• Genetic transformation: The term genetic transformation is defined as the directed desirable transfer of gene from one organism to another along with the subsequent stable integration and expression of a foreign gene in the genome.
• The gene that is transferred is termed as ‘transgene’ and the organisms that are formed after a successful gene transfer are termed as ‘transgenics’.
• Plant transformation: It is defined as the method of insertion of the DNA from other organism, usually a plant into the genome of the plant of interest.

Why do we use plant transformation technology?

1. To develop variety that are resistance to pests:
   • It is now possible to introduce foreign genes that impart resistance to insects into the plant genome with the advent of genetic engineering techniques based on recombinant DNA technology.
   • Combined with plant tissue culture, recombinant DNA technology has helped to develop innovative methods for the economic management of various types of biotic stresses, including insect pests.
   • In reducing the losses incurred by biotic stresses, including insect pests, such innovations will be of tremendous benefit.
   • Transgenic plants that display insecticidal Bt proteins alone or in combination with herbicide resistance proteins are revolutionizing agriculture.
2. For Quality enhancement of the plant variety:
   • In improving plant productivity and improving the quality of plant products, plant transformations/transgenics have tremendous utility.
   • Improvements may be related to improving the nutritional value of the plant or to improving the functional properties of the production or consumption process.
   • In order to block the development of certain metabolites, transgenic plants can be used by regulating the over-expression or inhibition (antisense expression) of some of the essential enzymes as shown.

What are the biological requirements for plant transformation?

• For the gene transfer to produce transgenic plant, the biological requirements are listed below:
• There must be a target tissue that consists of competent cells for plant regeneration.
• A technique for the introduction of DNA into these regenerable cells.
• A procedure for the selection and regeneration of transformed plants at an optimum frequency.

What are the physical requirements for plant transformation?

• Highly efficient, economic, reproducible, and should readily produce several transformants for testing.
• Should be safe for operators.
• Should be simple technically including a minimum of demanding or inherently variable manipulations, like protoplast production and regeneration.
• Tissue culture should be completed in minimum time as to reduce the related costs and to avoid unnecessary somaclonal variation.
• Stable, if vegetatively propagated then uniform transformants and if sexually propagated species then, fertile germline transformants.
• Integration patterns should be simple and the introduced genes should have low copy number, so as to reduce the probability of undesired gene disruption at insertion sites.

Plant transformation methods:

Physical gene transfer methods:

1. Electroporation:
   • In this method, electric pulse of high field strength is used inorder to form pores in the cell membrane.
   • If DNA is present at an appropriate concentration in the buffer solution, it will be taken up via these pores.
   • Plant materials is incubated in a buffer solution that contains DNA and exposed to high voltage electric pulse.
   • Plant materials is incubated in a buffer solution that contains DNA and exposed to intense electric pulse.
   • Plant materials is incubated in a buffer solution that contains DNA and exposed to intense electric pulse.
   • Advantages:
     • It is possible to transform both intact cells and tissue.
     • The transformation efficiency depends on the materials of the plant.
   • Disadvantages:
     • DNA is obtained by 40 to 50 percent of incubated cells.
     • Near to 50 percent of the cells transformed will survive.
2. Biolistic gene gun / particle bombardment:
   • It is also termed as microprojectile bombardment.
   • To deliver DNA into cells, foreign DNA is coated with high-velocity gold or tungsten particles.
   • This method is popularly being used for its ability to transfer foreign DNA into the mammalian cells and microorganisms.
   • Advantages:
     • All plant species can be transformed using this process.
     • All plant species can be transformed using this process.
     • The protocol for transformation is relatively simple.
   • Disadvantages:
     • Difficulty in acquiring single copy transgenic events.
     • High costs for microcarriers and supplies.
     • Intracellular target is irregular or random (cytoplasm, vacuole, nucleus, plasmid etc.)
     • Transfer DNA is not protected.
3. Microinjection method:
   • A direct physical approach involving the mechanical introduction of the desirable DNA into a target cell is microinjection.
   • The microinjection technique involves transferring the gene into the cytoplasm or nucleus of a plant cell or protoplast through a micropipette.
   • The most important application of this is the introduction of DNA into animal oocytes and embryos, either in the study of transient expression or in the generation of transgenic animals.
   • The key disadvantages of microinjection are that it is long, costly and needs to be carried out by trained and certified workers.
   • Advantages:
     • This method does not need protoplast.
     • The device is easy and inexpensive.
     • Methods may be helpful for the transfer of genes into cereals that do not easily regenerate from cultured cells.
     • Technically straightforward.
   • Limitations:
     • Less specific
     • Less efficient
     • The transformation frequency is low.
4. Liposome mediated transformation:
   • Liposome-mediated transformation includes liposome adhesion to the surface of the protoplast, its fusion at the attachment site, and the release of plasmids inside the cell.
   • Liposomes are lipid spheres that are used to bring molecules into cells.
   • These are artificial vesicles that can behave as delivery vehicles for exogenous materials including transgenes.
   • They are regarded as sphere of lipid bilayers encircling the molecule to be transported and encourage transport after fusing with the cell membrane.
   • Cationic lipids are those with a positive charge that are used for nucleic acid transfer.
   • Liposomes can interact more readily with the negatively charged cell membrane than uncharged liposomes.
   • Fusion between the cationic liposome and the cell surface results in the transmission of DNA directly through the plasma membrane.
   • Advantages:
     • High reproducibility degree.
     • Stability for the long term.
     • Low toxicity level.
     • Nucleic acid protection from degradation

Chemical mediated gene transfer methods:

1. PEG mediated gene transfer:
   • Polyethylene glycol (PEG) disrupts the plasma membrane of protoplasts in the presence of divalent cations (using Ca2+) and makes it permeable to naked DNA.
   • Polyethylene glycol (PEG) disrupts the plasma membrane of protoplasts in the presence of divalent cations (using Ca2+) and makes it permeable to naked DNA.
   • The protoplastic culture is taken into a tube and 40 percent PEG 4000 (w/v) dissolved in mannitol is added to this tube and calcium nitrate is added slowly and incubated for a few minutes.
   • Advantages:
     • A large number of protoplasts can be transformed concurrently.
     • A wide variety of plant species can be used successfully.
     • Limitations:
     • The DNA is prone to rearrangement and degradation.
     • Random integration of foreign DNA into genome may lead to unfavorable characteristics.
     • A challenging task is the regeneration of plants from converted protoplasts.
2. Calcium phosphate co-precipitation:
   1. The DNA is permitted to mix with calcium chloride solution and isotonic phosphate buffer to form DNA-calcium phosphate precipitate.
   2. The cells are transformed when the actively dividing cells in the culture are exposed to this precipitate for several hours.
   3. The efficacy of this technique depends on the high DNA concentration and the protection of the complex precipitate.
   4. The addition of dimethyl sulfoxide (DMSO) enhances transformation efficiency.
3. DEAE-dextran mediated transfer:
   1. With a high molecular weight polymer diethyl amino ethyl(DEAE)dextran, the desirable DNA can be complexed and transferred.
   2. This approach’s main drawback is that it does not generate stable transformants.

Biological method of gene transfer:

Agrobacterium-mediated gene transfer in Plants:

• Agrobacterium tumefaciens is a widely occurring soil bacterium that is responsible for causing crown gall, and has the capability to integrate new genetic material into the plant cell.
• T DNA is a term given for the genetic material that is situated on a Ti plasmid.
• A Ti plasmid is a common circular fragment of DNA found in almost all bacteria.
• Agrobacterium-mediated transformation is highly efficient and hence is most usually used method for plant genetic engineering.
• In the course of transformation, various components of Ti plasmid plays a role in effective transfer of the gene of interest into the plant cells. They are:
• T-DNA border sequences that are responsible for demarcating the T-DNA to be transferred to the plant genome.
• vir genes that are necessary for the transfer of T-DNA region to the plant but they cannot be transferred by themselves.
• Modified T-DNA region where the genes that are responsible for the formation of crown gall are eliminated and replaced with genes of interest.

Steps for Agrobacterium-mediated Plant Transformation Process:

• The Agrobacterium-mediated transformation process comprises of several steps. They are:
• Isolation of the gene of interest from the source organism.
• Development of a functional transgenic hybrid involving the gene of interest, promoters to drive expression; codon modification, if required to enhance successful protein production; and marker genes to favor tracking of the introduced genes in the host plant.
•  Insertion of the transgene into the Ti-plasmid.
•  Integration of the T-DNA-containing-plasmid into Agrobacterium.
• The transformed Agrobacterium is mixed with plant cells to permit transfer of T-DNA into plant chromosome.
• Regeneration of the new plantlets that are genetically modified.
• Examination for characteristic performance or expression of transgene at lab, greenhouse and field level. 

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• Nematodes are ubiquitous, found in almost all habitats. Mostly nematodes are parasitic in nature infecting humans, animals, insects and other invertebrates.
• Some other nematodes are plant parasites which can cause economic damage to cultivated plants.
• Nematodes are abundantly present in marine, freshwater, and in soil.
• Soil is an excellent primary habitat for nematodes. Due to microscopic nature of most nematodes, 100 grams of soil contains several thousands of nematodes.
• In soil most of the nematodes exhibit role with agriculture significance and those nematodes are plant parasite nematodes as well as free soil nematodes.
• Free soil nematodes plays significant role in decomposition of organic matters in soil and are beneficial to plants.
• Soil also contains human, animals and insects parasites as in juvenile larval form and mostly parasitic eggs.
• Most of the soil nematodes are present in plant root region in rhizosphere. The soil surrounding the plant root where root exudate migrate and microbiological activity is exceptionally high is called rhizosphere and the surface of root is called rhizoplane.
• Examples: Globodera pallida, Caenorhabditis elegans

Types of nematodes found in soil:

Most nematodes studied on the soil parasitic and they are classified on the basis of their feeding habits. The most common groups of nematodes present in agricultural soil are the bacterial-feeders, fungal-feeders, plant parasites, predators, and omnivores.

• Predatory nematodes feed on protozoa and other soil nematodes whereas Omnivores feed on different foods depending on environmental conditions and food availability
• Omnivorous nematodes primarily feeds on protozoans and other small nematodes as predators. But in the absence of their primary food source, they can feed on fungi or bacteria.

Important role of nematodes in Agriculture and health:

1. Nematodes as biological Pest control:

• Some predator nematodes attack and kill a range of pests such as borers, grubs, thrips and beetles with negligible effects on other organisms.
• These nematodes are known as ‘entomopathogenic’ nematodes.
• Nematodes generally feed on smaller organisms like protozoa, bacteria, fungi and other nematodes.
• Some nematodes live in association with specific bacteria that can infects wide ranges of insect pests. When such nematodes infects insects, it releases bacteria that multiplies in host insect and kill them

2. Improve soil fertility; Nitrogen cycle

• Nematodes directly helps in nutrient mineralization through their feeding interactions.
•  For example, bacterial-feeding nematodes consume Nitrogen in the form of proteins and other N-containing compounds in bacterial tissues and release excess Nitrogen in the form of ammonium (NH4+), which is readily available for plant use.

3. Decomposition of organic matters:

• Free-living nematodes in soil are very important and beneficial in the decomposition of organic material and the recycling of nutrients in soil.
• Bacteria and fungi feeding Nematode do not feed directly on soil organic matter, but feed on the bacteria and fungi which decompose organic matter.
• The presence of theses nematodes and their feeding activity accelerate the decomposition process.
• Nematodes feeding recycles minerals and other nutrients from bacteria, fungi, and other substrates and returns them to the soil where they are accessible to plant roots.

4. Plant parasitic nematodes:

• Some plant parasitic nematodes infects roots of plants and damage crops. Eg. Globodera
• The mouthpart of plant parasitic nematodes is a needlelike stylet which is used to puncture cells during feeding.
• There are two types of plant parasitic nematodes; Ectoparasitic nematodes which remain in the soil and feed at the root surface and Endoparasitic nematodes which enter roots and can live and feed within the root.

5. Nematodes as bioindicators of soil health or condition:

• Population of nematodes depends on condition of soil, climate, seasons, crops etc. Therefore indicates the condition of soil health.
• Analysis of the diversity and complexity of nematode communities in the soil is a valuable tool, which indicates soil biological fertility, or soil health.

6. Nematodes dispense microbes for infection:

• Nematodes carrying live and dormant microbes on their surfaces and in their digestive systems and help in distribution of bacteria and fungi throughout soil and also in rhizosphere region.  
• Sometimes nematodes acts as a vector for plant viruses. Eg.  Nepo viruses, Tobraviruses.

7. Human and animal pathogenic nematodes;

• Nematodes are pathogenic to animals and human.
• Eg. Ascaris,

Role of nematodes in Agriculture: Importance of nematodes in soil

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• Cellulose is relatively resistant polysaccharide, found in cell wall of plant cell.
• Cellulose is a linear polymer of β-D-glucose in which glucose units are linked together by β-1,4-glycosidic bond.
• It is the most abundant organic matter found in nature. In plant it occurs in association with lignin and hemicellulose.

Mechanism of cellulose decomposition:

• Pathway of cellulose decomposition follows series of enzymatic reactions.
• Enzymes responsible for cellulose decomposition is cellulase.
• Cellulase is a complex of three enzymes (ie. C1 enzyme, β-1,4-glucanase and β-1,4-glucosidase).
• Series of enzymatic reaction occurs outside the microbial cell in which complex cellulose is decomposed into free glucose molecules by extracellular enzymes.

Step I: hydrolysis by C1 enzymes:

• C1 enzyme hydrolyses native cellulose polymer to form smaller fragments.
• C1 enzyme is only found in true cellulolytic microorganisms.

Step II: hydrolysis by β-1,4-glucanase enzyme:

• β-1,4-glucanase hydrolyze the smaller fragments of cellulose to form even smaller fragments such as disaccharides, tri-saccharides etc.
• There are two types of glucanase ie. Endo-glucanase and Exo-glucanase.
• Endo-glucanase randomly cuts the fragments somewhere in the middle whereas exo-glucanase sequentially release glucose molecule from one end of the fragment.
• Some free glucose unit as well as disaccharides, tri-saccharides and other oligosaccharides are produced by the action of β-1,4-glucanase.

Step III: hydrolysis by β-1,4-glucosidase enzyme:

• β-1,4-glucosidase hydrolyses di, tri and oligosaccharides to form free glucose molecules.

Step IV: metabolism of glucose:

• Free glucose molecules then enter into microbial cell and metabolized by glycolysis to form pyruvate.
• Depending upon types of microorganisms and the condition of environment, pyruvate is converted into CO2 and water or ethanol or any organic acids

Examples of Cellulolytic microorganisms; Cellulose decomposers

1. Bacteria: Bacillus, Cellulomonas, Clostridium, Cytophaga, Polyangium, Pseudomonas etc
2. Fungi: Aspergillus, Alterneria, Fomes, Fusarium, Myrothecium etc
3. Actinomycetes: Micromonospora, Nocardia, Streptomyces, Streptosporangium etc.

Factors affecting cellulose decomposition in soil:

• Various environmental and other factors affects rate of cellulose decomposition in soil by microorgansims, some of them are;
• i. Addition of available Nitrogen:
  • Additionof inorganic nitrogen compounds such as ammonia, nitrite or easily decomposable nitrogen compounds like aminoacids and proteins increase the rate of cellulose decomposition by microorganisms.
  • Microorganisms require both carbon and nitrogen for biosynthesis of their cellular materials. Therefore, microbial decomposition of cellulose cannot occurs without nitrogenous sources.
• ii. Temperature:
  • Cellulose decomposition can occurs from temperature near freezing to above 65°C because both psychrophiles and thermophiles are involved in cellulose degradation.
  • But rate of cellulose decomposition is maximum in mesophilic range of temperature of 25-30°C because most cellulolytic microbes are mesophiles.
• iii. Aeration:
  • In anaerobic soil, anaerobic bacteria like Clostridium decompose cellulose and in aerobic soli mainly fungi and aerobic bacteria take part in decomposition of cellulose.
  • Rate of cellulose is higher in aerobic soil.
• iv. Moisture:
  • Excessive moisture brings anaerobic condition in soil. Therefore, rate of cellulose decomposition is slower in water logged soil.
• v. pH:
  • In neutral to alkaline soil, bacteria and actinomycetes mainly take part in cellulose decomposition.
  • In acidic soil, fungi are dominant cellulose decomposers.
  • Rate of cellulose decomposition is slightly higher in acidic soil than alkaline and neutral.
• vi. Addition of organic matter:
  • Addition of easily decomposable organic matters increase the rate of cellulose decomposition.
  • If only cellulose is present in soil, microorganisms cannot multiply fast so that the rate of decomposition become slower.
  • If small amount of easily decomposable organic matter is added initially in soil, microorganisms rapidly multiplies and grow their numbers. Furthermore cellulose is degraded rapidly when the easily decomposable organic matter is exhausted.
• vii. Lignin:
  • Lignin slows the rate of cellulose decomposition.
  • Lignin itself is not toxic to cellulolytic microbes. Inhibition of cellulose decomposition by lignin is due to its close association to cellulose in cell wall.

Cellulose decomposition; microbial decomposition of cellulose in soil

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Difference between Rhizosphere and Phyllosphere

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• The soil surrounding the plant root where root exudate migrate and microbiological activity is exceptionally high is called rhizosphere.
• The surface of root is called rhizoplane.
• Plant root produce and release various exudates containing sugar, aminoacids, organic acids, fatty acids, vitamins, nucleotides and other organic matters that promotes growth of microorganisms.
• Therefore rhizospheric soil is characterized by greater number of microorganisms than soil away from plant roots.
• The intensity of rhizospheric effects depends on the distance to which root exudates can diffuse. The number of microorganisms decreases continuously as the distance from the plant root increases.
• The term rhizosphere to soil ratio (R:S) indicates number of microbes in rhizospheric soil divided by number of microbes in soil free of plant root.
• R:S ratio is greater for bacteria (20:1) and less for fungi and actinomycetes.
• Effects of rhizosphere is almost negligible for algae and protozoa. It is because algae are photosynthetic and do not depends upon organic matter present in root exudates.
• On the other hand most bacteria cannot utilize relatively resistant to organic matter of soil and depends on easily available decomposable matter of root exudates. Therefore number of bacteria is exceptionally high in rhizosphere.

Examples of rhizospheric microorganisms:

• Large number of bacteria, fungi and actinomycetes are found in rhizosphere.
• i. Bacteria:
  • Many nitrogen fixing bacteria and phosphate solubilizing and other bacteria are found in rhizosphere.
  • For examples: Pseudomonas, Arthrobacter, Azotobacter, Agrobacterium, Flavobacterium, Cellulomonas, Rhizobium, Clostridium etc.
• ii. Fungi:
  • Some fungi are found associated with root forming mycorrhiza and other occurs freely in soil.
  • For examples: Marticella, Cephalosporium, Tricoderma, penicillium, Gliodadium, Gliomastix, Fusorium etc.
• iii. Actinomycetes:
  • Frankia, Derxia etc

Factor affecting rhizospheric microorganisms:

Various factors affect rhizospheric microbes and some of them are;

i. Proximity of soil to root:

• The number of rhizospheric organisms is greater near the root and their number continuously decreaseswith increase in distance from the root.
• It is because concentration of organic matter released by root in exudates decreases with increases in distance from the root.

ii. Temperature and light intensity:

• Low temperature and low light intensity decreases the rate of exudate secretion from the root so that number of rhizospheric organisms decreases.
• On the other hand number of microbes in rhizosphere increases when temperature and light intensity increases as multiplication rate is high.

iii. Type of soil:

• Types of soil also influences rhizospheric effects.
• For example; R: S ratio is very high in sandy soil and low in clay soil. It is because sandy soil contains very little or no organic matter and the root region is the only place where organics matter is available and microorganisms can grow. Therefore, number of microbes is high around root in sandy soil.
• On the other hand in fertile soil such as clay soil, organic matter is sufficiently available for growth, so microorganisms need not to depend on the root exudates for growth.

iv. Age of plant:

• With age of plant, rate of exudates secretion is altered so that number of rhizospheric microbes changes.

v. Types of plant and location of root:

• Location of root affects number of rhizospheric microbes.
• Root cap and regions of root from where lateral root arises are primary sites of exudate secretion. Therefore, number of microbes is comparatively high around these locations.
• Amount and type of exudates secretion differs with species of plant that influences growth of rhizospheric microbes. For example; some plant root release antimicrobial chemicals such as glycosides, hydrocyanic acids and several antifungal agents that inhibits rhizospheric microbes.

vi. Depth of root:

• In general number of rhizospheric microorganisms decrease with increase in depth of root, which is mainly due to anaerobic condition.

vii. Root respiration:

• Plant root release carbon-dioxide during respiration that make the soil acidic.
• Acidity of soil decrease number of rhizospheric bacteria.

viii. pH of soil:

• pH of rhizosphere become acidic due to root respiration and by oxidation of sulphur caused by Thiobacillsu spp. Acidification of rhizospheric soil decrease number of microorganisms.

ix. Pesticides and antibiotics:

• Spray of pesticides and antibiotics on agriculture crops decreases the number of rhizospheric organisms.

Role of rhizospheric microbes:

• Rhizospheric microorganisms are important for plant growth. They promote plant growth by various ways as given below;
• Some rhizospheric bacteria such as Rhizobium, Azotobacter, Clostridium etc. fix atmospheric nitrogen and make it available for plant gowth.
• Many phosphate solubilizing microbes such as Bacillus polymyxa found in rhizosphere release free phosphate from inorganic salt of phosphate. Free phosphate is important nutrient for plant growth.
• Several rhizospheric microbes (Azotobacter, Arthrobacter, Pseudomonas, Agrobacterium) produce growth hormone such as Gibberllin, Indole acetic acid (IAA) etc that promote plant growth.
• Many rhizospheric fungi are associated with plant root in the form of mycorrhiza. Mycorrhizal fungi promote plant growth by various ways.
• Rhizospheric microbes induce development of lateral root, root hairs development and mucilage secretion from plant root.
• Some rhizospheric microbes produces antibiotics and other antimicrobial chemicals that inhibit plant pathogens. Some time it may inhibit beneficial N2 fixing and phosphate solubilizing bacteria.
• Microorganisms also increase rate of exudate secretion. Exudate secretion from plant root helps in formation of soil aggregate that improve soil fertility.
• Some rhizospheric microbes eg Pseudomonas produces Siderophore. Siderophore is a chelating agent that tightly bind iron and make it unavailable for growth of pathogenic microorganisms.

Effect of plant root on rhizospheric microbes;

• Plant root usually promote growth of rhizospheric microbes. Sometimes plant root give minor unwanted effect to microorganism. Some of them are;
• Plant root produce exudate containing carbohydrate, aminoacids, nucleotide, vitamins etc that serves as food for growth of rhizospheric microbes.
• Some plant root produces antimicrobial chemicals such as glycosides, Zhydrocyanic acids and antifungal agents that inhibits growth of rhizospheric microorganisms.
• Plant root release CO2 during respiration that make habitat acidic and anaerobic.
• Some plant root produce chemicals that bring fungistasis. Fungistasis is referred to the inability of spore to germinate. For eg. Root of Allium produce alkylcystein sulfoxide that inhibit germination of sclerotia (spore) of Sclerotium capivarum.

Microbiology of rhizosphere and Rhizosphere effect

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• The surface of leaf is called phylloplane and the zone on leaf inhibited by microorganisms is called phyllosphere.
• The plant leaves are exposed to dust and air current that result in the establishment of typical flora on the surface of leaf.
• Cuticles, wax, leaf appendages and leaf exudates help in anchorage of microorganisms on the surface of leaf. These microorganisms may die, survive, multiplies on leaf depending on extent or influence of leaf exudates on microorganisms.
• Leaf exudates contains amminoacids, glucose, fructose and sucrose that provides nutrition for growth of phyllospheric microorganisms.
• Phyllospheric microorganisms also promote growth of plant by various ways.

Examples of phyllospheric microorganisms:

• The surface of leaf contains large number of bacteria, fungi, actinomycetes and algae as given below;
• Bacteria:
  • Beijerinckia, Erwinia, Azotobacter, pseudomonas, Pseudobacterium, Phytomonas, Serratia etc
• Blue green algae
  • Anabaena, Calothrix, Nostoc, Scytonema, Tolypothrix
• Fungi:
  • Alternaria, Cladosphorium, Cercospora, Penicillium, Saccharomyces,
• Actinomycetes:
  • Streptomyces

Factors that influences phyllospheric microorganisms:

• Various environmental factors and other conditions affects growth of microorganisms on the surface of leaf. They includes;

1. Leaf exudates:
   • Leaf exudates contains many microbial growth factors such as amino acids, glucose, fructorse, sucrose etc that facilitates growth of phyllospheric microorganisms.
   • Moisture released during transpiration provides water for growth of microorganisms on leaf.
2. Position of leaf:
   • Position of leaf also affects phyllospheric microorganisms.
   • The surface of leaf which is directly exposed to sunlight contains relatively low microorganisms than the other surface of leaf which is shaded. It is due to antimicrobial effect of UV light
   • Furthermore, surface of leaf which is directly exposed to sunlight is relatively dry and moisture is less available for the microbial growth.
3. Leaf appendages:
   • Leaf appendages such as trough and veins affect growth of phylospheric microorganisms.
   • Trough represents shallow depression on the surface of leaf. Microorganisms attach easily and grow in trough. Therefore the number of microorganisms is relatively higher on depressed area on the leaf.
   • Veins present in leaf form depression in one surface and hence influence number of microorganisms.
4. Stomatal cavities:
   • In stomata water is in the form of drops that provides moisture for growth of phyllospheric microorganisms. Therefore number of microorganisms is relatively high in and around the stomatal cavities.
5. Age and types of plants:
   • With age of plant, rate of secretion of exudates is altered that affect growth of phyllospheric microorganisms, number of stomata and hence rate of transpiration is different in different plant species that affects growth of phyllospheric microorganisms.
6. Environmental pollutants:
   • Environmental pollutants deposited on the surface of leaf in the form of dust influence growth of phyllospheric microorganisms.
   • For examples; cement and fertilizers are anti-microbial and they decrease number of phyllospheric microorganisms.
7. Insecticides, Antibiotics, herbicides etc:
   • Insecticides, antibiotics, herbicides and other chemical sprayed on leaf on agricultural crops are antimicrobial and decreases the number of phyllospheric microorganisms.
8. Phytoalexin:
   • Phytoalexins are the chemicals produced by plants leaf that inhibits phyllospheric microorganisms as well as other microorganisms.
   • Nature of phytoalexin differ from plant to plant.
   • Phytoalexin is produced by plant in response to certain stimuli called elicitor.
   • Elicitors may be biotic or abiotic factors.
   • Biotic elictors includes various chemicals produce by pathogenic and other phyllospheric microorganisms.
   • Abiotic elicitor includes Uv light, cold, tissue damage etc. When phytoalexin is produced by leaf, number of phyllospheric microorganisms decreases.

Role of phyllospheric microorganisms:

• Phyllospheric microorganisms are important for plant growth. They promote growth of plants by various ways as given below;
  • Some phyllospheric microorganisms such as Cyanobacteria and nitrogen fixing bacteria such as Azotobacter fix atmospheric Nitrogen and provide it to plant growth.
  • Phyllospheric microorganisms produce various plant growth hormones such as indole acetic acid (IAA), which is used by plant for their growth.
  • Phyllospheric microorganisms provide stimulus for production of phytoalexin by plants. Phytoalexin is the defensive chemical produced by plants that kill pathogenic as well as other microbes.
  • Phyllospheric microorganisms decompose leaf and help in formation of humus after falling of leaves from plant.
  • Some phyllospheric organisms have antagonistic effects against fungal pathogen and hence protect the plants from fungal diseases.
  • The phyllospheric microorganisms colonize the surface of leaf forming a layer. Therefore these organisms compete with pathogenic microorganisms for habitat and nutrients.
  • Some phyllospheric microorganisms degrade wax and cuticle of leaf and damage it.

 Effect of plants on phyllospheric microorganisms:

• Surface of leaf provides natural habitat for growth and multiplication of phyllospheric microorganisms. Various leaf appendages as well as wax, cuticles and leaf exudates help in attachment of phyllospheric microorganisms to the surface of leaf.
• Aminoacids, glucose, sucrose etc present in leaf exudates provides nutrition for growth of phyllospheric microorganisms. Similarly water released during transpiration provides moisture for growth.
• Plants usually support growth and activities of phyllospheric microorganisms. However sometimes plant produce phytoalexins that kills many phyllospheric microorganisms.

Microbiology of Phyllosphere

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