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Abstract

Pigeon pea is an important pulse crop of India and its production is greatly affected by wilt disease. Pigeon pea wilt is a very destructive soil borne disease caused by Fusarium udum. In present research work plant extracts (Garlic, Neem, Tulsi), bio-agents (Trichoderma viride and Pseudomonas fluorescens) and fungicides (Vitavax, Bavistin and Thiram + Carbendazim) were tested against Fusarium udum. Among them fungicides were found most effective in inhibiting mycelial growth of Fusarium udum recorded 100% inhibition followed by garlic bulb extract (52.12 to 56.33%). Among bioagents, Pseudomonas fluorescens inhibit the growth up to (46.00 to 51.79%) followed by T. viride (44.32 to 48.79%) amongst all treatments except untreated.

Keywords: Pigeon pea, Fusarium udum, bio-agent, fungicide

Pigeon pea (Cajanus cajan L. Millsp.) is one of the major pulse crops grown in the tropics and subtropics belonging to family Leguminaceae. It has wide adaptability and low input requirements mostly grown in kharif season.

Abstract

Pigeonpea (Cajanus cajan) is an important pulse crop of India and its production is greatly affected by wilt disease. Pigeonpea wilt is a very destructive soil borne disease caused by Fusarium udum. In the experiments, ten treatments were applied viz.; Neem leaf extracts (T₁), Garlic extracts (T₂), Tulsi leaf extracts (T₃), bio-agents Trichoderma viride (T₄), Pseudomonas fluorescens (T₅), Polymer coating (polycot) @ 4 ml diluted in 5 ml of water per kg of seed (T₆), Polymer coaating + Trichoderma viride @ 6.0 g per kg seed (T₇), Polymer + Pseudomonas fluorescens @ 6.0 g per kg seed (T₈), Polymer coating (polycot) 4 ml diluted in 5 ml of water per kg seed + Vitavax power @ 3g per kg seed (T₉) and T₁₀ served as untreated. The observations were recorded six times at 10 days intervals and started from 40 days. These polymer alone @ 4.0ml per kg and in combination with bioagents and fungicides were applied to seed were tested against Fusarium udum. The highest per cent disease control and per cent wilt disease incidence were recorded 96.09 to 64.09 and 2.74 to 33.32, respectively in T₉ (Polymer + Vitavax power @ 3g per kg seed) at 10 days intervals followed by T₈ (Polymer + P. fluorescens) gave 94.46 to 53.06 per cent inhibition, T₇ (92.39 to 52.74) and T₆ (90.97 to 49.73) however, the lowest per cent disease control was noticed as 76.19 to 40.13 per cent in T₁ (Neem leaves extract) amongst all treatments except untreated.

The concept of gene has undergone several refinements since its discovery in 1866 by Mendel. Most genes encode one polypeptide and can be operationally defined by the complementation test. The complementation or cis-trans test provides an operational definition of the gene. It is used to determine whether mutations are in the same gene or in different genes. Intragenic complementation may occur when a protein is a multimer containing at least two copies of one gene product. The concept of gene has evolved from a bead on a string, not divisible by recombination or mutation to a sequence of nucleotide pairs in DNA encoding one polypeptide chain. The unit of genetic material not divisible by recombination or mutation is the single nucleotide pair. Historically several experimental developments have occurred to facilitate understanding of the structure and function of gene and these are briefly described in a chronological order as follows to have better understanding of the gene concept as it evolved. However, before discussing evidences supporting various gene concepts, it is better to summarize important stages in the evolution of the gene concept.

Regulator Genes

Bacterial cells are capable of producing many kinds of enzymes in relatively large quantities but the kinds and amount of enzymes actually produced at any given time are usually those which are required at that particular time in response to the prevailing environment. Inducible enzymes are ordinarily absent or present in very small quantities until a specific compound appears. This compound is called as inducer. A typical example of this is production of b galactosidase (an enzyme that splits lactose into galactose and glucose, Fig. 44.1) once the lactose is introduced into the medium on which E. coli cells are grown. Here lactose acts as inducer for the enzyme b galactosidase.

Biochemical reactions are mediated by enzymes and all enzymes are proteins. Proteins are polymers of subunits (monomers) called amino acids which are often termed as ‘residues’. Each amino acid has an amino group (NH ) at one end and a carboxyl group (COOH) at the opposite end. 20 different kinds of amino acids occur naturally in proteins. Each enzyme consists of a certain number of amino acids in a precisely ordered sequence. The blueprint for making proteins is coded in the nucleotide sequence of DNA. The number of nucleotides which code for an amino acid is termed a codon. There are 20 common amino acids but only four different nucleotides. Obviously a singlet code (one nucleotide coding for one amino acid) could code only for four amino acids. A coding sequence of two nucleotides for one amino acid or a doublet code would produce 4₂ = 16 possible coding combinations or codons and this arrangement is not sufficient to take care of 20 amino acids. A codon size of three nucleotides for one amino acid or triplet code seems more likely as it produces 4₃ = 64 possible codons as illustrated in Table 43.1 (Strickberger, 1968).

In transcription, it has become clear as to how there is the transfer of genetic information stored in the sequence of nucleotides-pairs in DNA to the sequence of nucleotides in m-RNA molecules which carry that particular information from the nucleus to the sites of protein synthesis (ribosomes) in the cytoplasm. Further, there is the requirement of the process by which genetic information stored in the sequences of nucleotides in m-RNAs is used to specify the sequences of amino acids in the polypeptide gene products. This process known as translation takes place in the cytoplasm on complex workbenches called as ribosomes. With exception of water, proteins are by far the most prevalent component of living organisms in terms of total mass.

Proteins (= enzymes = genes) are composed of polypeptides and every polypeptide is encoded by a gene. Each polypeptide has a long sequence of amino acids linked together by covalent bonds. There are 20 different amino acids (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine, serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine) present in most proteins. All amino acids except proline contain a free amino group (NH₂) and a free carboxyl group (COOH) as illustrated below:

According to the central dogma of molecular biology, genetic information flows (i) from DNA to DNA during its transmission from generation to generation and (ii) from DNA to protein during its phenotypic expression in an organism (Fig. 41.1).

Watson and Crick based on their double helical DNA model, offered to explain DNA replication by a method where each single strand of DNA is template or mold for its complement and a new helix has one old strand and one newly synthesized strand. This type of DNA replication is called as semi-conservative as in this method one old strand is retained and one new complementary strand is synthesized. In contrast to semi-conservative mode of DNA replication, two more modes of DNA replication were proposed. One is conservative type where two new strands are synthesized in form of a double helix while the old double helix remains unchanged. The third type of DNA replication proposed has been dispersive mode of DNA replication which amounts to breaking down of double helical strands into small pieces along the length, their replication and random assemblage with newly synthesized pieces to form a patchwork single string of dispersed old and new pieces. These three modes of replication of DNA molecule are shown in Fig. 40.1.

The search for the chemical basis of genetic material began about one and half century ago. Soon after discovery of Mendelian principles of segregation and independent assortment in 1865, Friedrich Miescher (1844-1895) published a method for separating cell nuclei from the cytoplasm and from these cell nuclei, he extracted an acid material termed as “nuclein” which had unusually large amount of phosphorous. Miescher concluded that this substance was unique and was just not comparable with any other group known at that point of time. Later on nuclein, now named as “nucleic acid” was found to be associated with various proteins and the combined entities were termed as “nucleoproteins”.

In 1868, Johann Friedrich Miescher, a young Swish medical student got excited with an acidic substance that he isolated from pus cells obtained from bandages used to dress human wounds. To obtain large quantities of pus for his experiment, Miescher began purchasing used bandages from surgical clinics in Tuebingen, Germany. He separated pus cells from bandages and treated the cells with pepsin, a proteolytic enzyme that he isolated from stomach of pigs. After pepsin treatment, he recovered an acidic substance that was named as “nuclein” by him. This nuclein was unusual in the sense that it was rich in nitrogen and phosphorous, two elements known at that time to coexist only in certain types of fat. Miescher’s paper on this investigation was published in 1871. At that time, importance of Miescher’s nuclein was not fully appreciated. Even the existence of polynucleotide chains, the key component of the acidic material in Miescher’s nuclein was not documented until 1940s. The role of nucleic acid in storing and transmitting genetic information remained un-established until 1944 and the double-helix structure of DNA was not discovered until 1953 by James Watson and Francis Crick. Even in 1953, many geneticists remained skeptical to accept the role of nucleic acid as the genetic information carrier rather than proteins as nucleic acids exhibited less structural variability than the proteins (Snustad et al., 1997).

So far, we have seen the examples in which mode of inheritance of a particular character is tied to the behaviour of the nucleus or more specifically to the chromosomes. In other words, transmission of a character and its appearance in the individuals can be predicted from the knowledge of chromosome segregation and independent assortment. This analogy gets further strengthened on the ground that the DNA has been considered as the basic genetic material and practically all the cellular DNA is localized in the chromosomes. It has also been seen that DNA does not produce an organism alone but obviously depends upon an already existing medium in order to function. Thus, genotypic effects may considerably get modified by the environment. In the cell itself, one important source of environmental effect is the cytoplasm immediately surrounding the nucleus. The components of the cytoplasm may show wide variation between the individuals and therefore it cannot be ruled out that a genotype in one cytoplasm may function somewhat differently than the same genotype placed directly into another cytoplasm. It is well known fact that the cytoplasmic contribution of one of the parents in form of an egg is considerably larger than the cytoplasmic contribution of the other parent in form of sperm. For example, cross-fertilization between two strains capable of producing both sperm and eggs, leads to contribution of ‘A’ genotype and ‘a’ egg cytoplasm in one case and of ‘B’ genotype and ‘b’ egg cytoplasm in the other case. Thus, the cross-fertilized diploid zygotes have two possible genotypic-cytoplasmic combinations, namely, AB-a, and AB-b depending upon which strain provides the egg. If the egg is contributed by the A parent, then the genotype of the progeny shall be AB-a, and if the egg is contributed by the B parent, then the genotype of the progeny shall be AB-b. If each such cytoplasm produces a unique phenotype, then AB-b would have the same b phenotype as BB-b individuals. Such effects produced by the egg cytoplasm are termed as maternal effects.

Translocations

Chromosomes occasionally undergo spontaneous rupture or can be induced to rupture in high frequency by ionizing radiation. The broken ends of such chromosomes behave as though they were ‘sticky’ and may rejoin into non- homologous combinations giving rise to translocations. The most common type of translocation is reciprocal translocation which involves the exchange of segments between two non-homologous chromosomes. Translocations change only the arrangement of genes in the chromosomes, not the quality or quantity of genes. For this reason, they are sometimes referred to as chromosomal rearrangements. Individuals carrying such rearrangements should be phenotypically entirely normal unless the relations of a gene or genes to adjacent genes affect the phenotypic expression (position effect). The first translocation was discovered by Bridges in 1923 in D. melanogaster.

The genetic techniques for detecting and studying translocations will be more easily understood when the cytological phenomena produced by translocations are known. Suppose two chromosomes having respectively the genes ABCDEF and GHIJKL exchange sections and give rise to translocation chromosomes ABCJKL and GHIDEF. The individual thus formed receives from one of the parents the normal and from the other parent the translocation chromosome. Such an individual is a translocation heterozygote (Fig. 36.1).

When there is indication of linkage from F or testcross data through significant deviation from 9: 3: 3: 1 and 1: 1: 1: 1 ratio, it is implied that independent assortment is not taking place and this is taken as an indication of linkage. The inverse of linkage strength is the recombination value and is often symbolized by ‘p’ whose maximum value is 50% because out of four strands of a tetrad, two are always parental types and two are recombinant types when all the cells show crossing over.

Linkage

When two or more genes are located on the same chromosome, they are said to be linked. Genes on different chromosomes are distributed to the gametes following the law of independent assortment. However, genes located on the same chromosome, tend to stay together during gamete formation. For example, results of test-crossing dihybrid individuals will produce different results depending upon whether the genes are linked or are located on the same chromosome. This is illustrated as follows:

In most of higher organisms including animals, the aspect of sexes has been normally confined to males and females which exist separately or even in the same individual. An individual possessing both male and female reproductive organs is usually referred to as hermaphrodite. In plants, where staminate (male) and pistillate (female) flowers occur on the same plant, the term used is monoecious. The most common plants under monoecious form are cucurbits. However, majority of the flowering plants have male and female parts within the same flower and this situation is referred to as hermaphrodite. There are relatively less numbers of plants having male and female flowers located on different plants and this is termed as dioecious condition. Among the common cultivated crops known to be dioecious are asparagus, date palm, hemp, hops and spinach.

Phenotype is a result of gene products brought to expression in a given environment. Genes specify the structure of proteins. All known enzymes are proteins. Enzymes perform catalytic function, causing the splitting or union of various molecules. All of the chemical reactions which occur in the cell constitute the subject of intermediary metabolism. These reactions occur as stepwise conversions of one substance into another, each group being mediated by a specific enzyme. All of the steps which transform a precursor substance to its end product constitute a biosynthetic pathway. Several genes are usually required to specify the enzymes involved in even the simplest pathways. Before gene-enzyme relationship could be postulated, soon after rediscovery of Mendelian principles of inheritance, it was discovered that genes were not merely separate elements producing distinct individual effects, but they could interact with each other to produce completely novel phenotypes. The simplest example of gene interaction could be the fact that allele A could be characterized by phenotype A, and the allele B by the phenotype B and an organism having both alleles A and B at the same time might show phenotype C. The occurrence of such interactions means that the phenotype we observe is not present in the genes themselves but arises from a complicated developmental process resulting from a biosynthetic pathway as illustrated below (Stansfield, 1969)

Historically the first and still the most conclusive evidence for the existence of genes comes from the phenomenon of segregation of traits observed in the offspring of hybrids between individuals or strains that differ in some recognizable respect. The principle of segregation was formulated by Gregor Johann Mendel in 1866 under such peculiar circumstances that the scientific world failed to recognize it until after a lapse of 34 years. In the first place Gregor Mendel was not primarily a biologist but a Monk in the Augustinian monastery at Bruenn, Austria (now Brno, Moravia in Czech Republic). During his childhood, Mendel worked as a gardener and studied beekeeping. He became part time monk because it enabled him to obtain an education without having to pay for it himself. From 1840 to 1843 he studied Practical and Theoretical Philosophy and Physics at the Philosophical Institute of the University of Olomouc. Upon recommendation of his teacher, Mendel entered the Augustinian St. Thomas Abbey in Brno and began his training as a Priest. Born Johann Mendel, he took the name Gregor upon entering religious life. Mendel worked also as a substitute High School teacher. In 1850, he failed the oral part of his examination and could not become a certified High School Teacher. In 1851, he was sent to the University of Vienna to study further so that he could get formal education. Mendel returned to his Abbey in 1853 as a Physics teacher. In 1856, he took the examination to become a certified teacher but again failed the oral part.

Chi-Square

The Chi-square test (c₂) proposed by Karl Pearson in 1899 is used to measure goodness of fit of observed and theoretical or expected data and is a continuous distribution.

Formula for Chi-Square

1. General Formula

The general formula for Chi-square is as follows

Cell

Cells (small, membrane-bounded structures filled with a variety of chemicals in an aqueous solution that interact to give life) are the basic unit of life. Cells are capable of acquisition and use of energy, have the capacity to reproduce and respond to the environment and are able to carry out a variety of chemical reactions. They maintain a relatively constant internal environment despite changes in the external environment. There are two types of cells, namely, prokaryotes and eukaryotes.

The prokaryotic (pro = before, karyote = nucleus) cells are the simple form of cell types which include the various types of cells of bacteria and their relatives, all belonging to kingdom Monera. They possess a cell wall constructed of peptidoglycan, a substance unique to prokaryotes. Beneath the cell wall, there is a plasma membrane that encloses a cytoplasm containing DNA, RNA, proteins and various small molecules.

Genetics and Gene

Genetics is defined as the branch of biology dealing with heredity and variation. The hereditary units which are transmitted from one generation to the next are called genes. Genes have been defined in various ways. These are units of inheritance located in linear order on chromosomes. The genes are considered as hereditary determinants of a specific biological function located in a fixed place on the chromosome. Chemically, gene is a segment of DNA (deoxyribonucleic acid) encoding one polypeptide and defined operationally by the cis-trans or complementation test. Gene is the functional unit of inheritance (a sequence of DNA) which is located on a certain position (locus, plural loci) on a chromosome. The locus is sometimes used interchangeably for gene. DNA, in conjunction with a protein matrix, forms nucleoprotein and becomes organized into structures with distinctive staining properties which are known as chromosomes. The chromosomes are located in the nucleus of the cell. Since genes are located on the chromosomes, in many ways behavior of genes is parallel to the behavior of the chromosomes. Two or more alternative forms of a gene are called as alleles. All genes on a chromosome are said to be linked and they all belong to the same linkage group.