Introduction to Soil and Agricultural Microbiology 9789350432839, 9789350243107

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INTRODUCTION TO SOIL AND AGRICULTURAL MICROBIOLOGY Dr. G. Prabakaran, M.Sc., Ph.D., P.G.D.M.P. Head, P.G. Department of Biotechnology,

A.V.S. College of Arts & Science, RamaJingapuram, Salem - 636 106.

TAMllNADU.

HI! GflimalayaGpublishingGflouse MUMBAI • DELHI • NAGPUR • BANGALORE • HYDERABAD

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Pioneers of Soil Microbiology

Martlnus Beljerinck (1850 -1931)

Sergei Winogradsky (1856 -1953)

"This page is Intentionally Left Blank"

Dedicated to

my beloved wife Mrs. Latha Prabakaran,

M.A.. B.L..

and My Family Members

"This page is Intentionally Left Blank"

CONTENTS

1.

2.

3. 4. 5. 6. 7.

Lithosphere Microbial Flora of Soil (i) Soil Microorganisms (ii) Factors Influencing Soil Microbiological Population (iii) Microbiological Test for Soil Fertility Phytohormones Bio-Geo-Chemical Cycling Biological Nitrogen Fixation Microbial Interactions Phytopathology (i) PhytopathogeniC Fungi (ii) Phytopathogenic Bacteria (iii) Phytopathogenic Viruses (iv) Phytopathogenic Nematodes (v) Plant Disease Control

8.

Biocide~

9.

Biofertilizers Agricultural Biotechnology - Further Reading -Appendix - Glossary -Index

10.

1 17

37 46 58

73 87

123 129 140 147 149

153 157

"This page is Intentionally Left Blank"

LITHOSPHERE The solid component of earth is called Lithosphere. It is composed of three layers which are core, mantle & crust. Core is at the centre of the Lithosphere. Having a diameter of about 2,500 kms from the centre, it is in vapourised form. Mantle extends about 2,900 kms above the core. It is in molten state. Crust is the outer-most solid zone, which is about 8-40 kms above the mantle. Jt is very complex and consists of all types of living organisms. The word soil is derived from the latin word 'Solum', which means earthy material. [Pedology (Pedos-earth) or Edaphology (edaphos-soil)].

Soil and its Properties Soil is a stratified mixture of both inorganic and organic material, whicQ is basically derived either from parental bedrock or from dead and decaying organic matter. Air and water are normally present in the soil.

Composition of Soil Soil serves as a natural medium for the growth of plants. The soil system is not only chemical and geological but also biological and physical. Fertile soil (the term fertility refers to the inherent capacity of the soil to supply nutrients to plants in aoequate amounts and in suitable proportions) consists of four major components. These are:

Introduction to Soil and Agricultural Microbiology

2

(a) Mineral Materials (b) Organic Matter (e) Soil Water (d) Soil Air The soil is composed of about half solids and half pore space. The solid space consist of mineral material and organic matter which is 45% and 5% respectively of the total soil volume. The pore space is divided roughly into half. It consists of water and air which constitute 25% and 25% respectively of the total soil volume.

~ Organic Matter 5%

KKd

Mineral Materials

~=~] Soil Air •

45%

25%

Soil Water

25%

Fig. 1.1 Composition of soil

(a) Mineral Materials The soil consists of various mineral materials or nutrients, which are inorganic by nature. These nutrients are very essential for normal growth and development. There are two types of nutrients: (a) Macro elements C, H, 0, N, P, K, Mg, Ca, S (b) Micro elemeots __Mn, 80, Mo, Zn, Cu, Fe, CI Both macro elements and micro elements (trace elements) are required in large quantities, and these are obtained from various sources. Through photosynthesis, elements like C,H,Q build up tligher molecules like carbohydrates. If anyone of these elements is present in lesser amounts, the plant-growth will b9 retarded.

(b) Organic Matter Organic matter is not readily decomposed by micro-organisms. lndeed the living micro-organisms and the dead remains are of such a large magnitude that they contribute significantly to the organic matter

3

Lithosphere

of the soil. Nitrogen is present in organic combinations as proteins, amino acids, etc. Phosphorus is present as phospt,ates and in nucleic acids. Sulphur is present in mineral complexes such a gypsum, pyrite and in organic matter. The absorption of nutrients, by roots, from the soil is a very complex process influenced by numerous factors in the plant and soil. Solutes are transferred across the cytoplasmic membrane by means of Active transportation. The minute network of plant root and root hairs are in intimate contact with soil particles and colloids.

(c) Soil Water Soil water is classified into hygroscopic, capillary and gravitational waters. Hygroscopic water: Hygroscopic water is that water which is absorbed by a dry soil sample when exposed to water vapour. Capillary water: Minute soil particles are in the form of continuous capillary tubes and are capable of retaining a certain quantity of moisture against the gravitational forces. Gravitational water: The water which moves out of the soil due to gravitational pull is known as gravitational water. As the water moves into the soil, several elements go into the solution or are suspende~ in it.

(d) Soil Air Soil air is either absorbed by colloidal particles or dissolved in soil water. When there is an excess of water, the humidity percentage of' soil air may be as high a~ 100. The high humidity of soil air may encourage microbial activity. This comprises nearly 30% of the pore space in normal soil. The gaseous phase of soil consists of CO 2, O 2 nitrogen. Some of the gases like CO2 normally dissolve in water.

Soil Reaction Soil reacts in three ways, acidic, neutral and alkaline. Acidity denotes an excess of H+ ions over OH- ions, whereas alkalinity denotes an excess of OH- ions over H+ ions. At neutral reaction, the H+ and OH- ions concentrations are equal. Soil reaction is customarily expressed as pH, which is the negative logarithm of the hydrogen ion concentration. It may be shown as follows, assuming (H) is the concentration of the hydrogen ion. pH

1 = logH+

Introduction to Soil and Agricultural Microbiology

4

Range in pH The pH of mineral soils varies from values of 3 or less in very acid soils of some coastal areas to more than 10 in alkali soils of some arid and semi-arid areas.

Soil Formation (or) Pedogenesis (Genesis of Soil) The inorganic or mineral constituents of soil are derived from the parental material, the soil-forming rocks by weathering, while organic components of soil are formed either by decomposition of dead remains of organisms or by metabolic activities. Soil-forming rocks are basically classified into three types. These are: (i) Igneous rocks: Examples of igneous rocks are granite and diorite. (ii) Sedimentary rocks: Examples of sedimentary rocks are limestone and sand stone. (iii) Metamorphic rocks: Examples of metamorphic rocks are slate and marble.

Soil formation takes place by various processes. These are :

(a) Weathering 'Jf soil forming rocks. (b) Mineralization and Humification. (c) Formation of organo-mineral complexes.

(a) W,athering Process Parental rocks are dis-integrated by certain agents. These are physical, chemical and biological agents. By means of these agents, soil-forming parental rocks are broken down into smaller particles, called Regoliths. These regoliths are finally converted into mature soil. Weathering of soil-forming rocks includes the following :

(i) Physical weathering (ii) Chemical weathering (iii) Biological weathering

(i) Physical Weathering: Physical weathering agents are basically climatic in nature. [Wall Work (1970)]. The following agents are involved in climatic weathering of rocks : (a) Temperature (b) Water (c) Ice (d) Gravity (e) Wind

Lithosphere

5

(a) Temperature: Temperature causes the break-down of some heterogenous type of rocks, due to differential expansion and contraction coefficient. The temperature of the soil depends on the extent of energy absorption and loss. Mineral particles expand when the temperature is very high, and contract when the temperature decreases. Due to internal tensions, cracks are formed in between the rocks, leading the parental material to break into smaller particles. (b) Water: Water exists in various forms like rain water and wave water. It causes the mechanical weathering of soil-forming rocks. (1) Rain water: The natural water falls on the rock surface either by rain drops or hail storm. Due to the beating effect, the rocks are converted into finer particles. (2) Torrent water: The rocky materials are converted into smaller particles by water rolls. The rolling torrent water grinds them into finer particles. (3) Wave action: It takes place in sea shores. The rapidly striking water waves dislocate the solid particles of varying diameters. The debris is settled at the bottom and is finally converted into marine soil. (c) Ice: Ice is a highly effective physical weathering ag~nt. It converts rocks into smaller particles. Rocks are modified as a result of the glacial movements. (d) Gravity: Rocks are fragmented by abrasion caused by gravitational activity. Earthquakes convert rocks into finer particles. (e) Wind: It carries suspended sand particles, causing the abration of exposed rock. It acts like a mechanical carrier. Due to the wind-action, fine suspended particles are transported over long distances. (ii) Chemical Weathering: It occurs simultaneously with physical weathering and continues much beyond. During the process of Chemical weathering, parental mineral materials are converted into new mineral complexes by decomposition. For example, Feldspar ~ Clay Chemical weathering requires conditions like moisture and air. So, it is not effective in dry places (desert) The process of chemical weathering takes place through following reactions. (a) Solution (b) Hydrolysis (d) Reduction (c) Oxidation (f) Hydration (e) Carbonation

Introduction to Soil and Agricultural Microbiology

6

(a) Solution: Solution helps in the removal of water soluble minerals from weathered rocks. For example, soluble rocks like gypsum, limes through solvent action of water which increases in the presence of CO 2 and organic acids formed by decay of organic remains of plants and animals. (b) Hydrolysis: When the water molecules react with strong bases it releases some hydroxides. For example, Calcium, Magnesium, aluminium, Iron etc. These elements are absorbed by the plants in the soil. Orthoclase ~ Kaolin (c) Oxidation: When oxygen reacts with minerals, it produces oxides. It normally takes place in well-aerated soil. For example, ferrous oxide reacts with oxygen resulting in the formation of ferric oxide.

4FeO + D2

~

2Fe203

(d) Reduction: It occurs normally in deep zones of the earth's crust. During this process, oxygen is removed from the minerals. For example, Ferric oxide reduced into ferrous state

2Fe2D3

~

4FeO + 02

(e) Carbonation: In carbonation, water molecules combine with CO2 to form carbonic acid, which again reacts with some hydroxides of calcium and magnesium to form carbonates and bicarbonates. (a) C02 + H20 ~ H2C03 (Carbonic acid) (b) Ca(OH)2 + C02 ~ CaC03 + H20 + C02 ~ Calcium Hydroxide

Calcium Carbonate

Ca(HC03)2 Calcium Bicarbonate

(f) Hydration: In Hydration, water molecules attach themselves to particular rock materials. Due to this chemical reaction, the volume of . parental material increases and it becomes soft and easily weatherable.

2Fe203 + 3H20 ~ 2F9203· 3H20 Hematite

Limonite

(iii) Biological Weathering: It is carried out by some living organisms like bacteria, fungi, protozoans, nematodes, lichens and mosses. They initially transform the rocks into a dynamic system. Later, they alter the physical structure and mineral composition of the rock. For example, Lichens.

Lithosphere

7

The over-growth of lichens may cause cracking or flaking in the rocky surface. It also extracts some nutrients like P,S,K, Ca, Mg, Na, Fe and AI.

(b) Mineralization and Humification During the early stages of pedogenesis, the organic content of the primary soil or embryonic soil is not very high. With the decomposition of inorganic matter, followed by organic matter, the minerals and organic compounds become readily available in the soil. These compounds can be easily absorbed and utilized by the plants. The minerals are normally decomposed by living organisms especially by micro-organisms. Complex molecules like car.bohydrates, lipids, proteins, waxes and resins are broken down into simple molecules like carbon dioxide, minerals, water and salts. This process is known as Mineralization. The residual amorphous, incompletely decomposed, black coloured organic matter which undergoes mineralization is called humus. The process of humus formation is called humification. The soil is composed of minerals, organic matter and living organisms. The organic matter compris"es residues of plant and animals at different stages of decomposition. It is found mostly in the upper layers of the soil and it influences the physical and chemical properties of soils. The primary source of organic matter is plant tissue. Animals are the secondary sources of organic matter. Humus can be defined as a ligno - protein complex or an amino acid - lignin complex containing approximately 45% lignin compounds, 35% amino acids, 11 % carbohydrates, 4% cellulose, 7% hemi cellulose, 3% fats, waxes and resins and 6% miscellaneous substances including plant growth substances. Muller (in the year 1879), has recognized two kinds of humus, Mor and Mull Mor humus is acidic in nature. It supports an abundant fungal growth and low number of bacteria.

Introduction to Soil and Agricultural Microbiology

8

Pathways in Organic Matter Decomposition Living Organisms (Plants, Animals)

1 1 Carbohydrates, Proteins Organic Residues

Lignin, Fats, Waxes, Resins etc. Processes (Mineralization, Immobilization) 1Microbial Humus

Mull humus is neutral or slightly alkaline in nature and it supports an abundant microflora of bacteria. The Modern humus is an intermediatory between the two types of humus. It contains richer and varied fauna. To emphasize the role of living plants and animals in soil formation, Taylor proposed a formula in the year 1930, which goes as follows:

I S =M (C + V + VA + A) t + 0 S C

A

= = = =

Soil

M

Climate

V

Animals

VA

time

D

= = = =

Parent Material Vegetation (Plants) (Vegetation + Animals) Deposition

(c) Formation of Organo-mineral Complexes Some colloidal humus particles may bond with mineral particles to form organo-mineral complexes. Organa-mineral complexes are formed by two methods. They are :

(i) Electro-chemical bonding (ii) Cementing (i) Electro-chemical bonding: It is the aggregation of negatively charged colloidal clay, humus particles and metallic ions. (ii) Cementing: The action of the substances on the surface of soil particles effectively glues them together. The weathering process leads to formation of mature soil. The mature soil becomes a complex system of living and non-living materials.

9

Lithosphere

The mature soil can be classified into various types, namely; (i) Primary soil (ii) Secondary soil (iii) Residual soil The sril which is formed by weathering of soil-forming rocks is called primary soil. It is also known as embryonic soil. Soil is also transported from various places by gravity, water, ice and wind. This type of soil is called secondary soil. It is also known as transported soil. Soil material transported from one area to another, is known as eolian (loess) The soil which develops in situ above parental bedrock is called residual soil. It is also known as sedentary soil.

Soil Structure or Profile The vertical section of the earth's crust shows different layers or horizons. Each of the horizons vary in thickness, texture, structure, colour etc. In general, soils have the following four horizons, which are: (i)

Organic or 0 horizon

(ii) Mineral A horizon

(iii) Mineral B horizon

(iv) Mineral C horizon

Mineral A and B horizons form true soil. The R-horizon at the bottom is the consolidated bedrock. Each horizon is further sub-divided.

Profile of Soil Organic horizon or 0 horizon is the surface layer above the mineral layers and composed of fresh or partially decomposed organic matter. It consist of both Mor and Mull humus. Life is more abundant in this region. Carbon is also present. Organic horizon is sub-divided into 0" O2. 0 1 is the uppermost layer. It consist of dead leaves, branches, flowers, fruits and dead remains of animals. Below the 0 1 region is the O2 layer. The decomposition process starts in this region. Microbes like bacteria, fungi, actinomycetes ~re found in this region. Mineral horizons are of three types A,B,C. A-horizon: The A-horizon is sub-divided into AI, A2 and A3 .

10

Introduction to Soil and Agricultural Microbiology

AI is the region where the organic matter is very rich. This dark or brown coloured, amorphous organic matter mixes with mineral matter. A2 is the region where the mineral particles are larger in size and number, with small amounts of organic matter. Due to heavy rainfall, this mixture gradually turns into a light colour. B-horizon: It is also known as sub-soil. It consist of clay, iron, aluminium and humus. (It leads to formation of block or columnar structure.) The B-horizon is sub-divided into B" B2, B3 • C-horizon: It lies between the Band R region. It comprises of weathered materials known as regoliths, which are light coloured. R-horizon: It is the consolidated bedrock.

..•

•

•

.. .. .. I!-

01 O2

}

Organic Horizons

AI A2 A3 Bl

True Soil Mineral Horizons

B2 B3

-----t.~ C (Regolith)

•• •••• • • •• •••••• • •• •• • • •

- - - -..~~ R (Bed Rock) Fig. 1.2 Profile of soil

Morphology of Soil It includes the variations in texture, structure and colour. Texture: The texture of a soil is determined by the formation of different-sized soil particles. The soil particles have been classified into gravel, sand, silt and clay.

The gravel consists of coarse particles larger than 2.0 mm. Sand ranges from 0.02 to 2.0 mm in diameter. Silt consists of particles from 0.002 to 0.02 mm in diameter. The clay particles are ranged below 0.002 mm in diameter and are col!oidal in nature.

Lithosphere

11

Soil is a stratified mixture of various particles. Based on this, soils can be classified into five textural groups. These are : (a) Coarsely textured soils (b) Moderately coarse soils (c) Medium textured soils (d) Moderately fine textured soils (e) Fine textured soils. TABLE 1.1 SOIL TEXTURE

Common names

Texture

Basic soil Textural class names·

Sandy soils

Coarse

Sands, Loamy sands.

Loam soils

Moderately coarse Medium

Sandy loam, Fine sandy loam. Very Fine sandy loam Loam, Silt loam, Silt. Sandy clay loam, Silty clay loam, Clay loam.

Moderately fine Clayey soils

Fine

Sandy clay, Silty clay Clay".

* Common names are used to describe soil texture in relation to the basic soil textural class names.

** V.S. Department of Agriculture Classification System (NYLE. C. BRADY, 1984).

Sand, silt, clay soils are classified into various classes. For example, sandy soil, loamy sand, loam, silt sandy, clay loam etc. Structure: Soil particles like sand, silt, clay and gravel join together in various sizes to form peds. Peds are also known as aggregates. The arrangement of these aggregates in the earth's crust is called soil structure. Soil peds may be classified into granular, crumb-like, plate-like and prismatic. The structure of the soil is influenced by factors like micro-organisms, organic matter, air, moisture and root growth etc. Colour: The colour which is derived or inherited from parental material is known as Iithochromic.

The colour which is due to soil-forming processes is known as acquired or genetic colour.

The colour is sometime responsible for the functions of a soil. Colour also helps in identification of the various soil types.

Introduction to Soil and Agricultural Microbiology

12

Depending upon the organic matter, soil can be classified into dark coloured, light coloured, dark brown, red and yellow soils. Soil colour influences the soil temperature. Dark coloured soils absorb more heat than light coloured soils. .

Physical Properties of Soil Soil possesses many physical properties, like:

(a) Density: The average density of soil is 2.65 gms/ml. It may vary from place to place due to the weathering process. (b) Porosity: The percentage of soil volume occupied by pore space is called porosity. The spaces present between soil particles in a given volume of soil are called pore spaces. Pore spaces may be divided into -

(iJ (ii)

Micro-pore spaces Macro-pore spaces.

Micro-pore spaces are also called capillary pore spaces. They hold lots of water and restrict the free movement of water and air. Macropore spaces are non-capillary. They hold little water and allow free movement of water and air.

(e) Permeability: The movement of water through pore spaces is known as soil permeability. (d) Temperature: Due to solar radiation, the soil gets heat energy. Heat absorption varies between the different soil particles. For example Black soils absorb more heat than white soils. Sometimes, the soil is cool en because of water evaporation. (e) Water: Water is an important solvent and also acts as a transporting agent. According to Bouyoucos (1920), soil water is classified into the following types.

(i) Gravitational water: The accumulated excess water of large soil spaces is called gravitational water. When it further percolates down and reaches the parental rocky surface, it is called ground water. (ii) Capillary water: Due to the surface tension, water moves from one place to another. When the gravitational, ground water is drained, what remains is capillary water. (iii) Hygroscopic water: Soil particles retain some water so tenaciously that the plants cannot absorb it. It is called hygroscopic water,

13

Lithosphere

(iv) Combined water: Due to the chemical forces between molecules, combined water is formed. It is of no ecological significance. The total amount of water present in the soil is called holard. The amount of water that can be absorbed by plant roots is called chresard. The amount of water that cannot be absorbed by plant roots is called echard.

(f) Air: Soil air differs f~om atmospheric air in having more of moisture and less of oxygen. The soil air consist of three main gases, O2, CO2 and N2 • (Table 1.2). The composition of soil air and atmospheric air is as follows TABLE 1.2

Gases

Sr. No.

Volume in

1.

Oxygen

Soil Air 20%

Atmospheric Air 21%

2.

Nitrogen

78.6%

78.03%

3.

Carbon dioxide

0.5%

4.

Argon

0.9%

0.03% 0.94%

Chemical Properties of Soil Soil consists of various chemical compounds namely organic matter and inorganic elements. It exhibits certain significant chemical properties.

(i) Organic matter: The major constituent of organic matter is humus. It contains proteins, amino acids, purines, pyrimidines, hexose sugars, aromatic compounds, fats, alcohols, waxes, oils, lignin, tannions etc. (ii) Inorganic elements: The major constituents of inorganic compounds in the soil are Ca, Mg, K, AI, Si, Fe and Na. The minor constituents of inorganic compounds are B, Mn, Cu, Zn, Mo, Co, I etc. (iii) Colloidal properties: Soil is composed of crystalloids and colloides which exhibit absorption, coagulation, Tyndall phenomenon, Brownian movement etc. (iv) Soil pH: Soil pH varies from area to area. Some soils are neutral, some are acidic and some are basic. The pH value of soils normally ranges between 2.2 and 9.6.

Introduction to Soil and Agricultural Microbiology

14

In the high rainfall areas such as Kerala, Western Ghats, West Bengal and Assam the soils are acidic in nature. (The pH value of acidic soils is below 5.5/5.6.) The basic soils (The pH value of basic soils is upto 8.5) occur in Punjab, Bihar, Orissa, Chennai, A.P. and Delhi. The slightly acidic or neutral soils are suitable for the growth of most Iivinp organisms and plants because high acidic and high alkaline soils create injurious effects on plants and micro-organisms.

Major Soil Types Based on physical, chemical and biological properties soils are classified into various types. Geographically soils are divided into three major groups, which are: 1. 2.

Mature soils of peninsular India Alluvial soils of Indo-gangetic plain

3.

Scanty soil of Himalayas TABLE 1.3 CLASSIFICATION OF SOILS

S. No.

I.

Soil Order Zonal soils

Soil sub-order 1. Humid region

Soil groups (i)

Red yellow podozolic soils

(ii)

Alluvial soils

(iii) laterised red soils (iv) Non-Iaterised red soils 2. Arid region

II.

Interzonal soils

1. Calcimorphic 2. Holomorphic 3. Hydromorphic

Grey and clay soils

(i)

Dark soils

(ii)

Red Desert soils

(i)

Red soils

(ii)

Black soil

(i)

Saline soil

. (i) (ii)

III.

(v)

Organic soils Ground water soil

(iii) Planosols Azonal soils

1. Regosols 2. Lathosols 3. Alluvial soil

Lithosphere

15

In India the alluvial soils occur> in Punjab, Bihar, West Bengal, Godavari and Cauvery. The black cotton soils occur in Maharastra, Andhra, Assam, Orissa and Tamil Nadu. The Red soils occur in large areas of Mysore, Chennai and other regions. The black and acidic soils are mostly found in Kerala (Table 1.3).

Soil Taxonomy The latest comprehensive soil classification system is called Soil Taxonomy.The basis for identifying different classes in the system are the properties of soil. The properties could be physical, chemical and biological. The second significant feature of soil Taxonomy is the nomenclature. The names give a definite indication of the major characteristics of the soils. The other criteria used include moisture, temperature, colour, texture and structure of the soil.

Nomenclature An important feature of the system is the nomenclature used to identify different soil classes. The names of the classification units are mostly derived from Latin or Greek and are root-words in several modern languages. Since each part of a soil name conveys a soil characteristic or genesis, the classification name automatically describes the general kind of soil being classified. For example, soils of the order, Aridisol (Latin aridus, dry, and solumn, soil) are characteristic of arid or dry places. The names of orders are combinations of formative elements which generally define the characteristics of the soils and the ending, soil. The soils of the sub-order, Aquolls are the water soils (Latin aqua, water) of the mollisol order. Likewise, the name of the great group identifies the sub-order and order of which it is a part. The nomenclature as it relates to the different categories in the classification system might be illustrated as follows : Mollisol Aquoll Argiaquoll Typic Argiaquoll

-

Order Sub-order Great group Sub-group

Classification Categories There are six categories of classification in soil Taxonomy. These are: (a) Order, (b) Sub-order, (c) Great group, (d) Sub-group, (e) Family and (f) Series. These categories could be compared with those used for the classification of plants. A representative comparison is shown

Introduction to Soil and Agricultura~Microbiology

16

in Table 1.4, where white clover (Trifolium repens) and miami silt loam are the examples of plants and soils, respectively. The Trifolium repens identifies a special kind of plant, the miami series identifies a specific kind of soil. TABLE 1.4*

Plant Classification

Soil Classification

Phylum

-

Pterophyta

Order

-

Alfisols

Class

-

Angiospermae

Suborder

-

Udalfs

Sub-class

-

Dicotyledoneae

Great group

-

Hapludalfs

Order

-

Rosales

Sub group

-

Typic Hapludalfs

Family

-

Leguminosae

Family

-

Fine loamy

Genus

-

Trifolium

Series

-

Miami

repens

Phase""

-

Miami, eroded phase

Species

* Comparison of the classification of a common cultivated plant, white clover (Trifolium repens) and a soil miami series. (Courtesy Nyle C. BRADY 1984) ** Technically not a class in soil Taxonomy but used in field surveying.

aao

MICROBIAL FLORA OF SOIL The soil consists of a variety of microbial population. Microbial population in the soil shows a wide divergence in number, form and biochemical activities in response to various factors of the soil environment.

I. Soli Micro Organisms The numbers and kinds of micro-organisms present in the soil depend on many environmental factors. They are:

(i) Amount and type of nutrients available (ii) Availability of moisture (iii) Temperature (Iv) pH

The presence of roots and the extent of the root system in soil also affect the micro-organisms. The over-all influence of plant roots on soil micro-organisms is called the rhizosphere effect. Soil micro-organisms include bacteria, actinomycetes fungi, algae, protozoa and viruses. (Table 2.1).

18

Introduction to Soil and Agricultural Microbiology

TABLE 2.1 SOIL POPULAnON IN A FERnLE AGRICULTURAL SOIL

Type

Number per gram

Bacteria 'Direct count Dilution plate

2,500,000,000 15,000,000

Actinomycetes

700,000

Fungi

400,000

Algae

50,000

Prot6zoa

30,000

Bacteria Bacteria is found in large amount in the soil. Several billion bacteria are generally present in the soil. The bacteria includes both nutritional and physiological kinds of bacteria. Most of the soil bacteria are heterotrophic and bacillitype. Some of the bacteria have not yet been isolated and identified. Example of heterotrophic bacteria are Bacillus, Clostridium, Pseudomonas, Rhizobium, Azotobacter. Some of the bacteria are actinomycetes bacteria, for example, Streptomyces and Micromonospora. The actinomycetes bacteria can degrade many complex substances and improve the soil fertility. Some of them also produce antibiotics. The cyano bacteria and photosynthetic bacteria playa major role in the transformation of rock to soil. Bacteria does not occur freely in the soil but are closely attached to the different soil particles. They play a major role in organic matter decomposition, nitrogen fixation etc. They are the most dominant group of micro-organisms in the soil and are present in all types of soil. Bacteria live in soil as cocci (0.5J..1.), bacilli (0.5 - 3.0J..l.) or Spirilli. In the year 1925, Winogradsky classified the soil micro-organisms intc two broad categories. These are: (a) Autochthonous (Indigenous population) (b) Zymogenous (Fermentive organisms)

The indigenous population is always uniform and constant. For , example, Nocardia, Arthrobacter. The fermentive organisms require an external source of energy, for example Preudomonas and Bacillus. The most common soil bacteria come under the genera Arthrobacter, Sarcina, Pseudomonas, Corynebacterium, Clostridium, Micro coccus and Flavobacterium. Another type of bacteria like

19

Microbial Flora of Soil

Myxobacteria is also present in the soil. For example, myxococcus polyang;um, cytophaga and sporocytophaga (Fig. 2.1). In the soil, bacteria exist as mats, clumps and filaments called colonies on and around soil particles wherever food and other conditions are favourable. The jelly-like mixture of organic colloidal matter and inorganic materials makes an ideal medium for their development. Many of the soil bacteria are able to produce spores and similar resistant bodies, thus presenting both a vegetative and a resting stage.

(A) Pseudomonas Sp.

(8) Desu/fov;br;o Sp.

20

Introduction

to Soil and Agricultural Microbiology

(C) Clostridium tetanl

(D) Bacillus Sp. Fig. 2. 1 Some examples of Soil Bacteria.

Fungi The different kinds of fungi present on the soil surface, where oxygen is readily available. are Penicillium, Aspergillus, Rhizopus, Mucor and so on. A gram of soil contains thousands of fungi. The mycelium of fungi penetrates through the soil, forming a network. The major part of the total microbial bio-mass is occupied by soil Fungi. They derives nutrients for their growth from organic matters and living animals. The soil fungi can be classified into various types, like root infecting fungi, symbiotic fungi, mycorrhizal fungi, saprophytic fungi,

21

Microbial Flora of Soil

specialized and unspecialized parasites. (GarrAt 1950). Fungi are present in all soils and possess filamentous mycelium composed of individual hyphae. The hyphae may be septate or aseptate. The asexual reproduction of fungi takes place by spores of conidia, sporangia and oidia. Well-defined sexual reproduction also takes place by gametic fusion. Fungi muy be divided into three groups, which are: (a) Yeasts (b) Molds (c) Mushroom fungi Among the three groups, molds and mushroom fungi are an important component in soils. Yeasts are rare in such a habitat. Molds belong to the general purpose heterotrophic group of fluctuating soil . micro-organisms. They are distinctly filamentous, microscopic, or semi-macroscopic. Molds develop vigorously in acid, neutral, or - '. alkaline soil. In general, fungi can be classified into four classes. These are : (i)

Phycomycetes

Perfect Fungi

(ii) Ascomycetes

Perfect Fungi

(iii) Basidiomycetes

Perfect Fungi

(iv) Deuteromycetes

-

Fungi Imperfect

The Oeuteromycetes fungi produce abundant asexual spores and lack sexual stages. Exampies of Imperfect fungi are Cercospora, Colletotrichum, Alternaria and Fusarium. The other three classes of fungi have both sexual and asexual means of reproduction. The Phycomycetes fungi possess non-septate and unicellular mycelia and sporangia - containing spores. Examples of Phycomycetes fungi are Mucor, Rhizopus, phythium, Cytopus and Cunninghamella The Ascomycetes fungi possess septate mycelia and contain a definite number of Asco spores. Examples of Ascomycetes fungi are Aspergillus, Penicillium and Chaetomium. The Basidiomycetes fungi are characterized by specialized reproductive structures known as basidium , producing basidiospores.

(A) Mushroom

Introduction to Soil and AgriculturalMicrobiology

22

Examples of Basidiomycetes are Polyporusm, Boletus, etc. The primary function of filamentous fungi in soil is to decompose organic matter and help in soil aggregation. Some of the fungi are capable of forming ectotrophic associations on the root system of forest trees. Moreover, fungi function more efficiently than bacteria in soil.

, .\

(e) Rhlzopus sp.

(D) Altem"" $p.

23

Microbial Flora of Soil

Conidiophore

(E) Fuur/um Sp.

(F) Unicellular yeut

(G) Penicillium Sp. FIg. 22 Some 8XB111p/8s d SolI Fungi

24

Introduction to Soil and Agricultural Microbiology

Algae The population of algae is lesser when compared to that cf bacteria or fungi. The commonly occuring algae are green alga~ (chlorophyta) and diatoms (chrysophyta). Both of them are photo-autotrophic. Algae are commonly found on the soil surface. Algae combined with fungi (Lichens) can help to transform rocky material into soil particles. Cyanobacteria also provides nitrogen to certain plants. The cyanobacteria plays a very important role in Agriculture as biofertilizer. The prominent genera are Nostoc, Anabaena, Oscil/atoria, Calothrix and Scytonema. Most algae are chlorophyll - bearing organisms and like- higher plants, are capable of performing photosynthesis. They perform best under moist-to-wet conditions. Soil algae are'divided into four groups as follows: (a) Blue-green algae

-

Cyanophyta

(b) Green algae (c) Yellow-green algae ·

Chlorophyta Xanthophyta

(d) Diatoms

Bacilariophyta

The green algae are the commonly occuring soil algae especially if the pH is low. Soil algae in vegetative forms are numerous on the surface layers. In sub-soils, most algae are present as resting spores or cysts. Morphologically, algae are either unicellular or filamentous. Some of the green algae in soil belong to the genera, chlorel/a, chlamydomonas, chlorococcum and oedogonium (Fig. 2.3).

A. Chlorella

25

Flagellum

Contractile Vacuoles Nucleus

Stigma

Cell wall Pyrenoid Dead Cell

Adult (8) Chlamydomonas

FALSE BRANCH

Vegetative Cell

(C) Oscillatorla

(D) Scytonema

Introduction to Soil and Agricultural MicrobIoIOQY

26

~~~~.. CYANOPYCEAN \~

GRANULES

(E) NOIItoc Fig. 2.3 Some examples 01 Soil Algae

The blue-green atgae (cyanophyceae) contain ~ pigment known as phycocyanin, which imparts a special blue-green colour to these organisms. Some of the blue-green algae in the soil belong to the genera Oscillatoria, Nostoc, Chroococcus, Lyngbya, Anabaena, Cylindrospermum and Scytonema. Cyanobacteria like Nostoc and Anabaena possess specialized cells known as heterocysts which are implicated in nitrogen fixation. The blue-green algae are numerous in rice soils, and when such lands are flooded, appreciable amounts of atmospheric nitrogen are fixed or changed to a combined form by these organisms. Microscopic animal life in soils can be class.ified into two groups. These are: . 1. Nematodes;

~.

Protozoa

The Nematodes are commonly called threadworms or elworms. They are found in almost all soils, often in surprisingly large .numbers. These organisms are round and spindle-shaped.

27

Microbial Flora of Soil

On the basis of their food demands, nematodes can be distinguished into three groups as Those that live on decaying organic (a) Saprophytes matter. (b) Predators Those that are predatory on bacteria, algae and protozoa. (c) Parasites Those that are parasitic,attacking the roots of higher plants to pass at least a part of their life cycle. An example of Nematodes is Heterodera.

Protozoa The protozoa occur in rich soils. They range from a few hundreds to several thousand per gram. Most of them feed upon bacteria and some organic materials. Protozoa can be co-related with plant growth and soil nutrients (Griffin, 1972). The major role of soil protozoa is predatory. Protozoa are the Simplest form of animal life. They are considerably larger than bacteria (5-100 mm diam) and are unicellular. The protozoa are the most varied and numerous in the micro animal population of soils. More than 250 species of protozoa have been isolated. Sometimes ·as many as 40-50 different groups are found in a single sample of soil. Soil protozoa are divided into three groups (a) Amoebae (b) Ciliatas (c) Flagellates Pseudopodium

Nucleus

,EctooIasm

Endoplasm

vacuole {AJAmoeN

28

Introduction to Soil and Agricultural Microbiology

(8) 8odo

(e) Cerocobodo

(D) Ca/poda

(E) Plant Parasitic nematode Fig. 2.4 Some examples of Soil Protozoa

29

Microbial Flora of Soil

Of the three, the flagellates are the most numerous followed by amoebae and ciliates. The flagellated protozoa belonging to the class mastigophora are pre-dominant in the soil. Some .genera are Bodo, Cercobodo, Cercomonas, Heteromita, Monas, Spiromonas and Spongomonas. The class Sarcodina consists of different genera such as Amoeba, Biomyza, Trinema and Eug/ypha. It can move with the help of pseudopodia. The protoplasm may be nacked or encased in shells. The third group f soil protozoa belong to the class Ciliata. They are distinguished by the possession of minute hairs called Cilia around their bodies which help in locomotion. Examples of Ciliata are Co/pidium, Co/poda, Halteria, Vorticella and Uro/eptus (Fig. 2.4). As a source of food, p'rotozoa ingest bacteria and to a lesser extent, other microflora.· Examples- Bacillus, Micrococcus, Aerobacter and Pseudomonas. Protozoa are abundant in the upper layer of the soil and their numbers are directly dependent on bacterial population. The application of organic manures increases the number of protozoans in the soil.

Viruses Viruses occur sporadically in soil, tissues of dead plants and animals. Viruses includes both plant viruses and animal viruses. Some of the soil bacteria contain bacterial viruses (Bacteriophages). Some phytopathogenic viruses are also present in the soiL Certain animal and human viruses remain in the soil for long periods, causing damages to the host plant. (Fig. 2.5).

RNA

(A) Tobacco mosaic Virus - An electron micrograph of the negatively stained helical capsid

30

Introduction to Soil and Agricultural Microbiology

Head

Collar Core or tube _ _ _~-~~~~--(hollow) Helical sheath

Tail..-_-H fibers

Tail pins

(B) Bacteriophage Fig. 2.5 Some examples of Soil Viruses

Bacteriophages are the smallest inhabitants of the soil and they are known to attack the cells of bacteria. Bacteriophages can be seen only under an electron microscope because of their minute size. Viruses which attack actinomycetes are known as actinophages. The phages attacking blue-green algae are known as cyanophages. Viruses have also been observed in sections of fungal spores,! for example, penicillium spp.

Actinomycetes Actiriomycetes are soil organisms which have chs.racteristics similar to bacteria and fungi. Actinomycetes resemble molds in that they are filamentous, often profusely branched. Actinomycete~ fire similar to bacteria in that they ar~ unicellular and of about the same diameter. When they break up into spores, they closely resemble bacteria. On the basis of organization, actinomycetes occupy a position between true molds and bacteria. Actinomycetes develop best in moist,

31

Microbial Flora of Soil

well aerated soil. Their optimum development occurs at pH values between 6.0 and 7.5. The number of actinomycetes increases in the presence of decomposing organic matter -in the soil. Under favourable conditions, a biomass of more than 4,500 kilograms of actinomycete threads and spores might be present in a hectare~ The commonest genera of actinomycetes ~re Streptomyces, Micromonospora and Nocardia. (Fig. 2.6)

(A) Actinomyces sp.

(8) Nocardia Sp.

32

Introduction to Soil and Agricultural Microbiology

(C) Streptomycete CandIa arrangement . Fig. 2.6 Some examples of Soil Actinomycetes

II. Factors Affecting Soil Microbial Population The soil micro-organisms are influenced by various factors, as follows: (i) Ecological Factors (iii) Soil Moisture (v) Soil Air (vii) Inorganic Nutrients

(ii) (iv) (vi) (viii)

Soil Fertility Temperature Organic Matter Soil pH

(i) Ecological Factors: In the soil, the microbes are in dynamic equilibrium. The most pre-dominant organisms like fungi are capable of growing very fast. The stability of micro-organisms is influenced by various ecological factors. (ii) Soil Fertility: The microbes require some basic nutrients like nitrogen, phosphorus and potassium for normal growth. The same

Microbial Flora of Soil

33

nutrients are utilized by plants in the soil. The fertility level of soil in influenced by the availability of these elements. (iii) Soil Moisture: It is also an important factor for influencing the microbial population. The soil moisture varies, depending on the water-holding capacity of the soil. The water is very important component for various physiological process. The decomposition process is also determined by the nature of soil moisture. (iv) Soil Temperature: Micro-organisms have been found to exist under extreme temperature such as 60°C. In the same way, some of the microbes can grow at very low temperatures (-40°C). The temperature changes can also influence the micro-organism both quantitatively and qualitatively. (v) Soil Air: The soil consists of number of pore spaces, through which sufficient amount of O2 is supplied to the living organisms. A number of aerobic bacteria normally occur in such conditions. The high moisture level reduces the number of micro-organisms, particularly aerobic bacteria. So a large number of anaerobic bacteria are found. The N2 fixing bacteria are more active in aerated soil. (vi) Organic Matter: Organic matter is a major source of energy for most soil micro-organisms. The increase and decrease in the microbial population depends on the nature of organic matter. The organic matter influences the nature and properties of soil and affects the activity of micro-organisms in the soil. (vii) Inorganic Nutrients: The growth in microbial population also depends on the concentration of inorganic substances in the soil. The humus decomposes into several simple compounds like N,P,K, Na, Ca and Mg. Some amount of these nutrients are readily taken up by plants and micro-organisms in the soil. (viii) Soil pH: Soil pH plays a very important role in Microbial activity. The Hydrogen ion concentration of the soil is essential for plant growth and microflora. The fungi grow very easily under the pH 5-6 and the actinomycetes grow in pH 7. The high salt content may be toxic for some micro-organisms.

III. Microbiological Test for Soil Fertility The measure of soil fertility is the crop itself. The crop is influenced by the physical and chemical conditions of the soil and the presence of nutritive elements. The availability of the nutrients is affected by the activity of microbes. Soil fertility can be improved by keeping the soil well aerated, for the development of phosphate solubilizers, denitrifiers and incubation of Azotobacter.

Introduction to Soil and Agricultural Microbiology

34

The fertility of soil can be determined using two techniques. These are: 1. Phosphate solubilization 2. Denitrification

1. Phosphate Solubilization The cyclic movement of phosphorus between the living organisms and the environment is referred to as the phosphorus cycle. Phosphorus occurs naturally in the soil and water. It is also added to the soil in the form of chemical fertilizer and residues of dead plants and .animals. In the soil, the organic phosphate cannot be utilized directly by plants. Decomposers like bacteria and fungi convert the organic phosphorus to inorganic form. Both inorganic and organic phosphates occur in the soil. The inorganic forms are compounds of Ca, Fe, AI and P. The organic phosphorus containing compounds are derived from plants and micro-organisms and are composed of nucleic acids, phospholipids and phytin. Organic matter derived from dead and decaying plant debris is rich in organiC sources of phosphorus. Micro-organisms belonging to genera Pseudomonas, Mycobacterium, Micrococcus, Penicillium and Aspergillus are involved in the solubilization of inorganic phosphates into solutions. Many fungi and bacteria are potential solubilizers of bound phosphates as revealed by experiments in pure culture. Examples - Aspergillus, Penicillium, Bacillus and Pseudomonas. The main aim of doing phosphate solubilization is to determine the fertility of soil by detecting the number of phosphate solubilizers present in the soil.

Materials Required: Pipette, petriplates, soil sample, distilled water and Pikovskaya's medium. (For media composition Ref. Appendix - I) Experiment: One gram of garden soil was serially diluted using sterile distilled water. The diluted samples were incubated into pikovskaya's medium and then incubated at 37°C for 24 hours. Observation: After incubation, the plates were observed for the presence of phosphate solubilizers. Clear zones were observed around each colony denoting the presence of phosphate solubilizers. (Fig. 2.7).

35

Microbial Flora of Soil

Fig. 2.7 Phosphate solubilisation

2. Denitrification Nitrogen is the most abundant substance in the atmosphere. The conversion of nitrate into molecular nitrogen or nitrous oxide through microbial process is known as denitrification. Bacteria such as Pseudomonas, Bacillus, Micrococcus and Achromobacter are involved in the denitrification process. Soil bacteria like Thiobacillus denitrificans which are known to oxidize sulphur chemo-autotrophically also reduce nitrate to nitrogen. The source of energy is sulphur, and this energy is used to convert nitrate into molecular nitrogen. Materials Required: Sterile pipettes, petri dishes, soU sample, Nitrate broth tubes and Durham's tube. Experiment: Nitrate broth tubes were prepared and sterilized. 1 gram of soil sample was taken and was serially diluted using sterile water. 1 ml from each dilution was transferred into nitrate broth tubes containing Durham's tube. Then, they were incubated at 25°C for 2 weeks. (Fig. 2.8). Observation: On the 7th and 14th day of incubation , the presence or absence of air bubbles in the · Durham's tube were observed for this would indicate the presence of gas production. The presence of gas production was noticed, denoting the presence of denitrifiers in the soil sample.

36

mtroduction to Soil and Agricultural MictobioIogy

SOIL SAMPLE

10-'

10-2

10-3

10-4

10-5

,O~

Control

Nitrate broth tubes with diluted sample Durham's tubes

..

~.

-:0

B-

'

I" '6 .

U

-1' Control

Atter incubation: Gas ~uctjon in Durham's Tube except control

Control Fig.

2.8 DtJnlltification

000

PHYTOHORMONES Phytohormones These are growth-regulating chemicals. They are produced in the leaves of plants and translocated downwards. According to Pincus and Thimann (1948) a plant hormone is defined as "organic substances produced naturally in the higher plants, controlling growth or other physiological functions at a site remote from its place of production and active in minute amounts." Growth hormones are also known as growth promotors or growth retJulators. Phytohormones possess some special characteristics such as, (a) All hormones are organic in nature (b) They are present in traces (c) They are usually produced at the tips of roots, stems and leaves. (d) They are translocated from one part to another through phloem cells. (e) By using chemical methods, phytohormones can be easily isolated from the plant parts.

The hormones can be classified into various categories, natural and synthetic, like (i) Auxins

(N) Gibberellins

Introduction to Soil and Agricultural Microbiology

38

(iii) Cytokinins (iv) Abscisic acid (v) Ethylene (vi) Flowering hormones (vii) Phenolic substances (viii) Morphactins

(i) AUXINS Auxin is a generic term for compounds, characterized by their capacity to induce elongation in shoot cells. Auxins are produced at the tips of roots and stems and are transported to the region of elongation.

Examples of Auxins are: Indole acetic acid (IAA) Indole butyric acid (lBA) Napthalene acetic acid, (NAA) 2,4 - Oichlorophenoxy acetic acid (2,4-0), 2,4,5 - Trichlorophenoxy acetic acid (2,4,5-T) (A)

Q:JCH,-CH,-CH,-COOH H Indole-3-butyric acid

(l-

(C) hC' HC?' C--C -CH2-COOH

II

I

cO

CH2COOH

(8)

naphthalene acetic acid

(O)aq~

II

HC::::::C",,-C'N....- CH

H

H IAA

CI 2,4.5-trichlorophenoxyacetic acid (2,4,5-T)

(E)

ifH I

CI 2.4-dichlorophenoxyacetic acid (2,4-D)

Fig. 3.1 Types of Auxins Chemical Structure

39

Phytohormones

The chief plant auxin is indole acetic acid (IAA). It was isolated by Kogi et. al.. (1934) from corn germ oil and human urine. Later on, Thimann (1935) isolated heteroauxin from the fungus Rhizopus.

Bioassays of Auxins The term bioassay is used to describe the use of living material to test the e,fect of known and putative, biologically active substances. While dealing with biologically active substances such as phytohormones, it is necessary to have a means of measuring their biological activity. Bioassays are used for this purpose. The bioassays are based primarily on cellular enlargement. However, there are numerous responses that might be used in a bioassay for auxins. The following bioassays are applicable to the study of auxins.

Avena Coleoptile Curvature Test (Went Experiment) The oat (Avena sativa) plant possesses a thin protective covering over the newly formed plumule leaves, called coleoptile. This layer also Occurs in other monocotyledonous plants like grasses, maize etc. This has been proved by experiments on various plants by scientists like Charles Darwin, F.W. Went and Boysen Jensen.

COLEOPTILE SEED HUSK

EPIDERMIS

PRIMARY

ROOT

(A) L.S. of OAT Seedling

(8) T.S. of Coleoptile Fig. 3.2

40

Introduction to Soil and Agricultural Microbiology

The Avena coleoptile curvature test was developed by F.W. We.,t in the year, 1928. Preparation ( ) - - tip di~arded

first leaf exposed

Avena coleoptile

Results

agar block ---t;;Ji'U.. without lAA or other auxins

control

angle of coleoptile curvature ~T--

treated

Fig. 3.3 Went Experiment

agar block with 1M or other auxins

41

Phytohotmones

Response of Avena coleoptile to increasing concentration of IAA Standard CUI'\IEI

20

i ,. lS

t lM,ppm

He placed several decapitated tips of Avena coleoptiies on a thin block of Agar· agar. The block was cut into small square pieces and each piece was placed eccentrically on the cut ends of coIeoptiles. He observed the characteristic bonding of coleoptiles towards the side where the agar piece was not kept. The curvature is measured by recording the angle made by a vertical Une and a line drawn parallel to the curved portion of the stem. as shown in Fig. 3.3. A linear relationship betweer the concentration and the arnG'unt of curvature obtained exists within a certain range of concentrations of 1M. As shown in the Fig. 3.4. this range for 1M reaches an optimum peak at around O.2mg11. Redrawn from l.J. Audus, 1959. Plant Growth Substances. New York. Interscience Publishers. PHYSIOLOGICAL EFFECTS OF AUXINS -Auxins perform important functions, such as:

1. Cellular Elongation The main function of auxin in plants is to stimulate the cell elongation in shoots. The vertical growth of cells is known. as cell· elongation. Auxins decrease the osmotic potential of the cell. increase the permeability of the cell to water. induce the synthesis of RNA and proteins for wall components. and cause a reduction in wall pressure. This is how auxins stimulate cell elongation.

42

Introduction to Soil and Agricultural Microbiology

2. Root Initiation The concentration of auxins accelerates the growth of stems, but reduces the growth of roots. But the number of lateral branches in roots is increased. In general, roots are much more sensitive to auxins than stems.

3. Apical Dominance The influence of apical bud in suppressing the growth of lateral buds is called apical dominance. The apical bud of many vascular plants is very active in growth and lateral buds remain inactive. In the absence of the apical bud, active growth begins in the lateral bud. However, in a short time, the lateral bud closest to the apex will establish dominance over the remaining buds and cause them to become inactive again.

4. Parthenocarpy Pollination and fertilization are in some way connected with development of the fruit. The development of fruit in the absence of pollination is called parthenocarpic development, and the fruit that is formed is called parthenocarpic fruit. Parthenocarpy has been induced in fruits like banana, apple, orange, tomato, etc., by synthetic auxins like IAA, NAA.

5. Respiration Auxin stimulates respiration in many plants and there is a correlation between auxin - induced growth and an increased respiratory rate.

6. Prevention of Abscision Auxins control the falling of fruits and leaves. The leaves and fruits fall down from the plants only when an abscision layer is formed between petiole and stem at the point of attachment. Auxins prevent the formation of the abscision layer.

7. Callus Formation In tissue cultures where callus growth is normal, the addition of auxins is necessary to sustain the growth o! such callus. The amount of callus tissue formed is related to the c6ncentration of IAA applied.

8. Eradication of Weeds Certain auxins like 2.4,D and 2.4,5-T are used for the eradication .of weeds. Unwanted plants like Cyperus, Cynodon and Eichhomia can also be eradicated by using the auxins spray.

Phytohormones

43

9. Dormancy Example : Bulbs and Tubers Some auxins like a-Napthalene acetic acid (NAA) are used to prolong the dormancy buds. So that these buds could not germinate.

10. To Increase Cambial Activity Auxins promote the cambial activity when supplied from outside.

(ii) GIBBERELLINS The Gibberellins were first discovered by a J(lpanese scientist Kurosawa in 1926. It was observed in rice fields that a few plants were distinctly taller. seedless and pale in colour compared to normal rice plants. due to a disease called Bakanae or the foolish seedling disease. This disease had devastating effects on the rice economy of Japan during the nineteenth century.

Chemistry of Gibberellins So far fifty-two gibberellins have been isolated. In some cases as many as seven of these compounds have been found in one plant. For example. GA17 • GA38 • GA44 • GAg. GA20 • GA29• and GAS1 have been isolated from Pisum Sativum (Pea plant). Similarly. in the fungus Gibberella fujikuroi two crystalline forms of active material were isolated by Yabuta and Hayashi in 1939. These were named as gibberellin A and B. All gibberellins are almost similar in structure. They differ only in the number and position of OH and GH3 and GOOH groups at different carbon atoms of the gibbance ring.

Physiological Effects of Gibberellins Gibberellins perform some importantfunctions such as

t. Cellular Elongation:

The Gibberellins promote either stem elongation or cell division in plants.

2. Parthenocarpy: The development of fruit in the absence of pollination is called Parthenocarpy. Gibberellins have induced parthenocarpy in fruits like guava, orange, tomato, etc., 3. Bolting and Flowering: In many plants. leaf development is profuse and internode growth is retarded. This is called a rossette. Just before the reproductive stage, there is a striking stimulation of internode elongation. This is called bolting. The gibberellins help in flowering and development of fruits. 4. Increase the size and number of flowers

Introduction to Soil and Agricultural Mk:tobioIogy

44

S. Increase the cambial activity 6. Break the dormancy of seeds and buds 7. Increase the size and number of fruits (eg.: lemon, grapes) 8. Activate fermentation during wine formation.

(iii) CYTOKININS Kinetin is the cell-division inducing compound of kinetin (6-furfurylamino purine). Kinetin and closely - related chemicals are generally termed as cytokinins. Miller et.aL, isolated kinetin from the DNA yeast in the year 1955. Kinetin is also found in the liquid endosperm of coconut. Likewise, in .the year 1963, Letham isolated a compound namely 6 (4 hydroxy - 3methyl - trans - 2 butenyl - amino) Fig. 3.5 Structure of Zeatin purine, from the immature seeds of maize. It was also called Zeatin. Kinetin and Zeatin are the chief cytokinins.

Physiological Effects of Cytokinins 1. Induce the cell enlargement and stimulate cell division 2. Increase the rate of protein synthesis 3. Induces formation of tuber 4. Retains chlrophyll and delays senescence 5. Responsible for Bud development and shoot-growth

(iv) ABSCISIC ACID In the year 1961, Liu and Carns isolated asubstance in crystalline form from mature cotton fruit. The structure of the isolated substance was called abscisin I. Later on, in 1965, Ohkuma and colleagues isolated a similar substance from young cotton fruit and termed it abscisin II. Abscisin I and abscisin II are jointly known as abscisic acid ~N.

PhYSiological Effects of Abscisic Acid 1. Helps in the abscission of leaves, flowers and fruits 2. Inhibits cell-division and cell-elongation 3. Helps in redUCing transpiration rate

.

45

Phytohormones

4. Induces dormancy in seeds 5. Causes chlorosis

(v) ETHYLENE It is produced in plant cells as a result of metabolism. The chemical formula of ethylene is CH 2 = CH 2 It is the only plant hormone which occurs in the form of gas. Ethylene can be synthesized by the fatty acid, \inolic (Liebermann and Mapson in 1962, 1964).

Physiological Effects of Ethylene 1. Helps in the ripening of fruits like mango, banana, orange, etc., 2. Inhibits geotropism in stems 3. Accelarates apical dominance 4. Regulates the growth of cell-wall 5. Stimulates the formation of new roots.

(vi) FLOWERING HORMONES The hormones which induce flowering in plants are known as floringen. They are synthesized in the leaves and then transferred to buds, where they perform the function of differentiation of floral organs. Some environmental factors like temperature and light can play an important role in inducing flowering. The flowering in plants can be induced in any season by increasing or decreasing the duration of the day or night. (Photoperiodism).

(vii) PHENOLIC SUBSTANCES Phenolic substances like coumarin can also playa role in growth of plants.

(viii) MORPHACTINS They are synthetic growth inhibitors which contain a fluorenol (9-hydroxy-fluorene-9-carboxylic acid). They can inhibit the seed germination, growth of seedlings and apical dominance.

CJCJCJ

BIO-GEO-CHEMICAL CYCLING The cyclic movements of chemical elements between living organism and the environment (soil, air, water) is referred to as a Bio-geo-chemical cycle. Microbes play an essential role in the transformation of various chemicals like Carbon, Hydrogen, Oxygen, Nitrogen, Sulphur and Iron. Plants absorb N,P,K because these elements are readily available in the soil. They move from one biotic community to another through the food chain. Bio-geo-chemical cycles include two phases: (i) Organic phase (ii) Abiotic phase

Flowing of chemicals through the food chain is the organic phase. Abiotic phase is the major reservoir for all nutrients. There are two classes of abiotic phases in bio-geo-chemical cycles. These are (a) Sedimentary phase (b) Atmospheric phase , Bio-geo-chemical cycles that have dominant atmospheric phases are called atmospheric reservoir cycles; those whose sedimentary phases are dominant are called sedimentary reservoir cycles.

47

Bio-Geo-Chemica/ Cycling

In gaseous cycles, the main reservoir of nutrients is the atmosphere and the ocean. In sedimentary cycles, the main reservoir is the soil and sedimentary rocks. Both involve biotic and abiotic agents, both are driven by the flow of energy and are tied to the water cycle.

Water Cycle (Hydrologic Cycle) A cyclic movement of water between living organisms and the environment or Biosphere is referred to as the Water cycle. Water is not evenly distributed throughout the earth. Almost 95% of the total water on earth is chemically bound into rocks. Water forms a very significant part of environment and without the cycling of water, bio-geo-chemical cycles cannot exist, the ecosystem cannot function and life cannot be maintained. Water is a solvent; it is the medium by which nutrients are introduced into autotrophic organisms. The chemically-bound rock water does not cycle, but fresh water can move through the hydrologic cycle. The hydrologic cycle covers oceans, ice caps, fresh water lakes, saline lakes, rivers etc. Water circulates through evaporation, transpiration, surface run-off, evapo-transpiration and ground. Water is evaporated directly from any surface other than a plant. This process is referred to as -evaporation e.g., animal skin, soil surface, lake, etc. H20 Cycle

J, Evapotranspiration of water (land) from earth surface

J, Formation of clouds

J, Precipitation

J, Surface runoff (ground water)

J, Return of water

J, J, Sea

f-

via streams

48

Introduction to Soil and Agricultural Microbiology

The process of water evaporating from the surface of leaves of plants is called transpiration. Nutrients that have accumulated in the soil can be eroded by streams, removed altogether from a local eco-system and carried by ~oil seepage into surface water. Ground water saturates in either sediment or rock below the ~ater surface.

Gaseous Cycle It includes Carbon, Oxygen and Nitrogen.

Carbon Cycle Carbon exists in the fORn of inorganic and organic compounds. The concentration of CO2 in atmosphere is only 0.003% which is less that what is required to build the organic world. The carbon cyde involves the transfer of carbon dioxide and organic carbon in the atmosphere where carbon occurs as inorganic CO2 • Hydrosphere and lithosphere contains varying concentrations of organic and inorganic carbon compounds_ Sometimes CO2 can react with water to form carbonates and bicarbonates_ Carbon can be present in reduced forms for example methane (CHJ and organic matter, and in more oxidised fORns such as CO and CO2 • Carbon fixation occurs through the activities of photo-autotrophic and chemo-autotrophic micro-organisms. The CO2 is released into the atmosphere as a by-product of plants respiration. It is then used by plants in photosynthesis. Through the decomposition process, carbon is released back into the carbon cycle. The carbon compounds that are lost to the food chain after fermentation, such as methane, are readily oxidized to CO2 by inorganic reactions in the atmosphere. By microbial decomposition cellulose, lignin and Hemicellulose are most readily broken down by micro-organisms resulting in the formation of carbon compounds. Several other carbon compounds such as gum, inulin and related substances present in plant and animal tissues, eventually reach the soil. These substances are attacked by soil bacteria and fungi through ~heir co-enzymes which are also adaptive enzymes. Micro-organisms which are capable of utilizing pectiC substances as carbon and energy sources are abundant in soil and plant surface. Examples of such micro-organisms are Bacillus, Pseudomonas, Erwinia. These micro-organisms readily produce the pectinases.

Protopectin

Protopectinase ) Pectin

Pectinmethyl esterase ) Pectic acid

For example, starch serves as a storage product of plants e.g., tubers, bulbs, rhilomes. It contains two components namely amylose and amylopectin. Aerobically the microbes fully utilize the starch to produce'C02 and anaerobically (Fennentation) to yield methane, acetic acid, lactic acids. The organis!'fls like Bacteria and Fungi utilize starch as a carbon source for growth and multiplication. Starch

Amylase

~ Maltose (l-9luoosida~ Glucose

Some specific groups of bacteria are particularly effective in reducing carbon compounds to form lactic, butyric and acetic acids.

If glucose is acted upon by micro-organisms under aerobic conditions, then CSH120 6 + 60,2 Glucose is formed

If it is attacked anaerobically by yeasts and bacteria then CSH120 6

)

2.C~03 lattic acid is formed

An increase in CO2• as a result of decomposition of plant and animal residues added to the soil, leads to an increase in the ~ content of the. soil atmosphere. This results in a rise in the hydrogen ion concentration of the soil. The micro-organisms regulate the CO2 in the atmosphere and in plants. If they do not act on organic matter, the limited supply of CO 2 in the atmosphere will be exhausted and green plants will cease to manufacture carbohydrates. If the micro-organisms are over-active, then all the organic matter will be reduced to CO2 and the soil will not be fit for plant growth. Thus ·the micro-organisms do a balancing act. The carbon cycle is largely maintained by the balanced action of the micro-organisms in the soil. (Fig. 4.1). The fossil fuels, SlJch as coal and petroleum, are not actively cycled through the activities of micro-organisms. Burning of fossil fuels also adds CO2 to the atmosphere. This has led to a general rise in the concentration of atmospheric CO 2 resulting in the rise in global temperature. This phenomenon is known as the ,Greenhouse effect.

introduction to Soil and Agricultural Microbiology

50

Atmosphere

r;==============~_~__CO_2__~14+-------~~'r} ·~ c 0.0

1 I

C

0

Xl

p ~~

MICROBIAL

~

1:!a.

;p

f!

'5.

CElL5) ~

Methane Dissolves

.~

Combustion

CO2

PLANT

'iD c o

:0

'& E ~ .

ANIMAL

1

DECAY

.FOSSIl

FUELS~---------"----""~ _

Uthosphere (Soil)

--~

HYDROSPHERE (Water)

t

'

Fig. 4. 1 : The carbon Cycle

Nitrogen Cycle Nitrogen is the most abundant substance present in the atmosphere. Micro-organisms utilize N2 in the form ofNH:. NO; and NO; as well as organic nitrogen such as amino acids and proteins. The concentration of Nitrogen in the atmosphere is 79%. It is an essential constituent of proteins and chlorophyll found in organisms. The process of Biogeochemical cycling of N2 are : (a) Nitrogen Fixation

(b) Ammonification

(c) Nitrification

(d) Denitrification

51

Bio-Geo-Chemical Cycling

NITROGEN (Atmosphere)

DENITRIFICATION (PSEUDOMONAS)

NITRATE N03 ~ ~

PLANT, ANIMAL

ORGANIC CN)

NITRIFICATION (NiTROBACTER)

~

NITRITE N02

N2 FIXATION SYMBIOTIC (Rhizobium) ASYMBIOTIC (Azotobacter) )

DEAD ORGANIC WASTE

Fig. 4.2 The Nitrogen Cycle

(a) Nitrogen Fixation Nitrogen fixation is a process in which the gaseous form of nitrogen is converted into organic form (NH 3) by micro-organisms like bacteria and Cyanobacteria. Among bacteria the N2 fixation process is carried by Rhizobium (Symbiotic) and Azotobacter (Asymbiotic). Among Cyanobacteria the N2 fixation process is carried by Nostoc and Anabaena in both symbiotic and asymbiotic form. (Fig. 4.2). The bio-chemical reactions are as follows :

2H+

N2 (Dinitrogen)

~

2e

HN

= NH

(Oi-imide)

H2N - NH2 (Hydrazine)

(Ammonia)

(b) Ammonification The process of formation of Ammonia by micro-organisms, plants and animals, where organic amino nitrogen is converted into NH3 is known as Ammonification. The dead organic waste in the soil forms amino acids and is then converted into Ammonia. The enzyme, Deaminases transfers the nitrogen from organic to inorganic forms.

52

Introduction to Soil and Agricultural Microbiology

There are several factorS which influences the ammonification of proteins in the soil. Many organisms utilize urea to liberate ammonia. N2

) NH3

(organic amino)

(c) Nitrification The process of oxidation of ammonium ions (oxidation level =-3) to nitrate ions (oxidation level = +3) and subsequently to nitrate ions. +5) is known as Nitrification. Nitrification is an (oxidation level example of aerobic respiration. The process of Nitrification is carried out by two different types of Nitrifying bacteria.

=

In this process Ammonium is first converted to hydroxylamine (NH 20H) and then to nitrite by the bacteria Nitrosomonas, Nitrosococcus, Nitrosolobus The Nitrite is converted into Nitrate by Nitrobacter, Nitrococcus NH~

) NH20H

NO;

) NO;

)

NO;;

Normally nitrification is carried out by autotrophic bacteria. But there are some heterotrophic bacteria and fungi which also take part in nitrification. For example, Nitrosomonas, Aspergillus f1avus.

(d) Denitrification The microbial reduction of Nitrite to Nitrate with the liberation of molecular Nitrogen and Nitrous oxide (N2 + N20) is called Denitrification. It is carried out by bacteria such as Bacillus, Pseudomonas, Paracoccus, Chromobacterium etc. The biochemical reactions can be summed up as : 2NO;+10H

--~)

2NO; + 6H N20 + 2H

--~) N2 --~)

N2 + 4H20 + 20H+ 2H20 + 20H-

Nz +H 20

This is the reverse process of nitrification Le. nitrate is reduced to nitrites and then to nitrogen gas and Ammonia. Denitrification normally takes place in anaerobic soils. But some aerobic organisms such as Pseudomonas denitrificans also seem to reduce nitrate under certain conditions.

Bio-Geo-Chemical Cycling

53

Sedimentary Cycles There are different kinds of sedimentary cycles, depending on the kinds of elements, but two cycles are significant in a ecosystem, the Sulphur cycle and the Phosphorus cycle. (i) Sulphur cycle (ii) Phosphorus cycle

(i) Sulphur cycle: The cyclic movement of sulphur between the organisms and the environment is called the sulphur cycl,e. The sulphur cycle is both sedimentary and gaseous. The sedimentary phase of sulphur cycle is long-term and in this sulphur is tied up in organic and inorganic deposits. The gaseous phase of sulphur cycle is less pronounced and permits the circulation of sulphur on a global scale. Sulphur is an essential nutrient of plants and animals. It is most abundant in earth's crust in low concentration where it is unavailable to plants. Sulphur enters the soil as plant and animal residues, chemical fertilizers and rain water. Organic and inorganic sulphur compounds are microbiologically metabolised in the soil through different transformation processes such as mineralization, immobilization, sulphur oxidation and sulfate reduction. Initially, sulphur enters the atmosphere as H2S through several sources such as combustion of fossil fuels, volcanic eruption which quickly oxidizes into S02. Atmospheric S02' soluble in water, is carried back to earth through rainwater as weak H2S04• In its soluble form, sulphur mostly appears as sulphate (S04). It is absorbed by the plant roots and converted into certain organic molecules, (Aminoacids); The bacteria capable of oxidizing inorganic sulphur compounds could either be aerobic or anaerobic: ' For example, Thiobacillus thiooxidans. Several fungi and actinomycetes are also sulphur oxidizers. For example, Aspergillus, Penicillium, Microsporum Similarly, bacteria like DesulfoVibrio desulfuricans reduce organic sulphate into hydrogen sulphide. (Fig. 4.3)

(a) Oxidation S2 + O2 + 2H20 ~ 2H 2S04 H2S + 202 ~ S04 + 2H+

54

Introduction to Soil and Agripultural Microbiology

Hydrogen sulphide

(Oxidation) (Thiobacillus)

) Sulphate

(b) Reduction II)

I'

ATP

SO;2 ----+ SO;2

Iii) SO-32

I'

(Desulfovibrio) .

Sulfite

1

)-SSO;

Thiosulfate

The sulphur containing aminoacids are enzymatically attacked by micro-organisms to produce H2S. In an aerobic environment, the H2S is oxidised to sulphate by bacteria. The sulphate produced can be re-used by the autotrophs. SULPHUR COMBUSTION (FOSSil FUELS)

BACTERIAL OXIDATION

..Jz

::!o a:w~ GQ

c(x

IDO

MICROBIAL DEGRADATION

SUlPHATES IN SOil

Fig. 4.3 The Sulphur Cycle

(ii) The Phosphorus Cycle: The cyclic movement of phosphorus between the living organisms and the environment is referred to as the Phosphorus cycle. Phosphorus is an essential element in all living systems. It is present in the protoplasm of all living things. It is found in a huge amount as a calcium phosphate and in many minerals. The framework of plants and animals also consists of calcium phosphate.

55

Bio-Geo-Chemical Cycling

Phosphorus is found in organic form in the following substances (a) Nucleic acids (DNA and RNA) and nucleoproteins (b) Nuclear and cytoplasmic enzymes.

(c) NAD, NADP, FAD, FMN, ATP, GTP, UTP and other co-enzymes (d) Thiamine pyrophosphate, pyrophosphate (e) Phospholipids, Glycerophosphatides (f) Phosphorylated sugars (g) Phosphoproteins like phosphoarginine, phosphocreatinine etc., (h) Vacuoles and internal buffers as in organic orthophosphate. ORGANIC PHOSPHATES (Animals, Man) DEATH (Bones)

SOLUBLIZED INORGANIC PHOSPHATES

1

SOL-tJBLE INORGANIC PHOSPHATES

MICROBIAL ACTIVITY

ROCK PHOSPHATES

r

OCEAN

MICROBIAL DEGRADATION

INSOLUBLE INORGANIC PHOSPHORUS

Fig. 4.4 The Phosphorus Cycle

PhosphCJrus is rare in the atmosphere. It is present in the soil and water. Phosphorus has its physiological importance, when accumulated and liberated from energy during cellular metabolism. Phosphorus is added to the soil in the form of chemical fertilizers or resiques of dead plants and animals of phosphate ion PO; or HPO!or H2P04 and primary phosphates like NaH 2 P04 •

Introduction to Soil and Agricultural Microbiology

56

Micro-organisms are involved in the transformation of Phosphorus in four ways, .as follows:

1. Altering Solubility of Inorganic Phosphate In the Phosphorus cycle, the alteration is mainly between insoluble and soluble, inorganic and organic forms. Micro-organisms are involved in the solubilization of inorganic phosphorus. Species of Pseudomonas, Mycobacterium, Micrococcus, Penicillium and Aspergillus bring the insoluble inorganic phosphates into solutions. These micro-organisms produce acids like nitric and sulfuric acids. For example, Calcium phosphate is an insoluble inorganic phosphate. It reacts with H2S04 or HN03 to form calcium hydrogen phosphate in the followin:g reactions. Ca3 {P04 )2 + H2 S04 -----~) CaHP04 + CaS04 Ca3{P04)2 + HNO:l _.- - - - - - » CaHP04 + Ca{N03)2 Calcium hydrogen phosphate ionizes as follows: CaHP04

)

Ca; + HPO~ (ionic phosphate)

2. Converting Inorganic Phosphate into Organic Phosphate The plants absorb the soluble inorganic phosphate through the root system. The inorganic form is converted into organic form. For example, the inorganic phosphates react with ADP to form ATP. They also react with glucose to form glucose 6-phosphate. This is very important in bone formation of animals.

3. Mineralizing Organic Phosphates In the soil, the organic phosphates cannot be utilized directly by plants. Decomposers like bacteria, fungi convert the organic phosphor.us into inorganic form. Minerals and raw materials are released during this process. This phenomenon is known as mineralization. The process is catalyzed by Phosphatase enzymes. Micro-organisms like bacteria and fungi produce an enzyme called pytase. It releases soluble inorganic phosphate from phytic acid, (Inositol hexophosphate) Inositol hexophosphate + 6H 20 - - - - - - » Inositol + 6PO! The inorganic phosphate can be readily absorbed by plants for recycling. (Fig. 4.4).

57

BicrGeo-Chemical Cycling

4. Oxidation - Reduction Reactions A number of heterotrophic bacteria, fungi and actinomycetes utilize phosphite as the sole source of phosphorus in culture media. They oxidize the phosphite within the cell to organic phosphate compounds For e·~ample, Clostridium butyricum and E. coli.

Other Cycling Processes The Iron Cycle: The Iron cycle is very important in terms of microbial functioning. The cycling of iron compounds has a marked effect on its availability for other organisms. Iron is transformed 3 2 between the ferrous (Fe +) and Ferric (Fe +j oxidation states by micro·organisms. (Fig. 4.5) (Anaerobic) (Geobacter)

Iron (Oxi)

(AEROBIC)

Fe-(ANAEROBIC)

Iron (red)

Fig. 4.5 Iron Cycle

The major genera that carry out iron oxidations are Thiobacillus Leptospirillum and Sulfolobus. Iron reduction occurs under anaerobic conditions resulting in the accumulation of ferrous ions. Some of the micro-organisms reduce small amount of iron during their metabolism. Different microbial groups carry out the oxidation of ferrous ion, depending on environmental conditions.

000

BIOLOGICAL NITROGEN FIXATION Nitrogen is available in the atmosphere in the form of gas. It is converted into a combined form of organic compounds. Eukaryotic organisms like plants and animals cannot use Nitrogen directly. These organisms depend on the availability of fixed forms of Nitrogen for incorporation into their cellular bio-mass. The phenomenon of fixation of atmospheric Nitrogen by biological means is known as Diazotrophy or Biological Nitrogen fixation. The organisms such as bacteria, cyanobacteria involved in this process are called diazotrophs or Nitrogen fixers. The diazotrophs playa very important role in agricultural yield. The process of biological Nitrogen fixation has been classified into the following types. 1. Symbiotic N2 fixation

2. Asymbiotic (free living form) N2 fixation Both, symbiotic and asymbiotic process are carried out by bacteria and cyanobacteria. Symbiotic micro-organisms are those living in the root of plants. Non-symbiotic (Asymbiotic) micro-organisms are those living freely and independently in the soil.

59

Biological Nitrogen Fixation

Biochemistry of Nitrogen Fixation Nitrogen fixation depends on the Nitrogenase enzyme system which helps in the conversion of N2 to NH 3 • It consists of two components, component I and component II. Component I contains molybdo-ferro-protein (Mo-Fe-protein). It is also known as Dinitrogenase. It is a larger unit than component-II. Component II Dinitrogenase reductase contains ferrO-protein (Fe-protein). The enzyme system is a complex mixture of Fe-protein and Mo-Fe-protein. Nitrogenase is a complex, oxygen labile enzyme composed of two components. Dinitrogenase is composed of two dissimilar polypeptides, a2 and ~2. The Dinitrogenase protein contains two active metallo clusters, P clusters and Fe-Mo-Co (iron-molybdenum co-factor). The dinitrogenase reductase protein consists of two identical polypeptides. y2. Each contains two iron atoms. The main function of Fe-protein is to bind and hydrolyze mg ATP and transfer electrons to p clusters of Mo-Fe-protein. The P cluster acts as an intermediate electron acceptor and transfer the electrons to Fe-Mo-Co cluster. (Fig. 5.1 ) Dinitrogenose reductase (Fe protein)

Dinitrogenose (MoFe protein) a

H2 2NH3

FeMoco

Fig. 5. 1 Nitrogenase Complex

In addition to Nitrogenase, the N2 reducing system requires Mg ATP as a source' of energy and a reductant. Here ATP functions as carrier of energy. 'Whenever energy is needed, ATP undergoes enzymatic hydrolysis to form ADP + pi. Mg ++ functions as catalyst. The overall biochemical reaction for N2 fixation catalysed by Nitrogenase is as follows: +

N2 + 12Mg ATP + 6(H + e)

Nitrogenase ) 2NH3 + 12Mg ADP + pi Complex

60

Introduction to Soil and Agricultural Microbiology

The nitrogenase interacts with acetylene to form ethylene. HC == CH

2H . ) H2C Nitrogenase

= CH 2

The essential reactants in the bacterial N2 fixation process are:

(i) The Nitrogenase enzyme complex (ii) Reducing agents like Ferridoxin and Flavidoxin (iii) A regulatory system for NH3 production (iv) ATP.

1. Symbiotic Nitrogen Fixation by Bacteria There are some micro-organisms like bacteria which establish symbiotic relationships with different parts of plants (root, stem, leaf). A classic example is that of symbiotic association develope,d -oy Rhizobium species in about 13,000 leguminous plants. Leguminous plants are classified into three major botanical sub-families. These are -

,(a)

Papilionoideae

(b)

Ceasal pinioideae

(c)

Mimosoideae

Among leguminosae, the largest number of plants are in the Papilionoideae sub-family. Normally the legume plants are used as green manure.

Rhizobium and Root Nodulation Rhizobium is a free-living, gram negative non-sporulating, aerobic and motile rod-shaped bacterium measuring about (0.5-0.9 ).1m x 1.2-3 ).1m). It resides in the soil and is capable of fixing atmospheric Nitrogen.

Beijerinck was the first man to isolate and cultivate a ,bacterium from the nodules of legumes in 1888. He named it Bacillus radicicola. It is now place~ in the Bergey's manual of Determinative Bacteriology under the genus, Rhizobium. The term symbiosis ' denotes a mutually beneficial partnership between two organisms. In legume nodule symbiosis, the Legume is the bigger partner or macro-symbiont while the Rhizobium is the smaller partner or micro-symbiont. A cross-inoculation group refers to a collection of leguminous species that develop nodules on any member of that particular plant group. (Table 5.1)

61

Biological Nitrogen Fixation

TABLE 5.1 CROSS - INOCULATION GROUPS OF RHIZOBIUM AND BRADYRHIZOBIUM

Sr. No. I. 1.

2. 3.

4.

Rhizobium species Nodule forming bacteria R. leguminosarum R. phaseolus R. meliloti

Legume types

Crossinoculation group

Pisum Phaseoli

Vicia Bean group

Melilotu5, Medicago Trifolium

Alfalfa group

II.

R. trifoli Bradyrhizobium Spl

5. 6.

B. japonicum B.lupini

Glycine Lupinus, orinthopus

Soyabean group Lupine group

7.

Bradyrhizobium Spp

Arachis, Vigna, Cajanus

Cowpea group

Clover group

Nodu!e-forming bacteria can be · classified into Rhizobium and Bradyrhizobium.

Genus I : Rhizobium It consists of fast-growing and flagellated strains.

Genus II: Bradyrhizobium It consists of slow-growing and sub-polar flagellated strains. The development of nodules inside the host root is a complex process. A number of physiological events take place like recognition, infection of the host root, differentiation of nodules, prOliferation of bacteria and formation of bacteriods.

Physiological Events During the N2 fixation process,. the roots of legumes secrete a substance which attracts bacterium like Rhizobium. The bacterium also secretes some chemicals like Indole acetic acid. Microbial cells and their products together with associated microbial mucilages get infected. The infection occurs at the tip of young root hairs. It leads !o the formation of the infection thread (sheperd's cook). The root-hair curling occurs before the infection thread is formed by means of curling factors like cytokinins, etc. There is an involvement of some plant proteins like lectins and interaction of nod gene product in the incipient. This infection thread contains rod-shaped bacteria and it can multiply by means of rapid cell division. Finally it develops into a nodule.

62

Introduction to Soil and Agricultural Microbiology

Flow Chart

Normal Root Hairs J, ExJdation of Chemicals by Plant Roots J, Attraction of Rhizobium J, Secretion of IAA by Rhizobium J, Root Hair Curling J, Rhizobial Recofnition (Lectins) Invagif'lation of Root tfairs J, Infection Thread Formation J, Nodule Formation (Nodulin) J, Bacteriod Zone After the nodule formation, vascular bundles are formed and differentiated into a number of regions and get connected with the vascular system of the host root. The mature nodule constitutes the

Release of rhizobia

Fig. 5.2 (A) Spread of Rhizobium through infection thread

63

Biological Nitrogen Fixation

bacterioid zone, which is surrounded by several layers of cortical cells. The effective nodules are generally large and pink due to the presence of leghaemoglobin. The tetraploid chromosome nodules will become . enlarged and pleomorphic to form bacteriods. It appears as swollen, irregularly shaped, club-shaped, y-shaped or branched. (Fig. 5.2) During the nodule formation some proteins may be formed. These proteins are collectively known as nodulins. Nodulins may be divided into C-nodulins (Common) and S-nodulins (species specific).

Mechanism of Nitrogen Fixation

"ctero;d~_

~~@,~" (!) . . ~ ~

Symbk>.;ome

fi) ----

Peri bacteroid ......... membrane

~

Bacteroid . Symbiosome Fig. 5.2 (B) Bacteroids

Oxygen

J, LHb

J, OLHb

J, Bacterial Respiration

J, Energy Source

"'-+

-+

ATP + Mg -+ Electron carriers -+

MgADP + ip Nitrogenase

N2 ( NH3

64

Introduction to Soil and Agricultural Microbiology

The healthy plant roots possess a nodule which fixes nitrogen in a short duration when it is in the highest symbiotic relationship with the plant. The characteristic feature of the healthy root nodules of legume plants is the presence ' of a special red pigment - like haemoglobin called leg haemoglobin (LHb). It is found only in healthy nodules, unhealthy plants do not develop LHb. Normally LHb is present in the peri-bacteriod space. It regulates the concentration of oxygen and indirectly promotes the utilization of oxygen in bacteria, and favours nitrogen fixation. Nitrogenase is a functional enzyme which reduces nitrogen to ammonia in the presence of ATP molecules. It has two components, (a) Dinitrogenase (Mo-Fe-protein) (b) Dinitrogenase reductase (Fe-Protein)

These two components are essential for nitrogenase activity. The fixed nitrogen is used by the plants as the nitrogen source and in turn, carbohydrates produced by photosynthesis and aminoacids are provided to the bacteriods as carbon, energy, and nitrogen sources.

Factors Affecting Nodulation The following factors affect the nodulation process, namely; (a) Temperature (b) Light (c) Combined N2

(d) pH (e) Mineral Nutrition (f) Growth substances

(g) Ecological factors (h) Genetic factors

Symbiotic Nitrogen Fixation by .Non-Leguminous Plants Apart from legumes, the roots of some plants belonging to different non-leguminous angiosperms like Alnus, myrica, Casuarina, Discaria are nodurated by Frankia sp. Frankia species are actinomycetes(fitamentous bacteria) that form septated hyphae and numerous non-motile spores. In Frankia, a part of the hyphae becomes differentiated into specialized nitrogen-fixing cells called vesicles. The mode of entry into the host plant has been studied in some plant species and found to be similar to the entry of Rhizobium into the root hairs of some clover seedlings.

65

Biological Nitrogen Fixation

Mechanism of NH3 Assimilation in Diazotrophs Glutamic Acid + NH3

J.

(GS)

Glutamine + 2-oxoglutarate

J.

(GOGAT)

Glutamic Acid

J. Amino Acids + Proteins (GS) - Glutamine synthatase (GOGAT) - Glutamine oxoglutarate aminotransferase Maltose + CO2 ~ 2-oxoglutarate Maltose

~

glucose 6-phosphate

In cyanobacteria glucose 6-phosphate

~

Ribulose - 5 phosphate

in this conversion 6-phosphogluconate is a intermediate product. In bacferia (clostridium sp.) glucose 6-phosphate pyruvate

~

~

pyruvate

Acetate

Glutamic acid is the source of several metabolic products, such as amino acids, nucleotides, proteins.

Symbiotic N2 Fixation by Cyano Bacteria TABLE 5.2 SYMBIOTIC MICRO-ORGANISMS

Micro-organisms (Cyanobacteria) 1. Nostoc, Anabaena

Symbiotic structure Co/lema, Peltigera (Lichens)

2. Nostoc

-

3. Anabaena azollae

-

4. Anabaena cycadae 5. Nostoc

Cora/loid roots

-

MiCro/macro symbionts Ascomycetes Basidiomycetes (Fungi) Arithoceros (Bryophytes) Azolla (Pteridophytes) Cycas (Gymnosperms) Gunnera (Angiosperm)

66

Introduction to Soil and Agricultural Microbiology

The species of Nostoc, Anabaena, Tolypothrix develop symbiotic association with fungi (Lichens) bryophytes, pteridophytes, Gymnosperms, angiosperms. The genera of Lichens such as collema, peltigera, Lichina to fix Nitrogen symbiotically. (Table 5.2)

Azolla In India, the genus represented by Azolla pinnata is widely prevalent, free-floatin£ on the surface of water in road-side ponds, pools, lakes. These plants resemble the small and delicate moss gametophyte and cover the entire surface of the water. A species of Anabaena is associated with Azolla occuring in a ventral pore in the dorsal lobe of each vegetative leaf. It fixes atmospheric nitrogen. Azolla is being used as green compost for rice cultivation. An increase of 30 to 38% in grain yield has been reported from fields treated with Azolla.

Structure Azolla The sporophytes are small in size and are distinguished into stem, leaves and roots. The stem is thickly covered with leaves that over-lap each other. The roots arise from the under-side of the steam and are unbranched. The leaves are small in size and are divided into two lobes.

Reproduction The sporocarps are born on fertile leaves. They occur in pairs. Both microsporocarps (male) and macrosporocarps (female) are seasonally produced, decay and sink to the bottom of the pond. (Fig. 5.3 and 5.4)

Anabaena

Fig. 5.3 Azalia with Anabaena cells

Biological Nitrogen Fixation

67

(A) Sporocarp - Development and Megasporangium

(B) Microsporangiate sporo carp and A Microsporangium Hg. 5.4 Azolla

Microsporocarps are normally larger in size than macrosporocarps. Through the tin of macrosporocarps. the vegetative cells of Anabaena get into the plant and es~ablish symbiosis. Both male and female sporocarps are developed into micro and mq,cro gametophyte respectively. They produce sex organs like antheridium and Archegonium. Fertilization of egg cells with spermatozoid results in the formation of the embryo. Embryo

68

Introduction to Soil and Agricultural Microbiology

development normally takes place in the macrosporic complex. (Fig. 5.5)

t

t

EMBRYO (2n)

c ;

1

• SPOROPHYTE -+ SPOROCARP (2n) I MACROSPOROCARP ~ (Female)

,

MICROSPOROCARP (M,le)

,

~

if

ZYGOTE (2n)

en

FERTILIZATION _

~

MACROSPORE (n)

MACAIOG~~OPHYIE

i

L

" .

EGG(n).

ARCHEGONIUM (n)

SPERMATOZOIDS (n) 4

MICROSPORE (n)

!

MICROGAME TOPJHYTE (n)

ANTHERIDIUM (n)

Fig. 5.5 Life Cycle of Azalia

Cultivation of Azolla Azolla nurseries are raised in small plots (50-100sqm) or in concrete tanks with 5-10cm deep water (pH 7-8) containing superphosphate at 4-8kg P205/ha after seeding the plots with Azolla inoculum at the rate of 0.1 to 0.4kg per sq.m. These nurseries have to be planned several weeks ahead of the date set for transplanting rice seedlings. At the end of 2-3 weeks, when full growth of Azalia takes place, the water is drained and the Azalia growth is added into the rice fields by ploughing the mass (10-20 t/ha) into the puddled rice field. This is followed by transplanting of the rice seedlings within 7 days. (N.S. Subba Rao 1995)

Nitrogen Fixation by Free-living Bacteria The free-living bacteria capable of fixing molecular nitrogen belongs to three groups, (1) Obligate aerobic bacteria: For example, Azotobacter, Beijerinckia, Arthrobacter and Bacillus. (2) Facultative aerobic bacteria: For example, Aerobacter, Klebsiella, Pseudomonas (3) Anaerobic bacteria: For example, Clostridium, Chlorobium, Rhodospirillum, Rhodopseudomonas. The nitrogen fixation process is carried out by the enzyme This enzyme is responsible for the absorption and ree ,:;tion of N2 gas. The enzyme consists of two protein fractions,

ni~rogenase.

Biological Nitrogen Fixation

(a) Mo-Fe protein (b) Fe-protein

69

Dinitrogenase Dinitrogen reductase

-

The mechanism of N2 fixation required two steps,

(a) ADP molecule (b) Substrate reduction Normally the enzyme, nitrogenase is highly senl)ltive to oxygen. With the help of electron carriers like ferridoxins and Flavidoxin, N2 molecules are converted into ammonia. In addition to this the enzyme nitrogenase can also reduce certain other compounds. For example, C2H2 Acetylene

~

C2H4 Ethylene

Among the various nitrogen fixing bacteria, Azotobacter and Clostridium are the most investigated genera. Azotobacter is effective in increasing the yields of crops. It also synthesises some biologically active sUbstances such as IAA, vitamin-B and gibberellins. The amount of N2 fixed is measured by the well-known Micro Kjeldahl method.

Associative Symbiosis Besides the free-living form of N2 fixation, some of the organisms are capable of fixing N2 in rhizosphere region. For example,

Azotobacter paspa/i Spirillum Iipoferum

These organisms are associated with the roots of some grasses like paspalum, pennisetum, Digitaria etc. Ba9teria of the genus Azospirillum are N2 fixing organisms living in close association with plants in the rhizophere. For example, A. brasiliense, A. Ijpoferum Azospirillum promotes plant - growth and enhances crop - yield by 10-30% as shown in field experiments by Sumner in 1990. Inoculation of plants with Azospirillum has resulted in a significant change in growth characteristics such as

1. Promotion of mineral and water uptake

2. Root surface area 3. Root diameter and density 4. Length of root hairs (Hadas and Okon, 1987)

Introduction to Soil and Agricultural Microbiology

70

Azospirillum inoculation affects the concentration of free indole-3-acetic acid, indole-3 butyric acid as well as the respiration rates and activities of enzymes involved in the TeA cycle and glycolysis pathway in roots of maize and other plants.

Nitrogen Fixation by Free-living BGA : (CY ANO BACTERIA) Micro-organisms like BGA comprise a single-class cyanophyceae. The members of this class are considered to be the simplest living autotrophic plants. The BGA are predominantly fresh water forms, a few species are marine forms. They comprise unicellular forms and colonial forms. .. The plankton of lakes contains species of Nitrogen-fixing algae which are invariably heterocystous, such as Anabaena. Of the soil algae, the blue-green algae are the most important. They are capable of fixing atmospheric nitrogen in their bodies. Upon its release, nitrogen in usuable form increases soil fertility and improves the growth of crop plC'nts. In 1939, P.K. De conclusively proved that blue-green algae are the chief agents for nitrogen fixation in rice fields. They increase the fertility of the soil by fixing atmospheric nitrogen. (a) Unicellular forms: The thallus in some of species is a . unicell which may be spherical or oval. For example, Chrococcus, Anacystis. (b) Colonial forms: In most blue-green algae, the cells remain attached to their walls, after division to form a loose organisation of cells termed as a colony. The colonies may either be filamentous or non-filamentous.

(i)

(ii)

Non-filamentous colony: It may be cubical, spherical or irregular. For example, Microcystis, Aphanocapsa. Filamentous colony: It is the result of repeated cell divisions in a single direction forming a chain or a thread. It is known as the trichome. For example, Spirulina, Rivu/aria.

Most of the N2 fixing BGA belongs to the order, Nostocaies and Stigonematales under the genera, Anabaena, Anabaenopsis, Nostoc, cylindrospermum, Calothrix, Scytonema, Tolypothrix etc. In general, nitrogen fixation is associated with forms possessing heterocyst.

71

BiOlogical Nitrogen Fixation

Heterocysts Certain genera of Nostocales and Stigonematales produce enlarged, thick walled, pale yellowish specialized cells in addition to the vegetative cells. These large empty-looking specialized cells are called the Heterocysts. Heterocysts show a definite structural organisation. There are protoplasmic connections between them and the adjacent vegetative cells. In general, the heterocyst develops from an ordinary vegetative cell particularly a recently divided one. (Fig.

5.6)

Fig. 5.6 (A) Nostoc with heterocysts

Fig. 5.6 (B) Anabaena with heterocysts

7?

Introduction to Soil and Agricultural Microbiology

Mechanism of N2 Fixation Heterocyst provide a congenial environment for the effective functioning of the enzyme nitrogenase. Glutamate formed in the vegetative cells gets transferred to heterocyst cells. It reacts with NH 3 • The latter moves into the vegetative cells where it reacts with keto-glutarate to form glutamine.

t

OTHER METABOLITES

H

2

O

,,

l

FERREDOXIN •

:::j w

U

w

>

~w

(!)

w

>

1 /~

r

NH

I

HETEROCYST

I

GOGAT

I

GLUTAMATE

- - -.....~ GLUTAMINE _ _~.~ (GS)

Alpha KETO GLUTAMATE

' VEGETATIVE CELL

Fig. 5.7 Inte~action between Vegetative Cells and Heterocysts GS - Glutamine Synthetase GOGA T - Glutamine Oxoglutarate Amino Transferase

The glutamine is then converted into other metabolic products. During this process glutamine products are trans-located from heterocysts to vegetative cells. (Fig. 5.7)

DOD

MICROBIAL INTERACTIONS The environment consists of a number of micro-organisims. These interact among themselves, and with plants and animals. Such relationships provide them all with protection, nutrients and other benefits. This is known as symbiosis. On the basis of relative advantage to each partner the microbial associations can be classified into following types : 1. 2. 3.

Neutral Positive Negative

Neutralism Mutualism, Commensalism Antagonism TABLE 6.1 TYPES OF ASSOCIATIONS

2.

Types of Interaction Neutral Positive (beneficial)

3.

Negative (detrimental)

Sr. No. 1.

Example Neutralism Mutualism, Commensalism, Proto Co-operation Antagonism, Amensalism, Competition, Predation, Parasitism

74

Introduction to Soil and Agricultural Microbiology TABLE 6.2 TYPES OF INTER SPECIFIC INTERACTION (ODUM 1971)

Sr. No.

Effect of Interaction

Interaction Type

Population A

Population B

1.

Neutralism

0

2. 3. 4.

Commensalism Proto co-operation

+ + +

0 0

+ +

5.

Ammensalism

-

0

6. 7. 8.

Parasitism Competition

+ -

Predation

+

-

Mutualism

o = No effect (Population growth not affected) + = Positive effect (Population growth increased) - = Negative effect (Population growth decreased)

TYPES OF INTER SPECIFIC INTERACTIONS (Table 6.2) 1. Neutralism Neutralism is the most common type of inter-specific interaction. Neither population directly affects the other. For example, the normal microbiota of the human body. There is a large number of micro-organisms residing in the different body organs of humans such as skin, nose, eyes, respiratory treat, stomach and genito-urinary tract. (Refer Table No. 6.3) For example the micro-organism Staphylococcus, Streptococcus resides in the eye.

2. Commensalism (Latin, Com - together, Mensa - table) Commensalism refers to a relationship between organisms in which one species of a pair benefits; the other is not affected. (a) E. coli in man For example (b) Many cellulolytic Fungi

3. Mutualism (Greek, Sym - together, Bios - life) Mutualism is a symbiotic interaction where both the partners are mutually benefited. For example

(a) Legume - Rhizobium symbiosis (N2 fixation) (b) Fungal symbiosis mycorrhiza The organisms involved in the symbiotic relationship are called symbionts. The relationship may be a ecto-symbiosis (one organism

75

Microbial Interactions

remains outside the other) or endo-symbiosis (one organism is present within the other.)

Syntrophism Syntrophism is a kind of mutalistic association in which the nutrients are exchanged between two species. For example, Vitamins and amino acids

Proto Co-operation Proto Co-operation is a less extreme sort of interaction than mutualism in which the interaction is clearly beneficial to both species. In many cases, a mutually beneficial relationship may be obligatory for one species but not for the other. For example, the Nitrogen - fixing bacteria in the roots of legume plants could not survive without the host plant. However, the. host pla"t could probably survive without the bacteria.

Antagonism Any inhibitary effect of one organism on another organism is known as antagonistic effect, and this phenomenon is called antagonism. Antagonism is a balancing wheel of Nature. Antagonism has three facets, (a) Ammensalism (Antibiosis)

(b) Competition

(c) Parasitism and Predation (a) Ammensalism: In this interaction one microbial species is adversely affected by the other species, whereas the other species is unaffected by the first one. Antibiosts, for example, is a situation where the metabolites secreted by one organism inhibits the other organism. For example, Antibiotics, siderophores, enzymes etc., (Antimicrobial metabolites) Some organisms secrete cell wall lysing enzymes. For example, chitinase, 111, 3, gluconase. Siderophores are the other extra-cellular secondary metabolites. These are nothing but microbial iron-chelating compounds which are secreted by bacteria like Aerobacter, Pseudomonas, etc., (b) Competition: Competition refers to the type of interaction in which two species vie for a limited amount of food, water and other resources. Success of the species in competition is determined by ability and inoculum-potential of that species.

76

Introduction to Soil and Agricultural Microbiology

For example, Fungistatic substances produced by competitive micro-organisms.

(c) Parasitism and Predation: Parasitism is a type of interaction in which one organism consumes another organism. Predation is an apparent mode of antagonism where a living organism is mechanically attached by another with the consequences of death of the former. It is a destructive relationship. For example, bacteria like Bdellovibrios, a very small gram-negative bacteria, is capable of killing other gram-negative bacteria.

Parasitic Adoptations (1) Reduction of organs

(2) Hooks, suckers get attached to the body of the host (3) Blood sucking leeches and mosquitoes contain certain anticoagulant enzymes in salivary secretions. (4) Ingestion (cndoparasites)

Parasitodism Some diptera, Hymenoptera deposit their eggs in the immature stages of other insects. The larvae on hatching, feed on the host until they are fully grown. The host generally dies. This process is known as Parasitodism.

Characteristics of Predators (1) Hunting ability (2) Specialized and generalized predators

(3) No restriction in diet (4) Prey population reduces (5) Age, size and strength of prey· influence the direction that predation takes.

(6) Predators hunt only when it is necessary for them to procure food.

Some Specific Interactions (1) Plant - Microbe interactions (2) Animal - Microbe interactions (3) Microbe - Microbe interactions (1) Plant - Microbe Interactions The various portions of plants constantly interact with a large number of micro-organisms like, bacteria, actinomycetes, fungi, viruses and develop several types of inter-relationships.

77

Microbial Interactions

The interactions can be categorised into the following: (a) Rhizosphere

(b) Phyllosphere (c) Root Nodule bacteria with legumes and non-legumes (Ref. Chapter- 5 - P.No. 63-67) (d) Mycorrhizae (Fungal - roots association)

(a) Rhizosphere: The Rihizosphere is the region where soil and plant roots make contact. The kinds of micro-organisms in the Rhizosphere also differ from those in root-free soil. Bacteria are the predominant micro-organisms. The amino-acids and vitamins are also released from root tissue; these nutrients stimulate the growth of micro-organisms. The Rhizosphere region is a highly favourable habitat for the proliferation and metabolism of numerous types of micro-organisms. The dominant fungi of rhizosphere are Aspergillus, Fusarium, Cladosporium etc. There are also other bacteria like Arthrobacter, Bacillus, Pseudomonas, Agrobacterium, Rhizobium etc.

J

Flagellate

- - Non Pigmented Bacteria - - Amoeboid Protooza

~~~ 20

40

60

PLANT AGE (DAYS)

Ag. 6. 1 The ratio between the number of micro·organisms in the rhizophere (R) and the corresponding number of microbes in the soil (5) away from the root system (Rl5 ratio).

Actinomycetes are important constitutents of rhizosphere microflora. They include Actinomyces, Franikia, Nocardia, Streptomyces etc.

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Introduction to Soil and Agricultural Microbiology

RIS is the ratio between the total number of micro-organisms present in the rhizosphere to that present in the non-rhizosphere region. RIS r Total number of micro-organisms in Rhizosphere soil ra 10 = Total number of micrQ;-organisms in Non-rhizosphere soil The RIS ratio shown here depicts the rise and fall of bacterial and protozoan populations within the Rhizosphere during the PLANT root development of the Sinapis Root hair alba (mustard plant). At the beginning and end of plant - t - _ Cortex growth, the R/S ratio is very low, indicating that the population of ~ 0 {} I 0 oRhi~Plane micro-organisms in the soil Rhizosphere cind in the rhizosphere is of 0 0 0 I 0 nearly the same magnitude. 0 c> f Q L.) During the active growth () (1 I L::, phase, however, the a () , I a Root Cap microbial population is " 0 0 0 considerably higher in the 6 0 ',----' 0 Rhizosphere. 6 D 6,D 10 0 The phenomenon of 0 b. G O o ,,/1 loss of organic and inorganic () 0 ~ 6 Nonrhizospt)ere compounds from the root Fig. 6.2: A typical plant root showing mizosphere surface is known as root exudation. It contains carbohydrates, organic acids, enzymes, nucleotides etc. Apart from this some vitamins and growth .regulators (auxins) have also been identified in the exudates from a variety of plants in the trace amount. The Rhizosphere region can be divided into two zones (a) Irmer Rhizosphere . (b) Outer Rhizosphere The Inner Rhizosphere is close to the root surface, whereas the outer rhizosphere embraces the immediate adjacent soil. The overall influence of plant roots on soil micro-organisms is known as Rhizosphere effect. Certain bacteria called Rhizobacteria have the ability to colonize the rhizosphere, as shown in Fig. 6.2. (b) Phyllosphere: The leaf-surface is termed,as 'Phylloplane' and the zone on leaves inhabited by the micro-organisms is called

t--

79

Microbial Interactions

phyllosphere. The term phyllosphere was coined by the Dutch microbiologist, Ruinen. J. (1961). A Leaves-surface normally consists of some microbial population. The establishment of flora on the leaf-surface aided by cuticle, waxes and appendages helps in providing anchorag~ to the micro-organisms. Some common phylloplane micro-flora are Bacteria Fungi Yeast

Pseudomonas, Bejerinckia·etc. Aspergillus sp. Alternaria, Cladosporium Candida albicans, Saccharomyces cerevisiae

Phylloplane consist of several types of Saprotrophs, symbionts and pathogens. The phylloplane microflora are influenced by several factors, morphological and physiological. These include nutrition, radiation, temperature, relative humidity etc. The microbes are attracted by pollen grains, chemicals and nectors and therefore, a variety of microbes fall on floral parts. These could be both, beneficial and pathogenic micro-organisms. TABLE 6.3 SOME OF THE KNOWN PHYTOALEXINS

Sr. No.

1.

2. 3. 4.

5.

Plant Soya bean (Glycine max) Potato (Solanum tuberosum) Pea (Pisum sativum) Tobacco (Nicotiana tabacum) Apple (Pyrus malus)

Incitants. Phytopthora sojae

Phytoalexins Hydroxyphaseolin

Phytophthora infestans

Solanine

Penicillium expansum Pseudomonas Solanacearum Venturia inaequaJis

Pisatin Scopolin Phloridzin

The presence of spores of a pathogen on the surface of leaves results in the formation of chemical substances. These are known as phytoalexins. The compounds which induce the synthesis of phytoalexins are commonly known as elicitors. The phytoalexins contribute to disease resistance. (c) Root Nodule bacteria with legumes and non-legumes: (Ref. Chapter 5, Page No. 63-69)

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Introduction to Soil and Agricultural Microbiology

(d) Mycorrhizae (Fungal- roots assOciation): MycorrhizQ is an apparent association developed as a result of a symbiotic association between fungi and plant roots. The term mycorhiia was first coined by A.B. Frank in 1885. During the interaction, clusters of small rootlets are formed, covered by close colourless hyphae. The exact relationship between hypae and root system is still a debatable question. Some physiologists consider the fungi to be merely a parasite. Mycorrhizae roots differ from normal roots (a) They are short and thick (b) They lack root caps (c) They are most extensively branched (d) They lack root hairs (e) They are covered with fungal hypae There are three kinds of mycrorrhizae, (1) Ectomycorrhiza (Ectotrophic) (2) Endomycorrhiza (Endotrophic) (3) Ectendomycorrhiza (Ectend6trophic)

1. Ectomycorrhizae The fungus completely encloses each feeder rootlet in a sheath. The hyphae penetrate only between the cells of the root cortex. The hyphae grow inter-cellularly and develop Hartig net in cortex.

(A) Ectomycorrhizae

(8) Ectomycorrhizae

Fig. 6.3 (A) Ectomycorrhizae

81

Microbial Interactions

Ectomycorrhiza predominates in Pinaceae, Myrtaceae, Betulaceae and in other tropical and temperate families. There are about 5,000 fungi of basidiomycetes and Ascomycetes involved- in forming ectomycorrhiza. Outside the root surface, fungal mycelia form a compact and multilayered covering known as mantle. (Fig. 6.3)

2. Endomycorrhizae The fungus lives within the cells of the roots. The hyphae grow intra-cellularly and establish direct connections between the cells of the root. It is normally found in all groups of the plant kingdom. Harley and Smith (1983) have divided the endomycorrhizae into five distinct types. (a) Vesicular - Arbuscular Mycorrhiza (VAM) . (b) Arbutoid Mycorrhiza (e) Monotropoid Mycorrhiza

(d) Ericoid Mycorrhizae

(e) Orchid Mycorrhizae (a) Vesicular - Arbuscular Mycorrhiza (VAM): The hyphae are coiled and intra cellular vesicles and arbuscules are present. Over 90% of the vascular plants of the world flora form VA Mycorrhizae. Arbuscules function as haustoria and are involved in the inter-change of materials between plant root and fungi. Vesicles are large and multi-nucleate in nature.

Spores

substending Hypha

For example, VAM fungis as shown in Fig. 6.4 (b) I'rbutoid Mycorrhiza: The short roots are covered with a well-defined sheath and a Hartig.

Glomus Fig.

6.4 : VAM Fungi

For example,.Arbutoidae (Arbutus unedo) (c) Monotropoid Mycorrhiza: Roots form a ball through which fungal mycelium encloses the mycorrizal roots of neighbouring green plants. For example, Monotropa hypopitys (d) Ericoid Mycorrhiza: The fungi attached to outer-most layer of cortical cells form dense intra cellular cells.

Introduction to Soil and Agricultural Microbiology

82

For example, Rhododendron (Eri coidae) Fungi - Ascomycetes (e) Orchid Mycorrhiza: Orchids germinate only with the infected endomycorrhizal fungi.

For example, the fungi from the genus Rhizoctonia.

3. Ectendomycorrhiza It shares the features of both ecto and endo mycorrhizae. They are normally found in both gymnosperms and angiosperms. They have less-developed external mantle. Hyphae are established intra-cellularly in cortical cells.

Mycorrhizosphere Effect Mycorrhizophere is influenced by the microbial community. This effect is said to be a mycorrhizosphere effect.

Uses of Mycorrhizae (1) They play a key role in the selective absorption of some elements in plants. (P, Zn, Cu, Ca, K, S, Fe) (2) VA mycorrhizal fungi enhance water uptake in plants ,

(3) It enhances plant growth (4) It increases resistance in plants (5) It plays a significant role in soil fertility.

(2) Animal-Microbe Interactions There are many kinds of micro-organisms that interact with different groups of animals and develop a variety of relationship. They are: (a) Cyanellae (b) Zoochlorellae and Zooxanthellae (c) Ectosymbiosis of protozoa, bacteria with insects (d) Endosymbiosis of bacteria and fungi with birds and insects. (e) Ruminant symbiosis (f) Microbiota of the Human body (a) Cyanelfae: Some protozoa such as cyanophora and paulinella are associated with cyanobacteria, the cyanobacterial endosymbionts of protozoa. (b) Zoochlorellae and Zooxanthellae: Many marine invertebrates like sponges, jelly fish and ciliates harbor endosymbiotic,

83

Microbia/Interactions

spherical algal cells. These are of two types, zoochlorellae and zooxanthellae. Algae belong to the genera chrysophyta, chlorophyta and Rhodophyta. In this association.,.-.the organisms exchange the gases (C02 , O2 ) through photosynthesis and respiration processes. (c) Ectosymbiosis of Protozoa, Bacteria with Insects: Animals such as termites, cockroaches, etc. cannot degrade cellulose and lignin. Therefore they develop an ectosymbiotic association with cellulose - lignin decomposing micro-organisms like N2 fixing bacteria that can degrade these substances. (d) Endosymbiosis of Bacteria and Fupgi with Birds and Insects: There is a group of birds belonging to the genus, Indicator. These are commonly known as honey guides. The birds feed on the remnants of the exposed honey comb, but cannot digest bees wax. Therefore, they harbor micro-organisms like Micrococcus and Candida albicans to carry out the digestion. (e) Ruminant Symbiosis: Ruminants are a group of herbivorous animals. They have a special gut called Rumen. Examples of ruminants are cattles, sheep, deer, goat etc. (Fig. 6.5) Initial food

Esophagus

\

: •••• - ...CUd . ~ ·· ..

\

~

Small Intestine

Fig. 6.5 Ruminant Stomach

Introduction to Soil and Agricultural Microbiology

84

These animals use plant cellulose, which are not digested in the normal gut. Some of the anaerobic cellulose - digesting bacteria such as RlJminococcus, Bacteirodes develop mutalistic association with ruminants and hydrolyse cellulose and other polysaccharides into simple forms. Apart from carbohydrates, bacteria can also digest proteins and lipids. These bacteria ferment proteins and lipids to produce gases. (C0 2 , H2 ), which in turn are converted into methane by bacteria known as Methanobacterium ruminantium. The cellulose is degraded in the rumen by anaerobic bacteria, Fungi and Protozoa. The main component of cellulose is carbohydrates. It is normally found in hay, straw and grass. The rumen and the reticulum are the two major sections of the rum inant stomach. The ruminant stomach provides ideal conditions for the growth of micro-organisms. The continuous supply of bicarbonate and phosphate in pH 5.8-7.3 is also responsible for the microbial conversion. The protozoa and bacteria like Ciliates, Dip/odinium and Entodinium are predominant micro-organisms in rumen. The rumen-specific bacteria are basically anaerobic. An example of fungi is Neocallimastix frontalis belonging to chytridiomycetes. It hydrolyses cellulose into glucose and ferments glucose to acetate and other components such as ethanol, CO 2 and lactate. (f) Microflora of the Human Body: A number of commensals, saprophytes and facultative pathogens are found in human beings. Humans harbour a wide array of micro-organisms which interact with each other. TABLE 6.4 MICROFLORA OF HUMAN BODY

The Micro-organisms present in the various regions of the body are listed below : Sr. No.

Bodysites

Micro-organisms

1. Eye

Coagulase-negative Staphylococci, Streptococcus

2. Ear 3. Small Intestine

Pseudomonads, Enterobacteriaceae

4. Large Intestine

5. 6. 7. 8. 9.

Mouth Nose Skin Stomach Urethra

10. Vagina

Lacto bacillus, Clostridium E.coli, Klebsiella, Proteus, Actinomyces Haemophilus, Candida, S. aureus S . Pneumoniae, Viridans streptococci Bacillus, S. aureus, Mycobacterium Lactobacillus, Peptostreptococcus Fusobacterium, Bacteroides Candida, Gardnerella vaginalis, Lactobacillus.

85

Microbial Interactions

(3) Microbe-Microbe Interactions There are different types of harmless and harmful relationships between different types of micro-organisms. The relationships are as follows: (a) Lichens (Algae and Fungi) (b) Antagonism - (Refer page no. 75) (c) Parasitism - (Refer page no. 76) (a) Lichens: The Lichens are small groups of dual or composite organisms. The two groups of organisms, fungus (mycobiont) and algae (phycobiont) live in close proximity and appear as a single organism. The fungal mycelium forms a close network that appears as a tissue -like mass. The algal cells are embedded in it. The algae belong to Cyanophyta or Chlorophyta.

For example, Nostoc, Rivularia and Stigonema (Cyanobacteria) Trebouxia (green algae)

The fungi belong to Ascomycetes and some genera of Basidiomycetes. In this symbiotic association, the two partners are mutually benefitted. The fungus supplies water and minerals to the algae, and the algae provide food to the fungus.

Classification The lichens can be classified into various types. On the basis of the nature of fungal element, lichens are classified into two groups, (a) Ascolichens (Ascomycetes) (b) Basidio lichens (Basidiomycetes)

On the basis of habitat, lichens are classified into three groups, (a) Saxicolous (growing on rocks) (b) Corticolous (growing on leaves and bark)

(c) Terricolous (growing on soil)

Taxonomic Position of Lichen Division Sub~division Class

(eg. Usnea comosa) Mycota Eumycotina Lichens

86

Introduction to Soil and Agricunural Microbiology

Ascolichens Gymnocarpae Parmeliales Usneaceae Usnea Comosa

Sub-class Series Order Family Genus Species

Lichen Thallus The body of a lichen is a Thallus. It is grey or green in colour and irregularly shaped. On the basis of the structure of thallus, Lichens are classified into three groups, (i)

Crustose lichens

(ii) Foliose lichens (iii) Fruticose lichens

(i) Crustose Lichens (Fig. 6.6): The thallus is flat, insignificant and without any lobe. Sometimes it is buried in the substratum. Crustose lichens normally grow on rocks, on stones, bank or land substrate. For example, - Haematomma puniceum

V, i", \ 0TI§J ~ Fig. 6.6

~-+-~-Apothecium

(ii) Foliose Lichens (Fig. 6.7): The Thallus is more striking, flat, broad - lobed and leaf-like. It is attached to the substrate by rhizoid-like outgrowths (rhizinae) For example, Parmelia, Peltigera

(iii) Fruticose Lichens (Fig. 6.8): The Thallus is complex, slender and freely branched. The branches are cylindrical or ribbon-like. It is attached only by a flattened disc. For example, Usnea, Ramalina

."lobes

Fig. 6.7

~~-

Fig. 6.8

DOD

I - Stone

Attaching disc

PHYTOPATHOLOGY Introduction The science which deals with plant diseases is known as phytopathology. The study of plant diseases is important as these diseases cause damage to plants and plant products. The plant diseases are generally classified into following types.

(i) Soil-borne diseases

(ii) Air-borne diseases (iii) Seed-borne diseases Based on how they are spread, the diseases are classified into epidemic, endemic and sporadic diseases. Based on the plant parts affected, the diseases are classified into vascular diseases, root diseases, foliar diseases and fruit diseases.

Disease A disease in plants, is any deviation from the healthy condition. It expresses either in physiological and morphological c!langes.

Pathogenesis Pathogenesis is the actual mechanism of disease development.

88

Introduction to Soil and Agricultural Microbiology

Pathogen Any agent which causes damage to the host is a pathogen.

Inoculum Inoculum is a portion of a pathogen which is transmitted to a host and is capable of infecting the host.

Inoculum Potential According to Hors Fall (1932), inoculum potential is the number of infective particles present in the environment of un infected host. These could later infect the host.

Symptoms The external signs on the plant which indicate disease, are called symptoms.

Incitant The organism which causes the disease is termed incitant (causal agent)

Penetration The invasion of the host tissue by the living micro-organism is called penetration.

Infection The establishment of the causal organism in the body tissue after penetration is termed infection.

Incubation Period The intervening period between infection of the host by a infective agent and the appearance of first symptoms of the disease is called the incubation period.

Parasite A parasite is on organism which lives on other living organisms. All pathogens are parasites, but all parasites may not be pathogens. GENERAL SYMPTOMS OF PLANT DISEASES

Based on the result of interaction between the host and the pathogen, the symptoms of a disease may be divided into two categories. 1. Symptoms due to the external appearance of a pathogen. For example, mildews, smuts, rusts

Phytopathology

89

2. Symptoms which are the visible effects induced by the parasite on the host. For example, Necrosis, hypoplasia and hypertrophy

Mildews These are a group of fungal diseases affecting the seed plants. The pathogens grow superficially on the host surface. The mildews are of two kinds. (i) Downy mildews - The superficial growth is downy, layer consists of numerous sporangia. (ii) Powdery mildews - The patches appear powdery with the formation of numerous white canidia.

Smuts These are the fungal diseases affecting cereals and other grasses. Smut spores develop mostly in floral organs (ovaries) and other parts like leaves, stem (or) roots. For example, Wheat is affected by Ustilago tritici.

Rusts These are fungal diseases affecting grasses and other plants. The rusts appear as small pustules, red, yellow or black in colour. For example, Wheat is affected by Puccinia graminis.

Necrosis The death of the host tissues induced by the attack of a pathogen, is called Necrosis. Necrosis causes severe damage to host tissues. For example, rusts, smuts, blights, cankers, cause necrosis.

Hypoplasia It is a sub-normal (below normal) cell production in response to an attack by a pathogen. It leads to stunted growth and dwarfing of the host.

Hypertrophy It is an abnormal (over-growth) increase in size of one or more organ of a host in response to an attack by a pathogen. This is nothing but enlargement of individual cells. For example, Crowngalls.

Hyperplasia It is caused by an increase in the number of cells as a result of rapid cell-division.

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Introduction to Soil and Agricultural Microbiology

Chlorosis When the green pigment is destroyed and the tissues become yellow due to discolouration, this condition is known as chlorosis. For example, Bacteria, Fungi.

Cankers Local necrosis also results in open wounds, often sunk in stems and surrounded by living tissue. These are called cankers. For example, Citrus canker.

Damping Off The stem is attacked near the ground level. The affected portion becomes weak and finally disintegrates. In vegetables, this disease is caused by Phythiurn (Fungus).

Blights There is a sudden death of plant-parts such as leaves, stamens, blossoms etc. The dead parts become dark or brown in colour. For example,

(a) Early blight of potato - Alternaria solani (b) Late blight of potato - Phytophthora infestans

Mosaics These are characterised by light green, yellow or white areas inter-mingled with the normal green of the leaves. For example, TMV.

Tumors rThese are gall-like structures developing on stems and roots. For example, Crown gal/s.

Witches Broom The leaves become very much reduced, with shortened internodes, leaves show a broom-like structure.

Root-knot It is a giant celf which contains many nuclei, and expands irregularly. For example, Roct knot nematode - Meloidogyne javanica Host - Tomato

Phytopathology

91

I. FUNGAL DISEASES (Phytopatogenic Fungi) The various kinds of diseases caused by fungal pathogens are:

(A) Late Blight of Potato It is a serious fungal disease affecting potatoes. It is worldwide in its occurence. The taxonomical (systematic) position of phytophthora is Division Mycota Sub-Division Eumycotina Class Oomycetes Order Peronosporales Family Pythiaceae . Genus Phytophthora Species Infestans The mycelium is profusely branched and consists of aseptate, hyaline, coenocytic intercellular hyphae with haustoria. It has finger-like protuberances, surrounded by extra-haustorial sheath. Aerial hyphae may also develop. Each sporangiophore bears a sporangium at its tip. The sporangiophores emerge only through the stomata (leaves). The sporangium is multi-nucleate, thin walled, oval and hyaline. It has an apical papilla. The mature sporangia are readily detached (Fig. 7.1 (a».

Aetiology The late blight disease is caused by the fungus, Phytophthora infestans, which is called the potato blight organism.

Symptomology The fungus first appears on the plant-tops, generally after the blossoming period. The early symptoms are small, dead, brownish or purple spots or lesions. These appear on the tips and margins of the leaflets, petiole and stem. Under favourable conditions the lesions rapidly increase in size and occupy entire areas of the leaves. The blighted leaves curl and shrivel in dry weather. Potato tubers are also infected. The size and weight of the infected tubers decreases and they are also invaded by the sporangia or by zoospores. The sporangia spread the disease in the subsequent season (Fig. 7.1 (b».

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Introduction to Soil and Agricuitural Microbiology \_~----Sporangia

Sporangio __ phores

Zoospores

H=-+£.....-- Stomata

(a) Sporangia

(b) Infected potato tuber Fig. 7. 1 Late blight of Potato

Asexual Stage Under favourable condition the sporangia germinates either directly or indirectly to form germ tubes or zoospores-formation. After maturation, the zoospores are liberated and swim actively on the surface of water and then come to rest. Each zoospore loses it's

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93

flagella and may encyst. The encysted zoospore develops into a germ tube. The germ tube produces appresorium and infection I'lypha, which enters the host tissues either through epidermis or by stomata. Apart from Asexual reproduction, Oogamous type of sexual reproduction also takes place. Sex organs like antheridia (male) and Oogonia (female) are formed. After the maturation of sex organs, fertilizaton takes place resulting in the formatio:1 of Oospores. Oospores pass out germ-tubes or germinating oospores directly form zoospores. The Zoospores germinate to give rise to the mycelium.

Disease Cycle The infected tubers are the main sources of primary infection. The dormant mycelium in the tubers becomes activated. Under favourable conditions, the mycelium pushes out branched, hyaline sporangiophores. The mature sporangia are detached and spread through stomata to a new potato plant, assisted by rain or air current. By means of Indirect germination, the sporangia germinates into zoospores. Sometimes by direct germination the sporangium act as a conidium. It directly converts into germ tube or an infection thread.

Control of the Late Blight Disease The late blight of potatoes is controlled by various methods. They are(1) Resistant Varieties: The cultivation of some disease-resistant varieties is an ideal method for controlling the late blight disease. (2) Spraying: The best method of control is foliage spray. Some fungicides like Blifox-50, Bordeaux mixture, Dithane M-22 are used for this purpose. (3) Sanitation: The proper disposal of the potato tuber rs another method of controlling the late blight disease.

DISEASE DESCRIPTION Name of the Disease Host Pathogen Genus Species Symptoms

Late blight of potato Potato plant Fungi Ph}1ophthora Infestans Purple spots, lesions

.

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Introduction to Soil and Agricultural Microbiology

(B) Early Blight of Potato The disease appears on young potato plants. It is common in cold as well as warm regions.

Taxonomic Position The systematic position of Alternaria is Division Mycota Eumycotina Sub-Division· Class Deuteromycetes Moniliales Order Family Dematiaceae Genus Alternaria Species' Solani The mycelium is short, septate and branched. The light brown colour hyphae is inter-cellular or intra-cellular in nature. The cells are multi-nucleate. The organisms can multiply asexually by the sporulation method. The asexual spores are normally in the form, conidia. Conidia are large, several- celled and dark coloured. Under favourable conditions, conidia germinate and form germ tubes.

Aetiology The early blight disease is caused Alternaria so/ani.

Symptomology' The disease appears in the form of scattered pale brown to dark spots on leaflets. The spot is usually by chorotic zone. When the disease increases the size of the spot also increases. In cases of severe infection, the leaves dry up and finally fall down to the ground. Tubers are also affected. Lesions are developed on the skin surface. (Fig. 7.2 (a»

Disease Cycle During primary infection, the germ tube enters into the lower portion of leaves by stomatal opening. Under favourable condition, it produces leaf spots. After maturation, conidia are detached and dispersed by water, insects or air current. During the development of conidia, some toxic substances like alternaric acid are produced. (Fig. 7.2 (b»

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Phytopathology

~--Conidia

/J7.-II--7 Digestive gland

l!J--+--Vulva

Int~ne --'lr-'""":

Overies Fig. 7. 14 Plant parasitic 'nematode

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119

Root-knot Disease The Root-knot disease is a nematodal disease. It is characterised by severe knotting of the root". It is dangerous to the crop plants like sugarcane, tomato, etc.

Aetiology The Root-knot disease is caused by the nematode, Meloidogyne javanica. The female lays eggs into the egg sac. Eggs develop a larvae. The larvae develop and then enter into fresh roots through the soil and cause root galls (giant cells). The adult female nematode lays eggs in the root tissues. Again the larvae moves to neighbouring cells and cause new infections. The entire life-cycle is completed within 30 days.

Symptomology The symptoms appear mostly in underground parts. Roots are severely knotted. In some cases, chlorosis also appears. The infected root surface swells up and develops into root galls on giant cells. These cells also affect the vascular system of the plant.

Control of the Root-knot Disease (1) Nematicides like Nemagon.

(2) Resistant varieties (3) Fumigation method (carbon disulphide) (4) Crop rotation.

V. PLANT DISEASE CONTROL: (Control Measures) There are various methods of plant disease control, as follows:

(a) Exclusion (b) Eradication (e) Protection

(d) Immunization

(a) Exclusion: In the Exclusion method, the pathogen 1s kept away from entering the area in which the host is growing. This can be achieved through regulatory methods like quarantine and others. (b) Eradication: The eradication of the pathogen is another method for the control of plant disease. This can be achieved by (i) Chemical eradication (ii) Sanitation

(iii) Crop rotation

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120

(i) Chemical eradication: In chemical eradication, seeds are treated with fungicides. The term, fungicide generally refers to the chemicals used in the control of fungal diseases. The use of bordeaux mixture and sulphur dust is an effective method for controlling plant diseases. (ii) Sanitation: Sanitation of the crop-area can also help in the control of plant diseases. Some fungal diseases like red-rot of sugarcane, powdery mildews of wheat and downy mildews of peas are controlled using the sanitation method. (iii) Crop rotation: Periodic rotation of crops over a given area is useful for the eradication of soil-borne diseases like mosaic and wilt disease. (c) Protection: Plant disease can be prevented by protecting the host from an attack by a plant pathogen. Protection can be achieved through the following methods, (a) Environmental manipulation

(b) Chemical prophylaxis Environmental conditions do favour diseases. Therefore, the alteration of the environment makes it unfavourable for disease development. Chemical prophylaxis also plays an important role in controlling planf diseases. It consists in spraying or dusting diseased plant parts with fungicides. Sulphur is a potent fungicide. It is useful for the eradication of powdery mildews and other diseases. The bordeaux mixture is also useful. (d) Immunization: Immunization can be achieved by - (i) Genetic resistance and (ii) Chemotherapy. (i) Genetic resistance: The genetic resistance of the host can be improved by using disease -resistant varieties. It is the most effective method of disease control. The disease resistant varieties are manipulated by some breeding and genetic engineering techniques such as mass selection, back crossing, pure line selection, tissue culture, protoplast fusion etc. (it) Chemotherapy: Chemical destroy pathogens. Fungicides act as eradicants and kill fungal pathogens on plant parts. This can be achieved by systemic chemotherapy and topical chemotherapy. Depending on the nature of agents employed to control the diseases, the plant disease control methods are classified into: (1) Physical methods

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Phytopathology

(2) Chemical methods

(3) Biological methods

1. Physical Methods Physic;:"1 agents like radiation and temperature are commonly used to cO.ltrol plant disease. Radiations like X-rays, a-particles, y-rays and uv-light are used to control various diseases of crop plants. The hot water treatment and refrigeration is also used. The soil is sterilized using high temperature. This is useful to kill the soil-borne pCl;thogens. The infected seeds are treated with hot water at 54°C for 10 minutes. The low temperature treatment or Refrigeration is also used for controlling plant pathogens.

2. Chemical Methods Plant diseases are controlled by using chemical compounds. The application of different types of chemicals on the diseased plant parts is a highly effective and most widely used method of disease eradication. This can be achieved by seed treatment, fumigants, sprays and dust. The following chemicals are commonly used. (a) Bordeaux mixture: It is the most widely used copper fungicide. The Bordeaux mixture is a combination of copper sulphate and calcium. The chemical reaction is CUS04 + Ca(OH)2 ~ CU(OH)2 + CaS04 It is beneficial to crop plants like potato and apple. Hexochlarobenzene (HCB) is used to control various soil-borne pathogens. (b) Benzene compounds: Fungicides derived from Benzene like PCNB (Penta Chloro Nitro Benzene), Dexon, Dinocap, Hexochlorobenzene (HCB) are used to control various soil borne-diseases. (c) Systemic fungicides: Several systemic fungicides like Benzimidazoles, Triazoles and pyrimidines are widely used to control plant pathogens. (d) Soil-fumigants: Some nematicides like vapam, vorl ex and chloropicrin are used as fumigants. They are highly effective against a variety of soil micro-organisms. (e) Antibiotics: An antibiotic is defined as a chemical substance produced by a micro-organism, which can inhibit or kill other micro-organisms.

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Many of the known antibiotics are used for plant-disease control. Some commonly used antibiotics are Streptomycin, Aureofungin, Griseofulvin, Tetracyclines, Cyclo heximide, Streptocycline etc. The streptomycin is very useful in controlling some plant pathogens like Fire blight of apple (Erwinia sp). Tetracyclines are highly active against many plant pathogens. Streptocycline is a combination of Streptomycin and Tetracycline. It is highly effective against different bacterial pathogens, for example Xanthomonas spp. The antifungal antibiotics such as Cycloheximide', Griseofulvin and aureofungin are used for controlling fungal diseases like powdery mildews. The citrus gummosis and blight 0f rice can also be controlled by using Aureo fungin.

3. Biological Methods (i) They can be used to eradicate the inoculum of the pathogen. (ii) Toxic metabolites secreted by some ptants control nematodes in the soil.

(iii) The mycorrhizae controls the different root pathogens. For example, Phytopthora (pine), Fusarium, (tomato) etc. (iv) Bacteria like Streptomyces sp, Pseudomonas sp, etc. protects plants from various diseases.

000

BIOCIDES Crop protection is a basic and important component of crop production. The crop plants are naturally affected by pathogens, weeds and insect pests. Extensive use of synthetic pesticides has led to pest resistance, pesticide - resurgence, environmental pollution and toxic effects on man, cattle and other organisms. Resistance of pests to organochlorine, organophosphorous and other synthetic pyrethroids have been reported.

Biocides Biocides are living micro-organisms where structural units or metabolites are used against a target organism to restrict or eliminate it's activity. Biocides are environmentally safe because they do not cause any environmental, socio-economic and political problems. Basically there are two type of biocides, (1) Microbial biocides (2) Botanical biocides

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Introduction to Soil and Agricultural Microbiology

(1) Microbi,al Biocides Microbial biocides are micro-organisms such as bacteria, fungi, viruses, etc. used as biocides for controlling plant pathogens. Microbial biocides can be classified into the following types, (1) Bacterial biocides (2) Fungal biocides (3) Viral insecticides (1) Bacterial Biocides: Certain bacteria are very useful to mankind and are used as bacterial biocides. Bacterial biocides are further classified into (i) Bacterial b·actericides (ii) Bacterial fungicides (iii) Bacterial insecticides (i) Bacterial bactericides: Some strains (k84) of Agrobacterium radiobacter are used as bacteriocides for controlling crown gall disease, which is caused by Agrobacterium tumefaciens. (ii) Bacterial fungicides: Some bacteria like Pseudomonas and Enterobacter are used to control funga.l diseases such as Damping of seedlings, which is caused by pythium sp. (iii) Bacterial insecticides: Bacterial species belonging to the genera Pseudomonas, Enterobacter, Bacillus and Proteus are used for controlling the insect pests of agricultural and forestry crops. Among the bacterial species, Bacillus thuringiensis (Bt) has been used extensively in Agriculture.

Bt is a gram positive, rod-shaped bacterium closely related to Bacillus cereus, which is a common soil bacterium. Bt is used to kill a wide range of insects like pest larvae, beetles, flies, aphids and cockroaches. This bacterium was first discovered by a Japanese scientist, Ishiwata in the year 1902. In the year 1912, a German microbiologist isolated this bacterium from infected insects. He named it Bacillus thuringiensis (Bt). The 8t. bacterium secretes two types of toxins, exotoxin and endotoxin. (Fig. 8.2 A and B) Exotoxins

ex - exotoxin

Endotoxins

a - endotoxin

13 - endotoxin

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Biocides

(A) Plate showing the morphological variation of larvae at 1% Alblzla amara treatment

(B) Plate showing the morphological variation of larvae at 1% Alblzla amara + 0.1% BtK· combined treatment

* Bacillus thuringiensis sub sp. KURSTAKI. (Courtesy, B.V. Pradeep, 2001, PGPCAS, NAMAKKAL). Fig. 8.1

These toxins are ingested by the insect larvae. The toxins are digested by the enzymes and they form active fragments. The active fragments bind to the epithelial membrane in the guts of the insect. Finally, they disturb the osmotic,equilibrium of the"cell. (Fig. 8.1 A and B)

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Introduction to Soil and Agricuffural MicrObiology

(A) Bt· showing endorspores

(B) yA purified crystal of Bt toxin

• Bacillus thuringiensis FI{1.8.2

The bacterial spores themselves play a role by causing septicaemia in the affected insect, leading to gut paralysis and shock. The toxic effects include reduced ingestion and digestion of food, change of the body colour to black, oozing out of the body contents and finally, de~th of the insect larvae. (2) Fungal Biocldes: The fungal Biocides are generally classified into the following types, (ii) Fungal herbicides (i) Fungal fungicides (iv) Fungal insecticides (ifi) Fungal nematicides (i) Fungal fungicides: Several types of fungal fungicides interact with fungal plant pathogens. These fungicides are also called myco fungicides.

For example, Trichoderma viridae are used to t~eat the fruit trees whose wounds are caused by Chondrostereum purpureum. (ii) Fungal herbicides: Several strains of fungal species belonging . to genera, Col/eotrichum gloeosporiodes, Phytophthora palmivora are used as fungal herbicides for eradicating milk weeds. These herbicides are also known as myco herbicides.

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127

(iii) Fungal nematicides: Fungal nematicides are used for controlling nematodes like Meloidogyne, Globodera, etc. Fungal nematicides are also called myco nematicides. For Example, Verticillium, Arthrobotrys (iv) Fungal insecticides: A unique characteristic of insect-pathogenic fungi is that they penetrate through the insect cuticle and so they do no have to be consumed directly by the insect. They depend a lot on proper environmental conditions like humidity and temperature. Some of the fungal species belonging to the genera. Metarhizium, Hirsulella and Beauveria are responsible for controlling insect pests. Fungal insecticides are also known as myco insecticides.

(3) Viral insecticides: Several types of viral strains have been used for controlling insect pests such as gypsy moth, budworm and pine caterpillar. Baculoviruses are double-stranded DNA viruses that are pathogenic to arthropods. The Baculoviridae belong to two genera, as determined by their structural properties, Nuclear Polyhederosis Virus and Granulosis virus. Viruses belong to three major groups. These

~re

:

(i) Nuclear polyhedrosis virus (NPV) (ii) Granulosis virus (GV) (iii) Cytoplasmic polyhedrosis virus (CPV) (i) Nuclear Polyhedrosis Virus (NPV): Baculoviruses are large, rod-shaped DNA containing pathogenic viruses. The rod-shaped viral particles are occluded in proteinaceous bodies (OBs), often called Polyhedral Inclusion Bodies (PIBs). After the ingestion of the virus along with food, the PIBs disintegrate in the alkaline gut pH of the insect and the rod-shaped virus particles are released.

The viral particles bind with specific receptors in the mid-gut, enter the haemolymph and reach the other target organs like trachea, gonads etc. They replicate in the nucleus of the cell. The new virus particles, thus formed, infect the other cells. The larvae gets elongated, skin becomes transparent and the body - contents ooze out. The larvae after infection, climbs the top of the tree and hangs upside down. This is a typical symptom called ''tree-top disease." NPVs are highly specific, pathogenic to insects but safe to man and other beneficial organisms. NPVs can be transmitted from generation, to generation through the egg (transovarial transmission). They can be safely integrated into Integrated Pest Management (IPM)

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programs involving botanicals, synthetic pesticides and other microbials. NPV infects American bollworm (He/icoverpa armigera), tobacco budworm (Helicoverpa Virescens) and Gypsy moth (Lymantria dispar). (ii) Granulosis Virus (GV): GVs have the same effects as NPVs and are lethal to insects. Insects of food crops like codling moth (Laspeyresia Pomonella) and Crucifer caterpillars (Pieris sp.) are infected- by the GV virus. (iii) Cytoplasmic Polyhedrosis Virus (CPV): CPVs are viruses which are pathogenic to a few group of insects and they multiply in the cytoplasm of the insect cell. They cause impaired reproduction, reduced fecundity and O2 consumption, decrease in the pupal and moth size resulting in higher mortality rate. The pine caterpillar is infected by CPV.

2. Botanical Biocides Botanical pesticides are plants used for controlling other plant pathogens. They are highly effective, bio-degradable, safe to man and the environment, cheap and are easily available. The chances of resistance - development are nil because of the multiple mode of action due to an array of chemicals present in them. Higher plants have also been used as potential biocides. The Indian neem tree, Azadirachta indica is used (A. Juss) for controlling several insect pests belonging to more than 200 orders such as grasshopper, colorado and potato beetle. Neem has anti-feedant, repellant, growth-regulatory (IGR), ovicidal and other metabolic disruption properties. The extracts of the crude plant possess anti-microbial properties. More than 100 compounds have been isolated so far. Some of the major constituents are azadirachtin, nimbin, nimbidin, Salanin and gedunin. Other botanicals such as Pongamia pinnata, Vitex negundo, Ocimum Sanctum, Allium sativum, etc. also have insecticidal properties.

000

BIOFERTILIZERS In developing countries like India, population growth is increasing day-by-day. The pressure on agriculture is also increasing. Most of our agricultural lands are short of various minerals, which are essential for the growth and development of plants. Nitrogen is a major element required by the plants for their growth. Nitrogen is provided to the plants in the form of chemical fertilizer, causing health hazards and environmental pollution. Chemical fertilizers are also expensive. Biofertilizers are therefore, being recommended instead of chemical fertilizers. . The term "biofertilizers" denotes all the "nutrient inputs of biological origin for plant growth" (Subba Rao, 1982). Bacteria and cyanobacteria fix atmospheric nitrogen. Both are widely used as biofertilizers. Biofertilizers are also referred to as microbial inoculants.

.

Biological nitrogen fixation is a natural process whereby the micro-organisms transform the atmospheric nitrogen into a form which plants can use for building proteins, amino acids etc. The biologically active products (microbial inoculants) of bacteria, algae and fungi help in nitrogen fixation. Some of the Nitrogen fixers are(i) Symbiotic nitrogen fixers (Bacteria) Rhizobium sp.

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(ii) Asymbiotic nitrogen fixers (Bacteria) Azotobacter (iii) Algae biofertilizers (Cyanobacteria) (iv) Mycorrhizae.

The Government of India launched the 'National Project on Development and use of Bioferj,ilizers' during the Sixth Five Year plan. Under this project, one national centre, six regional centres and 40 BGA production centres have been established. (P.K. GUPTA, 1994). Seed inoculation has been widely used because it results in early nodulation and forms prominent nodules, clustered mostly around the, crown of the root postulated to be crucial for nitrogen fixation in the early stages of crop development (Hardarson et al., 1989). Soyabean is an important oil seed crop. It is known as a natural fertilizer factory because of its high nitrogen-fixing potential. Rhizobium is widely used for improving production of legumes. Effectiveness of symbiotic Nitrogen-fixation depends upon the proper establishment of the inter-relationship between a particular legume and specific strain of Rhizobium, (Dart, et al., 1976). Many methods have been used for the estimation of nitrogen-fixation in soyabean. Each of them has, distinct advantages and disadvantages, based on the complexity and cost of analysis (Bergersen et al., 1970). Azolla, the freely - floating aquatic fern, besides being an excellent biofertilizer, is a protein-rich feed for fish, ducks, poultry and pigs. This makes it attractive to farmers.

Mass Cultivatfon of Microbial Inoculants The mass CUltivation of bacteria, cyanobacteria and mycorrhizas are very useful in agriculture. •

Mass Cultivation of Bacteria Bacteria can be inoculated in the soil as biofertilizer, it can be multiplied on media to harvest an a large scale and then supplied to farmers. Rhizobium, Azospirillum and Azotobacter are some of the bacteria which are ~ed to help Nitrogen fixation.

(a) Mass Cultivation of Rhizobium Legumes have played a major role in food production throughout history. In 1898, Hellriegel and Wilfarth demonstrated that legumes fix Nitrogen through the active participation of micro-organisms in root nodules (Berkum et al., 1980) -

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Biofertilizers

Seven groups of Rhizobia have been recognised to inoculate legumes in India. These are R. /eguminosarum, R. me/i/otii, R. tritoti, R. phaseo/i, R. Japanicum, R. /upinti and Rhizobium spp. The nitrogen-fixing ability of legumes inoculated with Rhizobia range from 50 kg to 150 kg per hectare. Mass cultivation of Rhizobium is carried out through the following steps, (a) Sterilization of the growth medium and inoculation of broth. (b) Incubation for 3-4 day at 30-32°C. (c) Test the culture for its purity. (d) Transfer of the culture_ to a large fermenter for 4-9 days. (e) Allow the bacteria to grow (f) Check the quality of broth (g) Blend the broth with the sterile carrier (h) Packaging (in polyethylene bags) (i) Check the quality after different durations

(j) Supply to farmers The various steps involved in seed inoculation with rhizobial culture are: 200g Gum arabic (10%) + 50g sugar solution (10%) .! mixed well Dissolved in water

.! Boil for 15 min .! Cool it Sticker solution .! mixed with Rhizobial culture

.! Inoculum slurry Transfer the inoculum slurry

.! Add seeds

.! Mix properly

.! Spread the seed in shade

.!

Seeds coated with rhizobial cells

.! Sow in the field

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10% sugar solution is mixed with 10% gum arabic. It gets dissolved in water after some boiling. This helps to stick the Rhizobium cells to the seeds. Mix the Rhizobium culture to form inoculum slurry and then transfer the inoculum slurry on to the seeds and mix properly. Spread the seeds in the shade, for drying. The seeds are then coated with rhizobial cells. Finally, sow the seeds in the field.

Effect of Rhizobial inoculants on crop yield The effect of Rhizobial"culture on the yield of soyabean is shown in Table 9.1. The study was a part of the All India co-ordinated Research project on soyabean under the aegis of Marathwada Agricultural University, Parbhani, India, Kurundkar et al., (1991). Productivity of soyabean mainly depends on optimum nodulation by efficient Bradyrhizobium japanicum strains. The yield of soyabean can be substantially increased through Rhizobial inoculation. Kurundkar et al., (1991) studied the effect of different inoculum levels of Bradyrhizobium on soyabean. The results shown in Table 9.1 indicate that except for the number of leaves and branches/plant, Rhizobial inoculations influenced nodulation, growth and the yield of soya bean significantly. The number and dry-weight of nodules was significantly more in all the rhizobial population/seeds at 30 and 60 days of crop growth. There was also an increase in the grain yield due to rhizobial seed inoculation. The beneficial effect of Rhizobial inoculations on the growth and grain yield of soya bean has been reported by earlier workers too (Sable and Khuspee, 1977; Ganachanya and Nirmal, 1978). Rao and Sharma 5 (1980) observed that soya bean inoculated with 2 x 10 Rhizobial/seed had the highest yield of dry mat~er. Besides Rhizobium sp, some free-living nitrogen fixing bacteria are also widely used. Azobtobacter and Azospiriflum are now being used for pre-treating many cereals, to help nitrogen fixation.

(b) Mass Cu!tivation of Azospirillum Azospirillum is an anaerobic bacterium which grows into a white, dense colony on the semi-solid medium. It fixes the atmospheric nitrogen in the soil. It invades the root-system of grasses like digitaria, panicum, maize and sorghum and forms symbiotic association with the root system. Azospirillum is an obligate anaerobic bacteria found in the inner and outer surfaces of the root system of plants. The Azospirillum can be isolated from the plant roots by the following method.

TABLE 9.1 EFFECT OF RHlZOBIAL LOADISEED ON GROWTH AND YIELD OF SOYBEAN Mean/Plant

I Treatment

30 'days of crop growth

Dry Weight

Nodules

No . . Nodules Root (Mg)

Control

0.9

(g)

60 days of crop growth

Dry Weight

Nodulu

Shoot (g)

No.

2.310 0.152

1.192

1.6

18.920 0.175

1.252

Nodules

Root

(Mg)

(g)

6.715

0.292

Grain [ncr1000yield/ ease in seed ha grain weight yield over control

Crop maturity Leaves Height Branches Pods

Shoot (g)

No.

Cm

2.870

7.0 14.25

No. 2.6

No. 9.1

Kg

489

(%)

-

(%)

96.632

102 Rhizobia/seed

7.8

16.1

75.912

0.332

3.430

8.1 14.80

3.1

12.3

567 15.95

111.972

103 Rhizobia/seed

10.0

25.630 0.232 ; 1.305

17.3

80.990

0.372

5.857

9.5 15.90

3.2

13.1

722 46.49

112.270

10· Rhizobia/seed

11.8

32.585 0.245

1.390

19.4

92.557

0.397

6.032

8.6 17.75

3.0

14.0

730 49.28

111.970

5

13.9

36.737 0.265

1.460

23.4

111.285

0.410

6.145 12.3 18.10

3.2

16.6

772 57.87

113.552

6

13.9

35.950 0.270

1.480

24.8

115.772

0.455

6.212 ,13.2 18.45

3.4

17.1

787 60.94

113.007

S.E. ±

0.2

0.743 0.005

0.020

0.3

1.856

0.005

0.031

1.2

0.24

0.2

0.2

13

-

0.800

C.D. at 5%

0.6

2.292 0.018

0.063

1.2

5.727

0.018

0.098 N.S.

0.74

N.S.

0.6

41

-

2.412

-

10 R.i:uZobia/seed 10 Rhizobia/seed-

C.V. (%)

Kurundkar et ai., (1991), Marathwada Agricultural University, Parbhani, India.

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Introduction to Soil sndAgricUlturaJ. Microbiology

134

A

\

a

b

c

d

(a) Combined inocuIun (b) Brady tfIizobium japanicum (c) Azospirftum btasiIense

B

Fig. 9.1 (A) Field - 10th Day (B) Soyabean flower (Courtesy, N. ·Satavanakumar, 2OO1)

(d) Control

Biofertilizers

135

(i) The plants are uprooted from the soil and their root-systems are washed with distilled water to remove the soil particles from the roots. (ii) The roots are cut into small pieces and inoculated in a semisolid medium. Soil samples are sometimes used for the isolation of Azospirillum. (iii) The culture is incubated at 28-30°C for about two days to facilitate better growth of bacteria.,

(iv) A dense mat of bacteria appears 1-2~ below the surface of the nutrient medium. The characteristic features of Azospirillum are: (a) It forms white, dense mass of cells in the semi-solid medium.

(b) It is a curved, rod-shaped bacteria containing fat droplets in its cells. (c) The cell is spiral in organisation and it shows spiral movements. (d) The colony of Azospirillum has nitrogenase activity. The bacterial colony having the above features are selected for mass cultivation. The procedure for preparation of inoculants on large scale is same as that for Rhizobium. The three species of Azospirillum namely A. brasilense, A. lipoferum and A. amazonense have the capacity to increase the grain yield of rice, sorghum, millet etc.

Effects of Azospirillum on Crop Yield Grain crops like rice, wheat and barley give high yield when their seeds are treated with Azospirillum inoculum. Grains like sorghum also give a high yield when they are treated with Azospirillum before sowing. The yield increases from 15.2% to 63.6%.

(c) Mass Cultivation of Cyanobacteria The blue-green algae and Azolla constitute a system. This is the main source of algal biofertilizer in several countries. The mixture of several cultures of cyanobacteria such as Nostoc, Anabaena, ./ Plectonema, Oscil/atoria, Cylindrospermum and Tolypothrix have been found to be more effective than a single strain. The following methods are used for mass cultivation of cyanobacteria,

Introduction to Soil and Agricultural Microbiology

136

(a)

Field method

(b)

Polythene lined pit method

(c)

Cemented tank method.

Among these the polythene-lined pit method is more suitable for the preparation of cyanobacterial biofertilizer.

Mass Cultivation of the Blue-green Algae using the Pet Method Mass cultivation of cyanobacteria is carried out through following steps, (a) Prepare the cement tank, shallow trays of iron sheets in an open area. (b) Transfer 2-3 kg soil with 100g superphosphate. Add 2 ml of insecticide to protect the culture from insects. (c) When the water becomes clear, sprinkle 100g culture on the surface of the water. (d) The water level is always maintained to about 10 cm during the peribd. (e) After drying, the algal mat is separated from the soil, forming flakes. (f) The flakes are collected, powdered, packed and then supplied to the farmers.

(d) Mass Cultivation of Azolla Azolla is an aquatic fern. It harbours Anabaena azollae in its leaf cavity, providing symbiotic association. The following six species of Azolla are widely used for mass cultivation, A. pinnata, A. rubra, A. caroliniana, A. filiculoides, A. mexicana and A. microphylla. The common species of Azolla in India is A pinnata. 2

For mass cultivation, the microplots (20m ) are prepared in nurseries with 5-10 cm deep water. 4-20 kg P20sfha is added for good growth. Optimum pH(7-8) and low temperature should be maintained. The microplots are inoculated with Azolla at the rate of O~ 1 to 0.4 kg per sqm. At the end of 2-3 weeks, an Azolla mat is formed. The mat is harvested and the same micro-plot is inoclliated with fresh Azolla. The mat is dried to use as green manure.

. 137

Biofertilizers

Mass Cultivation of Azolla

2 Microplot (20m )

J. Added Water (5-10cm) Add P20S (4-20 kg/ha)

J. Inoculation of Azolla

J. (0.1-0.4 kg/sqm) Formation of Azolla Mat

J. Harvesting

J. Inoculation of Fresh Azolla

J. Green Manure

(e) Mass Cultivation of Mycorrhizae Mycorrhiza is a product of the symbiotic association between . specific Fungi and plant roots. (For further details Ref. Chapter VI, Page No. 82) The method of inoculum production of VAM fungi differ with respect to their nature and types. For Ectomycorrhizal Fungi, basidiospores, sporocarp, mycelial culture and fragmented mycorrhizal roots can be used as inoculum. The VAM Fungi can be produced on a large scale by the pot culture technique. The host plants, natural soil and mycorrhizal fungi are used for the inoculum production. The spores of VAM fungi can be isolated from the soil by wet sieving and decantation technique. (Gerdeman and Nicolson, 1963) The collected mycorrhizal spores are immediately sterilized on the surface. The sterilized sand and soil (1:1 w/w) with a little amount of moisture is used as pot substrates. The VAM spores are isolated from soil and sterilized with streptomycin for 15min, followed by successive washing in sterile water. Then, VAM spores' are transfered ta the sterile soil to grow in the host seeds.

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Introduction to Soil and Agricultural Microbiology

Method of Inoculum Production of VAM

Sterile soil + VAM spores

,I, Mix well

,I, Grow host seeds ,1,550 (Maize, Sorghum) Young seedlings

,I, Removal of seedlings

,I, VAM spores

,I, (Microscopic observation) Starter inoculum

,I, Chopped roots) Inoculated large pots

,I, Removal of seedlings

,I, (After 3-4 months) Bulk of Inoculum Pelleted seeds

,I, (Packing) Sow it in the fields Keep in the glass houses. After a few weeks, the seedlings are gently removed. Check the VAM spores microscopically. Then, chop the roots at starter inoculum and inoculate the larger pots. After 3-4 months, remove the seedlings and macerate the roots. The packed materials are used by the farmers.

The Benefits of Bjofertilizers (1) They are free from pollution hazards. (2) They can be used by small and marginal farmers. (3) They increase soil fertility (4) They increase the crop yields

•

139

Biofertilizers

(5) Some of the bacterial and cyano bac!erial biofertilizers secrete growth regulators and antibiotics. (6) They are easy to apply to the fields (7) The mycorrhizal biofertilizers provide certain elements to the host plants. (8) They are cheap when compared to chemical fertilizers. (9) They help the plant to grow by increasing the biological activity in the root region. (10) Quality control measures are also important in the interest of farmers.

Some Important Commercial Biofertilizers Nodin, Rhizoteeka, Nodosit, Rhizonit, Agro-teeka and MycoRhiz are some commercially available biofertilizers.

ClClCl

AGRICULTURAL BIOTECHNOLOGY Introduction Biotechnology is the fast-developing applied aspect of modern biology. Biotechnology is ''the science of applied biological process." Plant cell, tissue culture, transfer of nif genes of eukaryotes and Ti plasmids (vectors) are major biotechnological tools in agriculture, horticulture, forestry and industry. The tissue culture technology contributed to the production of plants by clonal propagation, to obtain pathogen-free plants. The Ti plasmid is used as vector for gene cloning in plants. Biotechnology has caused a revolution in agriculture through somaclonal varients, disease resistant plants, Nif genes and Nod gene transfer. Micropropagation and development of mycorrhizal fungi also play an important role in horticulture and forestry. The use of biofertilizers has become an alternate tool for synthetic chemical fertilizers. Biofertilizers are non-toxic. Plant protoplast, and tissue cultures has become an important tool for crop improvement and commercial production of natural compounds. Transgenic plants also play an important role in Agriculture. The genetically engineered species of cotton known as Killer cotton is highly toxic to boll worm. Biological Nitrogen fixation is utilized for crop improvement. This method enables the transfer of the Nif genes responsible for Nitrogen fixation in legumes and non-legumes.

Aagricultural Biotechnology

141

The gene bank is a complete collection of cloned DNA fragments which comprise the entire genome of an organism (Dahl et.aL, 1981). With the gene bank or germplasm bank, it is now possible to conserve the gene pool of economically and ecologically important plant species.

Tissue Culture It is the process whereby small pieces of living tissue (explants) are isolated from an organism and grown aseptically for indefinite periods on a nutrient medium. There, they grow into an undifferentiated mass known as callus. The process of reversion of mature cells to the meristamatic state, leading to formation of callus, is known as De differentiation. The component cells of callus have the ability to form a whole plant. (Re differentiation)

Gene Cloning Gene cloning can be defined as "Changing of genes by using invitro processes" (Dubey, 1995). Gene cloning or genetic engineering is a part of biotechnology and plays a significant role in agriculture. A cloning vector is simply a DNA molecule possessing an origin of replication (ori) and which can replicate in the host cell of choice. Some cloning vectors normally used are plasm ids and bacteriophages. Plasmids are the extra-chromosomal, autonomously replicating, covalently-closed circular molecule.

Ti Plasmids Recently, studies by plant pathologists on crown gall (an economically important plant disease) have revived interest in plant genetiC engineering. Crown gall is a plant tumour caused by the bacterium, Agrobacterium tumefaciens. Crown gall tissue represents true oncogenic transformation. Crown gall tumours have long been considered as a model of plant cancer. In studies on the pathogenicity of A. tumefaciens, scientists discovered that the genes responsible for inducing tumour are carried by a plasmid called Ti. The Ti plasmid becomes stable when incorporated into the genome of the infected plant cells. The Ti plasmid is a circular double stranded DNA molecule containing upto 200,000 base pairs organised into several genes. The size of Ti-plasmid ranges between 180-250 kb. Ti plasmid can be classified into three opine types, called Octopine, Nopaline and Agropine. One or more unusual amino acid derivatives encoded by DNA is known as opines. The gene sequence in Ti plasmid (T-DNA) is

Introduction to Soil and Agricultural Microbiology

142

divided into two types, TL-T-DNA (Leftside) and TR-T-ONA (Right side). (Fig. 10.1) auxin biosynthesis (= shoot inhibition)

cytokinin biosynthesis (= root inhibition)

virulence regionli (control plasmicf DNA transfer frbm becterium to plant cell and Integration of T-DNA into plant cell chromosome, also control host range of bacterium)

origin of

controls conjugative plasmid transfer between bacteria opine cataboliSm

.Incomoatibility gene

FIg. 10.1 Ti Plasmid

The genes responsible for biosynthesis are located at the different regions. The tra gene help in the transfer of T -DNA from one bacterium to other for example, onc gene is responsible for oncogenecity, ori gene for the origin of replication. The metabolism of opines is a central feature of crown gall disease. The type of opine produced is normally determined by the bacterial strain. When the callus tissue is cultivated invitro in the absence of bacterium, it retains its tumorous properties - the ability to form an overgrowth when grafted OlltO a healthy plant. The specific segment of Ti plasmid DNA is integrated into the plant nuclear DNA and is transcribed. The process of DNA transfer and tumour formation are distinct. The genes that induce tumour formation are very similar to that of transposons. The synthesis of auxins and cytokinins is controlled by some specific genes. Integration of T-DNA can occur at many different sites in the plant genome. The mechanism of integration of T -DNA is not clearly understood. However, it closely resembles bacterial conjucation. Ti-plasmid infect only dicotyledonous plants, monocotyledonous plants. Some attempts have been made to use Ti-plasmids as plant vectors. But these were fruitless because the plasm ids are normally larger in size. Under such circumstances, intermediate vectors can be used. These vectors can be manipulated invitro, transformed into E.coli and then to Agrobacterium tumefaciens. (Fig. 10.2)

Aagricu/tural Biotechnology

143 T-ONA

n plasmid sequence

~

Gene insertion /" , -. . .. into T-ONA, •

r

region

t

Transform AgrobadeniJm and infect tobacco plant

Firefly··' luciferase gene and promoter

1

1tf'1:1i"'.R_~ " and Plant T-ONA Plant genome

luciferase 'gene

genome

Fig. 10.2 Gene Transfer - Formation of Cloning vector and its use in Transformation

Structure of Nif Genes In nitrogen fixing organisms, the synthesis of the enzyme nitrogenase is controlled by the gene called nif gene. Dixon postgate (1968) has studied the structure of -nif gene According to Subba Rao (2000) the nif genes of K. pneumoniae has complete nif clusters consisting of 21 genes, nif JCHDKTYENXUSVWZMFLABQ in which T, Wand Z are the three potential new genes. The 21 st gene, C though proposed is probably a part of J gene. M

•

K. pneumoniae is widely distributed in water, soil grain and intestine of mammals. It has been used for detailed genetic analysis of the genes involved in nitrogen fixation.

144

Introduction to Soil and Agricultural MicrobiOlogy

All nif mutations are located near the his (histidine) operon. There are atleast 17 nif loci in the cluster. No non-nifgenes appear to be present within the nif region. The genes are organized in seven distinct operons, all of which transcribe towards the his genes. The nif region is about 24 kilobases (kb) long.

The Nif Genes of Klebsiella Pneumoniae

--_.

nif - Q

-® -A - CD - R - ® -w - [EJ - (YJ - @] -@) -@ -@ -00 -(Q) -(8) - J ~--

• Regulation

....1 - - - -_ _ _ _ _ _ _ _

7 Operons (Transcription units) 24 K b - - - - - - - - -...

Fig. 10.3

Fun~tions

of Nif Genes

(i) • The gene Nit H encodes the proto Fe protein. This is then processed by the Nit M gene. (ii) The gene Nit

a uptakes the molybdenum.

(iii) Nit B codes the enzyme involved in synthesis ot Femo-co. (iv) Nif D codes the a sub-unit ot dinitrogenase.

(v) Nif A regulator protein activates Nif operons. It is also responsible for the purpling of 6 cyanopurine, an analogue of adenine. (vi) Nif L inhibits activation of Nif operons. (vii) Nit RLA proteins are called regulatory proteins. (viii) The genes Nit Wand Nit U are not essential for nitrogen fixation. (Ix) The Nif F is responsible for transport of electron to dinitrogenase reductase. (x) Nit S is responsible tor the modification ot nitrogenase. (xi) Nit V is involved in the synthesis ot Femo-co. (xii) During aerobic growth, no nitrogenase is synthesized by K pneumoniae. Oxygen rapidly turns off all Nit-encoded proteins except RLA proteins.

Genetic Engineering of Nif Genes One of the greatest benefits of genetic engineering is the introduction of nif genes and their capacity to fix nitrogen in the plants.

Aagtiaultural Biotechnology

145

Eukaryotic: cell

E. coli

Chromosomal DNA

~""?J :_i;~;~~'2~): O .. ~

.:

's_

5

.:

/~ o O l E. coli

plasm ids

+

Fig., 10.4 Genetic Engineering of Nif Genes

s~

nifgenes

Introduction to Soil and Agricultural Microbioiogy

146

Steps Involved in Integration (of Nif genes with yeast cells) (i) Plasm ids from Ecoli and a yeast cell were cleaved and then fused to form a single hybrid plasmid. (ii) The nif genes were isolated from the chromosome of K. pneumoniae. (iii) Another Eeoli plasmid was cleaved. (iv) The isolated nif genes were introduced to form a hybrid plasmid.

(v) The yeast cell

re~ognized

the hybrid Eeoli plasmid.

(vi) The plasmid was then integrated into the yeast chromosome.

Difficulties in the Integration Process (a) The genes for nitrogen fixation were first inserted into the eukaryotic cell. (b) The yeast cells carrying the nitrogen fixation genes were not able to fix atmospheric nitrogen. This may be due to difficulties in gene functioning after transfer to foreign cells. (e) The transcribed RNA must be able to translate proteins on the yeast ribosomes. (d) Enough iron must be available in the yeast cell for incorporation into the nitrogenase molecule.

000

FURTHER READING Alexander, M., (1997) Introduction to Soil Microbiology, John Wiley and Sons, New York. Devlin and Withan. (1986) Plant Physiology, (4th ed.), CBS Publishers and Distributors, New Delhi - 110 032. Dubey, R. C. (1998) A Textbook of Biotechnology, S. Chand and Company Ltd., New Delhi. Dubey, R. C. and D. K. Maheshwari. (1999) A Textbook of Microbiology, (1st ed.), S. Chand and Company Ltd., New Delhi - 110 OSS. Gupta, P. K. (1944) Elements of Biotechnology, Rastogi Publication, Meerut-2S0 002 Pelczar J. C. S, Chan, Noel Kriedg (1993) Microbiology (sth ed.), Tata McGraw-Hili Publishing, New Delhi. Kurundkar, B. P., Thombre, P. A. and B. R. Kawale (1991) Effect of Different Inoculum Levels of Bradyrhizobium Japanicum on Soybean. Legume Research, 14 (4); 201-204. Nyle C. Brady. (1998) The Nature and Properties of Soils (9thed.), Eurasia Publishing House (P.) Ltd., New Delhi. Old, R. W. and S. B. Promrose, (1998) Principles of Gene Manipulation, (Sth ed.), Blackwell Scientific Publications. Powar and Daginawala, (1997) General Microbiology, Vol. II, (2"d ed.), Himalaya Publishing House, Mumbai - 400 004. Prescott, M., John. P. Harley and Dorald A. Klein. (1999) Microbiology, (4th ed.), WCB. Primrose, S. B. (1991) Molecular Biotechnology. Blackwell Scientific Publications.

(2nd ed.), Oxford

Primrose, S. B. (1994) Molecular Biotechnology, (2nd ed.), Blackwell Scientific Publications.

148

Introduction to Soil and AgricuUural Microbiology

Rangaswarili, G. (1993) Diseases of Crop Plants in India, (31d ed.), Prentice-Hall of India Pvt. Ltd., New Delhi. Rangaswami, G. and D. J. Bagyaraj, (1993) Agricultural Microbiology, (2nd ed.), Prentice-Hall of India Pvt. Ltd., New Delhi. Rangaswami, G. and A. Mahadevan, (1999) Diseases of Crop Plants in India, (4 th ed.), Prentice-Hall of India Pvt. Ltd., New Delhi. Ronal, M. Atlas. (1997) Principles of Microbiology, (2nd ed.), WCB. Senthil Kumar, N. (1998) Combined Effect of Nuclear Polyhedrosis Virus and Azadirachtin on the Feeding, Growth, Development, Biochemical and Histopathological Changes of He/icoverpa Armigera (Hubner) (Insecta: Lepidoptera; Noctuidae) Ph.D., Thesis, Submitted to Bharathiar University, Coimbatore (p. 129). Sharma, P. D. (1996) MicrobiologyandPlantPathology(2 nd ed.), Rastogi Publications, Meerut-250 002. Subba Rao, N. S. (1995) Soil Micro-organisms and Plant Growth, (31d ed.), Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi. Subba Rao, N. S. (2000) Soil Microbiology, (4th ed.), Oxford and IBH Publications, New Delhi. Verma,S. K. (1997) A Textbook of Plant Physiology and Biochemistry, (2nd ed.), S. Chand and Company Ltd., New Delhi. Wheeler, B. E. J. (1976) An Introduction to Plant Disease, ELBS and John Wiley and Sons Ltd.

ClQQ

APPENDIX Some Common Media for Culturing Micro-organisms ~edia for isolating Soil Micro-organisms

I.

BACTERIA

1.

Nutrient Agar:

3.0 g Beef extract 5.0 g Peptone Agar 15.0g Distilled water 1000 ml Heat until agar and peptone dissolve. Adjust pH to 6.6 to 7.0 using bromothymol blue as indicator. 2.

Soil Extract Agar Soil extract Glucose Dispotassium phosphate

100.0 ml 1.0 gm 0.5 g

900ml Tap water Soil extract is prepared by heating 1000 g of garden soil in 1000 ml of tap water in an autoclave for 30 min/at 151b pressure. A small amount of calcium carbonate is added and the soil suspension isjiltered-through a double paper filter. The turbid filtrate should be poured. Bottle and sterilize the extract in 100 ml quantities (pH 6.8)

3.

MacConkey Agar Peptone Protease peptone Bile Salt Mixture

17.0g 3.0 g 1.5 g

I

Introduction to Soil and Agricultural

150

Sodium chloride Agar

Neutral red pH

4.

.,)bi?/ogy

5.0 9 13.5 9

0.03 9 7.1

Pikovs Kaya's Medium Glucose

10.0 9

Tricalcium phosphate S04 (NH 4 4 )2S04 KCI

5.0 9

0 MgS04.7H 2O MnS04

0.1 9 trace

FeS04 Yeast extract

0.5 9

Agar Distilled water

0.5 9

0.2 9

trace

15.0 9 1000 ml

Clearing around the bacterial growth indicates P04 solubilization.

5.

Winogradsky's Medium for Clostridium pasteurianum 10.0 9 Sucrose Calcium carbonate 2.0 9 Sodium thioglycolate 1.0 9 Dipotassium phosphate 1.0 9 1.0 Magnesium sulphate 0.2 9 Sodium chloride 0.02 9 0.02g Manganese sulphate 0.02 9 Ferrous sulphate 0.02 9 Ammonium molybdate 0.001 9 Yeat extract water (10 per cent) 0.1 9 Agar 1.0 9 1000 ml Distilled water

6.

Yeast Extract Mannitol Agar (Rhizobium) Mannitol K2 K2 HP04 4 MgS04. 7H 20 MgS04.7H

10.0 9 0.5 9 0.2 9

Appendix

151

NaCI

1.0 9

Yeast extract Agar Distilled waibl

7.

1000 ml

Nitrate Broth Beef extract

3g

Peptone

5g

Potassium nitrate Distilled water pH

8.

0.1 9 20.0g

. Azospirik ''1 Semisolid MediuI1I K2HP04 KH 2P04 MaS 0 4 NaCI

19 1000 ml 7 ±0.2

0.1 9 0.4 9 0.2 9 0.1 9

CaCI 2

0.02 9

FeCI3

0.01 9

Na2MOO4 Sod. Malate Bromothymcol blue (0.05% Ethanol)

0.002 9 5.0 9 5.0ml

Vitamin solutions are sterilized by filtration through sintered glass funnel and added to the medium after sterilization.

II.

FUNGI

9.

Sabouraud's Dextrose Agar Mycological ~eptone

10.0 9

Dextrose

40.0g

Agar pH

15.0 9 5.6 ± 0.2

10. Potato Dextrose Agar Potatoes, peeled and sliced D-Glucose Agar Distilled water

200g 20 9 15 9 1 Litre

Introduction to Soil and Agricultural Microbiology

152

Boil 200 g of peeled diced potatoes in a litre of water for 1 hour. Filter and make up the volumes upto one litre. Add glucose and agar and steam until agar is dissolved.

11. Yeast Malt Extract Agar

30.0 g 5.0 9 20 9 1000 ml

Malt extract Mycological peptone Agar Distilled water

5.4

pH

To inhibit bacterial growth, 10% sterile lactic acid solution can be added to the molter medium just before pouring so as to bring down the pH to 3.5.

12. Coon's Medium (For Fusarium) 7.20 gm 3.60 gm 1.23 gm 2.72 gm 2.02 gm 15.00 gm 1000 ml

Sucrose Dextrose Magnesium sulphate Potassium diphosphate Potassium Nitrate Agar Water

13. Czapek's Agar 30.00 gm 2.00 gm 1.00 gm 0.50 gm 0.50 gm 0.01 gm 15.00 gm 1000 ml

Sucrose Sodium nitrate Dipotassium phosphate Magnesium sulphate Potassium chloride Ferrous sulphate Agar Distilled water

aaa

GLOSSARY Acid soil

-

A soil with a pH value < 7.0

Acquired colour

..

The colour which is due to the soil-forming process. Some viruses can attack actinomycetes.

Actinophages Adsorption

-

The attraction of ions or compounds to the surface of a solid. Soil colloids adsorb large amounts of ions and water.

Alkaline soil

-

Any soil that has pH> 7.0.

Ammonification

-

The organic amino nitrogen is converted into ammonia.

Antagonistic effect

-

Any inhibitory effect created on an organism by an other organism.

Apical dominance

-

The influence of apical bud in suppressing growth of lateral buds.

Autochtonous

-

The indigenous population, uniform and constant.

Biocides

-

A living organism as such, its structural unit or metabolite used against a target organism to eliminate its activity.

Bio-degradable

-

A material subject to degradation by 'biochemical process. All the nutrient input of biological origin for plant growth.

Biofertilizers Bio-geo-chemical cycling

-

The cyclic movements of chemicals between living organisms and environment.

Biomass

The amount of living matter in a given area.

Bio-technology

The science of applied biological process.

Introduction to Soil and Agricultural Microbiology

154

Callus

Undifferentiated mass of meristamatic cells.

Chlorosis

The discolouration of green pigment.

Chresard

The total amount of water present in the soil.

Crown gall

A plant tumour caused by the bacterium, Agrobacterium tumefaciens.

Cyanellae

The cyanobacterial endosymbionts of protozoa.

Dedifferentiation

The callus formation is known as dedifferentiation.

Diazotrophy

The process of biological nitrogen fixation.

Disease

Disease in plants is any deviation from the healthy condition and is expressed either by phYSiological changes or morphological changes.

Echard

The amount of water which cannot be absorbed by plant roots.

Elicitors

The compounds which induce the synthesis of Phytoalexins.

Eolian

Soil materials are transported from one area to another.

Evapo-transpirati on

The combined loss of water from a given area, and during a specified period of time, by evaporation from the soil surface and by transpiration from plants.

Gravitational Water

-

The water which moves out of the soil due to the gravitational pull.

Heterocysts

The enlarged, thick walled, pale yellowish, specialized cells.

Holard

The amount of water

Humification

The process of humus formation.

Hydrological cycle

-

~bsorbcd

by plant roots.

The cyclic movements of water between living organisms and environment.

Hypertrophy

It is an abnormal increase in the size of one or more organs of a host in response to the attack of a pathogen.

Hypoplasia

It is subnormal cell production in response to the attack of a pathogen.

Incitant

It is an organism which causes a disease.

155

Glossary

Infection

The establishment of the causal organisms in the body tissue after penetration.

Inoculum

A portion of a pathogen which is transmitted to a host and is capable of infecting the host.

Uchens

A small group of dual or composite organisms.

Uthosphere

The solid component of the earth.

Mineralization

The complex molecules are converted into simple molecules.

Mutualism

A symbiotic interaction in which both partners are mutually benefitted.

Mycorrhizae

The symbiotic association between fungi and plant roots.

Necrosis

Disease causing dead of host tissue& by the attack of a pathogen.

Nitrogen fixation

The process in which the gaseous form of N2 is converted into organic forms (NH 3)

Parasites

Micro-organisms that live on other living organisms.

Parthenocarpy

Fruit formation without fertilization.

Pathogen

An agent which causes damage to the host.

Pathogenesis

Mechanism of disease.

Permeability

The movement of water through pore spaces.

Phosphorus cycle -

The cyclic movement of phosphorus between the living organisms and the environment.

Phyllosphere

The zone on Micro"organisms.

Phytoalexin

The compounds contributing to disease resistance in response to injury.

Phytohormones

The growth regulatory organic substances.

Phytopathology

The science which deals with plant diseases.

Primary soil

The soil which is formed by weathering of soil forming rocks.

Protozoa

The simplest form of animal life.

Redifferentiation

The component cells of callus having the ability to form a whole plant.

leaves

inhabited

by

the

156

Introduction to Soil and Agricultural Microbiology

Rhizosphere

The region where soil and plant roots make contact.

Saprophytes

Micro-organisms that live on dead and decaying organic matter.

Soil science

The science which deals with the study of soil.

Soil Taxonomy

The comprehensive soil-classification system.

Symptoms

The external signs on the plant which indicate disease.

Syntrophism

A mutalistic association in which the nutrients are exchanged between two species.

Tumors

Gall-like structures developing on stems and roots.

VAM fungi

Vesicular arbuscular mycorrhizae fungi.

Zymogenous

Fermentive organisms.

000

INDEX Organism Index

Bacillus cereus

48

221-224

Agrobacterium

247

B. thuringiensis

A. radiobacter

220

Beijerinckia

138

248,251

Cercospora

172

161

Cercospora arachidicola

172-173

A. tumefaciens Alternaria solani Anabaena

115,31

Cercospora personata

175

Chondrostereum purpureum

224

89 67

Colletotrichum

225

Cytoplasmic polyhedrosis virus

227

E. coli

129

Erwinia

199

Erysiphe graminis hordei

197

Aspergillus

37

Aspergillus flavus Avena sativa Azatobacter

121

Azolla

240

Azolla pinnata

114

Azospirillum A. brasilense

135,139,240 121,237

Azospirillum lipoferum

121

Fusarium

185

Azotobacter paspaJi

121

Fusarium oxysporum

185

Bradyrhizobium japonicum

105

Granulosis virus

227

Helocoverpa armigera

105

227

B.lupini

158 158

Introductionto toSoil Soiland andAgricultural AgriculturalMicrobiology Microbiology Introduction

H. virescens virescens H.

227 227

Klebsiella Klebsiella pneumoniae pneumoniae

251,252 251,252

laspeyresia Laspeyresla pomonella pomonella

227 227

Meloidogyne javanica javanica Meloidogyne

211 211

Nocardia Nocardia

33, 134 134 33,

Nostoc Nostoc

124,151

Nuclear polyhedrosis Nuolear virus virus

226

Oscillatoria Oscillatona

41,42

Penicillium Penioillium

37

Phythium

161,221

R. phaseloli phaseloli R.

105 105

R. trifoli trifoli R.

105 105

Ruminococcus Ruminocoocus

148 148

Streptomyces Streptomyces

218 218

Sulfolobus Sulfolobus

99 99

Thiobacillus Thiobacillus thiooxidans thiooxidans

92 92

Tricchoderma viridae TMV

224 207,208

Ustilago avenae

181

U. hordei

181

U. scitaminea

181

Phytopthora infestans 145, 161, 163

U. tritici

159, 182

Plasmopara Plasmopara viticola

189

U. zeae

Potato Potato spindle spindle tuber tuber virus virus

206 206

Verticillium

181, , 225

Pseudomonas Pseudomonas Puccinia Puccinia graminis graminis Rhizobium Rhizobium

199,33 199,33 159, 159, 176, 176, 180 180 104,230 104,230

R. R. leguminosarum leguminosarum

105 105

R. R. meliloti maliloti

105 105

Xanthomonas Xanthomonas campestris.pv.oryzae campestris.pv.oryzae

201,202 201,202

Xanthomonas Xanthomonas campestris.pv. citri citn campestris.pv.

199,200 199,200

Yellow vein vein mosaic mosaio Yellow virus virus

206 206

QQQ 000

Index

159

Subject Index Ammensalism Ammonification Antagonism Antibiotics Auxins

131 88 130 217 64-72

Ectendomycorrhiza Ectomycorrhizae Endomycorrihizae Environment Eradication Ericoidmycorrhizae

Bacteriophages Biocides Bio-fertilizers Bio-sphere Bio-technology BLB Blight Bollworm Bolting Bordeaux mixture Botanical biocides Canker Chemical prophylaxis Chlorosis Commensalism Competition Conidia Crown gall disease Cytokinin

50 219 229-244 80 245 201,202 161 227 75 216 228 161,199 216 160 129 132 167 160, 162 76-77

Exclusion Florinogen Fungal diseases Gene cloning Genetic engineering Gibberellins Green manure Griseofulvin Haplophase Haustoria Hetrocysts Humification Hydrologic cycle Hyperplasia Hypertrophy Hypoplasia Immunisation Infection

Damping off Denitrification Denitrogenase reductase Diazotropy Dikaryophase Disease Early blight

161 61,62,90 110

Inoculum Iron cycle IPM

100 177 156 161,171

Late blight Leaf spot LHb Lichens and types

145 142 143 31 212 145 212 78 162-198 246 253 74-75 241 217 178 177, 189 124, 125, 126 13 80-82 160 160 160 214,215 157 156 98-99 227 162 172 108 151-154

160

Introduction to Soil and Agricultural Microbiology

Phytoalexins

Uthosphere

Phytopathology Microbial biocides Microbial inoculants Mildews Mineralization Mosaics Mutualism Myco-insecticides Mycoplasmal diseases Mycorrhizae

220 231 158, 188 12 161 129 224 203-205

PIBs Rhizosphere Rhizosphere effect Rhizoteeka Ruminant symbiosis Rust (Black, Yellow) Sheperd's cook

141,242

Smut Soil profile, types

Necrosis Neutralism Nif genes Nimbin Nitrification Nitrogen cycle Nitrogen fixation Nitrogenase Nod gene Oospores Orchid mycorrhiza

159 128, 129 51-253 228 89 87 88 102, 103, 120 106 165 145

Sporangia Streptocycline Sulphur cycle Symbi6sis Syntrophism Ti plasmid Tissue culture Transovarial transmission VAM Fungi Viral diseases

Parasitism Parasitodism Parthenocarpy Pathogens Pedogenesis Pedology Phoshorus cycle Phyllosphere

132 133 72 156 6 1 94-97 138

Viral insecticides

139 155-218 226 134-138 138 244 147 159, 175 1006 181 16-27 165 218 91-93 127 130 246-249 246 227 143,242 206-209 225-227

Witch's broom

6 185 162

Zoospores

164

Weathering Wilt

CJCJCJ