variability for fibre quality traits
2.7 Molecular marker studies

2.1 Screening for disease resistance cotton genotypes
Lakshmanan and Vidhyasekaran (1990) screened 1000 collections of cotton germplasm
under natural field conditions for their resistance to R. areola (R. gossypii). Of these, 166 exhibited
high levels of resistance and were tested again under artificial infection conditions in the greenhouse.
Sixteen genotypes exhibited a high level of resistance and 58 showed resistant reactions.
Genetic mapping generally corroborated classic predictions regarding the number and
dosage effects of genes conferring Xcm resistance studied by Wright et al. (1998). One recessive
allele (b6) was a noteworthy exception to the genetic dominance of most plant resistance alleles. This
recessive allele appeared to uncover additional QTLs from both resistant and ostensibly susceptible
genotypes, some of which corresponded in location to resistance (R)-genes effective against other
Xcm races. Among the six resistance genes derived from tetraploid cottons, five (83%) were mapped
to D-subgenome chromosomes.
Inheritance of bacterial blight disease resistance genes in cotton were studied by crossing the
resistance x resistance, resistance x susceptible and susceptible x susceptible by Sajjid et al. (2007).
These F1, F2 and back crosses indicated monogenic inheritance of resistance suggesting that
pedigree breeding would be adequate for transferring the resistance in the susceptible genotypes.
Biochemical studies were carried out on non-Bt and Bt genotypes of cotton by Hosagoudar et
al. (2008). The results indicated that non-Bt genotypes recorded high amount of total protein (7.19 to
9.18%) as compared to Bt genotypes, but total phenol ( -17.79 to -18.39%), total sugar (-12.17 to -
12.82 %) and reducing sugar (-4.51 to -19.05%) were recorded in lower amount compared to Bt
genotypes. Further, the decrease in total protein (-18.43 to -19.85%), total phenol (-32.76 to -38.63),
total sugar (-31.00 to -33.36%) and reducing sugar (-27.99 to -40.86) was more in infected plants of
Alternaria blight disease.
Vimala (2008) A total of 176 cultures/ varieties of cotton three varieties (SVPR 3,MCU 3 and
Deltapine-45) showed resistant reaction,60 entries were found to exhibit moderate resistance and the
remaining entries showed susceptible or highly susceptible reaction.
Among the one hundred ninety six cotton genotype studied by Chattannavar et al. (2009), 64
variety/ hybrid/ genotype showed immune reaction, 4 were highly resistant, 49 were moderately
susceptible and 15 were highly susceptible to bacterial blight.
One hundred forty-one cotton genotypes/ hybrids/ varieties in different Gossypium species
were screened by Chattannavar et al. (2010) under field conditions. Three (CCA 4, FDK 172 and FDK
173) of the G. arboreum genotypes showed moderately resistant reaction to Alternaria blight and
remaining genotypes showed moderately and highly susceptible reaction.
With respect to the bacterial blight, all the G. arboreum and G. herbaceum genotypes showed
immune reaction to bacterial blight, and six G. hirsutum genotypes (RAH 332, BS 79, SCS 404, NDLH
1905, LC and NDLH 1938) showed moderately resistant reaction.
Among 63 cotton genotypes studied by Jagtap et al. (2013), 3 genotypes showed moderately
resistant, 31 showed moderately susceptible and 2 showed susceptible reaction against bacterial
blight of cotton. Disease severity at 60 DAS ranged from 2.42 to 27.5 per cent. PH 1009 (2.42 per
cent) and Paig 29 (2.42 per cent) had shown lowest disease severity. Disease severity at 90 DAS
ranged from 9.63 to 58.6 per cent. NH 633 (9.63 per cent) had shown minimum disease severity
followed by PH 1062 (9.91 per cent) and PH 1031 (10.37 per cent). Disease severity at 120 DAS
ranged from 11.63 to 68.94 per cent. The lowest PDI was recorded by NH 633 (11.63 %) followed by
Paig 265 (13.26%) and NH 637 (13.55 %). Mean disease severity (PDI) of cotton genotypes was
recorded in range 9.71 to 51.68 per cent.
2.2 Screening of sucking pest resistant/ tolerant cotton genotypes
Malik et al. (1986) studied infestation of jassids and Pectinophora gossypiella on 5 lines
of Gossypium arboreum and 35 lines of G. hirsutum and concluded that the incidence of A.
devastans was less in the G. arboreum lines (average 8.7-12.4 nymphs/15 leaves) than in the G.
hirsutum lines (12.4-16.2).
Imtiaz et al. (2002) studied the resistance level of ten cultivars to sucking pest complex i.e.,
jassids, thrips and white flies and identified the resistance level of the genotypes.
Syed et al. (2003) investigated the relative resistance of 20 cotton varieties against sucking
pests viz., jassids, thrips, white flies and mites. They recorded the highest and least pest population
on each genotype.
Abida et al. (2004) investigated forty cotton varieties registered for cultivation in Punjab
province for their relative resistance against some insect pests i.e., jassids, aphids Helicoverpa
armigera (Hub), Earias vittella (Fab.) and Earias insulana (Boise). At the stage of square formation of
the cotton genotypes they were categorized into different classes based on pest reactions.
Muhammad et al. (2004) studied seven cotton strains for their resistance against bollworm
complex under sprayed and unsprayed conditions and concluded about the resistance level and yield
performance of the genotypes in both sprayed and unsprayed conditions.
Ali et al. (2007) tested eight genotypes of cotton for their resistance against whitefly, thrips,
and jassid. Genotypes did not show significant differences for jassid and thrips population that ranged
from 1.40 to 1.82 and 10.59 to 12.85 per leaf, respectively. Maximum population of whitefly (7.55/leaf)
and jassid (2.26/leaf) was recorded, while thrips (28.16/leaf) were maximum.
Attaullah et al. (2007) investigated 6 cotton strains for resistance against sucking pests (jassid
Amarasca devastance, whitefly Bemisia tabaci and thrips Thrips tabaci) and bollworm complex
(Helicoverpa armigera and Earias spp.) under unsprayed conditions. Recorded the genotypes
reaction to both sucking and boll worm pest complexes and also compared the yields of genotypes.
Jindal et al. (2007) investigated the relative resistance of 346 cotton genotypes against
sucking pests i.e. jassid and whitefly. The jassid injury grade, jassid and whitefly population/leaf was
recorded on each genotype.
Muhammad et al. (2009) conducted studies on five cultivars of cotton for resistance against
whitefly, thrips, jassid, and aphid. The FH-634 was found to be the most resistant genotype to the
sucking pest complex, whereas, FH-682 was found to be most resistant line to the jassid. Jassid and
whitefly populations almost remained above economic threshold level throughout the season.
Muhammad (2010) studied the seasonal dynamics of cotton jassid, whitefly and thrips were
compared on transgenic Bt cotton line, “IR-FH-901” expressing Cry1Ac insecticidal protein with its
parent non-transgenic cotton cultivar, FH-901 and concluded that, there is no difference in transgenic
Bt and non-Bt cotton for jassid, whitefly and thrips attack and application of suitable insecticide is
required to theses pests on transgenic cotton.
Salman et al. (2011) studied the resistance levels of six cotton varieties viz. MNH-635, NIAB-
86, SLH-257, CIM-446, CIM-482 and NIAB Karishma to sucking pest complex i.e. thrips, jassids, and
whiteflies. The genotypes were evaluated to know the sucking pest reactions.