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ACHARYA N G RANGA AGRICULTURAL UNIVERSITY Rajendranagar, Hyderabad 500 030

Practical Manual

for Insect Ecology and Integrated Pest Management

Course No. Ento.231 (2+1) (2012-13)

Editor G Raghavaiah

Professor & Head Department of Entomology

Agricultural College, Bapatla

Co-Editors M S V Chalam P V Krishnayya

T Madhumati P Seetha Ramu

K Sridevi T Sridevi

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INDEX

S. No.

Title of the Exercise Between Pages

Signature of the Teacher

Remarks

1 Study of distribution patterns of insects in crop ecosystems

2 Sampling techniques for the estimation of insect population and damage

3 Pest surveillance through light traps, pheromone traps and forecasting of pest incidence

4 Acquaintance of insecticide formulations

5 Calculation of doses/ concentrations of different insecticidal formulations

6 Compatibility of pesticides with other agrochemicals and phytotoxicity of insecticides

7 Acquaintance of mass multiplication techniques of important predators Cheilomenes, Cryptolaemus and Chrysoperla

8 Acquaintance of mass multiplication techniques of important parasitoids egg, larval and pupal parasitoids

9 Acquaintance of mass multiplication techniques of important entamopathogenic fungi

10 Acquaintance of mass multiplication techniques of Nuclear Polyhedrosis Virus (NPV)

11 Study of insect pollinators, weed killers and scavengers

12 Extraction of nematodes from soil and roots -preparation of temporary and permanent slides

13 Identification of different types of nematodes

14 Identification of different mite species

15 Identification of different non-insect pests: rodents, birds, crabs, snails & slugs, squirrels and other mammalian pests

16 Identification of different non-insect pests-house hold and veterinary insect pests

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Acharya N. G. Ranga Agricultural University Department of Entomology Agricultural College, BAPATLA

CERTIFICATE

This is to certify that this is a bona-fide record of practical work done by

Mr./Ms._______________________________ I.D. No. ___________ in the Course

No. ENTO: 231 Insect Ecology and Integrated Pest Management during ---

Semester 20 20

Signature of the Course in-charge

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Ex No. 1 Date :

STUDY OF DISTRIBUTION PATTERNS OF INSECTS IN CROP ECOSYSTEMS

* * *

Objective: To get familiarized with distribution patterns of insects in crop ecosystems

Study of distribution patterns of insects in a crop-ecosystem is most important to validate methods for population estimation and to understand the pest population. Information on distribution or dispersion of pest species provides a valid base for developing a sound sampling plan and gives information about behavior of the species. The dispersion of species is influenced by social instinct such as breeding, protection against natural enemies and heterogeneity of the environment. Individuals of a population arrange themselves in a manner that is specific to each population and these arrangements in space appear to be of considerable importance in the study of dynamics of ecosystem.

Dispersion

The manner in which members of pest population are distributed in space is the dispersion or the distribution pattern of the species.

The internal distribution patterns are important which are related with some characteristics of a population. Individuals in any population may be distributed according to three basic patterns.

1. Regular / Uniform distribution

2. Random/poison distribution

3. Clumped / aggregated / over-dispersed/contagious distribution

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To understand the distribution/Dispersion of insect species one need to calculate,

A) Mean ( x ) = fx ------

n

B) Variance (S2) = fx2 (fx)2 /n ----------------------------

n-1

Where, f= Frequency of number of plants/branches x= Number of insects per plant/branch

n= Total number of plants

C) Variance - Mean ratio (VMR) = S2 --------

x

D) Index of David and Moore (IDM) = S2 ------- -1

x

E) Index of Lexis = S2/x

F) Charlier Coefficient = S2 - x x

1) Regular / uniform distribution:

It may occur where competition between the insects is severe due to physical factors. Here, the variance is less than Mean ((S2< x ). Hence, the variance-mean ratio (VMR) is less than one, IDM is less than zero, Index of Lexis is more than one and the Charlier Coefficient is less than zero.

x x x x x x x x x

x x x x x x x x x

x x x x x x x x x

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2) Random or Poison distribution:

It is relatively rare in nature and occurs where the environment is very uniform and there is no tendency to aggregate. Each insect has equal probability of occupying any point in space and the presence of one individual does not influence the distribution of another. Here the variance-mean ratio (VMR) is always one (S2= x ), IDM is equal to zero, Index of Lexis is unity and the Charlier Coefficient is zero.

3) Aggregated/Clumped / Aggregate/Negative binomial distribution:

This is the most frequently observed pattern and individuals show varying degree of clumping together due to attraction or instinct as in case of some insects. Large scale clumping helps to evade possible danger of predation, climate or diseases. Bees are able to exist in cold climate by increasing the input of heat among them thus modifying the environments. Usually the environment decides the degree of aggregation of clumped patterns. Here the variance-mean ratio (VMR) is more than one (S2> x ), IDM is more than zero, Index of Lexis is more than one and the Charlier Coefficient is more than zero.

The aggregation may also be due to

1. Response of microclimatic differences and daily and seasonal changes in weather.

2. As a result of reproduction or social attraction

3. Characteristics of the species i.e. degree of sociality

x x x x x x x x x x

x x x x x

x x x

x x xx x x xx x x x x x x x x x x x x

xxx x x x x x x x

x x x x

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Aggregation always leads to intra-specific competition for food, space and reproduction etc. The degree of aggregation as well as the overall density, which results in optimum population growth and survival, varies with species and conditions.

Record Work:

Workout the mean, variance and different indices to conclude the Distribution/Dispersion patterns Spodoptera litura (Fab.) based on the following larval population data on cabbage.

Number of

Insects Per Plant

x

Plant Frequency

f

fx fx2

0 22

1 3

2 5

3 8

4 12

5 11

6 9

7 23

8 7

9 6

10 4

n= fx = fx2 =

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Ex No. 2 Date :

SAMPLING TECHNIQUES FOR THE ESTIMATION OF NSECT POPULATION AND DAMAGE

* * *

Objective: To understand sampling techniques for the estimation insect pest population and their damage

Estimation of Insect Population

Population studies are helpful in pinpointing the factors that bring about numerical changes in the natural population and also in understanding the functions of the life-system of the pest species. Extensive studies over a large area are needed to understand the distribution patterns of a pest population, to predict the damage it is likely to cause to initiate control measures and to relate changes in the pest population to certain climatic or edaphic factors. The type of population estimation will depend on the objectives in view. The estimates are of three types.

I) Absolute estimates II) Relative estimates III) Population indices

I) Absolute estimates

The total number of insects per unit area (acre or hectare) is the absolute population. The numbers per unit area of the habitat (per plant, per shoot or per leaf) indicates the density of population. The estimates of absolute population and population density are useful to know and analyze the key mortality factors.

The following methods are commonly employed for knowing the absolute estimates.

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1. Quadrate method

Small areas or quadrates will be chosen at random from a large area which contains the population. The area of the quadrate relative to the whole area is estimated and also population in the quadrate is known exactly. From a quadrate the insects can be counted or collected directly and their number can be correlated directly with the field population. The reliability of estimates made from this method depends on how representative the quadrates are of the whole population and how close one gets to count the numbers.

2. Line-transect method

In this method a person will walk in a straight line at a constant speed through a habitat, the number of individuals encountered can be counted. The data based on such encounters can be used in estimating the absolute population of locusts and grasshoppers. The number of encounters in influenced by the speed of person, the speed of individuals comprising the population the distance over which they can be perceived and the density of the population under studies.

3. Capture, Marking, Release and the Recapture Technique

The number of flying insects can not be assessed by any of the methods described earlier. The capture recapture method can be used for these studies. The losses or gains in a population over a period can be determined with the help of this method.

For the effective application of capture-recapture technique in population estimations the following conditions must be satisfied.

1. The marking of individuals should not lead to changes in their behavior or longevity and marks do not get lost easily.

2. The marked individuals after being released becomes completely mixed up with the unmarked individuals of the population.

3. The population is sampled randomly with respect to its mark status.

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4. The method of marking should be such as to distinguish between different dates of capture.

a) Group marking methods

The most important pre requisite in the technique is that there should be no influence on the longevity or behavior and the natural camouflage of the marked individuals.

The different marking methods are as follows :

i. Paints and solutions of dyes: Different colours of paints can be used for marking moths, locusts and grasshoppers, beetles and many other insects. Florescent pigments with gum Arabic glue can also be used.

ii. Dyes and fluorescent substances in powder from: The hairy insects can be marked by dusting them with various dyes.

iii. Labels: The locusts and butterflies have often been marked by attaching small labels as a part of their wing.

iv. Mutililation: Clipping the wings of lepidopterans, damaging the elytra of beetles by incising edges or cutting small holes are some of the examples.

v. Radioactive isotopes: There are two methods of marking with isotopes. Those may be used as labels outside the organism or alternatively can be fed and incorporated in the tissues radioactive metals such as, cobalt (Co60) and Tantalum (Ta182) are widely used. In recapture studies the marked individuals can be detected by the use of Geiger Muller tube or by autoradiography.

b) Individual marking methods: In these methods individuals are marked singly and they provide good information on the longevity and dispersal of marked individuals. In involves the use of small labels which may be attached to wings or by having a combination of spots in various positions on the body and by the use of different colours.

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II. Relative estimates

In these estimates the population is measured in indeterminate units which allow comparison. The relative estimates are obtained by catch per unit time by using various traps. Various types of collection nets are available for use in different habitats and the sweep net is most widely used for sampling the insects from vegetation. Only those individuals on the top of the vegetation and those that do not fall off or fly away on the approach of the collector can be caught with a sweep net. Various traps like, flight aquatic traps, pit fall trap, light trap can be used to collect insects and their trap catch can be correlated with the actual population existing in the field.

III. Population indices

In this type the bio products of pests such as exuviae, nests, webs, frass or their effects (damage to the host) are considered instead of pests themselves.

Estimation of Insect Pests Damage

A species that interferes with activities of plant and cause damage to yield is known as pest. The total yield losses by different pests to all agricultural crops at global level is estimated to be 42.1% of attainable production.

Estimation of crop losses caused by insects to economic crops are exceedingly difficult because,

1. They variable in nature of damage.

2. Insect population fluctuates both in time and space.

The nature of damage caused by insect pests of crop plants is a function of pest population. So it is mostly insect capacity to increase in number rather than the nature of damage.

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The following four points should be kept in view to estimate the losses.

1. Any insect which cause some kind of the damage to crop can become pest when its population increase above a critical level. The critical level depends upon the nature of the damage caused by the insect.

E.g. In case of leaf feeders, the leaf eaten is near index of the losses caused by caterpillars.

In case of insect vectors of virus of disease a very small population of infective individuals can spread the disease to whole crop.

2. The losses caused vary both in time and space from 0 to 100%.The estimation is fairly easy at these two extremes, but there are large numbers of factors which tend to invalidate any estimation in between these extreme limits.

3. The loss may be either quantitative or quality. In case of quantitative loss reduced yield is observed, where as in qualitative loss, quality may be affected.

E.g. In case of wheat bug (Eurygastor integriceps) is known to affect adversely the baking quality of wheat.

4. Insect losses in terms of money are also objected. That the selling price of the commodity would be reduced, if insect infestation were to be greater extent.

The measures generally followed for estimating the losses caused by insect pests are based on either growing a crop as free from insect infestation as possible and then comparing its yield with that of check crop in which insect activity has been normal, or by making use of differential infestation and comparing the yield.

The above ones are used in the following methods for estimating the crop losses. The methods are as follows,

1. Mechanical protection of crop from insect pest damage

2. Chemical protection of the crop

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3. Comparison of yields in different fields having different degrees of pest

infestation

4. Comparison of average yield of healthy plant with that of infested plants

5. The average amount of damage caused by individual insect

6. Manipulation of natural enemies

7. Simulated damage

1. Mechanical protection of crop from insect pest damage:

The crop is grown under the enclosures of wire gauze or cotton cloth. These enclosures keep the pest away from the crop. Then, the yield of crop under such enclosures is compared with the yield obtained from the infested crop under similar conditions.

This technique has been used with that various modifications for estimating the losses caused by leafhoppers and whitefly to cotton

Flaw:

a. The limitation in the case of enclosures is that the plants generally become pale and weak due to changes in micro environment.

b. This technique can not be adopted on an extensive scale because it is very time consuming and impracticable on a field scale.

2. Chemical protection of the crop:

The crop is protected from pest damage by best scheduled chemical recommendation of pesticides. Then, the yield of treated crop is compared with that subjected to normal insect infestation.

This technique has been very widely used and it can be adopted on a large scale in cultivators field.

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Flaw:

The crop treated with the chemical protection can also be physiologically affected for better or worse because of the effect of the chemical protectant.

3. Comparison of yields in different fields having different degrees of pest infestation:

The yield is determined per unit area in different fields having different degrees of pest infestation. The correlation between the yield and degrees of infestation is worked out to estimate the loss in yield.

Israel (1959) estimated the damage by stem borer at two stages of plant growth i.e., dead hearts in early vegetative growth phase and white ears produced at flowering stage. Then, the following equations were obtained

Y=2110.85-5.935X1 ------ Dead heart stage. Y =22 - 3.41 X2 ---- White ears stage Where,

Y= Yield of paddy, X1 and X2 = Degree of pest incidence

There was 0.28% loss in yield per unit increase in dead hearts and 0.624% loss in yield per unit increase in white ear heads. This technique can be used for estimating crop loss due to different pests and diseases over a large area.

Flaw:

The yield in different fields can also be influenced by the soil heterogeneity

4. Comparison of the average yield of healthy plants with that of attacked plants:

In this process individual plants from the same field are examined for the pest incidence and their yield is determined individually. The loss in yield is estimated by comparing the average yield of healthy plants with that of plants showing different degrees of infestation. The same data can also used for working out the correlation between the yield and infestation on the basis of infested individual plants.

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Pradhan and Prasad worked out the correlation between damage by Chilo partellus and the yield of sorghum in the following equation;

Y = 6.6204 X1- 0.9257X2 -27.17

Where,

Y = Yield of sorghum grain per plant X1= Number of ears per plant X2= Percentage of stalk length infested Advantage:

The advantage of this technique over the above method is soil heterogeneity factor is considerably reduced

Flaw:

a. The different plants showing various degrees of infestation in itself is a proof that plants differ from one another in some unknown factors due to which they carried different degrees of infestation. This factor may be genetic or physiological or it may be mere soil heterogeneity in the same field.

b. It is a time consuming and involves lot of labor.

5. The average amount of damage caused by individual insect:

For this method, the preliminary information is obtained from studies on biology of the pest species. The details regarding the amount of damage caused by different stages or stages of the insect, and the exact nature and amount of loss caused are then worked out.

E.g. It has been estimated in the case of phadka grasshopper (Hieroglyphus negrorepletus). It consumes on average 42 grams of green leaves of maize during its life time. It was estimated that this insect would cause 18% loss in yield of maize at a population level of 10 grasshoppers per square yard.

Flaw: It is very difficulty to use this technique over large area and it is time consuming.

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6. Manipulation of Natural Enemies:

The manipulation of natural enemies of a pest species offers a means of evaluating plant damage. This technique has not been widely used. The pest is controlled by introducing predators or parasites into the field and the yield of such crop is compared that on which no such pest control measures have undertaken.

This method is feasible only in small plots and is not practicable on field.

7. Simulated damage:

Many investigators have attempted to simulate pest injury by removing or injuring leaves or other parts of the plant. The simulated damage may not always be equivalent to the damage caused by an insect. Insects may persist over a period of time or inject long acting toxins rather than producing their injury. Feeding on margins of leaf may not be equivalent to tissue removal from the centre of the leaves. Insect feeding is usually extended over a period of time and is difficult to incorporate the concept of rate of injury.

E.g. simulated damage studies have been conducted on the spotted boll worm on cotton. Detopping of cotton plant reduces the monopodial branches by 9.69%. The number of sympodial branches, squares and flowers increased by 14.5, 47.8, 2.54 %. Shedding of fruiting forms was greater in detopped plant that is 83.5% than control plant 71.4%. This results in 12.8 to 22.1 reduction in boll formation and yield of cotton.

Record Work:

Visit the near by crop field, observe and record the populations of available insect pests, their nature and extent of damage.

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Ex. No.3 Date :

PEST SURVEILLANCE THROUGH LIGHT TRAPS, PHEROMONE TRAPS AND FORECASTING OF PEST INCIDENCE

***

Objective:

To understand the concept of insect pest surveillance through light and

pheromone traps

Light traps:

Light acts as a source of attraction for some insects has been deployed to catch insects in suitable traps. Most of the insect species are nocturnal and are positively photorophic. The phototrophic behaviour is altered and modified by temperature, humidity, moisture etc. this phenomenon has been utilized by the entomologists to capture adult insects in a device called light trap.

In agriculture, light traps are important tools in insect ecological research and pest management. They are used for the following purposes.

Determining the presence or absence of insect species in an area.

Obtaining quantitative estimates of population density, species composition, age and sex.

Providing early warnings of crop infestation and oviposition.

Determining economic threshold levels to assist insecticide application.

Suppressing population to help managing the pest.

Detecting migration.

Collecting specimens for taxonomic purposes or for establishing laboratory cultures.

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An ideal light trap should be cheap, durable and robust. It should be serviced easily by personnel of little training, should be highly efficient and attract a large number of insects of different species.

Trap design : Light traps vary in design but generally consist of three components viz., 1) a light source 2) arrangement of baffles around the source 3) a catch container with a killing agent.

1. Light Source : Various types of light sources have been tested and used. Numerous studies have shown that the shorter visible and near ultraviolet wavelengths between 320-600 nm are most attractive to a wide variety of insects. The most efficient sources are the mercury vapour lamp and fluorescent tubes.

2. Baffles : Most light traps are fitted with baffles, which surround the bulb. They are often four, arranged perpendicular to each other to help retain these insects and thus greatly increase the catch of a trap. They may be made from an opaque material such as galvanized iron, or from a transparent material such as acrylic.

3. Container : In most traps, insect attracted to light fall into a funnel fixed below which opens into a killing and holding container. Dichlorvos is commonly used as a killing agent. Kerosene and water mixed with some detergent also serve as killing agent in many local light traps.

Some of the commonly used light traps are

The Chinsura light trap

Robinson trap

Bamboo light trap

Mercury vapour lamp

Modified Robinson light trap

Rothamsted trap

The Pennsylvania and Texas trap

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The new Jersy trap

The aquatic light trap

Incandescent lamp

Advantages :

1. Both male and females are attracted and there is possibility of using them as a control measure.

2. It is an eco-friendly measure of control since it is non-insecticidal.

3. It is compatible with any other methods of control in IPM.

4. It is simple, cheap and can be handled even by a less trained person.

5. Technical know-how requirement is less except that identification of desired target species is essential.

Disadvantages :

1. Beneficial non target insect species may also be trapped.

2. Light trap data vary with the weather conditions as well as moon light phases.

3. Expensive and bulky.

4. Availability of power source restrict trap installation.

5. It is not specific to a particular species of insect and therefore cumbersome to work with mixtures.

6. It should be operated in a large area and useful for strong flying insects.

Pheromone traps :

Pheromones are semiochemicals that are secreted into the external environment by insect which elicit a specific response in receiving individuals of the same species. These chemicals are also called as ectohormones. Pheromones are identified by extracting them from the insects and later synthesized artificially. The

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synthesized product is then impregnated in rubberized septa and used in integrated pest management.

Pheromones are used in integrated pest management (IPM) programmes for, .

a) Population density surveys

b) Forewarn regarding outbreaks of important insect pests

c) Male confusion Mating disruption

Pheromones have greater direct behavioural control usefulness in surveys to determine the presence/abundance of insect species so that other control measures can be exercised. Several models of pheromone applications and traps are available.

Modes of Pheromone Application:

1. Rubber septa sulphur free

2. Hollow fibres: Small thermo-plastic tubing sealed at one end & filled with pheromone. Pheromone release depends on evaporation through open end . Effectiveness is controlled by adjusting the length.

3. Twist tie ropes: 15 cm long plastic tube containing pheromone sealed at both ends is attached to crop manually. High concentration of pheromone provides relatively long persistence of release.

4. Laminated flakes : Two layers of vinyl sandwiching central porous layer with pheromone. Flakes are applied with sticker and thickening agent - through special equipment or by hand. Emission rate from flakes - controlled by layer thickness & chemical concentration

5. Micro capsules : Micro encapsulation of small drop lets of pheromone - done by using polymer can be easily manufactured on large scale. Readily applied over a large area with conventional sprayers.

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Modes of Pheromone Application

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Types of Pheromone Traps:

1. Delta trap : It is a rigid and durable plastic trap using a replaceable sticky insert. The insert on its top consists of a non-drying adhesive. It can be removed by opening one end of the trap. Pheromone lures are placed in the centre of the sticky inset. Catch inspection is possible without the need for dismantling the trap. The dispensers and sticky inserts should be replaced every six weeks. Traps should be inspected once in every two to three weeks.

2. Funnel trap : Robust trap made of moulded plastic with a large base and removable cap, for housing a pheromone dispenser. Kits may be supplied with an optional killing agent (insecticidal strip) or an insecticidal spray may be used inside the trap for control purpose. Flying insect pests are lured into the trap by the pheromone attractant. Insects once enter the trap, unable to escape and are exposed to the insecticidal strip.

3. Probe trap : This is used in grain storage silos. It is an acrylic cylindrical tube with small angled holes drilled on the upper 2/3rd of its length. Lower part of the tube contains a removable collection tube. At the top of the trap, there are two holes to pass a card to fix a marker, which lies on the to of the grain. The tap is vertically buried in the grain 0.5 to 1 m below the grain surface. The trap should be kept at 10-35 m distance. The crawling insects enter the tap through holes and fall into the specimen tube through funnel. The collection tube is coated with a substance, which prevents insects from crawling out.

4. Omni directional pheromone trap : It is exclusively used for monitoring Earias. It consists of an aluminium vessel of 30 cm diameter. Holes are provided on the sides of vessels. The trap contains a septum on the inner side and hung in the field.

5. Bait trap : The olfactory stimuli from the food source attract insects and are manipulated in the pest management.

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6. Poison bait trap: This is used to trap and collect the larvae of Spodoptera litura. Poison bait consists of 500 g of molasses, 5 kg of rice bran and 500 g of carbaryl 50 wp/acre. The pelleted baits are kept along the irrigation channels during evening hours to attract the caterpillars. Upon ingestion of the bait containing carbaryl 50% wp, larvae will be killed.

Advantages of pheromones in pest control :

1. Pheromones are safe to environment.

2. They are species specific.

3. They are safe to natural enemies.

4. Pheromones need small doses.

5. Compatible with other pest management programmes.

6. They are economical than other control techniques.

Disadvantages :

1. Basic behaviour of most of important insect pests like pheromone reception, migration etc. was not fully understood.

2. The pheromones of a few insects were only identified, a large number of them are to be identified.

3. If the crop is affected by more than one pest and when pheromone trap is placed for major pest, there are chances of secondary pest outbreak.

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Types of Pheromone Traps

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Fore Casting of Pest Incidence

Fore casting is the systematic monitoring of pest population, dispersion and dynamics in different crop growth phases to fore warn the farmers to take up timely crop protection measures needed. An advance knowledge of probable pest infestations (out breaks) in a crop would be very useful not only to plan the cropping pattern in such a way as to minimize the damage but also to get the best advantage of the pest control measures.

Insect forecasting service is useful in the following ways:

1. To predict the forth coming infestation level of the pest, which knowledge is essential in justifying use of control measures mainly insecticidal applications.

2. To find out the critical stage at which the applications of insecticides would afford maximum protection.

3. To assess the level of population and damage by pest during different growth stage of crop

4. To study the influence of weather and seasonal parameters on pest

5. To fix up hot spot, endemic and epidemic areas of the pest

6. To fore warn the farmers to make decisions in timing of control measures.

To forecast of any pest or pest infestation must be related to the economic threshold of the pest. The fore casting will be made by conducting the following studies.

1. Population studies

These studies should be carried out several years using appropriate sampling methods to find out the seasonal range, the population variability and geographic distribution. The seasonal counts should be related to the climatic and topographical data.

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2. Studies on the pests life history

The possible number of generations and the behavior of the different larval instars under controlled conditions in the insectary, fecundity and the length of life cycle both in the field and in the laboratory can be related to range of environmental factors such as temperature, humidity etc.

3. Field studies on the effects of climate on the pest and its environment

Climatic factors usually influence pest numbers and also its parasites and predators either directly or indirectly. The spread of pest from area to area is largely determined by wind currents. A nationwide pest observatory work over entire country is essential to note the systematic occurrence of insect pests in many places and this would contribute to an effective pest forecasting service.

Types of pest fore casting

a. Short term fore casting

It may cover a particular season or one or two successive seasons only and is usually made employing insect trapping methods or some other sampling procedures for the pest with in the crop. Such predictions can also be based on the rate of emergence of the pest observed by rearing in the laboratory.

b. Long-term fore casting

Long term forecasts cover large areas and are based mainly on the possible effects of weather on the insect abundance or by extrapolating from the present population density into the future.

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Various methods of pest forecasting

Forecasting based on environmental factors

Environmental factors such as temperature and rainfall are the two most important factors on which forecasting can be made.

Forecasting based on climatic areas

The areas where critical infestations are likely to occur can also be forecasted for some pests. The principal factors may be biotic, topographic or climatic. Combinations of temperature and rainfall, temperature and atmospheric humidity or in the soil insects, soil temperature and soil moisture content are the most important.

Predictions from empirical observations

Attempts have been made to forecast the population of the insect pests in the forth coming season by counting the pest numbers in the previous season. This can however be usually successful in case of static pests only.

Record Work:

1) Draw neat labeled diagrams of different light and pheromone traps, and different pheromone application method given in the photographs.

2) Record populations of known insects trapped in the light/pheromone trap present in the department

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Ex No. 4 Date

ACQUAINTANCE OF INSECTICIDE FORMULATIONS

* * *

Objective:

To get familiarized with the different formulations of insecticides.

It is essential that the toxicant must be amenable to application in an effective manner so as to come into direct contact with the pest or leave an uniform and persistent deposit upon the plant surface. The toxicant is to be made available in a diluted form or in a form easily distributed. Therefore the compound containing the toxicant must be formulated in a form suitable for use as a spray, dust or fumigant.

Common formulations of pesticides are: 1. Dusts: In a dust formulation, the toxicant is diluted either by mixing with or by

impregnation on a suitable finely divided carrier. The carrier may be an organic flour or pulverized mineral or clay.

2. Granular: In a granular formulation, the particle is composed of a base as an inert material or vegetable carrier impregnated or fused with the toxicant which is released from the formulation in its intact form or as it disintegrates giving controlled release. Granules are prepared in three ways:

i) Impregnation technique ii) Agglomeration technique iii) Stick-on technique

3. Wettable powders (WP) or water dispersible powders (WDP): It is a powder formulation which yields a rather stable suspension when diluted with water. It is formulated by blending the toxicant with a diluent such as attapulgite, an auxillary material and surfactant.

4. Water soluble powder (SP or WSP) : It is a powder formulation readily soluble in water. Addition of surfactants improves the wetting power of the spray fluid. This

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formulation usually contains a high concentration of active ingredient and therefore convenient to store and transport.

5. Emulsifiable concentrate (EC): The formulation contains the toxicant, a solvent, an emulsifying agent and other spray additives. It is a clear solution and yields an emulsion of oil-in-water type when diluted with water to spray. When sprayed the solvent evaporates quickly leaving a deposit of toxicant from which water also evaporates.

6. Suspension concentrate or Flowable (F) : Tis formulation is developed when an active ingredient is insoluble in either water or organic solvents. The active ingredient is milled with a solid carrier (e.g. inert clay) and subsequently dispersed in a small quantity of water.

7. Solution concentrate (SC): The liquid formulation contains the active ingredient in a water miscible solvent. When mixed with water during spraying the solvent dissolves in water leaving the active ingredient alone. Addition of a surfactant provides wetting power.

8. Concentrated insecticide liquids: The technical grade of the toxicant at highly concentrated level is dissolved in non-volatile solvents. A more volatile solvent is also added to enable solution and drop formation.

9. Microencapsulation: Particles of a pesticide, either liquid or dry, surrounded by a plastic coating are with water and applied as a spray, the toxicant is released slowly.

10. Aerosols: The toxicant is suspended as minute particles ( of size from 0.1 to 50 microns) in air as a fog or mist. The toxicant is dissolved in a liquefied gas under pressure.

11. Fumigants: A chemical compound which is volatile at ordinary temperatures and sufficiently toxic is known as a fumigant. Fumigants are mostly used against storage pests and in confined enclosures.

31

Record Work: 1. Observe and note down the different formulations of insecticides displayed in the

laboratory. Also observe the toxicity level and other instructions on the cover. 2. Collect the information on recent and new formulations that are available in

market along with the brochures.

Group of Insecticide

Trade name Common name Formulation

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Ex No: 5 Date :

CALCULATION OF DOSES / CONCENTRATIONS OF DIFFERENT INSECTICIDE FORMULATIONS

* * *

Objectives: To calculate the dosages of given insecticides for field application To calculate the strength of the spray solution and To calculate the amount or quantity of insecticide required to apply in a given cropped area.

All insecticides are toxic not only to insects but also to warm blooded animals including man. Active ingredients of pesticides are generally available in concentrated forms with primary producers. They have to be diluted and formulated as per recommended concentration or strength and it varies with the nature of pest and the type of crop to be treated. The preparation of recommended spray or dust in the field should be known to a plant protection worker and should get acquainted with the calculation of quantities of diluents and formulated insecticides required to prepare the same.

C. Dilution of dusts: The dilution of dusts requires a certain quantity of talc to be mixed with to reduce the concentration of the dust and for dilution, rectangular method is generally used, where in the centre of the rectangle, the percentage of required concentration of dust is given and on the right hand side top corner, highest concentration of the dust available and on the right side corner bottom, lower concentration to be given. The values are diagonally subtracted and represented on left hand side top and bottom corners.

33

Example:

A chickpea farmer has advised to dust 6% Malathion dust for control of gram pod borer in his 3 acres of field at the rate of 10kg/acre. Calculate the quantity of talc he has to purchase from the market for diluting the 10% malathion dust available with him.

a) 6kg of 10% dust has to be mixed with 4 kg of talc to get 10kg of 6% dust. b) For getting 10kg of 6% malathion dust, the requirement of talc is 4kg and for getting 30kg (for 3 acres) of 6% dust, the requirement of talc is 4/10 x 30 = 12kg. c) For getting 10kg of 6% dust, the requirement of malathion is 6kg and for getting 30kg (for 3 acres) of 6% dust, the requirement is 6/10 x 30 = 18kg.

B. Dilution of spray formulation: The liquid formulations have to be diluted to ensure the minimum outreach of the active ingredient that is sufficient to act against insect pest.

C. To find out the quantity of insecticide required for treating an area at a required strength, the following formula may be adopted:

Total quantity of spray Concentration of the solution required x spray solution desired

Strength of the formulation

Quantity of insecticide

10%

0%

6

4

6%

=

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Example: C. A paddy farmer was advised to spray 0.05% chlorpyriphos (20% EC) for the

control of rice leaf roller in his 4 acres of cropped area. Calculate the cost of the chemical when the spray fluid required is 300lt/acre and the cost of the chemical is Rs. 500/lt.

Quantity of chemical required per acre =

For 4 acres = 0.75 x 4 = 3lt Cost of the chemical = 3 x 500 = Rs.1500/-

2) To obtain the strength/concentration of a finished spray solution, when a known quantity of chemical is added to a known quantity of water, the following formula may be adopted:

Quantity of Strength in percentage formulation used x of the formulation

Quantity of finished spray solution required

Example: For the control of groundnut leafminer, farmer has mixed 300ml of chlorpyriphos 20

EC in 300lt of water and sprayed in his field. Find out the concentration of spray fluid he has applied in his field.

Three hundred (300) ml of chlorpyriphos 20% EC is added to 300lt of water. The strength/concentration of chlorpyriphos in the spray liquid is

0.3 x 20 300

= 0.02%

300 x 0.05 20

Concentration of spray fluid

=

= 0.75lt

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c. Calculation of Granular formulations: Example: Carbofuran 3G at the rate of 1kg a.i/ha was recommended for the control

of root grub in groundnut. Calculate the cost of the granules for applying in 4 ha of cropped area, when cost of the chemical is Rs.30/kg.

For getting 3kg a.i. of carbofuran 3G, the requirement of formulation is 100kg

For getting 1kg a.i., the requirement is = = 33.33kg/ha

For 4 ha the chemical requirement is 33.33 x 4 = 133.32 Kg The cost of chemical is 133.32 x 30 = Rs. 3999.60

Record Work: 1. Calculate the dosage of insecticides with given strength and recommendations. 2. Calculate the amount of insecticide required to apply in given cropped area

1 x 100 3

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Ex No. 6 Date

COMPATIBILITY OF PESTICIDES WITH OTHER AGRO CHEMICALS AND PHYTOTOXICITY OF INSECTICIDES

* * *

Objective: To get acquainted with the compatibility and phytotoxicity of pesticides

Compatibility of spray chemicals: When two chemicals are brought together in a single spray mixture, due to reaction, a compound differing from either parent may be formed. Knowledge of the effects of such compounds on the plants when applied is essential to avoid improper use.

The different types if incompatibility are: i) Chemical incompatibility : Different compounds are formed due to

reaction of various chemicals as in synthetic organic compounds with an alkaline material which causes injury to plants.

ii) Phytotoxic incompatibility: The component parts though themselves are not injurious to the plants and do not show any chemical reaction when mixed, the mixture causes injury to plants.

iii) Physical incompatibility: The chemicals change their physical form to one that is unstable and hazardous for application.

Phytotoxicity of insecticides: The adverse effect of insecticide on the plants due to the treatment is known as

phytotoxicity. These effects on plant may either be permanent leading to death of the plant or temporary leaving scorching behind and allowing the plant to recover after some time. Many of the chemicals, which are non-phytotoxic at normal doses may prove to be phytotoxic at higher doses.

Record Work: 1. Observe the physical compatibility by mixing any two insecticides. 2. Spray a chemical with hand sprayer to demonstrate phytotoxicity

37

Ex No: 7 Date :

ACQUAINTANCE OF MASS MULTIPLICAITON TECHNIQUES OF IMPORATNT PREDATORS - Cheilomenes, Cryptolaemus and

Chrysoperla ***

Objective: To acquaint with the mass multiplication techniques of predators viz., Cheilomenes Sp., Cchrysoperla Sp. and Cryptolaemus Sp

Lady bird beetles (Coccinellidae: Coleoptera) are the most important group of predatory insects. The majority of them are predaceous on injurious insects like aphids, mealy bugs, scales, thrips, leafhoppers, mites and other soft bodied insect pests of field and horticultural crops. They can be employed to effectively regulate notorious insect pest populations in integrated pest management.

Rodolia cardinalis (Mulsant), Cryptolaenus montrarieri Mulsant and Corinus coeruleus Mulsant have been successfully used in classical biological control. Indigenous coccinellids like Chilocorus nigrita (Fabricius), Cheilomenes sexmaculata (Fabricius), Coccinella septempunctata Linnaeus, Scymnus coccivora Ayyar and Pharoscymnus horni (Weise) have potential for exploitation in augmentative biological control.

Production procedure for Green lace wing : Chrysoperla carnea

Larvae are important predators of insect pests viz., aphids, mealy bugs, eggs and smaller larvae of various insects of agricultural importance and mites. Each larva has potential to feed on average 12 aphids/day or about 120 aphids during the entire developmental period.

Equipment and facilities required :Black musclin cloth, cotton rolls, plastic jars (20x15 cm), Chrysopa cage, glass tubes (10x2.5 cm), camel hair brush, honey, pollen, laboratory host, Corcyra, refrigerator etc.

38

Biology (When reared on Corcyra) : Larval period : 6.50 days

Pupal period : 7.50 days

Adult longevity (male): 27.50 days Adult longevity (female) : 27.50 days Fecundity : 34.28

Incubation period : 3.00 days

Rearing :

Newly hatched larvae are reared in individual vials or plastic cages containing hexagonal cells. On an average 1 cc eggs of the laboratory host C. cephalonica are required for rearing 5-6 larvae of the predator. In each hexagonal cell a larva along with sufficient prey is placed and covered with a layer of white paper sheet. Finally the cage is covered with lid and shifted to the room at 27 20C and 60 5% RH for further development. If needed, food may be added after 3 to 4 days in each cell by sprinkling from the top and left undisturbed till the cocoon formation. After one week the cocoons may be collected and kept in separate jars for emergence. Newly emerged 10 pairs of C.carnea caged in a jar are provided with a swab of honey (20%) and pollen grains of castor. Jars are covered with wet black cloth. It prefers to lay eggs on black cloth used for securing the adults, hence these eggs are removed daily from the cloth by cutting their base by a sharp blade or scissors.

Precautions :

1. Always wear larvae either in individual vials or hexagonal cells of the Chrysopa cage due to cannibalistic habit.

2. Flowers containing pollen grains of castor or maize enhances its fecundity by many folds, hence may be offered to the adults along with prey.

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3. If viability of eggs of the predator starts deteriorating through generations on laboratory host. Shift at least for one generation of larval rearing on natural prey (aphids).

4. Freeze laboratory hosts at least for 6 hours at 40C to 60C in order to kill its embryo.

5. Handle adults preferably with the aspirator.

Mass multiplication of Creptolaemus montrouzieri :

After 15 days of infestation of pumpkins with Planococcus citri, they are exposed to a set of 100 beetles for 24 hr and kept in cage. The beetles during the period of exposure feed on mealy bugs and deposit their eggs singly or in groups of 4-12. The grubs are visible in such cages within a week of exposure. The young grubs feed on eggs and small mealy bugs but as they grow they become voracious and feed on all stages of mealy bugs. For facilitating the pupation of grubs, dried guava leaves or pieces of paper are kept at the base of the cage. The first beetle from the cage starts emerging on 30th day of exposure to C. montrouzieri adults. The beetles are collected daily and kept in separate cages for about 10-15 days to facilitate completion of mating and pre oviposition. The beetles are fed on honey agar medium. From each cage about 175 beetles are obtained.

Production procedure for Cheilomenes sexmaculata

Cheilomenes sexmaculata is a very important, polyphagous predator of aphids and other soft bodied insects. It has been recorded in most crop ecosystems, particularly where aphids are serious pests. It has been produced in the laboratory and used for the suppression of A. craccivora on groundnut. C. sexmaculata is also used in cotton, citrus and sunflower crop-ecosystems.

Production procedure :

The initial culture is started by collecting unparasitised pupae of C. sexmaculata from the field. The parasitized pupae look dark black to brown and are brittle to touch, whereas, healthy pupae are bright yellow coloured with black markings and are soft to

40

touch. The freshly emerged adults are fed with A. craccivora for ten days in which period mating may occur. The days old, pre-fed beetles are released on pumpkins bearing colonies of 25 days old Ferrisia virgata. Dry leaves of Bauhinia purpurea Walls are arranged on the periphery of the pumpkins in a slanting position. Any other plants leaves can also be used as an oviposition substrate. The leaves should be broad and should remain stiff after drying. These leaves act as oviposition substrate and also help the beetles to climb over the pumpkin. Honey (50%) soaked in absorbent cotton swab is provided as an additional carbohydrate source. The beetles are allowed to feed on F. virgata in the same cage for 25 days. The pumpkin is replaced with another infested pumpkin if necessary. Dead adults are removed regularly and replaced with fresh ones to ensure regular supply of eggs. The honey swabs are changed on alternate days. The eggs deposited on the leaves are collected and used for further multiplication. Eggs thus collected are kept separately in glass vials, as the grubs are highly cannibalistic. The grubs are released on cowpea seedlings infested with A. craccivora, grown either in plastic cups or glass jars. Twenty five grubs can be reared for 8-9 days in one jar containing more than 6500 aphids. Four jars are required for rearing 100 grubs up to the pupal stage. The adults emerging in 5-6 days from pupae are used either for further multiplication or for field release. The adult beetles survive for 50-70 days on mealy bugs. A regular decline in fecundity is observed 3-4 weeks after emergence and the beetles stop laying eggs during the last week of their life. Hence, the beetles should be discarded after the fourth week as they become less productive and rearing becomes uneconomical.

Record Work: 3. Observe and note down identification marks of different insect predators in the

laboratory and draw neat labeled diagrams of the specimens. 4. Collect different insect predators and arrange them with labels in the insect box

given.

41

Ex No: 8 Date:

ACQUAINTANCE OF MASS MULTIPLICATION TECHNIQUES OF EGG, LARVAL AND PUPAL PARASITOIDS

* * *

Objective: To acquaint with the mass multiplication techniques of egg, larval and pupal parasitoids viz., Trichogramma Sp., Bracon Sp. and Tetrastichus Sp., respectively

Egg parasitoid : i) Trichogramma Sp :

Fy : Trichogrammatidae Ord : Hymenoptera

Trichogramma sp are of common occurrence and distributed through out the world. They parasitise eggs of Lepidopteran mainly but are also reported from Coleoptera, Neuroptera and Diptera. In India it is commercially available for the pest suppression of sugarcane, cotton, sorghum, maize and paddy borers.

a) Biology : Incubation period : 24-36 days

Larval period : 2-3 days

Pupal period : 3-4 days

Adult longevity

Male : 5-7 days

Female : 5-20 days

Fecundity : 35-300 eggs

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b) Equipment and facilities required :

Refrigerator, B.O.D. incubators, eggs of Corcyra cephalonica (rice meal moth), glass tubes, sieve (40 mesh size), paper cards, UV lamp, table lamp etc.,

c) Rearing :

In India, Trichogramma sp are reared on the eggs of rice meal moth. Freshly collected eggs of Corcyra are cleaned of the scales, mites and other foreign matter associated with these and are glued on the Trichocard with uniformly thin layer using 2 per cent gum Arabic in distilled water (W/V). The sprinkling of the eggs is done either with camel hair brush or a fine sieve which does not allow more than one or two eggs to pass through its hole at a time. Thus 18000-72000 (1 ml) frozen host eggs are glued on a trichocard (15x7.5 cm). If the eggs were not frozen the trichocard should be exposed to UV lamp for about 10 minutes. The card is further divided through punching into 6 strips each of 7.5x2.5 cm size which can be easily pressed and separated. A strip containing glued eggs on it was inserted into a glass tube (10x2.5 cm) having newly emerged adults. The adult parasitoids are provided with honey streaks (50% honey dissolved in water) drawn on inner side of the tube and secured tightly with muslin cloth and rubber bands. The card is changed after 24 hrs and replaced with fresh card. Thus continuity of change over is maintained for 3 to 4 days or till female survive and remain productive. The host eggs oviposited by female turns black after 3 days of parasitization. The parasitoid completes its life cycle in 7-9 days at 27 20C and 75 5% RH.

Precautions :

1. If host eggs are not frozen/treated with UV rays to kill the embryo, the moths larvae may hatch out from the unparasitised eggs. These larvae should be brushed out gently since they eat away the unparasitised eggs.

2. Avoid super parasitism either by exposing host eggs upto 8 hrs or providing 6 eggs for one parasitoid.

43

3. Maintain pure species of different species of Trichogrammatids through proper handling and regular examination.

4. Do not offer frozen eggs to T. japonicum as it does not develop well on such eggs.

5. Do not rear T. brasiliensis at the temperature exceeding 260C where undesired male formation is more.

6. Do not cold store parasitized eggs at 5-100C for more than 15-20 days as beyond this storage biological attributes of the parasitoids are affected.

7. Use healthy eggs of host for healthy parasitoid.

8. Do not put excess gum while sprinkling the host eggs.

9. Do not rely on super parasitized parasitoids as they are normally weak and unfit for the production of healthy progeny.

Larval parasitoid : ii) Bracon hebetor :

Fy : Braconidae Ord : Hymenoptera

It is a well known external, gregarious larval parasitoid of several of the lepidopterans.

a. Biology

Egg period : 24-36 hrs

Larval period : 4-6 days

Pupial period : 3-7 days

Adult longevity : 15-25 days

Average fecundity : 130-200 eggs

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b. Equipment and facilities required :

B.O.D. incubator or air conditioned room, larvae of rice meal moth, room humidifier with humidistat, oviposition cages, glass plates, rubber bands, forceps etc.

c. Rearing :

The adult parasitoids soon after emergence are ready for mating and hence are held in a glass jar (10x7.5 cm) and fed with honey (5%) as food. The female parasitoid lays 8 to 12 eggs on the ventral surface of the host larvae after paralyzing it through stinging. The mouth of the jar containing the parasitoids is covered by a marking cloth over which 80-100 full grown larvae of Corcyra are kept in position with the help of rubber bands. After one day, the parasitized larvae are removed gently with the help of forceps and kept on paper plates (3x8 cm) as to avoid falling of the eggs from the host body and left undisturbed till cocoon formation. After cocoon formation, the remains of dead host larvae are removed. The cocoons are placed in glass jar (19 x 19 cm) for emergence. The optimum rearing conditions are 27 20C and 60 5% RH with a photoperiod of 12-14 hours.

d. Precautions :

1. Provide sufficient light and high RH (60+80%) to adult parasitoids to ensure proper mating.

2. Avoid infestation of mite, Pediculoides ventricosus in the culture.

3. Sterilize all equipment and cloths in hot air at 1100C for 1 hr. also remove the dead larvae regularly.

4. Do not cold store parasitoid cocoons for more than one month at 5-100C as beyond this period the biological attributes of the parasitoid are adversely affected.

45

Pupal parasitoid : iii. Tetrastichus israeli

Fy : Eulophidae Ord : Hymenoptera

The pupal parasitoid was observed to parasitise the pupae of Opisina arenosella and an average 90 adult parasitoids emerged from a single pupa under natural conditions. It can be mass reared on fresh pupae of S. litura, H. armigera, Plusia sp., or Ergolis sp. a. Biology Egg period : 20-24 hrs

Larval period : 4-6 days

Pupal period : 6-10 days

Longivity : 10-15 days Fecundity : 50-150 days

b. Equipment and facilities required :

B.O.D. incubator, A.C. room, glass tubes, fresh pupae of hosts, honey, rubber bands, markin cloth etc.

c. Rearing :

Mass rearing of the parasitoid on Opisina is done in some places. The adult parasitoids (newly emerged) are fed with honey offered as fine droplets on waxed paper and held in glass vials. Fresh pupae (0-1 day old) are offered at the rate of 3-4 females/big pupa or 1-2 females for smaller pupa. The parasitoids are allowed to parasitise and die in the vials and the parasitized pupae are kept for development at 27 +2 and 65+5% RH.

46

d. Precautions :

1. Provide fresh host pupae.

2. Some times it may realize breaking the outer shell of host pupa for easy emergence of parasitoids.

3. Maintain cleanliness.

Record Work: 1) Observe and note down identification marks of different parasitoids in the

laboratory and draw neat labeled diagrams of the specimens. 2) Collect different parasitoids and arrange them with labels in the insect box

given.

47

Ex No. : 9 Date :

ACQUINTANCE OF MASS MULTIPLICATION OF IMPORTANT ENTAMOPATHOGENOIC FUNGI

* * *

Objective: To acquaint with the mass multiplication techniques of entomopathogenic fungi viz., Beauveria bassiana, Nomuraea rileyi

With the ever increasing awareness of the harmful effects of the chemical pesticides on man and his environment the immediate need for sustainable, eco-friendly pest management has been felt very strongly providing an impetus to research and development of microbial pesticides. Among the different microbial agents developed and tested, bacteria, viruses and fungi are considered promising for the control of insect pests. Microbial agents that kill insects can be exploited successfully only after a reliable method of production is developed. Problems that are to be addressed are identification of virulent isolates/strains, retention of pathogenicity, process development for productivity in terms of yield in the commercial production and storage mechanism of ensuring retention of the desirable features. Various basic techniques to be employed for the utilization of entomopathogenic fungi in insect pest control are discussed.

1. Multiplication on media Beauveria bassiana can be easily multiplied on Potato dextrose agar and Saborauds dextrose agar (SDAY) while Nomuraea rileyi has specific growth requirements and can grow well on rich media like Saborauds maltose agar (SMAY).

48

(A) Semisynthetic media Potato Dextrose gar

(PDA Saborauds dextrose agar

(SDAY) Saborauds maltose agar

(SMAY)

Potato 200g Agar 15 g Agar 15g

Dextrose 20 g Peptone 10 g Peptone 10 g

Agar 20 g Dextrose 40 g Maltose 40 g

Distilled water 100 ml Yeast extact 5 g Yeast extract 5 g

Distilled water 1000 ml Distilled water 1000 ml

For preparation of PDA, potatoes are peeled and sliced. 20 g of the potato pieces are boiled in water for 10 minutes and the extract obtained by filtration is made up with water to 1000 ml after cooling. The dextrose and agar are added to the extract, melted and dispensed into glass tubes (10 ml/tube), 25 ml conical flask and autoclaved at 15 psi for 20 minutes.

For SDAY and SMAY media, add all the medium components to the water and dissolve by heating. Add chloramphenicol @ 80 ml/l. Dispense into glass tubes (10 ml/tube), 25 ml/250 ml conical flask, Autoclave.

B. Sorghum

Crush the sorghum grains in a mixie to get broken pieces. Weigh 25 g of the crushed sorghum in a 250 ml conical flask and add 22.5 ml of 1% yeast extract solution. Soak overnight at 250C. Plug the flasks and autoclave. Immediately after cooling, break the clump of sorghum aseptically using a blunt forceps. Add dry spore of Nomuraea rileyi to the sorghum using a micro-spatula. Shake well to disperse the spore evenly in the medium and incubate the flasks at 250C in the dark.

49

C. Barley

5 g of crushed barley is taken in a 250 ml conical flask and 45 ml of distilled water containing 0.125 g of yeast extract. The flask is plugged with non-absorbent cotton and autoclaved. After cooling, the flasks are inoculated aseptically with dry spore of N. rileyi, mixed well using a sterile spatula. The flask is plugged and incubated at 250C in the dark till mycelia growth and sporulation is complete.

II. Mass Multiplication

Take 250 g of wheat bran in a polythene cover, add 250 ml of distilled water containing 10 g of molasses and 1.25 g yeast extract. Autoclave at 15 psi for 20 minutes. After cooling, cut one end of the cover (corner) in a laminar airflow, add aseptically dry powder of B. bassiana from a PDA slope, insert a sterile tube containing non-absorbent cotton in the cut end and seal it. Incubate at 250C. Mycelial growth appears on 3rd day and continues for 3 days. The substrate with the mycelium is transferred to a sterile plastic tray/tub and covered tightly with a polythene sheet using a rubber band. Aeration is given everyday in the laminar airflow. The sporulation starts on 6th and continues till 10 days. The tray is transferred to the refrigerator and kept for the week. The tray is then removed, the cover is opened and the tray kept in shade for drying. the substrate is dried and the dry spore is harvested by sieving through a muslin cloth. The spore is stored in the refrigerator and used when required.

III. Multiplication on larvae

Prepare a suspension of the fungal conidia (2 108 conidia / ml) in 0.02% sterile tween-80 solution containing 0.05% streptomycin sulphate. Wash the leaves and allow the moisture on the leaf surface to dry keeping only the stalks dipped in water. After drying keep the leaf stalk in moist cotton plugs to keep the leaves fresh for the least 48 h. Apply/spread the spore suspension on the upper leaf surface and when still wet, release 6-7 days old host insect larvae. Freshly molted larvae should be preferred. Alternately, 2-3 leaves can be kept in a small conical flask or bottle with the stalks dipped in water and can be sprayed with the spore solution. Transfer the leaves along with the larvae to a plastic tub/container that has been thoroughly disinfected. Allow the

50

larvae to feed on the treated leaves for 48 h and then transfer to fresh castor leaves. In case of H. armigera larvae, they should be maintained individually. Here, infection can be caused by surface treatment of diet pieces with the spore solution.

Mortality starts on 6th day generally. Take out the dead larvae (cadavers) and wash them with tap water followed by sterile tap water containing 0.05% streptomycin sulphate. Blot them dry on a blotting / tissue paper and transfer to a petri plate containing a thin layer of moist cotton covered by tissue paper. Incubate in the dark at 250C. Mycelial growth starts after 24 hrs and sporulation starts after 48 hrs. After 2-3 days, transfer the petri plates to the refrigerator and store. The larvae can be placed for sporulation in plastic tub on sterile moist mud covered with a news paper and cover the tub with a polythene sheet.

Record Work: Collect insect cadavers infected by entomopathogenic fungi.

51

Ex No. : 10 Date :

ACQUAINTANCE OF MASS MULTIPLICATION TECHNIQUE OF NPV

* * *

Objective: To acquaint with the mass multiplication technique of Nuclear polyhedrosis viruses (NPV) of Helicoverpa armigera and Spdoptera litura

Pathogens used in insect control include viruses, bacteria, fungi, protozoans and nematodes. Their utilization for insect control requires their mass production either or natural host insects or using synthetic media.

Viruses :

Among viruses of the group baculoviridae, nuclear polyhedrosis viruses are utilized for the successful suppression of various insect pests of many agricultural and horticultural crops.

Nuclear polyhedrosis viruses of Helicoverpa armigera and Spdoptera litura are highly specific to their respective live hosts for multiplication. So production of viruses for use as insecticides needs mass production of their hosts as a first step. Basic steps in the production of nuclear polyhedrosis viruses of any insect are

1. Mass culturing and maintenance of host insects 2. Host inoculation with viruses

3. Harvesting of viruses

4. Purification

5. Storage Host insects can be reared either on their natural host plants (foliage, pod, fruit

etc) or on artificial diet. Since natural host plants cannot be found throughout the year, maintenance of host insects on artificial diet has the advantages of rearing under sterile conditions, avoiding contamination, saving space, time and labour. Thus the use of artificial diet for mass production of host insects is economical and easy.

52

Mass culturing of host insects can be started either from field collected adults using light traps or from field collected larvae reared to adult stage in the laboratory. Production starts with the availability of males and females of the host insects that are allowed in a oviposition jar having 10% honey and water soaked cotton wads placed separately inside in lids as adults food and its top covered with muslin cloth pinned with few muslin cloth strips hanging inside the jar.

Eggs laid on cloth are collected daily and surface sterilized by soaking in 0.15% sodium hypochlorite for 5-10 minutes. Eggs settling at the bottom of the container are collected and incubated in petri dishes.

Rearing of newly hatched H.armigera larvae is done by transferring individually (since the larvae are cannibalistic) into rearing vials containing artificial diet. In case of S.litura, since the eggs are laid in masses and larval stages are not cannibalistic, first two instars can be reared in groups or castor leaves.

Preparation of artificial diet :

Ingredients

Quantity Kabuli gram flour : 105.00 g

Sorbic acid : 1.00 g

Methyl para hydroxyl benzoate : 2.00 g Ascorbic acid : 3.25 g Yeast tablets : :10.00 g Agar-agar : 12.75 g Formalin (10%) : 2.00 ml Vitamin E : 2 capsules Multivitaplex : 2 capsules Streptomycin sulfate : 0.25 g Water : 780.00 ml

53

Gram flour yeast tablets, methyl parahydroxy benzoate and sorbic acid are added to half the quantity of water in a blender and mixed for 2-3 minutes. Simultaneously agar-agar is boiled with remaining quantity of water and cooled down to 700C. Hot agar liquid is added to the blender and mixed with other ingredients. Finally multivitaplex, vitamin E, ascorbic acid and formalin are added and blended for about 2 minutes.

Hot liquid diet is dispensed into rearing vials/tubes and allowed for solidification at room temperature for about 20 minutes. Tubes/vials are arranged in plastic/aluminium trays. Single larvae should be introduced in each vial on the diet surface and closed with cotton plug. The trays can then be stacked on racks. While 20% of the larval population can be allowed to pupate for continuous maintenance of host insect culture, the rest can be used for virus production.

Mass production of viruses :

Infestation of 8 to 9 day old larvae is done by inoculating the surface of the diet poured n plastic cups (as against in vials/tubes used for healthy host insect culture), with a virus dose of 1.1x104 polyhedrol inclusion bodies per larva. Larvae are placed individually on virus contaminated diet in cups and capped. The containers are arranged in plastic trays and stacked on racks.

Observations on larval mortality is made daily and the moribund/dead larvae with viral symptoms (cuticle of the larvae becomes fragile and ruptures easily when touched; body colour changes to blue or bluish purple) are harvested and placed in conical flask.

The diseased/dead larvae are macerated in a mixture using sterile distilled water. The contents are allowed as such in conical flasks for several days at room temperature, during which time the polyhedra settle down as a white layer. Alternatively, macerated contents can be sieved through a double layer of muslin cloth and the filtrate is standardized for the PIB/ml using a haemocytometer for field use.250 larval equivalents (LE)/Ha; One LE = 6x109 PIB

Record Work: Collect NPV infected lepidopteran larvae from the field.

54

Ex No: 11 Date:

STUDY OF INSECT POLLINATIORS, WEED KILLERS AND SCAVENGERS

* * *

Objective: To understand the role of insects as pollinators, scavengers and weed killers in nature

Role of pollinators:

Pollination refers to the transfer of anther to stigma in flowering plants for sexual reproduction.

Insects and in cross-pollination in fruits, vegetables, ornamentals, cotton, tobacco, sunflower and many other crops.

Insect pollination helps in uniform seed set, improvement in quality and increase in crop yield.

Entomophily refers to cross pollination aided by insects

Pollination classes Type of insects Melitophily Bees Cantharophily Beetles Myophily Syrphid and Bombylid flies Sphigophily Hawk moths Psychophily Butterflies

Phalaeophily Small moths

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1. Honeybees as pollinators

All bee species aid in pollination

Value of honey bees in pollination is 15-20 time higher than that of the honey and wax it produces.

Per cent increase in yield due to bee pollination

Mustard - 43% Sunflower - 32-48% Cotton - 17-19% Lucerne - 112%$ Onion - 93% Apple - 44% Cardamom - 21-37%

2. Hoverflies Syrhus sp. (Syrphidae : Diptera) Brightly coloured flies

Body is striped or banded with yellow or blue

Resemble bees and wasps

Larval stage predatory, adults are pollinators

Crops pollinated carrot, cotton, pulses

3. Carpenter bee, Xylocopa sp. (Xylocopinae : Anthophoridae) Robust dark bluish bees with hairy body

Dorsum of abdomen bare, pollen basket absent

Adults are good pollinators

Construct galleries in wood and store honey and pollen

4. Digger bees, Anthophora sp. (Anthophoridae : Hymenoptera) Stout, hairy, pollen collecting bees

Abdomen with black and blue bands

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5. Fig wasp, Blastophaga psenes (Agaonitae : hymenoptea) Fig is pollinated by fig wasp only. There is no other mode of pollination. There

are two types of fig Caprifig and Symrna fig.

(i) Capri fig It is a wild type of fig-not edible

Has both male and female flowers

Pollen is produced in plenty

Natural host of fig wasp

(ii) Symrna fig It is the cultivated type of edible fig

It has only female flowers

Pollen not produced

Not the natural host of fig wasp

In fig wasps males are wingless, present in caprifig and females winged. Female wasp lays eggs in caprifig, larvae develops in galls in the base of the flowers. Male mates with female even when the female is inside gall. Mated female wasp emerges out of flower (capirfig) with lot of pollen dusted around its body. The female fig wasp enters Smyrna fig with lot of pollen and deposits it on the stigma. But it cannot oviposit in the ovary of symrna fig which is deep seated. It again moves to Capri fig for egg laying. In this process Smyrna fig is pollinated. Caprifig will be planted next to Smyrna fig to aid in pollination.

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6. Oil palm pollinating weevil : Elaeidobius kamerunicus (Curculionidae : Coleptera)

Aid in increasing oil palm bunch weight by 35 per cent and oil content by 20 per cent.

7. Other pollinators

Butterflies (eg (Deilaphila spp.) and moths (Acherontia spp.) Ants, flies, stingless bees, beetles etc.

Scavengers : Insects which feed on dead and decaying plant and animal matter are called scavengers.

Remove decomposing material and prevents health hazard

Convert complex material into simple substances

Eg : Rove beetles (Staphylinidae : Coleoptera) Adults and larvae feed on decaying matter

Chafer beetles (Scarabaeidae : Coleptera) Bark beetles (Tenebrionidae : Coleoptera) Nitidulids (Nitidulidae : Coleoptera) Water scavenger beetle (Hydrophilidae : Coleoptera) Daddy long legs (Tipulidae : Diptera) Muscid flies (Muscidae : Diptera) Termites (Isoptera) Ants (Hymenoptera)

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Weed Killers:

Quite many insects feed upon unwanted weeds just the same manner they do with cultivated crops. Because they damage the noxious and menacing weeds, these insects are considered helpful or friendly to man. In many cases the occurrence of these insects have contributed much towards eradication of the weed or at least keeping it in check.

What is a weed? A weed is a plant in the wrong place. They cause losses in many ways like (a) Yield loss due to competition (b) Increased cost of cultivation (c) Direct injury ot man, livestock or livestock products (d) Depreciation of watershed and wild life values (e) Serving as alternative hosts for insect pests or plant pathogens.

Insects as agents for weed control:

From the year 1902 when eight species of insects were introduced into Hawai from Mexico for the control of Lantana camera, insects have been principal agents used in biological control of weeds. These insects feed on various pats of the weed plants and destroy them. Important groups of insects which have been successfully used for weed control are;

Lepidoptera : Phycitidae, Trotricidae Homoptera : Coccidae Hemiptera : Coreidae, Tingidae Coleoptera : Cerambycidae, Chrysomelidae, Buprestidae,

Cuurculiionidae, Galeuricidae Diptera : Agromyzidae, Trypetidae

Action on weeds : Insects often destroy weeds through direct destruction of vital parts. Ex : Action of Cactoblastic cactorum on Opuntia. The weed may die quickly or die during the next season.

Insects also attack weeds indirectly through (a) creating favourableness to infection by plant pathogens (b) affecting the competitive advantage of the weed.

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Desirable attributes of a weed killer :

1. It should not itself be a pest of cultivated plants (as Orthezia insignis) and should not even at a later date turn to attack useful crops, which is often the case with weed killing insects.

2. It should be effective in damaging and controlling the weeds.

3. It should preferably be a borer or internal feeder of the weed. Leaf feeders have also been found to be equally effective in checking weeds.

4. If should be able to multiply in good number without being affected very much by parasitoids and predators.

Examples of biological control of weeds with insects :

1. Lantana weed Lantana camera : It is a perennial shrub, native of Central America; is used extensively throughout the world as an ornamental plant. But it became a pest of range lands, coconut plantations, field crops, etc. the search by Albert Koebela in Mexico and Central America for insects destructive to Lantana resulted in the introduction of eight species of insects into Hawaii of which a seed fly Ophiomyia lantanae was more effective. O.lantanae was introduced into India from Hawaii for the control of Lantana. But an introduced coccid (scale insect) Orthezia insignis besides its failure to effectively check the weed began to infest economic plants like citrus, coffee, cinchona and tomato.

2. Prickly pear, Opuntia spp : Various species of Opuntia have been transported by man around the world. In many places it has become a very serious weed occupying millions of cultivated area. One of the outstanding examples of use of insects in the biological control of weeds was the control of Opuntia in Australia. The prickly pear, Opuntia inermis (O.stricta) got accidentally introduced into that country by 1840. The cactus spread was so rapid that in the year 1925, 24 million hectares of cultivable land were rendered useless. Control of this weed by chemical and mechanical means was not feasible and was too costly. In 1925 the moth borer, Cactoblastis cactorum (Pyralidae : Lepidoptera) was introduced from Argentina and the plants were killed by

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damaging them into papery structures. Within few years the weed population was reduced to a very great extent that Opuntia was no more a problem.

In India, Opuntia dilleni was wrongly introduced in 1780 in the place of O.cccinellfera for the cultivation of the commercial cochineal insect Dactylopius coccus valued for its dye. The cactus got established and spread rapidly assuming a serious proportion as a noxious weed. Dactylopius tomentosus was introduced from Sri Lanka in 1926 and within two years the insect effected a striking control of Opuntia dilleni in about 1,00,000 acres.

3. Crofton weed Eupatorium adenophorum in Nilgiris and Palani hills was controlled by introducing an exotic Tephritid fly, Procecidochares utilis from New Zealand.

4. Water hyacinth (Eichhornia crassipes) was successfully controlled with Neochetina eichhorniae, N.brunchi and mite Orthogalumna terebrantis (Curculionidae) in Kerala and Karnataka.

7. Water fern Salvinia molesta was successfully controlled with Cryptobagus cingularis (Curculionidae) in India.

8. Control of Siam weed Chromolaena odorata by release of Parenchaetes pseudoinsulata (Arctiidae) has been found promising in Kerala and Karnataka.

9. Parthenium hysterophorus (Congress grass, carrot weed, white top) has been successfully controlled in Karnataka by Mexican beetle, Zygogramma bicolorata (Chrysomelidae).

Classical biological control of weed is relatively inexpensive when applied in proper situation. Ex : Opuntia triacantha was controlled in the Island Nevis, West Indies between 1957 and 1960 at a cost of $1,500. This would have cost $30,000-40,000 annually with herbicides. A successful biocontrol project offers long term control with little or no additional costs after initial implementation costs.

Record Work:

Collect, observe and identify different insect pollinators, scavengers and weed killers from the fields.

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Ex No: 12 Date:

EXTRACTION OF NEMATODES FROM SOIL AND ROOTS, AND PREPARATION OF PERMANENT AND TEMPORARY SLIDES

* * *

Objective: To acquaint with the technique of extraction of nematodes from soil/plant samples and to prepare their temporary and permanent slides.

Materials Required

1. Sieves - 20-mesh (833-m aperture), 200-mesh (74-m aperture), 325-mesh (43-m aperture) and Coarse sieve (1 cm aperture)

2. Two stainless steel bowls or plastic buckets 3. Beakers 250 ml & 600 ml 4. Coarse spray water bottle

Procedure

1. Mix soil sample and pass through coarse sieve to remove rocks, roots, etc. 2. Take a 600 cc subsample of soil: pack lightly into beaker for uniformity. 3. Place soil in one of the buckets or pans; half fill with water. 4. Sieving and decanting process (various combinations of the following):

a) Mix soil and water by stirring with hand or paddle; allow standing until water almost stops swirling.

b) Pour all but heavy sediment through 20-mesh sieve into second bucket; discard residue in first bucket; discard material caught on sieve.

c) Stir material in second bucket; allow standing until water almost stops swirling.

d) Pour all but heavy sediment through 200-mesh sieve into first bucket; discard residue in second bucket.

e) Backwash material caught on 200-mesh sieve (which includes large nematodes) into 250-ml beaker.

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f) Stir material in first bucket; allow standing until water almost stops swirling.

g) Pour all but heavy sediment through 325-mesh sieve into second bucket; discard residue in first bucket.

h) Backwash material caught on 325-mesh sieve (which includes small to mid-sized nematodes and silt material) into 250-ml beaker.

5. Sample in 250 ml beaker will probably be too dirty for direct viewing; sample may be placed on Baermann Funnel or subjected to sucrose-centrifugation. The combined procedure allows extraction of nematodes from larger volumes of soil.

Extraction of Nematodes by the Baermann Funnel Technique

Materials Required

1. Glass funnels (12.5 cm diam.) 2. Wire-mesh baskets (10 cm diam., 5-10-mm aperture) 3. Rubber tubing 4. Kimwipes or facial tissue 5. Ring stand 6. Tubing clamps 7. Coarse sieve (1 cm aperture) 8. 250 ml beakers 9. 50 ml beakers 10. Two water bottles, coarse and fine spray

Procedure

Attach 10-cm length of rubber tubing to funnel stem and clamp tubing. Mount funnel on ring stand. Fill funnel two-thirds full with water. Place wire-mesh basket on top of funnel and use it to support tissue. Mix soil sample and pass through coarse sieve to remove rocks, roots, etc. Spread soil subsample (50 cc soil) evenly on tissue; fold in edges of tissue. Complete filling funnel with water so that water level is about 5 mm above wire-

mesh; do not let water and soil lose contact during extraction period - add water as needed.

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Maintain temperature at 22-25 o C so that it is conducive to nematode movement. Note nematodes move through tissue and settle in funnel; only active stages are recovered.

After 48 h, recover extracted nematodes by releasing 20 ml of water from stem of funnel into a counting dish.

Killing Nematodes Extracted nematodes need to be killed as quickly as possible to allow it to

assume a specific Death Position

Nematodes can either be killed first and then fixed or killed and fixed in one processing

Nematodes can be killed by adding equal volume of boiling water to nematode suspension or keep the nematode suspension in near-boiling water for 1-2 minutes.

Fixing/Preserving Nematodes Pipette 2-3 drops of formaldehyde (Formalin) into 7 ml of heat killed nematode

suspension

Pour 2 ml of hot fixative into 2 ml of nematode suspension

Fixatives Following are the most suitable fixatives that keep off the nematodes from drying-out.

Plant tissue that contain sedentary nematodes can be preserved in lactoglycerol ( equal volumes of glycerol, lactic acid and distilled water) or lactophenol (equal volumes of glycerol, lactic acid and distilled water+ 1% phenol)

Staining Gently wash plant material and gently on paper towels

Cut or slice thick roots or tubers into small lengths

Place them in muslin cloth and tie-up the corners and label the bag

Bring the stain solution (Lactoglycerol+0.1% cotton blue or 0.05-0.1% acid fuchsin) to near boiling on hot-plate and dip the plant martial containing muslin bag for three minutes

Open the muslin bag rinse in running water and place it in clearing/destining solution (equal volumes of glycerol and distilled water + few drops of lactic acid)

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Phloxine B (15 mg/ 1 lit water) is used to stain gelatin matrix that surrounds Meloidogyne eggs and adult females in the roots.

Slide Preparation Takeout the required heat killed and preserved nematode or stage of the

nematode on to a clean slide and attend the staining and distaining processes

Dissect out the nematode or its stage if they are embedded in plant material after the staining and distaining.

Blot-out excess fixative/stain/distaining solutions from the slide