The biopesticide sector is rapidly experiencing a significant growth by introducing biopesticidal product in the global market. The number of micro-organisms is included in the taxonomy of Microbial Biopesticides which include baculoviruses, fungi, nematodes, and bacteria. Few decades ago, with the use of microbial species, and entomopathogenic bacterium a new microbial species have been discovered with particular virulence factors and toxins.
It has been recorded that fungi comprised of versatile strains which include Verticillium, Hirsutella, Paecilomyces, Isaria, Anisopliae, Brongniartii, and metarhizium. In fact, several bacteria are included in the classified group (Saccharopolyspora, Streptomyces, Burkholderia, Pseudomonas, Serratia, etc.) of bacterial entomopathogens. Most of the farmers are using pesticide (chemicals) to control the disruption which has been made by the pests. In the report, it has been analysed how bio-pesticides are used for controlling pests and health-related negative aspects.
To study the DNA sample of fungi the genus namely Coniothyrium minitans and Trichoderma virens, an experiment has been conducted and their DNAs are studied in detail using Polymerase Chain Reaction (PCR). Both the classified categories of fungi namely Coniothyrium minitans and Trichoderma virens for the amplification of DNA were passed through the DNA amplifier/PCR machine/Thermocycler. All the images which have been obtained were viewed over the Gel Doc systems named as Gel Imager. In the experiment, large bands have been analysed in Trichoderma virens in comparison to Coniothyrium minitans. In this report, various samples of different fungi were selected and deep driven analysis has been performed in the report. This research report is intended for the analysis of the frequency of resemblance and difference among the selected fungi classification. From the Phylogenetic tree, it has been concluded the seven fungal organisms are having a significant relationship with each other.
In the modern era, to improvise the growth of the crop, most of the farmers are using chemical compositions like pesticides and fertilizers. But the question is why they are not concerned about the adversity of chemicals? It has been recorded that the toxin rate in food due to pesticides is quite high (Refer Appendix). Due to intensive toxicity in food, most of the people are suffering from various chronic and acute health-related diseases. Not even confined to this much of information it has been also analysed that pesticides and chemical substrates in agriculture adversely impact the sustainability of the environment as well. All these chemicals are not soluble due to which these chemicals and the chemicals unfortunately went into the oceans which ultimately harms the aquatic life as well. Thus, the report aims to discover sustainable methods and approaches which reduce the need for chemicals and spray in the field of agriculture. A new method has been discovered in the report which improves the productivity of the crops and protect the crops from pests with the use of biotechnology. According to UNSDGs goals, all countries should need to take conserve for the purpose of achieving the sustainability of the environment.
It will be imperative for the farmers to understand the crop cycle periods so that they will be able to take proactive steps for the preservation and protection of crops from insects and pests. By taking protective measures they resist the pests and destruction that they cause to the crops as well. It has been analysed that the environmental factors (soil fertility, temperature, the PH level of soil, temperature, and climate) affect the growth of pests significantly. Although, it has been analysed that pests spread diseases to mankind and it also harms the sustainability of the environment in an adverse sense too. In order to control the pests, it will be the prime accountability of the farmers to employ pest control measures because pest degrades the life span human health directly. There is a various type of pests (insects, arthropods, microbial organism, weeds, mollusks, termites, beetles, flies, spiders, bacteria, snails, slugs, shipworms, etc.) that effective the sustainability of the crops. There is a question that why to control the pests? In order to acquire a clean, and disease-free environment we need pest control measures. To control the pests most of the farmers are using pesticides (Chemically alter substrates). But these chemicals infect the human body adversely. No doubt that pesticides control the growth of pests but it also led to a number of health-related diseases.
It is well known that of the crops, that farmers grown are free from chemicals then it will be sage and alternatively healthy for us. It has been analysed that most of the countries are starving from food just because of the lack of quantity of the food. Even, number of people are number of health-related disease because they are not getting sufficient nutrients from the food. Pests reduces the quality and quantity of the food, even due to pests’ large crop in India get infected. All countries are trying their level best to for the achievement of sustainable energy. It has been analysed that with the use of pesticides or chemicals the future generation of the countries will get inversely infected.
By conducting the experiments, it has been proved that with the use of Fungi, the disease and pests would be controlled. This is a biological solution for controlling the pests. In order to prevent diseases and pests, fungi: pathogens are used. With the use of fungi namely Trichoderma virens and Coniothyrium minitans, the pests and disease in the field of agriculture would be intensively controlled because both the pathogens will act as insecticides and pesticides. Even, both the pathogens are bio actively driven which implies that these pathogens will not adversely impact the quality and quantity of the food. With the help of DNA amplification (detecting the mutations of the pathogens) the tendency for preventing the disease is identified. In addition to this, the sample of both the pathogens are amplified with the help of DNA amplification PCR technique.
From the studies, it has been analysed that T. virns and C minitans both the pathogens act as a biological pesticide which can be used for the prevention of crops from insects and pests. Both the pathogens act as a biologically alert agent which will be used as a pesticide and insecticides. Although, the major advantages of using these pest control agents are- theses pathogens are biologically active agents. Moreover, with the use of these pathogens, farmers would be able to prevent their crops from pests and insects which ultimately leads to better quality of food. In experimental analysis, it has been identified that C. minitans fungi have the ability or tendency to control the pests and insects but it has been also analysed that residual substrate of these fungi’s impacts the environment in an adverse sense.
With the help of DNA amplification and sequencing, the structure of the gene of both the bacteria will be effectively understood, even the factors which make these bacteria a biological agent can also be analysed. By using genes of pathogens, the plant will get the potential to fight agents the pathogenic diseases effectively. This proves that with the help of biotechnology, quality of life among people will be improved to a greater extent. Even, it has been analysed that with the use of Pathogens, farmers can able to reduce the chemical effects. In short with the use of biologically activated agents as pesticides and insecticides, farmers would be able to reduce toxicity of the crops. The prime aim of this report is to understand the DNA composition of both the bacteria so that the adverse effects of pesticide and insecticides can be reduced and the quality of crops can be improvised at a greater rate.
Extraction of DNA
Practically procedure of activities which have been performed while conducting the experimental analysis. Initially, 2ml culture tubes have been taken and both the pathogens namely T.virens, and C. minitans has been taken into the tubes. Thereafter, the sample of both the pathogens are centrifuged at 14000 Rpm. The centrifugation activity is performed for approx. 8minutes. After centrifugation, a solid pallet has been formed which could be extracted into the biological waste tube. In the culture tube, water of quality 1ml has been taken and quaked with the solid pallet. Further, the mixture (semi-solid) has been centrifuged again, for approx. 5 minutes at 14000rpm speed. After the centrifugation process, the liquid over the pallet has been discarded and the pallet is used for the extraction of DNA. With measuring device, the quality of the pallet was measured and by analysing the quantity of pallet and equivalent among Chelex 100 resin was taken into the test tube for the purpose of removing the inhibitor compounds from the Pallet. For breaking out the Fungal cells, clean sand with moulded granules was added into the mixture. Make sure that the quality of sand should be similar to that of pallet quantity. In both of the test tubes take 500 ul of Milli Q water. For crushing the sample, the micro pestle was used. Micro pestle grinds the fungal pallet until in fungal material no clumps have been seen. After 15-20 minutes, DNA of pathogens starts releasing. The test tubes in which the samples were taken get incubated for 10 minutes at the temperature range of 70°C. Thereby, again the sample was centrifuged for 8mins at 14000 Rpm. The DNA sample which has been obtained was transferred into the fresh and clean tube and the sample was stored at 4°C.
Amplified DNA gel Electrophoresis
In the electrophoresis tank, 1% of agarose gels and Ethidium bromide has been poured. Thereby, after 10 minutes, again pour 1% of agarose. In the first lane, carefully inject the 5 µl of the DNA size marker. In the leaving gaps, inject 5 µl of the PCR products (C.minitans, T.virens, and negative control). For 45minutes, the electrophoresis tank has been connected to the source of electricity. For the visualisation of results, Bio imager has been used and the image of each sample was captured and used for recording the results that have been obtained from the experiment.
Discussion and results
To compare the DNA size of both the bacteria’s C minitans and T. Virens, the DNA of both the fungi were put on the Gel wells of DNA size marker. By using this method, the band size of the both the DNA’s were examined. From the underlying Images, it has been analysed that the gel wells are having space in between. In addition to this, the DNA estimation band has been noticed with the confined negative control which appears in between the band of T virens DNA and C minitans. Look at the figure which has been given below, a number of bands have been analysed during the experiment. From the image, it is clear that there is a little deviation between the size of DNA of both the nominated Fungi. It has been analysed that the development DNA evidence is the slow casting procedure. The main purpose of conducting this experiment is to analyse the DNA band size of various Microorganisms.
Degree of homology
The DNA sequencing of a number of fungi has been analysed (Refer Appendix) for the sake of examining the homological characteristics between them. To analyse the homological level, DNA of Microorganisms like Trichoderma harzianum ITS1, Clonostachys rosea ITS1, Beauveria bassiana ITS1, Metarhizium anisopliae, Verticillium lecanii, Colletotrichum gloeosporioides, Coniothyrium minitans, and Trichoderma virens has been grouped. The DNA group arrangement of all these organisms are shown in the Appendix part of the report. With the help of homological characteristics of all parasites, the similarities some all the micro-organisms have been analysed. Various indicators are used for the determination of likeliness which includes red ink, green ink. High likeliness among the parasites can be shown with the help of red-inked region. Meanwhile, the green inked region shows the similarities but up to a small extent. With the help of ICT region, we can examine the level/ degree of homology between all the seven micro-organism species.
In this section of the report, with the help of the identity matrix, the comparison in the context of percentage has been shown and explained. With the help of an identity matrix, the relationship between the Seven classified microorganisms has been examined. It has been analysed that some of the microorganisms out of nominated seven microorganisms show similar properties due to the inherent genius stereotype and same metabolic functioning.
In the given figure percentage of similarities among all the species has been shown. From the table it has been clear that the relationship between C.minitans with T.virens and B.bassiana is quite resemblant.
- C.minitans and T.virens
- C.minitans and B.bassiana
There is a number of reasons which states a similar characteristically nature among C.minitans with T.virens and B.bassiana. The growth pattern of C.minitans various with the environmental variants. Some of the authors stated that the similarities between the microorganism is due to oblige mycoparasites. While examining the relationship amidst C.minitans and B.bassiana, it has been analysed that due to differentiable biocontrol factors both the microorganisms show similar properties. It has been analysed that B.bassiana is an entomopathogenic fungus. While C.minitans is having a capacity to survive in dense soil for a long period of time. Moreover, it has been analysed that the sclerotial is one of the functionality of the C.minitans that enables pathogens to survive in complexified environment. the biological factors are the major reason that shows sort of uniqueness. While from the table it has been observed that M.anisopliae did not show relationship factor with any of the Pathogen specious. Along with this, M.anisopliae is different from the T.virens because such a taxonomy of pathogens restricts the growth of pathogens. Thus, it has been clear from the table that some microorganisms show a similar characteristical nature between other organisms due to genus stereotype and same metabolic functioning.
hylogenic relationship and analysis
With the help of polygenic tree, the connection among all the nominated seven microorganisms has been established. The main purpose of this tree is to showcase similar characteristics among the microorganisms in the context of the term genealogy. From the experiment, the lines show the similarity and differentiable factors among the microorganisms. From the tree, T.virens microorganism shows a unique behaviour in comparison to all of the microorganisms. This is the only microorganism that shows a unique behaviour when compared with other bacteria. Due to remarkable characteristics, T.virens did not show any sort of similarities with any of the other microorganism. In the above paragraph, the relationship and connection between the M.aniopilae and C.minitans has been analysed which showed in the tree. When it comes to Llecanii and C.gleoesporiodes both the microorganisms are having a similar trait which shows that most the bacteria is having a strong relationship with each other. In addition to all this, the difference between the bacteria has been also analysed. It has been analysed that due to biocontrol operator the microorganisms show differentiable properties from each other.
In nutshell, it has been concluded that DNA characteristics of various microorganisms are analysed in the report through which the similarities between all the nominated bacteria have been analysed and evaluated. The genes between the various specious has been analysed in the report. By analysing the genealogy of the microorganism, we can efficiently examine the relationship between the microorganisms. In addition to this, from the experiment, it has been analysed that the nominated bacteria would be used as an insecticide that restricts the growth of weeds and the entrance of insects and pests. It will be imperative for the biotechnologists to conduct more and more experiments so that they will generate a greater scope of innovations like in this research the use of microorganisms has been investigated.