30.Basics of phytopathology. The main diagnostics methods of plant diseases

Phytopathology or plant pathology is the study of disease in plants. Diseases caused by pathogens which attack plants are studied by phytopathologists, along with damage caused by environmental factors. Because humans rely heavily on plants for food, this branch of the biological sciences is very important, and specialists in the field can sometimes command very high fees for their services.

Learning to identify plant diseases is one aspect of phytopathology, as is learning the terminology associated with the description of plant diseases to ensure consistency. Most researchers are also interested in learning how to manage specific outbreaks of disease, and in preventing future outbreaks. Phytopathologists can work in labs, examining samples from diseased plants and growing test crops, and they can also work in the field for agricultural companies, government agencies, and nurseries.

In terms of pathogens, there are a number of things which can cause damage to plants, including fungi, bacteria, viruses, and nematodes. Just like human pathogens, plant pathogens can be handled in a variety of ways, with disease control methods such as quarantine, destruction, or spraying. Some plant diseases are extremely common, leading to active efforts to prevent outbreaks and to control them quickly when they do occur. Breakthroughs inphytopathology often focus on the development of new approaches to perennial problems.

DNA-Based Diagnostic Kits

DNA diagnostic kits are based on the ability of single stranded nucleic acids to bind to other single stranded nucleic acids that are complementary in sequence (referred to as homologous).

The tool used in DNA diagnostic kits is the Polymerase Chain Reaction (PCR). There are 3 steps involved in PCR. The DNA is first unwound, and its strands separated by high temperatures. As the temperature is lowered, short, single-stranded DNA sequences called primers are free to bind to the DNA strands at regions of homology, allowing the (Taq) polymerase enzyme to make a new copy of the molecule. This cycle of denaturation-annealing-elongation is repeated 30-40 times, yielding millions of identical copies of the segment.

The primers in PCR diagnostic kits are very specific for the genes of a pathogen, and amplification will occur only in diseased plants. 

he primers in PCR diagnostic kits are very specific for the genes of a pathogen, and DNA amplification will occur only in diseased plants. (Figure 1)

Several PCR-based methods have successfully been adapted for plant pathogen detection. Real-time PCR (RT PCR) follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified, using fluorescent dyes, as it accumulates in the reaction mixture after each cycle. It offers several advantages over normal PCR, including: reduced risk of sample contamination, provision of data in real time and simultaneous testing for multiple pathogens. Real-time PCR protocols are among the most rapid species-specific detection techniques currently available.

DNA microarrays are also of great use for simultaneous pathogen detection. This is important, as plants are often infected with several pathogens, some of which may act together to cause a disease complex. Microarrays consist of pathogen-specific DNA sequences immobilized onto a solid surface. Sample DNA is amplified by PCR, labeled with fluorescent dyes, and then hybridized to the array 

PCR-based diagnostics is very sensitive compared to other techniques; detection of a small amount of DNA is possible. PCR can also help farmers detect the presence of pathogens that have long latent periods between infection and symptom development. Moreover, it can quantify pathogen biomass in host tissue and environmental samples, and at the same time detect fungicide resistance. PCR-based detection, however, is expensive compared to protein-based diagnostic methods, and also requires costly equipments.

So far, PCR kits have been developed to detect black Sigatoka disease in bananas, Phytophthora infestations in potatoes, and Fusarium infection in cotton.

Protein-Based Diagnostic Kits

The first step in a defense response reaction is the recognition of an invader by a host’s immune system. This recognition is due to the ability of specific host proteins, called antibodies, to recognize and bind proteins that are unique to a pathogen (antigens) and to trigger an immune reaction 

Protein-based diagnostic kits for plant diseases contain an antibody (the primary antibody) that can either recognize a protein from either the pathogen or the diseased plant. Because the antibody-antigen complex cannot be seen by the naked eye, diagnostic kits also contain a secondary antibody, which is joined to an enzyme. This enzyme will catalyze a chemical reaction that will result in a color change only when the primary antibody is bound to the antigen. Therefore, if a color change occurs in the kit’s reaction mixture, then the plant pathogen is present, (Figure 3b).

The enzyme-linked immunosorbent assay (ELISA) method makes use of this detection system, and forms the basis of some protein-based diagnostic kits. ELISA kits are very easy to use because test takes only a few minutes to perform, and does not require sophisticated laboratory equipment or training. There are already numerous ELISA test kits available on the market. Some of them detect diseases of root crops (e.g. cassava, beet, potato), ornamentals (e.g. lilies, orchids), fruits (e.g. banana, apple, grapes), grains (e.g. wheat, rice), and vegetables. ELISA techniques can detect ratoon stunting disease of sugarcane, tomato mosaic virus, papaya ringspot virus, banana bract mosaic virus, banana bunchy top virus, watermelon mosaic virus, and rice tungro virus.

One of the first ELISA kits developed to diagnose plant disease was by the International Potato Center (CIP). It can detect the presence of all races, biovars, and serotypes of Ralstonia solanacearum, the pathogen that causes bacterial wilt or brown rot in potato. They also developed a kit that samples for the presence of any of the following sweet potato viruses: SPFMV (sweet potato feathery mottle virus), SPCSV (sweet potato chlorotic stunt crinivirus), SPMSV (Sweet potato mild speckling virus), SPMMV (Sweet potato mild mottle virus), SwPLV (Sweet potato latent virus), SPCFV (Sweet potatochlorotic fleck virus), SPCaLV (Sweet potato caulimovirus), and C-6 (new flexuous rod virus).

31 Gametogenesis in animal. Gametogenesis is a biological process by which diploid or haploid precursor cells undergo cell division and differentiation to form mature haploid gametes. Depending on the biological life cycle of the organism, gametogenesis occurs by meiotic division of diploidgametocytes into various gametes, or by mitotic division of haploid gametogenous cells. Animals produce gametes directly through meiosis in organs called gonads. Males and females of a species thatreproduces sexually have different forms of gametogenesis: spermatogenesis (male), oogenesis (female). Oogenesis is the creation of an ovum (egg cell). Oogenesis consists of several sub-processes: oocytogenesis, otidogenesis, and finally maturation to form an ovum (oogenesis proper). Oogonium —(Oocytogenesis)—> Primary Oocyte —(Meiosis I)—> First Polar Body (Discarded afterward) + Secondary oocyte —(Meiosis II)—> Second Polar Body (Discarded afterward) + Ovum. Oogenesis starts with the process of developing oogonia, which occurs via the transformation of primordial follicles into primary oocytes, a process called oocytogenesis. Oocytogenesis is complete either before or shortly after birth. When oocytogenesis is complete, no additional primary oocytes are created, in contrast to the male process of spermatogenesis, where gametocytes are continuously created. The succeeding phase of ootidogenesis occurs when the primary oocyte develops into an ootid. In late fetal life, all oocytes, still primary oocytes, have halted at this stage of development, called the dictyate. These cells then continue to develop, although only a few do so every menstrual cycle. Meiosis I of ootidogenesis begins during embryonic development, but halts in the diplotene stage of prophase I until puberty. As a result of meiosis I, the primary oocyte has now developed into the secondary oocyte and the first polar body. Immediately after meiosis I, the haploid secondary oocyte initiates meiosis II. However, this process is also halted at the metaphase II stage until fertilization, if such should ever occur. When meiosis II has completed, an ootid and another polar body have now been created. Folliculogenesis. Synchronously with ootidogenesis, the ovarian follicle surrounding the ootid has developed from a primordial follicle to a preovulatory one. Both polar bodies disintegrate at the end of Meiosis II, leaving only the ootid, which then eventually undergoes maturation into a mature ovum.. Spermatogenesis is the process by which male primordial germ cells called spermatogonia undergo meiosis, and produce a number of cells termed spermatozoa. The initial cells in this pathway are called primary spermatocytes. They gives rise to two cells, the secondary spermatocytes, and the two secondary spermatocytes by their subdivision produce four spermatozoa. In mammals it occurs in the male testes and epididymis in a stepwise fashion, and for humans takes approximately 64 days. It starts at puberty and usually continues uninterrupted until death. Spermatocytogenesis results in the formation of spermatocytes possessing half the normal complement of genetic material. In spermatocytogenesis, a diploid spermatogonium which resides in the basal compartment of seminiferous tubules, divides mitotically to produce two diploid intermediate cells called primary spermatocytes. Each primary spermatocyte then moves into the adluminal compartment of the seminiferous tubules and duplicates its DNA and subsequently undergoes meiosis I to produce two haploid secondary spermatocytes, which will later divide once more into haploid spermatids. This division implicates sources of genetic variation, such as random inclusion of either parental chromosomes, and chromosomal crossover, to increase the genetic variability of the gamete. Each cell division from a spermatogonium to a spermatid is incomplete; the cells remain connected to one another by bridges of cytoplasm to allow synchronous development. It should also be noted that not all spermatogonia divide to produce spermatocytes, otherwise the supply would run out. Spermatidogenesis is the creation of spermatids from secondary spermatocytes. Secondary spermatocytes produced earlier rapidly enter meiosis II and divide to produce haploid spermatids. During spermiogenesis, the spermatids begin to grow a tail, and develop a thickened mid-piece, where the microtubules gather and form an axoneme. Spermatid DNA also undergoes packaging, becoming highly condensed. The resultant tightly packed chromatin is transcriptionally inactive. One of the centrioles of the cell elongates to become the tail of the sperm.