STUDIES on INSECT FEEDING DETERRENTS with SPECIAL REFERENCE to the FRUIT EXTRACTS of the NE` M TREE, Azadirachta Indica A.Juss

STUDIES ON INSECT FEEDING DETERRENTS WITH SPECIAL REFERENCE TO THE FRUIT EXTRACTS OF THE NE` M TREE, Azadirachta indica A.Juss

A Thesis submitted by

JARNAIL SINGII GILL M,Sc.(Agric.), D.I.C.

for the Degree of Doctor of Philosophy in the University of London.

Imperial College of Science and Technology, Siiwood Park Field Station, Sunninghill, Ascot, Berkshire. March 1972. ABSTRACT

Twenty-eight candidate anti-feedants were evaluated by feeding

tests with Schistocerca. The most effective was azadirachtin, a pure

chemical isolated from the fruits of the Neem tree, Azadirachta indica.

Systemic activity of Neem derivatives is reported for the

first time. Soil-applied Neem formulations were readily taken up by

the roots of several plant species including woody ones, and translocated

throughout the aerial parts, to inhibit feeding of Schistocerca. Foliar

sprays applied to upper, lower, or one-side of potted bean plants protected

the unsprayed parts against locust attack, indicating translocation through

both xylem and phloem vessels. Phytotoxic effects on young bean plants were not observed following foliar sprays of 10% Neem seed aqueOus extract,

or soil applications of 2%, will, of Neem seed dust. Seed and seedling dips protected young plants for 1-2 weeks. The effects of rain, soil type, and storage on the efficacy of Neem products were studied; the active chemical(s) in Neem extracts proved to be stable and persistent.

In general, the protection given to bean plants by high volume sprays of

0.1% Neem kernel suspension, or soil applications at rates of 100 kg seed dust equivalent per acre lasted for 2-3 weeks: higher doses gave protection over longer periods of up to 8 weeks, using hungry Schistocerca test animals.

In addition to the rejectant action, other results suggest that

Neem extracts possess growth retarding properties. The larvae, or eggs of several insect species (Pieris, Musca, Stomoxys, Trialearodes,

Anopheles, Dysdercus) failed to develop normally when ex osed to Neem treatments.

The possible value of Neem preparations under peasant farming conditions in the tropics is discussed. CONTENTS

Page

ABSTRACT 2 GENERAL INTRODUCTION 8

PART I. Comparative Efficacy of Candidate Feeding Deterrents

INTRODUCTION 20

MATERIALS AND METHODS 29

1. Test Insects 29 2. Test Chemicals 29

(a) Commercial compounds 29 (b) Neem derivatives 30 3. Test Techniques 35 (a)Feeding methods 35 (b)Application of chemicals to taste receptors • . 37 RESULTS

A. Development of a Testing Technique . . . @ . . . 41 1. Feeding Experiments 41 (a)Feeding single test papers 41

(b)Feeding multiple test papers 45

2. Direct Touching of Receptors with Chemicals 45

(a)Palpi 45

(b)Labrum region 47

B. Evaluation of Candidate Rejectants 50 1. Primary Evaluation of 28 Chemicals 51

2. Secondary Evaluation of Ten Chemicals 54

3. Evaluation of Miscellaneous Preparations of Neem . . 59

4. Efficacy of Extracts of Three Batches of Neem 62 seeds Page

C. Habituation of Schistocerca to Neem Applications • • •• • • • 63 D. Interaction of a Deterrent and a Phagostimulant 66

DISCUSSION 68

PART II. The Systemic Action of Neem Derivatives

INTRODUCTION 76 MATERIALS AND METHODS 78 1. Test Insects 78 2. Breeding Cages 78

3. Soils 78 4, Neem Products 79

5. Plant Species 79

6. Spraying Apparatus . •. . . • 79

7. Assessment of Damage 80

8. Statistical Treatments 80

RESULTS

A. Soil Applications of Neem 82

1. Bean Plants 83

(a) Young plants 84

(b)Old plants 84

2. Other Crop Species 89

(a) Wheat, barley, tomatoes 90

(b)Rice, 'Cotton 90

(c)Cabbage,'sugar—cane, grass 92

(d)Chrysanthemum, spindle tree 92

B. Foliar Applications of Neem . . . * . 93

1. Beans 93

2. Barley 93 Page

C. Seed Soaks and Seedling Dips 95 1. Seed Soaks 95 (a)Bean seeds 95 (b)Barley seeds 95

2. Seedling Dips 96 (a)Rice 96 (b)Beans 96 (c)Tomatoes 96

D. Absorption, Translocation and Persistence of Neon Applications 100

1. Absorption and Translocation 100

(a) Immersion of parts of plants in neem 100

(b) 11 f " leaves in neem 101

(d) Application to parts of bean plants 107

2. Persistence of the Protective Effect of Neem. . •

(a)Grass 109

(b)Soil 109

E. Certain Other Aspects of Neem Applications 112

1. Phytotoxic Effects 112

2. Possible Growth Stimulating Effects on Root Development 114

3. Effects of Rainfall 115

4. Effects of Soil Types 126 5. Possible Effect of U.V. Light 129 6. Shelf Life and Storage 130 7. General Feeding Behaviour of Schistocerca on Neem Treated Foliage 132

Pane

F. Studies concerning Persistence of Neem in the Field 135

1. Applications under Semi-Field Conditions 135

(a)Preliminary applications, 135

(b)Confirmatory applications . • 139 (i)Pre-sowing 140 (ii)Pre-emergence 141

(iii)Post-emergence 143 2. Applications under Field Conditions 144

(a)Pre-sowing application 145 (i)Dust broad-cast 145 (ii) Spraying the seed bed 145

(b)Post-germination application 147 (i)Dust 147

(ii)Spray 147

DISCUSSION 150

PART III. Preliminary Studies of the Effects of Neem Applications apinst Several Insect Species

INTRODUCTION 161 MATERIALS. AND METEODS 162 1. Test Species 162 2. Experimental Conditions 162

RESULTS 164 1. Pieris brassicae 164

(a)Foliar applications 164

(b)Soil applications 167 2. Muses. domestics 173

(a)Effects on adult flies 173

(b)Development of larvae on food medium containing neem 175 3. Stomoxys calcitrans 176 (a)Effects on larval development 176 (b)Incubation of eggs on papers treated with neem 178 4. Ano-theles stephensi 179 (a) Larval development in medium contaminated with neem 179

5. Trialeurodes vaporariorum 182 (a)On cabbage 182

(b)On cotton ...... 185 6. Schistocerca gregaria 187 (a) Development of hoppers on grass containing small quantities of neem 187

7. The Nematode, Erslixl...m2kis spp. 191 8. Probing Experiments against 192

(a)Plutella xylostella , • • • •

(b)Dysdercus cigulatus 193 (c) /lyzus persicae 194

(d) 3griotes spp. 194

DISCUSSION 196

CONCLUDING REMARKS 208

SUI,MARY 218

ACENOWLEDGEMENTS 225

BIBLIOGRAPHY 226

APPENDICES •. . . 0 . 246 8.

GENERAL INTRODUCTION

In the war against hunger and disease the use of modern, synthetic insecticides has made a substantial contribution. The supression of the vectors of the insect-borne diseases, notably malaria, has brought about a significant improvement in public health in many tropical countries. The introduction of high yielding hybrid varieties of wheat, rice, and maize has ushered in the 'green revolu- tion', raising hopes of millions. Many of these varieties are, however, highly susceptible to insect pests and diseases and the use of pesticides is essential to ensure their success. In the developed countries higher yields and high quality of agricultural produce are also in part dependent on the adoption of chemical methods of pest control. As the standard of living rises, so also does the demand for unblemished food. The use of insecticides is, therefore, likely to continue unabated in the near future.

Nevertheless, extensive use of pesticides constitutes one of the major sources of environmental pollution and the need to reduce the quantities of persistent toxic chemicals applied to the environment is appreciated by scientists and policy makers alike. The dilemma they are faced with, however, is the choice, in many instances, between starvation and disease on the one hand and slow and possibly irreversible upset of natural balance on the other. Even enlightened agencies like the World Health Organisation continue to plead for the retention of the use of DDT. A major contribution to the solution would seem to lie in the development of alternative, non-hazardous methods of control of pests. Further research in the field of biological and selective control methods is urgently needed. Eventually, an integrated system of pest management based on the minimal use of chemicals and 9.

maximal interplay of the natural regulatory forces of the eco-

system will have to be developed to achieve a degree of stability

commensurate with maximum production of food and fibre. To develop

such a system is a challenging task for the scientists.

Increasing apprehension about the pollution risks inherent

in the use of the conventional pesticides has helped to focus greater

attention on the development of selective methods of control. This

is well illustrated by the fact that in the fiscal year 1965, more than

70% of the grants of Entomology Research Division, Agricultural Research

Service, USDA, were channelled on to selective control methods (Asquith

et al.,1966). The shift in the emphasis from conventional to selective

control in USDA is further evident from Table 1: the support for

research of conventional insecticides dwindled from 66% in 1955 to 21%

in 1967, while that of other methods of control correspondingly

increased from 17% to 42% during the same period.

TABLE 1. Showinz_the changing trends of research efforts in the Entomolou- Research Division, RS USDA, estimates for the

Area of research Percentage of total effort 1955 1957 1962 1967

Conventional insecticides 66 60 32 21

Biological and specific chemical 17 21 35 42 methods

Fundamental entomology 17 19 33 37

(after Ilansberry, 1960

As a result of the vastly expanded research effort, there have been some significant advances in the fields of sterilisation, fungal and viral agents,'pheromones, ecdysones and other similar 'third

generation' category of pesticides (e.g., Kilgore et al., 1967; Ascher, 10.

1971; Jacabson and Crosby, 1971). The present investigation on.

the use of feeding deterrents is to be viewed in this context.

Feeding Behaviour and Host Selection

Feeding involves complex behaviour patterns correlated

with basic aspects of host plant relationships. Different behaviour

patterns have evolved in connection with the utilisation of different

kinds of food by different species but in every case the behaviour

involves a means of quality control arida regulation of quantity (Dethier,

1966). How this control of quality and quantity is achieved forms the

subject matter of the three different though allied fields of sensory

physiology, insect dietetics, and host selection. That insects detect

chemicals through the senses of taste and smell is well known, but the

basis of specific diet preferences , and the means by which the

preferences are implemented are still poorly understood.

The mechanism probably consists of a complex and interacting

sequence of responses to a variety of stimuli culminating in ingestion

to repletion (Thorpe et al; 1947; Dethier, 191i7). The nature of the

components of the sequence may vary from species to species and may

indeed be interpreted differently by different authors (Thorsteinson,

1958; Hamamura et al., 1961; Goodhue, 1963). Nevertheless, it is

generally agreed that the integrated behaviour consists of the following:

locomotion bringing the insect to its food, cessation of locomotion on

arrival, biting or its equivalent (probing, sucking, etc.), continued

feeding, termination of feeding (satiation). Each successive step

in the sequence may involve sensory perceptions which constitute a check

on quality.

The main concepts on feeding behaviour and host selection

are described by Thorsteinson (1960), Fraenkel (1969), Dethier (1966, 1970), 11.

Kennedy (1965), and Beck (1965). Important reviews of this field

include the proceedings of the Symposia on (i) 'the physiological

relations between insects and their host plant' held at the International

Congress of Entomology in Amsterdam, 1951; (ii) 'Insect and host plant'

held in Wageninzen, 1969; (iii) 'Insect behaviour: Royal Entomological

Society symposium No.3, London, 1966.

Nutrient and non-nutrient factors

The degree of selection exercised by species varies widely from polyphagous insects which eat a wide range of plants to oligophagous insects which are more restrictive in diet, while some species are almost' monophagous.

According to some workers, host selection in many species depends upon the presence of necessary nutrients in the right proportion, coupled with the absence of repellents - a view emphasised, but not exclusively, by Thorsteinson (1960). Greater stimulation of feeding by sucrose than other sugar is attributed to its nutritional importance. Less stimulating compounds, such as amino-acids, may act as synergists with other compounds. Other nutrient and presumably nutrient substances are known to enhance feeding in some species.

For example, ascorbic acid and thiamine for chorthippus (Thorsteinson,

1958), lecithins, phosphatdyl inositol and other phospholipids for

Melanoplus (Nayar and Thorsteinson, 1963) and unsaponifiabfe extracts of bran, olive, and corn oil, and wheat germ oil for Schistocerca

(Chauvin, 1951; Dadd, 1960), lecithin, alanine and serine for

Leptinotarsa (Hsiao and Fraenkel, 1968).

The contrary view has been well expressed by Fraenkel (1969) who concluded that host selection is principally guided by the presence and/or absence of secondary substances, and that qualitatively and 12. quantitatively nutrients can play only a very minor part in this context.

All plants are, more or less, equally nutritive and could well serve as food, provided the insects ate enough of them and no adverse chemical or mechanical factors were operating. These secondary, non-nutritive factors could act as attractants, feeding stimulants, repellants, deterrents, hormono-mimetics, and defence agents for insects.

The relative importance of nutrients and secondary plant substances as feeding stimulants and deterrents, no doubt, varies widely in different species. But certainly the presence of distasteful naturally occurring compounds and hormono-mimetic substances in certain plants calls for investigation of their possible use in pest management practice.

Definition of a Feeding Deterrent

The study of deterrents is essentially one of stimulation and response. According to Beck (1965), chemical stimuli may be classified according to the successive stage of feeding:

Stimulus Positive gativeNe

orientation - distant attractant repellent

orientation - contact arrestaut repellent

biting or piercing incitant suppressant

continued feeding stimulant deterrent

These terms are confusing. The term repellent is probably best restricted to compounds which repel insects (i.e., cause them to move away) when acting in the vapour phase, that is, on olfactory organs.

In order to distinguish compounds which cause rejection of potential food when acting upon chemoreceptors, the organs of taste, the terms feeding deterrent (Dethier et al., 1960), rejectant (Lewis, in Fraser,

1965), and anti-feedant (fright, 1963; Ascher, 1964) have all been used.

Suppressant mid repellent (in relation to contact) in Becks (1965) classification are also synonymous. Therefore, a chemical which 13. inhibits feeding without killing or eliciting evasive reaction on the part of the test species may be termed a feeding deterrent, rejectant, anti-feedant, and gustatory repellent. The four terms have been used as synonyms in this thesis.

Possible Advantages and Disadvantages of Feeding Deterrents in Crop Protection.

The main advantages claimed by Wright (1963, 1967) are their selective action on target species and the comparatively low toxicity of the few known compounds. In some circumstances (e.g., against

Schistocerca) deterrents may limit pest damage at the point of application more effectively than conventional pesticides, which require a certain amount of time before the toxicant can act, during which- time the invading insects can continue feeding. In general, the larval stages of insects are more destructive_ than adults (some adults, of course, do no direct damage) but they are also relatively less mobile.

If large areas are treated with a deterrent, such relatively slow moving populations, e.g., lepidopterous caterpillars, may be starved to death by the use of feeding deterrents before they reach untreated crops.

Alternatively, anti-feedants might force insects on to 'catch crops' to be sprayed with toxicants. According to Lewis (in Fraser, 1965), "anti- feeding compounds acting systemically offer more promise than has been realised, but the empirical approach has been unrewarding. More fundamental work needs to be done on factors governing the feeding responses of pest insects and the plant constituents responsible."

On 'repellents for phytophagous insects' a category into which, in a wider sense, the anti-feedants have to be placed, the

Environmental Pollution Panel (1965) of the U.S.President's Science

Advisory Committee commented: "Repellents for plant eating insects do not appear to offer any special advantage over conventional insecticides. 14.

They require the same exhaustive study and development in order to prove their toxicological safety and have the same inherent problems as to application and confinement to the treated area.m

The greatest factor suppressing commercial interest may well be the lack of a really good example of an anti-feeding compound, so far. The few reported compounds required relatively high dosage to ensure complete foliar coverage and possessed limited spectrum of activity.

Presentation of Thesis

The thesis is presented in three parts. Part I deals with the comparative efficacy of 28 feeding deterrents against Schistocerca.

Part II reports the discovery of the systemic property of the active ingredient of neem, its uptake, translocation and persistence in soils and in plants. The effect on persistence of soil type, rain, plant species and mode of application is also considered. Part III is concerned with the activity of neem extracts against several insect and other animal species. Evidence for a hormone-like, growth disruptive effect of neem on the larval stages of some insects is presented.

The Locust Problem

Much of the experimental work to be described in the following pages is concerned with Schistocerca gregaria Forskal, which was used as the principal test species. The Desert Locust is one of the oldest and most damaging insect pests. Locust plagues affect

29 million square kilometers in sixty countries and territories and cause losses of several million pounds annually (Sullen, 1969; Haskell, 1970).

(Precise information on losses caused by Schistocerca is not available; one estimate puts annual expenditure on locust control by the affected countries at 20 million dollars; actual and potential losses must, therefore, be many times more.) A number of insecticides can be used 15.

effectively for the control of locusts (McCuig, 1968). However,

considerable damage is likely to occur before the toxicant canses

paralysis and death of locusts and prevents their feeding. This may

he too inadequate a protection for many valuable crops, such as

horticultural and plantation crops, orchards and plant breeders'

materials.

The pattern of agriculture in many developing countries which

lie in the 'locust belt' is changing from one of subsistence to commercial

and intensive cultivation.. There is greater use of agricultural inputs,

such as improved seeds, fertilizers, pesticides, and machinery, most of

which are imported and, hence, expensive. The risks of the farming

community are far greater now than in the past. A locust attack can

devastate the entire vegetation of an area almost overnight. One of the

possible options to avoid such losses lies in the use of inexpensive

feeding deterrents so that the locusts are prevented from eating the

crops. The development and use of feeding deterrents has, therefore,

a special relevance in locust research.

Feeding Behaviour of Locusts and Grasshoppers

The large literature on feeding in Orthoptera has been reviewed by Mulkern (1967). The role of nutritional factors as phago-

stimulants in feeding by Acridoidea was stressed by Dadd (1963). In

some grasshoppers non-nutritional factors play an equally important role

(Thorsteinson and Maya'', 1963; Harley and Thorsteinson, 1967). A direct role in the reproductive biology of Schistocerca exercised by certain plant constituents was described by Ellis et al., (1965), and

Carlisle et al., (1965). Thomas (1966) and Chapman (1966) provided information on the type and distribution of chemoreceptors on the mouth parts of Schistocerca and Xenocheila zarudnyi. It is suggested that there might be a basic pattern in Acridoidea. 16.

According to Thomas (1966), the sense organs of the mouth parts of Schistocerca are distributed as follows: (i) the maxillary and labial palps both bear sensilla scattered on their whole surface with a dense array of sensilla on the tips; (ii) the inner surface of the clypeo-labrum has four paired restricted areas of sensilla: two paired groups near the proximal end, the third elongated paired group at the distal end, and the fourth pair near the torma of clypeo- labrum, (iii) the hypo-pharynx also bears a paired group of sensilla near the dorsal end, opposed but slightly post-terior to the two paired groups of sensilla near the proximal end of clypeo-labrum.

The role of these chemoreceptors in the feeding behaviour of Locusta was studied by Haskell and Schoonhoven (1969) and in that of

Schistocerca by Haskell and Luntz (1970). It has been shown that the receptors on the palps act as the first line of selection, being the first to receive stimuli, while the receptors on the distal end of clypeo-labrum have a role in the detection of deterrents. If a set of receptors was removed by electric cautery, its role was taken over by another set of receptors indicating compensatory substitution mechanism. Blaney and Chapman (1971) have shown that as hunger mounts the receptors on palps no longer inhibit feeding.

Biological Activity of the Neem Tree Extracts

Although several anti-feeding compounds, including alkaloids, carbamates, organotins, plant growth regulants, saponins, triazenes, and general repellents, are described in Part I, the evaluation of the extracts of the neem tree forms the major part of this investigation.

Besides possessing remarkable anti-feeding activity against Schistocerca, such extracts have been in domestic use in India for various other insecticidal and medicinal purposes. It may, thus, not be out of place to describe some of these traditional uses of neem extracts. 17.

The neem tree, Azadirachta indica A.Juss, is widely distributed throughout the Indo-Malayan region and is also found in tropical Africa. In India, the various parts of the tree have been used for insecticidal and medicinal purposes since ancient times.

Neem oil is reported to be in use in the nth century B.C., being perhaps the oldest known non-edible medicinal oil (Shama Sastry, 1929). The oil is said to be useful for bleeding gums and pyorrhoea and is efficacious in a variety of skin diseases. Some women take this oil with their food during pregnancy to have healthy babies (MacMillan,

1935). An ounce of neem oil given to women in labour is said to help in immediate delivery. Oil is also used in the hair to kill vermin.

Infusions and decoctions of leaves and other parts of neon are used for washing wounds, as poultices, and are put in bathing water as an antiseptic. Fermented preparations are taken with honey to cure jaundice. The bark is considered to be tonic, astringent and antiperiodic. Green twigs are chewed to brush teeth for mouth hygiene.

Medicated toothpaste and soap containing neem are available on the market. A paste of bark or seed is smeared by mothers around teats of their mammary glands to wean persistent children off milk. The name neem is supposed to have been derived from the Sanskrit word nimba, meaning 'relieving sickness'.

The timber of the neem tree is comparatively immune to the attack of termites and other wood boring insects and is 'durable even in open situations' which is perhaps indicative of its resistence to weathering and rot. It is, therefore, preferred for building materials, such as door panels and rafters, furniture, agricultural implements, and toys.

The Hindus use it for making idols. The protection is popularly attributed to the oil contents of the wood. The above and similar other 18.

statements about the quality of neem timber appear in the works of

many writers (Kanjilal, 1901; Brandis, 1906; Troup, 1909; Warden,

1921; Rodger, 1936; Trotter, 1940).

It is customary to put shade-dried leaves of neem in stored

grains and woollens to protect them from insect attack. The odour

produced by the burning of powdered leaves is said to be fatal to insects.

(No experimental evidence is, however, available for this property.)

The seedcakes left after extraction of oil and leaf-compost are used

as fertilizer for their 'supposed insecticidal properties'. Neem

preparations taken internally act as anthelmintic. Many other uses of

neem are described by Mangunath, 1958, and Mitra, 1963 (see Appendix:3 & 4 •

1 PART I. EFFICACY OF CANDIDATE FEEDING DETERRENTS. 20.

INTRODUCTION

Many phytophagous insects are restricted in host range because of the presence of naturally occurring distasteful chemical substances in otherwise acceptable plants. In the search for possible substitutes for toxic pesticides, however, comparatively little attention has been directed towards the application of non-toxic anti-feeding compounds or rejectants as a means of deterring insects from attacking crops. A good feeding deterrent should possess the following properties:-

(i) First of all, it must be distasteful to pests at low concentra-

tions.

(ii) It should be non-toxic to parasites and predators which control

the pest, to some extent, by natural means.

(iii) The chemical should have low mammalian toxicity and be non-toxic

to human beings and to other higher animals.

(iv) It should be persistent for some time under different environ-

mental conditions.

(v) Most important of all, it must be systemic so that it can be

readily absorbed by the plant from the leaves or the roots and

translocated to the growing points. Otherwise the new plant

growth developing after treatment, being uncontaminated, will

be selectively attacked while the older treated leaves remained

distasteful.

(vi} It should be easily available at competitive prices and in

formulationswhichcoulti be applied without sophisticated

equipments. 21.

Some of the important anti-feeding compounds which are reported to have passed the initial laboratory screening tests are reviewed below.

Non-Botanical Anti-Feedants

1. Triazenes

This group of chemicals includes compound 24,055 (4-dimethyl triazino-acetalnilide) of American Cyanamid, the deterrent properties of which have been extensively tested outside the laboratory (Wright, 1963).

The compound was effective against insects with chewing and biting mouthparts, e.g., cabbage worms, asparagus beetles, and bean beetles, but was not effective against insects with piercing or sucking types of mouthparts, e.g., aphids, leaf-hoppers, and squash bugs. Control of up to seven days was obtained depending on.dosage applied. The chemical possessed a limited systemic activity; leaf surfaces treated on one side with the chemical were not eaten when the other non-treated side was offered for feeding to the Mexican beetle larvae which feed primarily by rasping the surface of the leaf. However, the product did not prevent the feeding of new plant growth and was ineffective against internal borers. It was concluded that compound 24,055 did not warrant the cost of development and registration involved in the commercialization of a compound, and further work with it was terminated (Wright 1963),

2. Organoletallics

Most of the recent work on anti-feedants has dealt with organotins and other organometallics. These chemicals are normally used as fungicides and some are chemosterilants. Fentin acetate (Brestan) and fentin hydroxide (Du-ter) are the most widely investigated representa- tives of this group of compounds. Their anti-feeding activity has been shown against a number of insect species, but the chemicals are not 22.

systemic: Ptodenia Mural Phytophthora infestans, Leptinotarsa

decemlineata (Ascher and hones, 1964); P. litura, Agrotis ypsilon,

Gnorimoschema operculella, (Ascher and Sarah, 1964); Mamestra, Agrotis,

Aphis fabae, Cercospora beticola - combined aerial spray of Brestan,

phosphamidon and fentin (Pivar et al., 1965, 1968); Carpocapsa pomonella

and Grapholitha molesta (Marfurt and Toscani, 1968); Hylobius pales,

(Thomas 1969).

Field trials on cotton to control the American boll worm,

Heliothis zea,with fentin acetate and hydroxide were carried out by

Findlay in South Africa in 1968. Economical control of the pests was

not obtained and yields were vastly inferior to those with conventional

insecticides. Although the larvae were controlled satisfactorily,

fentin acetate and, to a certain extent, the hydroxide tended to retard

growth of the crop and proved to be phytotoxic at certain concentrations.

Similarly, Wolfenbarger et al., (1968) found that on cotton fentin

hydroxide reduced the number of boIls damaged by the Heliothis by 60% in

field cage trials, but the reduction in the number of damaged squares in a

field trial, in which 5.6 kg/ha fentin hydroxide was applied, was only

10-15%.

Several other organotins, such as•Deca-fentin (Cela, 1968),

fentin chloride, bis-(fentin)-oxide, and tricyclohexyltin hydroxide

(Ascher and Meisner, 1969), hexamethylditin or Pennsalt TD-5032 (Ascher

and Moscowitz, 1969; Ascher et al., 1970) are reported to possess anti-

feeding properties. Hexamethylditin is reported to possess systemic

action. 23.

3. rhenovethanols

Another group of systemic fungicides with anti-feeding properties has been reported by Jermy and Matolcsy (1967); they found that derivatives of phenoxyethanol and phenoxyacetic acid were good anti-feedants for insect species of Phytodecta fornicata, Ceutorrhynchus macula-alba Hyphantria cunea, Plutella maculipennis, Pandemis dumetana,

Athalia rosae. The results are based on laboratory studies in feeding tests using 15 nun leaf discs painted on both sides with 1yo solution or suspension of these compounds. No field trials were reported by the authors.

4. Plant growth regulants

Tahori et al., (1965) reported that several plant growth regulants at relatively high rates inhibited the feeding of cotton leaf- worm by foliar applications and by immersion of the leaf petiole in solutions of the compounds. Phosfon (2,4-Dichlorobenzyltributyl ammonium chloride) was the most effective; leaf-dip in 4,000 ppm solution inhibited feeding by 89',i1.

5. Alkaloids and Saponins

A number of alkaloids including gramine, veratrine, lobeline, lupinine, etc., were shown to inhibit the feeding of grasshopper,

Melanoplus bivittatus by Harley and Thorsteinson (1967). A list of alkaloids, glucosides, sterols and saponins recorded from plants within the geographical range of M. bivittatus is also given by the authors.

The saponins, digitonin and diosgenin, when fed to the grasshopper mixed in a synthetic diet, besides being anti-feedants, also proved toxic to the nymphal stages. 24.

6. Carbamates

It has been shown that a large number of phenyl carbamates with alkyl or alkoxy substituents on the ring prevent feeding of the salt marsh caterpillar, Estigmene acrea thurberiella, at dosages of about one-tenththelethal rates (Georghiou and Metcalf, 1962).

7. Miscellaneous repellents

Several other chemicals have shown deterrent activity in passing. Dethier (1947) stated that strong antiseptics, like copper stearate and copper resinate, although comparatively non-toxic, seemed to inhibit the feeding of tent caterpillars, Malacosoma americana, and that mercuric chloride acted as a gustatory repellent to Phormia.

Other insect repellents, such as Indalone, MGK repellent 874, Metadelphene,

Rutgers 6-12, and dimethyl phthalate, were found to inhibit the feeding of the larvae of Affriotes spp., in feeding tests using filter papers immersed in different concentrations (Griffiths, 1969).

Neem and Other Botanicals

The anti-feeding activity of neem extracts has been observed by several workers Onssain, 1928; Volkonsky, 1937; Chauvin, 1946;

Pradhan et al., 1963). (A detailed review of the deterrent activities of Neem is given in Part III of the Thesis.)

Three derivations of Neem, i.e., (i) azadirachtin, a pure chemical isolated from neem seeds; (ii) an alcoholic extract, about 4% by weight of seeds; (iii) a simple aqueous formulation prepared by grinding and sieving of seed kernels, were included in these tests.

Azadirachtin also served as the standard chemical for comparison with other deterrents.

Several other examples of the presence of anti-feeding factors in plant extracts are reported in literature. Soo lIoo and Praenkel (1964) 25.

showed that solvent extracts, and extracted leaf powders from the fern,

Nephrolepis exaltata schott, deterred the feeding of the southern army worm, Prodenia aridania. The active factor was water soluble. A substance that deterred feeding by the adults of Anthonomus Aundis Boh.

was found in an extract of tung-meal (a by-product of the process of

extracting oil from the seeds of Aleurites fordii) and in more concentrated

form in tong oil. A 2 to 15-fold decrease occurred in the number of

feeding punctures made by the weevils when cotton buds were dipped in

a filterate of the tung-meal (Harder and Davick,1966). Filbert et al.,

(1967) found that Scolytus multistriatus would not feed on the benzene

extract from the bark of non-host, Carya ovata alone or when mixed with

the benzene extract from the bark of host plant, Ulmus americano, an otherwise stimulatory extract. Subsequent fractionation indicated that

Juglone (5-hydroxy-1, 4-nephthoquinone) was the chemical constituent responsible for this deterrent effect. The findings from further studies

(Gilbert and Norris, 1968) showed that when 'Juglone' was extracted from the non-host plant the 'residual' twig was equally acceptable to the bark beetle as its host plant, U. americana, confirming that 'Juglone' was responsible for deterrency. Another species of bark beetle,

S. quadrispinosns, tolerated the chemical and was not deterred.

An anti-feedant alkaloid 'isoboldine' has been extracted from the fresh leaves of Cocculus trilobus by Wada and Munakata (1968).

Leaf disc tests with Abraxas miranda prevented complete feeding at 200 ppm or greater.

Another chemical, 'ecdysterone', which applied to cabbage leaves at 100 ppm inhibited feeding of Pieris brassicae completely is reported by Ma (1969). Additional anti-feedants still being investigated at the laboratory stage by Japanese workers are reviewed by Munakata (1970). 26.

The purpose of this discussion is to point out:-

(i)the lack of a good feeding deterrent for insects amongst

either botanicals or non-botanicals;

(ii)although the presence of anti-feeding factors in plants is

well established, no extracted chemicals suitable for pest

control have been reported, apart from azadirachtin, the

subject of the, present Thesis.

(iii)plant extracts, which have been examined, have not been tested

for a possible systemic action.

It appears that studies with plant extracts have been undertaken more to establish host plant relationships than to develop deterrents.

Techniques for Testing of Deterrents

The methods of screening rejectants have been adapted largely from those used to test simulants. Some techniques have been adapted from those used for stomach poisons. The main methods used to test stimulants and rejectants are reviewed below.

Aqueous extracts of the preferred plants were tested for their attraction by offering them to insects in an inert medium, such as elder-pith (Chauvin, 1952), filter paper discs (Yamamoto and Fraenkel,

1959; Augustine et al., 1964) or plain agar medium (Maxwell, et al.,

1963; Simons, et al., 1968). The amount of feeding was adjudged in various ways, such as by recording ( ) the number of biting marks,

(ii) the amount or area of filter paperzor leaves eaten, (iii) the number of faecal pellets produced, (iv) weight loss, and (v) the number or frequency of insects present on or near the food. The quantitative determinations of these criteria have been made graphically, plani- metrically, photographically, photometrically, or gravimetrically. 27.

Sucrose impregnated filter paper discs treated with a

rejectaut were fed to Schistocerca confined in cages (Butterworth and

Morgan, 1968; Haskell and Mordue, 1970). Several commercially

available repellents were evaluated in feeding tests with larvae of

Agriotes spp. by Griffiths (1969). The test chemical was applied to

one edge of the filter paper disc dipped in a nutrient solution and the

number of bites on the treated and non-treated areas were counted to

give an index of deterrency.

Harley and Thorsteinson (1967) fed the test chemicals to

Melanoplus mixed in a chemically defined synthetic diet made into wafers.

Ascher and Moscowitz (1968) tested the anti-feeding activity of several

triphenyltins against houseflies incorporating them into larval rearing

medium.

Woodbury (1943) developed a method (used earlier by McGovern,

1940, to test stomach poisons) for comparing the deterrent action

of compounds against leaf-feeding insects. A large flat seed leaf was cut from a bean plant and flattened by pressing it on stretched cheese cloth. It was then treated by spraying or dusting with the chemical

in the appropriate apparatus. After spraying, the leaf was laid flat

on damp sand, with the petiole inserted into a vial of water to maintain

turgidity. A batch of five 4th instar larvae of the test species was placed on the leaf and confined under a small cage. After a suitable interval, the leaf was examined and the amount eaten was measured.

Jenny (1966) compared the deterrency of various non-host plants to the larvae of a variety of beetles and caterpillars by (0 feeding leaf discs

of the host and non-host plants mounted on separate pins, and (ii) by offering sandwiches of the non-host plant in two layers of the host plant. 28.

Relative acceptance or rejection was recorded on an arbitrary scale.

Similar or slightly modified methods of feeding the test insects on leaf discs treated with organotins were used by Ascher and associates

(Ascher, 1970).

To test the deterrent action of lime sulphur, about 200 honey bees were fed on a series of concentrations in vials arranged in a Latin square design. Sucrose was used as a stimulant and the amount of liquid consumed served as the index of inhibition (Butler et al., 1943).

The drinking preferences of the grasshopper, Melanoplus bivittatus, for several chemicals were studied by applying aqueous solutions or emulsions to the distal segments of maxillary and labial palpi by Harley and

Thorsteinson (1967). Similar manipulation of chemoreceptors with chemical films were used earlier by Frings and Frings (1949) to determine loci of contact chemoreceptors in insects.

To test the _systemic deterrent action of natural products, pairs of cotton seedlings were put in test tubes, one containing a nutrient solution and the other nutrient mixed with the substance under test.

The protection of seedlings against the boll weevil of cotton was observed to indicate systemic action (Matterson, et al., 1963). 29.

MATERIALS AND METHODS

A. TEST INSECTS

The stock of Schistocerca gregaria F. used as the test species in these experiments was obtained in the form of egg pods from the Centre for Overseas Pest Research, London. The locusts 3 were reared in 0.3 m cages in a constant temperature room at 27°C and a relative humidify of 65%. Each cage contained a 60 watt bulb which provided an additional heat gradient, raising the temperature to approximately 30°C at a distance of two feet from the bulb. The locusts were fed on green grass, and wheat bran was given as a protein supplement.

B. TEST CHEMICALS

The following chemicals were tested for their anti-feeding activity against Schistocerca on the basis of discussion in the

Introduction.

Azadirachta indica (neem) derivatives

1. Azadirachtin (a pure chemical isolated from the seed). 2. Alcohol extract of seed. 3. Kernel suspension.

Alkaloids

4. Gramine. 5. Rordenine. 6. Hyoscyamine (sulphate). 7. Lobeline (hydrochloride). 8. Lupinine. 9. Veratrine (mixture).

Carbamates

10. Carbaryl (M-methyl-l-naphthyl carbamate). 30.

Ormanotins

11. Triphenyltin acetate or Fentin acetate (Drestan, 60% W.P.). 12. Tripheyltin hydroxide (Tinicide, 50% W.P.). 13. llexamethylditin (Pennsalt, TD-5032). 14. Stannous chloride.

Plant Growth Regulants

15. 2,4-dichlorobenzyl tributyl ammonium chloride (Phosphon). 16. N-dimethyl amino-succinamic acid (B-nine). 17. 2,4,6-trichlorophenoxy acetic acid (compound 19296 of KA K. Plainview, New York).

Saponins

18. Digitonin. 19. Diosgenin. Triazenes 20. Compound 24,055 4-(dimethyl - triazeno) acetanilide (American Cyamamid). Others

21. Copper stearate (copper content 10: ICI). 22. Copper resinate. 23. Mercuric chloride. 24. Indalone (alpha, alpha-dimethyl-alpha-carbobutoxydihydro-gamma- pyrone). 25. MGK. Repellent 874 (2-octylthio-ethanol). 26. Metadelphene (N, N-diethyl-meta-toluamide). 27. Rutgers 6-12 repellent (2-ethyl-1, 3-hexanediol). 28. Dimethyl phthalate. The above compounds were commercially obtained with the exception of Neem derivatives which were specially prepared.

2. Neem Derivatives

Three products obtained from neem seeds were tested for anti- feeding activity against Schistocerca. These were (i) azadirachtin, a pure chemical, isolated by Butterworth and Morgan (1968, 1971) by the method outlined below; (ii) an alcoholic concentrate,containing 3.8% by weight of the neem seed, and (iii) an aqueous suspension of neem seed kernels. Samples of (i) and (ii) were provided by Dr. Morgan of the Keele University, Staffordshire. The preparation of the three products is described as follows: 31.

(i) Azadirachtin and the alcohol extract

Seeds (2 kg) were ground with 95% ethanol (2.5 1.) in portions in a Waring Blender. The solution was filtered and the residue of the seeds was ground again with 95% ethanol (2.0 1.).

The combined ethanolic filtrates gave an oily residue (170 g.) which was partitioned between light petroleum (b.p. 40-60°C, 800 ml.) and an aqueous methanol (5:95, 800 ml.). The combined methanolic extracts gave a dark brown tacky residue (76 g.), which has been called the

'alcoholic extract' (abbreviated as ALC) in the present investigations.

The 'alcoholic extract' was dissolved in warm toluene

(300 ml.) and chromatographed on a column of Floridin earth (Florex XXS,

950 g.) prepared in toluene. The column was eluted with ether-acetone

(95:5) 200 ml. portions. Fractions 14-25 contained azadirachtin

(AZD for short) and these combined fractions (2.6 g.) were subjected to preparative thick-layer chromatography (PLC) on ten 40 x 20 cm. plates, eluting twice with ether-acetone (95:5). The strongly fluorescent- quenching band 5 cm. from the origin gave azadirachtin (1.5 g.) m.p.

149 to 150°C.

The compound is a highly oxidized t:riterpenoid insoluble in ether and light petroleum but very soluble in ethanol, methanol, acetone and chloroform. It is a colourless, amorphous powder which, dissolved in carbon tetrachloride, forms micro-crystals. The constitution of the chemical has yet to he determined. A more detailed account is given by

Butterworth and Morgan (1971).

In a simplified form, the extraction procedure is outlined below: 32.

Neem seeds (2 kg.)

ethanol extraction

Oily residue (170 g.)

partition, methanol

Light petroleum

'Alcohol Extract' (76 g.)

chromatography, Floridin earth

active fraction (2.6 g.)

PLC

azadirachtin (1.5 g.)

(iii) Aqueous 'suspension'

Seed kernels were thoroughly ground and extracted with distilled water until the material passed through a 100 mesh sieve.

To prepare this formulation, one gm of seed kernels (moisture content

5-7%) were first pounded to a powder with a pestle and mortar, and then made into a fine paste by adding 5 ml. of water. The material was ground further by adding more water and then passed through a 100 mesh sieve. The residue on the sieve was removed into the mortar and re-ground in the above fashion. The process was repeated 2-3 times until the residue yielded no more 'milky' material. Thus the preparation constituted a 'suspension cum emulsion' consisting of nearly the whole mass of the kernels except a small residue left in the sieve. When the 'suspension' prepared from one gm. of kernels was evaporated at 60°C, and the residue on the sieve was also dried likewise, the following quantities of dry solid mass were obtained, on an average: 33.

Solids from suspension 856.0

If residue 46.7

Losses (moisture and possible 97.3 volatilisation)

Total 1,000.0

On filtering the 'suspension' through a 11 cm. Nhatman,

qualitative I, filter paper and evaporating the solution, 258 of the

solids (220 mg.) were recovered as solute while 75,01. (636 mg.) were left

as residue on the filter paper. So, the preparation consisted of

about 86% of the kernels on the basis of dry weight, out of which

nearly 2510 was in solution and 75% in a suspended form.

For the purposes of this thesis, this formulation' prepared

from one gm. of seed kernels and made to 100 cc. with water has been

taken as 1.0%, w/v, preparation and named 'kernel suspension' or 'aqueous

extract', and has been abbreviated to 'KNL'.

3. Miscellaneous preparations from different parts of neem fruits The Neem tree fruits once a year and this necessitates

storage of seed over several months, and where stocks are large storage

space may present some problems. A reduction in stock bulk is,

therefore, desirable. A simple method which could be adopted by the

peasant farmers might be to obtain solids from an aqueous extract of the

crushed neem seeds by the process of open pan evaporation. In India,

the farmers are already familiar with this technique which is being

used to obtain 'gur' (jaggery or crude sugar) from sugar-cane juice.

The following other formulations were prepared from the neem

fruits: (a) Whole fruit (drupe) extract: The fruits were finely ground and

extracted with water by sieving through a 100 mesh sieve; the 34.

residue on the sieve (1), the aqueous extract (2), and the

solids (3) obtained by evaporating the aqueous extract at 60°C

(which redissolved into a fine suspension in water) were tested

for their deterrent action.

(b)Shell or husk extracts: The husk was ground finely and filtered

and the residue on the filter paper (4) and the aqueous solution (5)

were tested for deterrent action.

(c)Kernel extracts: The kernel was processed in three different ways:

(i)Ground and sieved: The kernel was finely ground and extracted

with water through a 100 mesh sieve. The aqueous extract was

evaporated at 60°C to obtain solids: the residue on the sieve (6),

the aqueous extract (7), and the solids (8) were evaluated for anti-

feeding activity.

(ii)Ground and filtered: The process was similar to (a) above,

except that the extract was filtered using a Whatman filter paper,

Quality I, and the residue on the filter paper (9), filterate

solution (10) and the solids (11) were tested for their deterrent

action.

(iii)Ground and centrifuged: The finely ground kernel aqueous

extract was centrifuged at 4,500 r.p.m. for three hours. The

mixture separated out into a whitish top layer of fat, a clear liquid

in the middle, and solids at the bottom. These were separated and

an aliquot of the middle liquid was evaporated to obtain solids.

The four products: the fat layer (12) which formed a whitish emulsion

with water, the liquid from the middle (13), the solids obtained from

the aqueous portion (14), and the solid residue from the bottom (15)

which, when suspended in water, formed a turbid preparation, were

tested as rejectants against Schistocerca. 35.

The extraction process is summarised by the flow-chart given below:

Extraction of different preparations from the neem seed

Fruit

Whole fruit - Kernel. Shell or husk - ground and sieved. ground and filtered.

1. Residue on sieve. 4. Residue on filter paper. 2. Aqueous extract. 5. Aqueous extract. 3. Solids from aqueous extract.

. I

Ground and sieved. Ground and Ground and centrifuged. filtered.

6. Residue on sieve. 9. Residue on 12. Fatty layer (top). 7. Aqueous extract. filter. 13. Aqueous ext. 8. Solids from aqueous 10. Aqueous ext. 14. Solids from ext. extract. 11. Solids from 15. Residue at the bottom. aqueous ext.

C. TEST TEGMIQUES

In preliminary experiments, two kinds of test techniques based on (1) feeding treated filter papers, (2) the application of chemical solutions mounted on wire-loop to taste receptors, were evaluated.

(1) Feeding filter. papers: It was found that 1.5 ml. sucrose solution

(0.1 M) was sufficient to moisten a filter paper disc (Whatman,

Qualitative I, 11 cm. diameter) without causing a run off, giving a

sucrose deposit of 0.54 mg/cm2. Sucrose solution was pipetted on

to a filter paper, placed on a petri-dish, and allowed to dry at

room temperature. The test chemical was then applied to the

sucrose impregnated filter paper, also at a standard rate of 1.5 ml.

solution per disc. The choice of the solvent depended on the

solubility of the test chemical. The solvent was allowed to

evaporate at room temperature. 36.

The test diets were presented to Schistocerca in two ways

(i)as single papers to small batches (5-7) of locusts in small cages, or

(ii)as an array of several papers having different treatments to a large population (150-200 locusts) in a single cage. Details of the presentation were as follows:

(a)Single papers in separate cages

The treated filter papers were offered for feeding to 5-7 Vth stage Schistocerca hoppers, confined to a single 11 cm. paper disc in a

2.5 litre glass jar. The mouth of the jar was secured with a piece of muslin, and a 2-inch wide metallic climbing strip was provided in each cage. There were three replicates of each treatment. The insects' were starved for 18 hours and then presented with the test feeding papers for 24 hours.

(b)Multiple choice of papers

Unlike the previous method, this technique involved several choices between several test papers treated with different concentrations.

Treated papers mounted on corks with drawing pins were presented for 3 feeding to 150-200 adult Schistocerca, 5-15 days old, in a 0.3 m cage.

The locusts were starved for 18 hours before being offered the treated papers for the next 24 hours. Usually, a series of 5-7 different concentrations with three replications each were tested in one test involving the presentation of 15-21 papers to the test population, including the control treatments. The filter papers were placed randomly on the floor, measuring 2' x 2.5', of the cage.

Percentage inhibition

The evaluation of the deterrent effects of the treatments was based on the assessment of the quantities of treated papers eaten by starved locusts. The treated papers were kept for 18-24 hours in a 37. dessicator containing silica gel, and weighed before being presented to locusts. At the end of the feeding period, the papers were removed from the cage(s) and again kept in the dessicator for 24 hours. The amount eaten was determined from the difference in weight before and after feeding.

To facilitate comparison, the amount of treated papers eaten by the locusts was expressed as a relative quantity '% inhibition', taking the quantity of sucrose treatment paper eaten as the standard.

% inhibition = 100 - (T/S x 100) where T and S were the amounts eaten of filter papers treated with the test chemical plus sucrose and sucrose only, respectively.

It should, however, be stressed that, in the present context, the term 50% inhibition (or ED50), for example, does not refer to t12e concentration at which 50% of the locusts are inhibited from eating; but the term, instead, represents a concentration, the application of which to sucrose treated filter papers reduced the quantity of paper eaten by the locusts by 50% as compared with the quantity eaten of

'sucrose only' treatments, when several papers treated with several concentrations were presented randomly to locusts for eating in a food choice experiment.

2. Ai lication of chemical solutions to taste receptors

The reaction of adult Schistocerca was tested by applying chemical solutions on a wire-loop to the following parts known to possess chemoreceptors: (a) maxillary and labial palpi; (b) labrum region (to stimulate receptors in the buccal cavity by ingestion of a small quantity of the chemical); (c) antennae; (d) tarsal region, and (e) ovipositor.

The locusts were starved for 18 hours, and then secured in 6 cm. long glass tubes with an internal diameter of 1.3 cm. (Piga). To condition 38.

them to their captivity the animals were left mounted in the tubes

for two hours before starting the test. They were then induced to

drink water to satiation from a syringe. The end-point of satiation

was indicated by the exudation of a water bubble from the mouth and

a refusal to accept Amor- more water. The insects were tested for

satiation of thirst before assaying each concentration of the chemical.

A receptor surface such as the dome of a palpus was touched with a

series of concentrations in a sequence starting with the lowest

concentration, and the production of a specific response, if any, was

observed.

When a stimulant, such as sucrose,was touched on the dome

of the palps at a concentration above the threshold value, the locust

was excited and moved its head and palps, making an effort to eat or bite.

A similar touching of domes with a deterrent like azadirachtin also

produced a movement of head and palps, but the response was an attempt

at evasion - palps were withdrawn and head was turned away, and no

attempt was made at biting. Strong deterrents, like the neem products,

induced the exudation of a darkish brown repugnant fluid from the mouth

in the form of a bubble (Fig.2). The colour of the bubble varied from a

very light brown to almost darkish brown depending upon the concentration,

apparently indicative of the degree of revulsion. Some chemicals, e.g.,

saponins, did not produce a response and were ignored by the locusts;

these were presumably inoffensive and did neither stimulate nor deter

the animal.

Azadirachtin was assayed using (i) revulsion reaction, and

(ii) inhibition of feeding (matching stimulant action of sucrose against

deterrent action of azadirachtin). In each case, the movement of the

mouth parts - head or palps - was taken as the threshold value. The Fig. 1 Showing Schistocerca enraged in glass tubes, and application of a chemical to•the palpi with a wire loop.

Fig. 2. — Showing exudation of a darkish fluid from the mouth of Schistocerca as a reaction to a deterrent. 40. reaction of three batches of 15 insects each (45 insects) was tested with different concentrations of azadirachtin. Further details are given under experimental results. 41.

RESULTS

A. DEVELOPMENT OF A BIO-ASSAY TECHNIQUE

1. Feedin Exeriments Usinm Filter Parser Discs

(a) Feedin} sin.gle test papers in separate cages The standard technique of offering sucrose impregnated

filter papers treated with the test chemical for feeding to small

batches of test insects in separate cages was evaluated in this

experiment. Azadirachtin at three levels of 10, 1.0, 0.1 ug/1

was applied at a rate of 1.5 ml. per 11 cm. paper, thus providing

doses of 0.16, 0.016, and 0.0016 ng/cm2. Each filter paper was

fed to five mid-instar, V stage hoppers of Schistocerca in 2.5

litre glass jars for 24 hours, the hoppers having been starved

earlier for 18 hours. The percent inhibition of feeding is given

in Table 1.1. As expected, the inhibition of feeding was related to dose;

the higher the dose, the smaller the quantity eaten. However,

there was a large day to day variation. For example, in the first

test, the inhibition at 10 ug/1 level was 95.3%, while, at the

same level of 'application in the fourth test the inhibition was

22.1% only. In some cases, greater inhibition was recorded at a

lower concentration; in the third test, azadirachtin at 0.1 ugh

induced 11.3% more feeding than sucrose treatment, and in the fourth

repeat of the test, a dose of 1.0 ug/1 inhibited feeding less than

a dose of 0.1 ugh (6.7% compared with 9.6%). 42.

TABLE 1.1. Day to day variability of the deterrent action of azadirachtin: single filter papers presented to batches-TT-3) of V stage Schistocerca.

Quantity (mg) of Filter Paper Eaten and % Inhibition of Feeding*

Cone. Repeats on Different Days applied Deposi (ug/l) (ng/cm I II III IV V VI

10.0 1.6 x 10- R 28 42 278 324 48 80 I R 23 42 146 429 24 164 2 11 23 44 248 425 33 278 3 Total 74 128 672 1178 105 522 95.3 76.9 16.3 22.1 89.4 68.3 (inhibition)

1.6 x 10-2 2 45 60 164 353 61 382 1.0 1 R 96 112 194 575 39 292 2 11 92 84 151 485 77 248 5 Total 233 256 509 1413 177 922 85.1 53.7 36.6 6.7 82.1 44.0 (inhibition)

0.1 1.6 x 10 B1 270 12 574 - 430 239 410 321 24 268 373 127 466 112 113 213 12 61 216 411 581

Total 804 48 903 1019 777 1457

48.7 91.3 +11.3 9.6 21.2 11.4 (inhibition)

Control 1Z.1 452 171 249 587 327 419 (0.1M 0.0 146 411 245 649 sucrose) 112 623 309 R 493 73 408 517 414 577 3 Total 1568 553 803 1515 986 1645

* % inhibition = 100-(T/S x 100), where T and S are the quantities eaten of fi,lpr papers treated with the test concentration plus sucrose,and sucrose?, respectively. ** Locusts were starved for 18 hours, followed by 24 hours feeding. 43.

Since the response of each individual is graded within

the sum totals of a group response, a greater variability was to

be expected than if the criterion of response had been locusts

eating' (all or no response for each individual). The variability was considered to be large enough for a need to develop an alternative method for routine testing of anti-feeding chemicals.

(b) Presenting multiple filter papers placed randomly in a single cam

One way of minimising variability due to the test population is to increase the number of animals per treatment. For numerous tests a large number of animals would be required and, in the case of Schistocerca with a life cycle of 6-8 weeks, this would entail heavy requirements for food and shelter. Another solution of the problem was to offer the test food to a large insect population in a single cage by placing the food randomly in the manner of a food choice experiment.

As described previously under 'Methods', several filter papers, treated with a series of concentrations including controls and placed 3 randomly at the floor of a large 0.3 m. cage, were fed to 200 adult locusts. A constant starvation period of 18 hours, followed by a constant test feeding; period of 24 hours was followed and the amount of feeding was determined by pre-,and post-feeding weighings of the filter papers. The percent inhibition of feeding in four repeats of a test with azadirachtin is given in Table 1.2.

The daily variability was much less when compared with the no previous method, whileltransfer of insects to separate cages was involved, which saved time. The insects were used between the ages of days 5-15 after fledging. To avoid a possible conditioning effect, test feeding the same population on consecutive days was avoided;

44.

Till3T,E 1.2. Day to day variability of the deterrent action of azadirachtin: multiple filter papers presented to 200 adult Schistocerca.

Quantity (mg) of filter paper eaten and inhibition of feeding

repeats on Different Days

Cone: applied Deposit I II III IV (ug/1) (n'g/cm Eaten Inhi- Eaten Inhi- Eaten Inhi- Eaten Inhi- paper bition paper bition paper bition paper bition (mg) 'A (mg) (10 (mg) % (mg) /Dc

500 8.0 0.0 100 - 0.0 100 + 100 1.6 0.0 100 0.0100 4 ± 1 94.7 2 - 1 98.8 + ± 50 0.8 10 ± 1 92.7 - - 4 ± 1 94.2 8 1 95.0 + 10 1.6 x 10-1 11 It 2 91.4 29 t 3 81.6 8 ±2 89.0 15 - 1 91.1 5 0.8 x 10-1 56 ± 12 57.6 80 ± 15 48.8 24 ± 68.1 38 ± 8 77'3 2.5 0.4 x 10-1 - _ - - - - 86 ± 9 49.2 ± 1.0 1.6 x 10-2 - - 88 11 43.7 39 ± 1 47.8 124 ± 18 26.5 0.1 1.6 x 10 3 - - 159 ± 16 2.1 - _ _ _ (+) -+ 0.001 1.6 x 10.- - 184 - 5 18.4 - _ _ _ (+) Control 132 ± 7 - 156 ± 8 - 75 ± 6 - 169 ± 26 - (0.1M sucrose •

*Average -standoaderror of three replicates/treatment; starvation period 18 hours followed by 24 hours feeding period.

***A inhibition: See Table 1.1. I45.

test feeding was interspersed with normal feeding on grass and

wheat bran. The same population could, thus, be used for 4-5

tests, and subsequently utilised for probing tests to fix the

upper and lower inhibition limits of a chemical. The method

was a marked improvement on previous test techniques and was employed

for all subsequent tests in the present investigations.

2. Stimulation of Taste Receptors by Direct Touch

Areas known to possess chemo-receptors on the following parts of the insect body were excited by touching them with graded concentrations of a test chemical and observing for a specific response.

(a) Palpi(revulsion response)

The touching of the domes of both maxillary and labial palpi

with a chemical film mounted on a wire-loop elicited a characteristic

response from adult Schistocerca; the animal was excited, moved its

antennae, mouth parts, and also the head making an effort at biting

in case of an attractant and - at avoidance in case of a rejectant.

The reaction was increased by pressing the film against the dome of

the palpus. The response of the animal to the chemical was,

however, not so great if the film only gently touched the dome;

when the palps.were alternatively touched by the film with slight

pressing a stronger reaction was produced.

Evaluation of azadirachtin by stimulating the receptors on

the dome by alternate touching of the left and the right maxillary

palpi of Schistocerca is given in Table 1.3. Azadirachtin was

dissolved in methanol to make a 0.1% stock solution which was further

diluted with double distilled, de-ionised water. No sucrose was 46.

TABLII1 1.3. Evaluation of the revulsive action of azadirachtin by touching the domes of the maxillary palp of adult Schistocerca with films of different concentrations of the chemical mounteu on a wire-loop.

Azadirachtin: Response and Concentration (mg/1) Animal no 0.5 0.75 1.0 1.5 2.2 3.3 7.5 1 + + + + + + 2 + + + + + +

3 _ _ - - - -1- 4 + + + + +

5 _ _ _ + + - + + + +

6 - + + + 8 + + + + +

9 _ + + + + + 10 _ _ _ _ + +

11 + + + + + +

12 + + + + + 13 + + + + 14 _ + + + + + 15 - - - + +

Rl 3 5 9 10 11 14 15

2 5 11 10 13 14 R2 9

11 (replicates) 3 7 9 12 14 15 3 Total 8 16 25 30 33 41 44

Revulsion 17.8 ' 35.6 55.5 66.6 73 3 91.1 97.7 47.

used to stimulate feeding so the reaction represents an evasive

dose was nearly reaction on the part of the animals. The ED50 1.0 ug/l% which is much higher than the ED50 determined by feeding experiments, which was 2.3 ng/l%. This was to be expected as the present situation represented 'revulsion' to an unpleasant chemical while the latter was 'inhibition' of an acceptable compound, sucrose.

(b) Labrum Region

When the region around the notch of the labrum was touched with a chemical film without actually breaking the film and without letting the animal ingest a large quantity of the chemical, the touch produced a positive or negative reaction similar to the one produced by the touching of palp-domes. Apparently, a small quantity of the chemical was ingested by the locusts, presumably stimulating the receptors in thebuecal cavity. Occasional biting and swallowing of the chemical film was sometimes unavoidable because of a very sharp reaction on the part of the test insect to a strong chemical.

Azadirachtin was tested using this method. A stock solution of 0.1 of azadirachtin was prepared in methanol. Each dilution was made up in such a way that it contained 0.1M sucrose dissolved in the diluent water. The labrum-labium was touched with the chemical film mounted on a wire loop. The results are given in

Table 1.4.

At the highest level of 10 ug/l, AZD revulsed the feeding

of 86.6% of the test animals. With progressive dilution of the dose used, the revulsion was overcome by the stimulant effect of

sucrose, so that at 0.03 ug/l, 56.6% of the animals were induced

to feed on the mixture, and 43.3% were inhibited off feeding.

48.

TABLE 1.4. Inhibition of feeding response of Schistocerca to 0.1M sucrose ifa7--727713:75Titin, determined by lorric.y.tnit- - parts (near labrum notch).

Response and Concentration (ug/i) Animal Sucrose no. 10 3 1 0.3 0.1 0.03 0.1M

1 (-) (-) 0 0 0 (+) (+) 2 0 0 (-) 0 0 0 (+)

3 (-) (-) (-) 0 0 (+) (I.)

4 (-) (-) o o 0 0 (+) 5 (-) 0 0 (+) (+) (+) (+) 6 (-) (-) (-) (+) (+) (+) (+)

7 (-) (-) 0 0 0 (+) (+)

8 (-) (-) (-) 0 0 0 (-0

9 (-) (-) (-) 0 (+) (+) (+) 10 (-) (-) 0 0 0 0

iii 9(-) 8(-) 5(-) 2(+) 3(+) 6(+) 10(+)

R0 (repeats) 8(-) 6(-) 6(-) 1(+) 4(+) 5(+) 10(+)

R 9(-) 7(-) 7(-) 2(+) 3(+) 6(+) 10(+) 3 Total 26(-) 21(-) 18(-) 5(+) 10(+) 17(+) 30(+)

Inhibition (A- 100 100 100 83.3 33.3 43.3 0.0 Revulsion 'A 86.6 70.0 60.0 0.0 0.0 0.0 0.0 Feeding (;) 0.0 0,0 0.0 16.6 66.6 56.6 100

(-) = avoidance reaction; (+) = feeding or biting reaction. 0 = neutral reaction, no avoidance or biting. (c)Antennae

When the tip of the flagellum was touched with a film of a

deterrent chemical, the animal moved the antennae away. Repeated

contact dulled the reaction and the insect stopped making the with-

drawals. However, such a manipulation did not result in a

sufficiently characteristic response to be used for bio-assay.

The response would appear to be more of a tactile nature rather

than that of contact chemoreception. Touching of the flagellum

in the middle portion or near the scape also produced no specific

reaction.

(d)Tarsal Region

No visible revulsive response was observed when the tarsal

region was touched with different chemicals. In Schistocerca,

this method, therefore, could not be used to bio-assay chemicals,

as was possible in some other insects, e.g., blowflies.

(09Yi22412E. Touching (or even dipping)the ovipositor area of Schistocerca

with chemical solutions did not result in a visible specific response.

However, when the abdomen was cut from the body and the ovipositor

region was brought into contact with a strong mercuric chloride

solution, a spasmodic movement of closing and opening of the valves

was observed. This might be due to irritation caused by the

corrosive action of mercuric chloride.

Conclusion

Although it was possible to evaluate chemicals into

acceptable or unacceptable compounds by stimulating certain taste

receptor areas of Schistocerca, the technique of feeding treated

filter papers in a large caje to about 200 hungry locusts gave

more consistent results and also more closely simulated field condit

conditions, and was therefore adopted for screaing of anti-feedants. 50.

B. COMPARATIVE UPICACY OF CANDIDATE REJECTANTS

1. Primary Evaluation

In the preliminary screening, candidate rejectants were

tested at three or four levels with a reduction factor of ten.

There were three repeats, each with three replicates per treatment.

The percent inhibition for the various test chemicals is given in

Table 1.5. The quantities of filter paper eaten in various treatments

are given in Appendix Table 1..

As the results in Table 1.5 provide data for a 3-4 point

assay, some results could be analysed statistically to determine ED50 values, but with rather large fiducial limits. However, for many

of the more effective deterrents, the points were scattered around 90%

inhibition and the information gained from such analysis would not be

of practical use. Therefore, it was decided to carry out a. more

detailed assay of ten of the most active chemicals.

2. Secondary Evaluation

When compared at the level of 1,000 mg/1 (15.8 ug/cm2), the

ten most potent deterrents in the descendant order were:

70 inhibition

(1)Azadirachtin 100 (2)Alcohol Ext. 100 (3)Kernel Ext. 100 (4)Veratrine 98.5 (5)Gramine 92.0 (6)Carbaryl 85.5 (7)TD - 5032 81.3 (8)Brestan 78.2 (9)Mercuric chloride 77.6 (10)Dal-ter 71.8 51.

TABLE 1.5. Deterrent effect of candidate anti-feedants: primary evaluation with adult Schistocerca.

rio Inhibition of Feeding on Treated Sugared Paper

Applied concentration 104 103 2 10 (mg/1) 10 Dose/cm2 (ug) 158 15.8 1.58 0.158 Neem products 1.Azadirachtin - 100 100 100 2.Alcohol Ext. (seed) - 100 100 100 3.Kernel cusp. (seed) - 100 100 95.3

Alkaloids 4.Gramine 100 92.0 88.9 5.Hordenine. 30.2 16.2 (+) 11.2 (+) 3.3. 6.Ilyoscyamine (sulphate) .32.8 39.2 19.7 11.1 7.Lobeline (hydro- - 36.9 9.9 (+) 11.3 chloride) 8.Lupinine 38.6 19.3 15.5 - 9.Veratrine 100 98.5 88.2 84.2

Sanonins 10.Digitonin - 23.8 12.4 5.7 11.Diosgenin . (+) 47.2 (+) 26.7 (+) 11.5 -

Organotins 12.Fentin acetate - 78.2 66.5 29.00 13.Fentin hydroxide - 71.8 69.0 14.5 14.11exa methyl di=tin - 81.3 61.2 8.1 15.Stannous chloride - 25.7 15.8 (+) 64.1 52.

TABLE 1.5 (Continued)

Applied concentration 4 2 (mg/1) 10 103 10 10 2 Dose/cm (ug) 158 15.8 1.58 0.158

Plant growth regulants

16. Dichloro benzyl 100 32.2 0.9 - tributyl ammonium chloride

17. Di methyl amino- (+) 37.8 (+) 31.2 (+) 11.5 - succinamic acid 18. Trichloro-.phenoxy 89.7 59.5 35.6 - acetic acid

Carbamates • 19. Carbanyl - 85.5 68.2 31.3

Triazenes 20. Compound 24,055 54.4 (+) 28.5 (+) 62.6 -

Miscellaneous

21. Copper stearate 20.1 17.7 15.5 2.9

22, Copper resinate 28.1 18.6 3.5 - 23. Mercuric chloride 99.7 77.6 18.5 - 24. Indalone - 34.0 (+) 12.1 (+) 0.7 25. MC-I. repellant - (+) 31.7 (+) 48.6 (+) 62.8

26. Meta-deiphene - (+) 66.1 (+) 37.6 (+) 33.0

27. Rutgers 6-12 - 28. Di-methyl phathalate 7.7 (+) 15.5 (+) 17.6 -

Figures are for average of three replicates; the amount of filter paper eaten by locusts in control treatment varied from 71 to 519 mg, with 18 hours starvation period followed by 24 hours feeding,

(+) indicates more feeding of the treated filter papers than 'sucrose only' papers. 53.

M.ercuric chloride which was a poisonous abrasive was substituted with the next best chemical, phenoxy acetic acid, and these ten chemicals were evaluated further to determine the ED50 values, i.e., the levels which induced 50p inhibition of feeding in adult Schistocerca on filter papers treated with 0.1M sucrose. The results are given in Tables1.6 and 1.7. The calculated regression lines are shown in Table 1.8 and Fig.3. The neem products were by far the most effective deterrents against Schistocerca.

The experimental design amounted to a choice feeding situation, in which the locusts were still hungry at the finish of the experiment. The variation within the replicates was not significant, while the variation between the treatments was highly significant (Fig.4).

The response curve was sigmoid in nature and to find ED50 value (the dose which will inhibit feeding by 50), the data were analysed by the technique of Probit Analysis (Finney, 1964), using a computer programme available at the Imperial College.

In all cases, a regression existed which was significant at

5% level, but there was also a significant heterogeneity resulting in 2 large x values. Accordingly, some of the fiducial limits have a wide range. However, there was a good agreement between the calculated and observed values of ED (Fig.3). 50 The technique of Probit analysis was used in the absence of a better alternative, though the analysis is primarily meant for use with quantal responses. 54.

TABLE 1.6. Inhibition of feeding ( 0 of adult Schistocerca on sucrose (0.1M) impregnated filter papers by different deterrents.

Inhibition of Feeding of sugared paper (quantity of paper eaten (mg)/replication given in brackets)

Applied Dose/cm2 Gra- Vera- Cone. (t1:9) (ug) TD-5032 2,4,6-T Brestan Du-ter mine trine Carbaryl 3.3 474 94.0 95.7 - - - - - (24) (25) 1.0 158 93.6 81.2 84.8 82.1 100 100 - -1 (25) (109) (86) (66) (0) (0) 3.3 x 10 48 86.2 64.1 73.9 77.2 92.8 98.2 - (55) (207) (147) (83) (31) (8) - 1 10- 16 73.0 27.5 67.9 71.8 84.9 96.8 91.4 (107) (418) (180) (103) (65) (17) (34) -2 3.3 x 10 4.8 63.5 27.3 49.2 46.5 69.2 91.5 83.0 (144) (420) (286) (196) (133) (38) (71) 10-2 1.6 26.1 17.6 25.1 28.1 46.0 76.2 79.9 (292) (475) (422) (263) (234) (107) (84) -3 3.3 x 10 0.5 8.2 12.8 - 30.5 56.6 72.3 (363) (503) - - (301) (194) (116) -1 10 3 1.6 x 10 - 15.5 41.54 27.9 - - - - (366) (261) (303) 3.3 x 10-4 0.5 x 10-1 - - - - 31.92 7.9 - - - - - (304) (387) Control (396) (577) (563) (366) (433) (447) (420) (sucrose 0.54 2 0.1M; mg/cm )

1. Average of three replications per treatment; starvation period 18 hours; feeding period 24 hours to 200 locusts. 2. Carbaryl killed locusts after ingestion of food.

TABTF 1.7. Inhibition of feeding () of adult Schistocerca on sucrose (0.IM) impregnated filter papers by neem Products

AzaCirachtin Alcohol Ext. Kernel Ext. Applied Dose cm2 Paper T Inhibi- Applied Dose cm2 !Paper Inhibi- Applied Dose am2 Paper Inhibi- conc. eaten tion conc. eaten tion conc. eaten tion (ug) (ug) (ug) (%) (mg) (%) (%) (mg) (%) (%) (mg) (%) 5 x 10-5 0.8 x 10- 0 100 5 x 1074 0.8 x 10- 0 100 5 x 10 0.8 0 100 10-5 1.6 x 103 4 98.9 10-4 1.6 x 10-2 31 93.8 103 1.6 x 101 5 98.6 5 x 106 0.8 x 10-3 17 95.0 5 x 10-5 0.8 x 10-2 140 72.2 5 x 10-4 0.8 x 101 55 84.1 10-6 1.6 x 104 30 91.2 2.5x 1075 0.4 x 102 179 64.5 2.5x 104 0.4 x 101 178 48.6 5 x 10-7 0.8 x 10-4 77 77.3 10-5 1.6 x 10-3 249 50.5 10-4 1.6 x 10-2 222 35.8 2.5x10-7 0.4 x 10-4 171 49.2 5 x l06 0.8 x 10-3 291 42.4 5 x 10-50.8 x 10-2 259 25.1 10-7 1.6 x 10-5 347 26.5 10-6 1.6 x 104 347 31.3 10-5 1.6 x 103 268 22.5 Sucrose 0.5 mg/cm2 337 0.0 Sucrose '0.54 mg/cm2 505 0.0 Sucrose 0.54 meom2 346 0.0 0.11 0.1M 0.311

Average of three replicates per treatment; starvation period 18 hours; feeding period 24 hours to 200 locusts.

TABLE 1.8. Regression equations and calculated ED values for certain 50 anti-feedants against adult Schistocerca. Fiducial limits 95% ED50 Relative Anti-feedant Regression equation Upper Lower Potency x2 DF (Applied % conc.) -2 1. Phenoxy-acetic y = 3.5172 - 0.8145 x 1.51 x 10-1 6.1 x 10-1 6.0 x 10 1.0 296 4 acid 2. Fentin acetate y = 3.0577 - 0.8180 x 4.2 x 10-2 7.9 x 10 2 1.8 x 1072 3.6 69 3 3. Fentin hydroxide y = 3.1061 - 0.7817 x 3.8 x 10-2 8.6 x 10-2 9.0 x 10-3 4.0 23 3 4. Hexa-methylditin y = 2.0927 - 1.1508 x 3.0x 1072 5.3 x 10 2 1.6 x 1072 5.0 133 4 5. Gramine y = 1.9770 - 1.0127 x 1.0 x 10-2 x102 9.0 x 10-3 1.5 x10 .9 4

6. Carbaryl y = 1.6500 - 0.8958 x 1.8 x 10-3 6.9 x 10-3 6.4 x 10-3 8.4 x 10 243 3 2 7. Veratrine y = 2.4042 -0.9124 x 1.4 x 10-3 2.0 x 10-3 9.4 x 10-4 1.1 x 10 53 5

8. Kernel Ext. y = 2.7120 - 1.1838 x 1.2 x 10-4 5.4 x 10-4 1.2 x 10-5 1.3 x 103 177 4 -5 -6 9. Alcohol Ext. y = 3.1689 - 0.8393 x 6.6 x 10-6 1.4 x 10 1.9 x 10 2.3 x 1d 68 4

10.Azadirachtin y = 1.7917 - 1.9485 x 2.3 x 10-7 3.1 x 107 1.5 x 107 6.6 x 1075 15 2

6 1)Phenoxy acetic acid

2)Fentin acetate ITS 3)Fentin hydroxide ROB Hexa methylditin

G P 5)Oramine

DIN 6)Carbaryl

FEE 7)Vera trine

`fa 8)Kernel extract 9)Alcohol extract 3 lo) Azadirachtin

Il.m•••• ••••••• •••••ki• ••••••••••

ITS .5 OB R P G IN

3 FEED 10 % 3

-3 -2 -1 0 - x Yo CONCENTRATION (LOG X 10 )

Fig. 3 - Probit regression lines for inhibition of feeding by certain anti-feedants against adult Schistocerca. laoeoo • • • o-oeo

Fig. 4 - A series of sucrose impregnated filter papers treated with three different concentrations (decreasing from left to right) and controls, loft after simultaneous exposure to 150 hungry Sehistocerca in the same cage. There were three replicates of each treatment; note the consis— tency of the results. 59.

3. Anti-feeding Activity of Miscellaneous Preparations from Different Carts of Neem Seed

The deterrent action of several preparations (described earlier at page 35 ) from different parts of the neem seed, namely, shell, kernel, and the 'whole' neem fruit was studied using a three- point assay with Schistocerca. Azadirachtin 0.01 mg/1 (1.6ng/cm2) was used as a standard treatment for comparison. The percentage inhibition of feeding of treated sugared paper by different preparations is given in Table 1.9. All the preparations inhibited feeding, though as expected, aqueous extracts of kernel and their solids were the most potent. Some of the preparations (residues on the filter papers and sieves) induced feeding at low levels. It may be concluded that although the kernels from the neem fruit seemed to contain the maximum amount of the active ingredient, other parts of the.fruit, such as the shell and the residues left after filtering of crude aqueous preparations also possessed appreciable anti-feeding activity. Thus preparations based on the whole fruit rather than the.kernels only will be. more useful under simple situations of use,i.e., like peasant farming.

4. Comparative Efficac of Three Different hatches of Neem Seed

Three batches of seed were obtained from India: batch A consisted of fully ripe and well stored seed; batch B contained some immature seeds, which were otherwise well dried and properly stored, and batch C had been stored wet and sent through the post as such.

Consequently, batch C was badly infected with rot fungus and only a few seeds had healthy kernels.

60.

TABTE 1.9. Deterrent action against adult Schistocerca of different formulations prepared from the various parts of the neem fruits, compared wiih a standard dose of azadirachtin.

'; Inhibition t Standard Error

Neem formulations AZD Applied concentration 1,000 100 1.0 0.01 (mg/1) Dose/cm2 (ug) 15.8 1.58 0.08 0.16 x 10-3

Drupe - Shell & kernel • - (ground and sieved) 1. Residue on sieve 85.9 ± 0.7 6.8 ± 3.5 25.9 ± 1.7(+) 66.3 ± 2.1 ± ± 2. Aqueous extract - 99.2 3.5 0.3 81.2 0.9 3. Solids of aq.ext. - 90.0 ± 1.6 6.3 ± 0.8 63.1 ± 5.6 Shell (ground and filtered) 4. Residue on filter 85.0 ± 5.6 11.3 I- 0.7 10.1 ± 2.3(+) 84.3 ± 1.45 5. Aqueous sol. 100 35.3 ± 3.7 15.3 ± 3.8(+) 84.3 ± 1.45 Kernel A. Ground and Sieved 6. Residue on sieve 98.3 ± 0.6 80.2 ± 8.8 43.5 t 5.6(-+-) 46.3 ± 5.8 7. Aqueous extract 100 100 45.1 ± 4.2 73.3 - .2 + 8. Solids (solution) 100 100 80.1 ± 3.6 91.5 - 3.3 B. Ground and filtered 9. Residue on filter 100 99.0 14.8 ± 3.3(+) 50.4 ± 3.3 10. Aqueous 861. 100 100 50.8 ± 1.5 - 11. Solids 100 100 72.9 ± 3.5 80.5 ± 2.2 C. Ground and centrifuged 12. Fat (top layer) 99.5 76,2 ± 4.5 44.7 ± 1.8 91.5 ± 4.5 4. 13. Aqueous extract .100 100 90.5 - 2.5 88.4 t 1.5 4- 14. Solids (aliquot) '100 100 37.1 - 2.7 0.5 - 2.2 ± + 15. Residue (bottom) 100 100 5.1 0.9(+) 80.5 - 2.2

- Average of 3 replicates per treatment. - indicates more feeding of the treated papers than the standard controls. 61.

The kernel aqueous preparations from the three batches of seed were tested by the technique of feeding the treated sugared filter papers to Schistocerca. The results are given in Table 1.10.

Batch C, the kernels of which had deteriorated almost completely seemed to induce feeding rather than inhibit it. There was a small non-significant difference between the other two batches, A and

B. The proper storage of seed was evidently important to preserve the deterrent action of the seed. 62,

TABLE 1.10. Effect of the quality of the fruit on the deterrent action of neem: comparative efficacy of the aqueous 2Leparations of three different lots of neem kernels in inhibiting feeding of adult Schistocerca on treated sugared papers.

Wt of filter paper eaten (mg)

Applied R R Total 1 2 cone. (ng/1) (replicates) (mg) Inhibition Batch A (mature and well-stored fruit)

1.0 13 11 14 38 88.3 0.01 95 83 116 294 9.3

Batch B (containing some immature seeds) 1.0 20 21 18 59 81.9 0.01 95 112 95 302 6.9

Batch C (fungus attacked seed)

1.0 88 92 135 315 2.9

0.01 185 140 152 477 47.2 (-0,

Azadirachtin 0.01 5 7 7 19 94.2

Sucrose 108 104 112 324 0.0 (0.1M) •

Treated sugared filter papers were fed to about 150 locusts randomly in a 0.3 m3 cage. indicates more feeding of the treated paper than 'sucroseonly'controls.

1.5 ml. of the applied concentration was2spread over 11 cm: filter paper, giving a deposit of 15.8 and 0.16 ng/cm for 1.0 & 0.01 mg/1 treatments respectively.

Azadirachtin was included'as a standard treatment for comparison. 63.

C. HABITUATION OF ADULT SC IISTOCEROA 10 THE DETERRENT h JJECT OF NUM

An adult population of 150 Schistocerca was fed continually

for nine days on filter papers treated with azadirachtin and neem

kernel suspension. To avoid death due to starvation, the animals

were fed on green grass daily for four hours (10.00 to 14.00 hours);

the food was then removed and the animals were starved for the next

6 hours (14.00 to 20.00 hours). They were fed on treated filters for

the next 14 hours (20.00 to 10.00 until the next morning). This daily

routine continued for nine consecutive days. At the end of this

period, the locusts were fed on green grass and wheat bran for the

next four days, and thereafter, on treated filter papers during the

remaining four days of the experiment. The percent inhibition of

feeding on treated sugared papers to constant levels of azadirachtin

(50, 10, 5, and 1.0 ug/1) and neem kernel suspension (1,000 ug/1)

over different days of the experimental period are given in Table 1.11

and shown in Fig.5.

There was a clear indication of habituation on the part of

adult Schistocerca in its inhibition response to azadirachtin and

kernel suspension. The waning effect was more pronounced at the

lower levels (5 & 1.0 ug/l). After four days feeding on green' grass,

the animals, which had become habituated, were again inhibited as

sensitively as before by the neem products, but habituation recurred

by the fourth day of the resumption of the daily feeding on treated filter

papers. There arc feW examples of quantitative data on habituation in

the literature. The present results provide a striking example and a

useful contribution in this context. The habituation response may

also pose some practical implications in the use of deterrents. 611.

TABLE 1.11. Habituation of adult Schistocerca to the deterrent action Of neem: continual daily feeding of the same population on neem-treated sucrose-impregnated filter papers.

Percent Inhibition of Feeding

Azadirachtin IWL Applied conc.(ugil) 50 10 5 1.0 1,000

ose -2 cmR (1g) 8.0 x 10-1 1.6 x -1 8.0 x 10 1.6 x 10-2 16 Day 1 100 99.3 94.0 56.3 100 2 96.9 98.7 77.2 23.8 96.9 3 96.8 97.9 83.1 32.6 99.2 95.0 88.8 10.4 .(+) 25.2 (+) 68.9 5 76.4 74.5 34.6 6.5 78.6 6 86.5 74.5 38.6 12.4 72.4 7 69.1 33.0 7.7 (+) 22.2 (+) 79.2 8 69.1 33.6 0.5 4.3 88.4

9 58.1 42.9 28.8 0.9 (+) 69.8

10 - 13 - fed on green grass before resumed feeding on neem over the next four days.

14 97.4 93.2 78.6 46.3 99.0 15 93.3 87.3 66.7 47.6 98.5 16 97.0 92.4 63.5 53.4 98.4 17. 85.4 63.3 49.1 15.5 (+) 96.5

- Average of three replicates per treatment. - Locusts were daily fed on green grass from 10-14 hours; starved from 14-20 hours, and fed on treated filters from 20 to 10 hours until next morning. 0

r=i 20

0 z H

H 100

4 6 8 ' Interval 16 17 of normal feeding DA YS Fig. 5 - Showing 'habituation' of Schistocerca to the deterrent action of azadirachtin. 66.

D. INTERACTION O1 A PHAGOSTIMULANT (SUCROSE) AND A DETERRENT (NEE M)

Different plant species may vary in attractiveness to a

pest species. Accordingly, the rate of application of an anti-feedant

may have to be adjusted to achieve the same degree of inhibition,

depending upon the attractiveness of the plant species (or stage of the

growth of the same species., To test this hypothesis by analogous

experiments filter papers were impregnated with different concentrations

of sucrose but were treated with a constant level of azadirachtin (5 ug/1).

The results of such a feeding test are given in Table 1.12.

The hypothesis that the application level of a deterrent

may have to be adjusted to produce a standard effect, depending upon the

attractiveness of the crop, is supported by the results of the experiment.

The level of azadirachtin (5 ug/l),which inhibited feeding of Schistocerca

by about 50$ when filter papers were impregnated in 0.12M sucrose, failed

to inhibit feeding on papers dosed with 0.5 and 1.0M sucrose. Thus,

the young germinating crops which are highly preferred by the locusts may

need comparatively higher levels of neem application. 67.

TABLE 1.12. Interaction of a phago-stimulant (sucrose) and a deterrent (neem): feedin of adult ScUg;tecercq on filter papers im- pregnatedwith different levels of sucrose but treated with the same level of azadirachtin.

Applied Concentration of Azadirachtin: 5 ughl (0.08 ug/cm2)

Quantity (mg) of filter papers eaten

R 1 R Total Sucrose R1 2 3 level (mg) Inhibition (M) replicates)

1,0 503 405 447 1352 -

0.5 88 74 83 245 - 0.25 48 39 47 136 32.3

0.12 38 28 30 96 52.2 0.06 25 28 28 81 59.7

* 0.01 58 78 65 201 - (standard)

* No Azadirachtin. xx. Treatments were fed to 150 adult locusts placed randomly in a 0.3 m) cage. 68.

DISCUSSION

Development of a Bio-assay Technique

To evaluate deterrent action, the technique of presenting food to small batches of Schistocerca in separate cages in the form of sucrose impregnated filter paper discs treated with azadirachtin was used by Butterworth and Morgan (1968) and Haskell and Mordue (1970).

The phago-stimulant used in the experiments of Haskell and Mordue was

0.1M sucrose. It was claimed that the resultant concentration of sucrose presented on dry filter paper to Vth instar Locusta hoppers gave near maximal food intake (Sinoir, 1968). It is also approximately the concentration of sucrose found in most grasses (the preferred food of locusts and grasshoppers) expressed as a percentage of dry matter

(Waite and Boyd, 1953). The method was, therefore, followed in the preliminary experiments. A large day to day variability was observed, and at times a higher dose inhibited feeding to a lesser degree than a lower dose. The following may be the reasons for such a variability:-

(i) The locust hoppers were not exactly of the same age, though

the age differences did not exceed 2-3 days;

Although the hoppers were allocated to each cage in a random

way, it was possible that the less active ones were picked up

first, thus causing further variation in feeding. Since there

were only five hoppers per replication (15 per treatment), a

biased distribution of hoppers might have resulted in a large

variation in the amount of feeding.

(iii) The comparisons were based on batch responses. There was not

only a feeding gradient amongst the batches but also between

the members of each batch. The heterogeneity in population at

(i) and (ii) above increased both the individual and group

variation. 69.

For a limited number of experiments hoppers of the same age could be arranged by collecting and rearing them in separate batches soon after moulting but to make available large number of animals of exactly the same age for routine testing would be both laborious and wasteful, and would entail a large scale breeding programme. Moreover, the hoppers had a peak period of feeding in the middle of the instar

(Hill and Goldsworthy, 1968), so to ensure a relatively uniform rate of feeding a particular batch of hoppers could be used for feeding tests once or twice only.

The alternative method, developed in the present work, of presenting multiple treated filter papers to about 200 adult Schistocerca gave more consistent results than the previous method. The papers were placed randomly in a 0.3 m3 cage in the manner of a food choice experiment.

No transfer of insects to separate cages was involved which saved time.

The method is considered to be an improvement over existing techniques and could also be used for similar tests with other relatively active moving insects.

The test technique,based on a quantal response of Schistocerca by activating the receptor surfaces on the domes of the palpi and other mouth--parts with chemical films mounted on a wire-loop couldbe used to classify chemicals into rejectants and stimulants in rapid screening tests.

Efficacy of potential deterrents

Of the 28 chemicals evaluated for their deterrent action against

Schistocerca, when arranged in a quartile distribution of inhibition rela-_ tive to a standard diet, ten chemicals (azadirachtin, alcohol and kernel extracts of neem, gramine, veratrine, fentin acetate, fentin hydroxide, hexa methylditin, carbaryl, mercuric chloride) fell in the range of

76-100L inhibition; seven chemicals (Hyoscyamine, lobeline, diosgenin, stannous chloride, phosphon, phenoxy acetic acid, indalone), in the range 70.

of 26-75% inhibition; five chemicals (hordenine, lupinine, digitonin, copper stearate, copper resinate) in the range of 1-25% inhibition, while the remaining six chemicals (B-nine, compound 24,055, MGK repellent

874, metadelphene, Rutgers 6-12 and dimethyl phthalate) stimulated feeding instead of inhibiting it.

There does not appear to be a definite correlation between chemical grouping and rejection; however, neem derivatives, organotins, and some alkaloids were markedly more active than the examples from other groups.

The neem products were by far the most potent deterrents against Schistocerca, azadirachtin being 6.6 x 105 times more effective than phenoxy acetic acid tested by the above method.

§2fcificity of deterrent action

According to Thorsteinson (1960), there was at that time no record of an alkaloid stimulating feeding. However, sparteine was stated to stabilize colonisation of the aphid, Acyrthosipho snartii (Kennedy and

Stroyan, 1959). Similarly, the phago-stimulant effect of mustard oil glucosides on cabbage insects is a well known example of a specific relationship. Specific stimulation of linamarin and lotaKistrin to

Mexican bean beetle, Epilachna varivestis (Nayar and Fraenkel, 1962) and of hyperiein to chrysomela brunswicensis (Rees, 1966) have also been reported.

In the present experiments, although the alkaloids suppressed feeding in general, hordenine and lobeline at low levels of 10 ppm stimulated feeding. In the experiments of Harley and Thorsteinson (1967), lobeline, hordenine and hyoscyamine inhibited the drinking response of

M. bivittatls by more than 75%, veratrine between 26-75% and gramine less than 15%. This is nearly opposite to the results obtained in the present findings - veratrine and gramine inhibited more than 90% of the feeding of

Schistocerca. 71.

Also, the saponins (digitonin and diosgenin) which were non-acceptable to Nelanoplus, stimulated feeding of Schistocerca.

It was suggested that ingestion of such steroid chemicals by insects interfered with metabolism and utilisation of essential sterols and might seriously influence survival and development. This was apparently not the case with Schistocerca. However, experiments on survival and growth in Schistocerca with intake of saponins would be necessary to clarify the issue.

The anti-feeding compound 24,055, which was reported to be generally effective against chewing and biting insects, in fact, stimulated feeding of Schistocerca (except at a high level of 1); likewise, the general repellents (items 24-28 of Table 1.5) acted as stimulants, although they were reported to inhibit feeding of larvae of Agriotes spp. by Griffiths (1969).

Anomalies in the reaction of different species to the same chemical are, however, not uncommon and are perhaps connected with the broader aspects of host selection and survival behaviour. Some of these problems have been discussed in detail in the review papers of

Thorsteinson, 1960; Dadd, 1963; Beck, 1965; Mulkern, 1967, and

Fraenkel, 1969 . The'aim of the present studies was to evaluate deterrents for practical use against the Desert Locust. Neem derivatives showed high promise; carbaryl and organotins might be useful under certain conditions. The role of alkaloids in imparting possible resistence to certain crops might be considered in evaluating plant breeding programmes.

Habituation

It is well known. that sensory systems can become less responsive after repeated stimulation. But the waning of response can 72.

arise from several causes, and the explanations of these stimulus specific decrements may vary from simple sensory adaptation to different forms of learning. Habituation may be described as a relatively permanent waning of response as a result of repeated stimulation which is not followed by any kind of reinforcement (Thorpe, 1963).

Habituation differs from sensory adaptation in its temporal characters and depends upon some form of plasticity in CMS. There remains much to be learned concerning the neuro-physiological basis of habituation, both in lower and higher animals, and there are few examples of quantitative data to show such selective decrements in the taxis mechanism.

In Schistocerca, the waning of response to the constant stimulus situation is clearly evident from the percentage inhibition of feeding on different days. At a dose level of 5 ug/l of azadirachtin, the inhibition of feeding decreased from 94.0 on day one to 0.5% on day eight; on resumption of testing after a break of four days, inhibition recovered to some extent at first, but waned again. Similar waning was observed in case of other doses under test. This interesting observation could be used to study the phenomenon of habituation further.

Since the use of anti-feedants has not progressed much beyond the laboratory stage, information as to the possible adaptation of insects to continued use of such chemicals is lacking. The experimental evidence presented here suggests such a conditioning is possible and could be rapidly induced. Wolfenbarger et al., (1968) obtained 705 control of aliothus complex in field cage trials with fentin hydroxide but the reduction in the number of damaged squares in a field trial using 5.6 kg/ha fentin hydroxide was'only 10-1Z. Similarly, Findlay (1968) 73. obtained satisfactory control of Heliothis zea with fentin acetate and hydroxide under laboratory conditions, but this control was not matched under field conditions. 'These comparative failures might be due to several factors including a poor coverage of young squares with spray deposits, but a possible adaptation to the deterrent may also have occurred.

Interaction of deterrent and phago-stimulus

When a constant dose of azadirachtin was applied to filter papers treated with different amounts of sucrose, feeding of Schistocerca was not inhibited at the higher levels of the stimulant. A five-fold increase in the concentration of stimulant resulted in a 22; increase in feeding and a ten-fold increase in a 500% increase relative to the standard treatment. This might though be expected.

Similarly, by analogy, the prevention of feeding by a deterrent depends upon matching its action against the stimulant effect of the plant.

Since young and growing parts of a plant are usually more attractive to insects, their protection would require higher levels of deterrent to achieve the same degree of inhibition. Laboratory trials might, therefore, be carried out with younger leaves or seedlings. Under field conditions levels of application might have to be adjusted according to the palatibility of the crop.

Deterrent activity of the different parts of the neem seed

Pradhan and Jotwani (1962) recommended the spraying of crops with 0.1$ neem kernel suspension at 100 gallons per acre to prevent damage from Schistocerca. To remove kernels from the fruit without the help of decortication equipment is, however, both laborious and tedious and has been in part responsible for the recommended practice by the peasant farmers in India not being adopted (personal information of 74. the author). The results of tests with preparations from different parts of the seed indicate no evident advantage in removing the kernels.

In fact, the husk contains a mucilaginous substance which should improve the wetting and sticking qualities of the preparation.

The experiments also showed that proper storage of seedis important to preserve the activity of the neem seed. The recovery of the active ingredient is also likely to be affected by other factors, such as maturity of seed, locality, and variety. These factors need to be investigated further.

Concluding remarks

The experiments reported in this part of the Thesis confirm that azadirachtin and crude neem preparations are effective deterrents for Schistocerca when directly applied. But, as discussed earlier, to be of practical value a feeding deterrent should be systemic in action.

There is no information in the literature on this point. Consequently, an investigation of the possible systemic properties of neem preparations was undertaken and is reported in Part II of the Thesis. PART II. THE SYSTEMIC ACTION OF NEEM DERIVATIVES. 76.

INTRODUCTION

Twenty eight chemical compounds were tested for their anti-

feeding activity against the Desert Locust, Schistocerca gregaria F.

in Part I, and several of them prevented feeding by the Locust (see

Table 1.8 for ED50 values of inhibition). Many of them, however,

were not suitable for an extensive use in crop protection. Phosphon

was a growth regulant which, besides being expensive, also dwarfed

plant growth and might be used in very special conditions only,(e.g.,on

ornamentals). Mercuric chloride was a poisonous corrosive unsuitable

for agricultural use. Organotins, Compound 2,4,6-trichlorophenoxy

acetic acid, and certain carbamates (sevin) were already in use as

fungicides and insecticides respectively with an additional bonus of

anti-feeding, but were relatively expensive for general use.

The Desert Locust is a pest of arid zones where subsistence farming

is still the rule and, with the added difficulty of the unpredictability

of the visitation of locusts, an inexpensive and locally available

plant product like the neem seed is likely to be used as an anti-feedant

in practice. The neem tree also has the advantage of improving soil,

providing wood, timber and shade.

That locusts did not eat neem leaves and were deterred by neem

extracts have been known for a long time (Hussain, 1928). Very

.little practical use; however, seems to have been made of this property.

The Indian Agricultural Research Institute, New Delhi, recommended

the spraying of crops with 0.1% neem kernel suspension at 100 gallons

per acre, and was also reported to have sprayed its own area with

this preparation in 1962, when a locust swarm did visit the farms of

the Institute and did not damage the sprayed crops. But, there were

still several hurdles to its use: water was a limiting factor in

many areas and difficult to obtain even for drinking purposes;

taking out the kernels from the seed without proper decortication 77. equipment was both tedious and laborious. The behaviour of the deposits of neem under field conditions was not known fully and there were con- flicting claims: the effect was reported to last for 15 days according to the Institute, while Venkatesh et al., 1970, reported it to last for three days only. There was an obvious need to investigate further the behaviour and persistence of neem deposits under both laboratory and field conditions.

Having confirmed that neem contained a deterrent, and for above reasons was of possible practical value, it was necessary (a) to determine its persistence on/in plants, (b) to investigate the possibility of systemic action. For unless the substance(s) showed systemic action, the growing points of the plant would be attacked irspiteA protection elsewhere. Loss of biological activity of a chemical such as a neem preparation might arise in one or more of the following three ways,

chemical change resulting from the absorption of ultra-violet or other x-rays in the atmosphere, ii. physical removal of material from the leaf surface by weathering elements like rain and wind, and iii. absorption and subsec.uent metabolism in a plant tissue. Experments are described to test the biolocical activity of neem,and some of the factors affecting such an activity,. 78.

MATERIALS JND METHODS

1. Test Insects

Adult Schistocerca gregaria were used for the feeding tests. The

eggs were obtained from the Centre for Overseas Research, London and

were incubated at 300C and R.H. 65%. The hoppers were reared on green

grass: wheat bran was given as a supplementary protein food. Adult

locust of 5-20 day were utilised for feeding tests after which they were

discarded and replaced by new. adults. There were 150-200 adults to a

cage of 0.3 cu. meter capacity, giving a population density of). 500 locusts

per cu. meter of space. The insects were starved for 6-8 hr before being

fed on the experimental plants.

2. Breeding Cages

Two wooden cages of 0.3 cu. meter capacity each were used for carrying

out the feeding trials. To facilitage observation from the outside, the

front of each cage had a large glass pannel and a small, removable wooden

door. The wooden door had a six inch square window closed by a sliding

lid. The window was large enough to permit the introduction of one Eg

pots to the cage,

The cages were kept in a 27°C CT room and were lighted internaly

'with a 60 watt bulb which also provided additional heat. The temperature 0 inside the cage was.about 30 C as recorded by a thermometer hung on the

hind -wall of the cage. There was a temperature gradient inside the cage

emnating from the light bulb. The cages were cleansed and disinfected

with 'petal' regularly: no disease epidemic occurred amongst the

test animals.

3. Soil

The pot soil used consisted of a seived and sterilised mixture of

three parts loam, two parts sand and one part peat, and was in eommon

use at Ashurst Lodge. Other types of soil used to test the effect of 79.

soil type, etc., are described under the respective experiments.

4. Neem Products

The three neem products, (i) azadirachtin, (ii) an alcohol extract, and (iii) water extract were used and are described fully under Part I.

The neem seed was stored at 4°C and azadirachtin and the alcohol extract were stored in a deep freeze. The following abbriviations have been used in the text for the various neem products to economise space:

AZD (azadirachtin); ALC (alcohol extract); ENL (kernel aqueous extract);

ERT (fruit or drupe aqueous extract). Similarly, the word neem has been used to connote neem formulations or the active ingredient(s) in such preparations, unless stated otherwise.

5. Plant Species The following plant species were used in these experiments:

i.Barley (Hordeum vul are L.).

ii.Beans (Vicia faba L.). Unless otherwise stated, broad beans of dwarf variety (Sutton's) were used. A regular supply was maintained by weekly seeding of about 100 seeds in a wooden box. The seedlings were grown in the greenhouse at Asharst.

iii.Cabbage (Brassica oleracea L.).

iv.Chrysanthemum C. indicum L.).

v: Cotton (Gossypinm hirsutum L.).

vi. Grass (Holcus mollis).

vii.Rice (Laza sativa L.).

viii.Spindle tree (Euonymus europaeus L.).

ix.Sugarcane(Saccharnm officinarum L.).

x. Tomato (Solanum lyso,3ersicum L.).

6. larAyla_42aliat2I The spraying of bean plants was carried out by a chromatographic all glass sprayer ( )- slightly modified by increasing the internal 80. diameter of the orifice to 0.02 mm. The droplet-size was not considered important in view of the systemic property of neem formulations, hence there was no need for a more sophisticated spraying apparatus. Usually ten cc of spray material per plant of beans was used which was sufficient to drip spray a medium sized full grown plant.

7. Assessment of Damage

A visual system of assessing the plant damage with the following ranking index was adopted: o - no feeding; 1 - slight nibbling, apical bud intact; 2 - leaves partly eaten, some damage to apical bud; 3 - apical buds and one-third of the plant eaten; 4 - only a few lower buds remained on the stem; 5 - all the buds and most or all of the plant eaten. This rank-index of five points is based on practical considerations of economic loss; for instance a rank of 0 to 1 would amount to no or . negligible damage; 2 to 3 would mean satisfactory protection and marginal damage (the crop would recover), while 4 to 5 would indicate serious or total loss with no hope of subsequent recovery of the damaged crop.

Moreover, estimates of damage based on a scale of 10% (ten point rank- index instead of the five point index) would have been subject to greater personal bias. Quantitative measurements of the leaf area before and after treatments would have permitted a better assessment and evaluation of the damage but would have been too time consuming for practical use. Such measurements were also difficult to make with potted plants.

8. Statistical Treatment The amount of feeding by adult Schistocerca on plants under test was assessed by a visual. ranking system as described above at item 7.

The ranks were related to economic loss and the differences between the

treatments were self-evident. Using this ranking system the results

obtained in replicate experiments were always highly consistent. The

control treatments (untreated plants) were consumed fully by the hungry 8i. locusts within a short period. A statistical analysis of the data was not likely to bring out extra useful information.

awever„ to test this hypothesis, five tables of the results

(nos. 1, 2, 18, 29, and30; a 16.25% sample of the total no of Tables) were submitted to the Kruskal-Wallis, one way analysis of variance by ranks (Kruskal, 19521and Kruskal and Wallis 1952) as described by

Siegal, 1956. (The Kruskal-Wallis test requires, at least, ordinal measurement of the variable, which the rank values of the present data are: otherwise the test is supposed to be one of the most efficient of the non-parameteric tests for 'k' independent samples.)

A sample analysis of one of the Tables (no. 18) is reproduced at Appendix 2 . As expected, the difference between the treatments was highly significant (P value less than 0.001). The results of the other

Tables appear at their respective places; here again, the difference between the treatments was highly significant. The data of the other tables was not, accordingly, analysed statistically, the significance of the results being self-evident. 82.

EXPERIMENTAL RESULTS

SYSTEMIC ACTION OF NEEM PRODUCTS

A simple way to test the systemic uptake was to immerse the roots of bean seedlings in aqueous preparations of neem and observe for a possible uptake and translocation by feeding the plants to Schistocerca.

Such a test indicated systemic action of neem: the plants dipped in neem products were not eaten by the locusts, while the control plants were eaten completely.

It was now necessary to quantify this effect. The plant, being a living system, is in a continuous state of physiological and bio- chemical flux, so that considerable variations occur not only between the species but also within the species, at different periods of growth.

Environmental factors such as soil, weathering conditions notabley rain affect the behaviour of systemic compounds. The effects of some of these factors were studied and have been described.

A. SOIL APPLICATION OF NLEM FORMULATIONS

1. 12Elication to Bean Plants

(a) Young bean seedlings: Three products were tested by feeding the bean seedlings to adult Schistocerca. These were i. azadirachtin

(AZD), a pure chemical, ii. an alcoholic concentrate (ALC), refined from

95y'o ethanol extract of neem seed and contained about 4% by weight of seed, and iii. an aqueous suspension (RNL) prepared by grinding seed kernels in water until the material passed through a 100 mesh sieve.

These products were diluted in 0.05% 'Teepol l and applied in a range of seven concentrations around young bean plants at the 6-8 leaf-stage.

The bean plants were transplanted singly to plastic cups containing 150 gm soil consisting of a sieved and sterilized mixture of 3 parts loam, 2 parts sand and 1 part peat, and the different treatments were applied in 10 ml as part of first watering. 83.

The efficacy and persistence of treatments were assessed by placing the trated plants, together with controls, randomly in wo.3 cu. meter cage with 200 adult Desert Locusts which had been starved for 8-10 hrs.

After 12 hr of exposure to the hungry locusts, the damage suffered by each plant was assessed using the visual ranking index system. The same treated plants plus fresh controls were exposed in this way to starved locusts for 12 hr after 1.5, 4, 7, 15, 21, and 25 days. The experiment was repeated three times using three replicates of each treatment and controls on each occasion. The locusts were still hungry at the end of each exposure and were ready to attack fresh control plants. The results obtained after 1.5, 15, and 25 days are given in Table 2.1.

The plants grown in the most heavily treated soilf10, 100, 1,000 ppm of AZD, ALC and ENL respectively were still only slightly damaged by the hungry locuSts (Plates 6,7, &8.). The data was analysed by using the Eruskal-Wallis one way analysis of variance by ranks: the difference between the treatments was highly significant (P less than 0.001).

(b) Old bean plants: Having obtained these results with young bean seedlings using one variety, the experiment was repeated to confirm the uptake, etc., of neem formulations in old plants and several varieties of beans, and several other plant species.

Applications of the three neem products - AZD, ALC, KNL - were made to the soil around old bean plants of flowering and fruiting stage, in the same manner as in case of young bean plants. Here too, nearly complete protection of beans against Schistocerca lasted for more than two weeks. The bean pods were also not eaten by the hungry locusts.

The results for 15 days are given Table 2,2.

(c) Varieties of beans: The following 12 varieties of beans were grown in 500 gm plastic pots, four seedlings to a pot (more seeds were

put in but only four seedlings were kept).

84.

TABLE 2.1. Deterrent effect of Azadirachtin and other neem preparations on Schistocerca attaching young bean plants. Rank-index of feeding on day 1.5, 15 and 25 Azadirachtin ALC Ext. KNL Ext.

PPM 1.5 15 25 PPM 1.5 15 25 PPM 1.5 15 25 10 0 1 1 100 0 1 2 1,000 0 1 2 1.0 .0 2 2 10 0 3 4 100 0 3 4 0.5 0 3 4 2.5 0 4 5 80 0 4 5 0.2 0 4 5 1.0 1 5 - 40 1 5 - 0.1 1 5 - 0.5 1 5 - 20 1 5 - 0.05 1 5 - 0.2 1 5 - 10 2 5 -

0.01 1 5 - 0.1 1 5 - 1 4 5 - Control 5 5 5 0.0 ti 5 5 0.0 5 5

Plant damage ranking index:

0, no feeding; 1, slight nibbling; 2, leaves partly eaten; apical bud damaged; 3, apical buds and 3 of plant eaten; 4, a few lower buds remaining on the stem; 5, all of the buds and most of the plant eaten.

Average of three repeats each of three replicates (± 1). Feeding test over 12 hours to about 200 hungry locusts: Kruskal - Wallis test: H.Ratio DF P value

Treatments 36.17 8 0.001 (less than)

Days 17.54 7 0.05 ( ft “ ) 85.

TABLE 2.2. Protection against adult SehistocrcA of old bean plants with soil a)plication of neem.

Azadirachtin ALC Ext. KNL Ext.

Day Day Day

PPM 2 7 15 PPM 2 7 15 PPM 2 7 15 10 0 0 0 100 0 0 1 1,000 0 0 1 2 0 1 1 20 0 2 3 200 0 1 2 1 0 2 3 10 1 3 4 100 1 2 3 0 4 4 5 0 5 4 5 0 5 5 4

Ranking Index: Same as in Table 2.1. Av. of two repeats of three plants of each (± 1). Feeding test over 12 hour exposure to 150-200 hungry locusts. Kruskal-Wallis test:

H ratio DF P value

Treatments 29.07 3 0.001 Days 4.81 8 0.8 n.s. 20

SUD SUSP:

Fig. 6 - Protection against Schistocerca of beans grown in soil treated with 'neem suspension'. Figures on the pots are ppm of kernel to dry soil weight. CRUDE EA7 :

Fig. 7 - Protection against Schistocerca of beans grown in soil treated with ?alcohol extract' of neem. Figures on the pots are ppm of crude extract to dry soil weight. Pig. 8 — Protection against Schistocerca of beans grown in treated soil with azadirachtin (ppm)..The illustration shows the survival of plants after 24 hour exposure to 200 adult desert locusts starved for 18 hours. 89.

Carter's

1.Claudia Aquadulce 8. Giant Windsor

2.White Leviathan Dobie's

3.Green Leviathan 9. Dread Naught

4.Mammoth Windsor 10. Giant Green Windsor

5.Dwarf White Fan 11. Giant White Windsor

Sutton's 12. Giant Seville

6.Dwarf Broad Beans

7, Broad Beans - Colossal

The three neem products of AZD, ALC, and KNL were applied

at 10, 100, and 1,000 ppm respectively by dry weight Of soil (w/w)

as part of watering to 4-7 leaf-stage seedlings. The plants were kept •

at 2000 during the period of the test. Feeding tests were carried

out t8 hr and 15 day post-treatment by placing the pots randomly in

a cage containing 150 locusts. There was no feeding in any of the treatments while control plants were completely eaten by the locusts.

Since some of the varieties grew very tall and became unmanageable

in the small pots, the experiment was not continued beyond 15 days.

However, the results proved that neem was taken up systemically by all

the varieties of beans.

2. Application to Different Crop Species

Species belonging to both monocot and dicot plants were tested

in a series of experiments for the uptake of neem formulations system-

ically. The tests were carried out according to the availability of

seed or plant material, and odd groups of crops (cereals with tomatoes, etc.)

had thus to be included in the same test. But, the main purpose of

the experiment - the uptake of neem products in different crops - was not affected in any way. 90.

(a) Wheat, barley and tomatoes: Wheat and barley seed were grown in small plastic pots containing 150 gm of soil; tomatoes were transplanted to similar pots. The neem treatments were applied to about 4 inch tall wheat and barley seedlings and tomato transplants of 3-4 leaf-stage, as part of first watering. The results of feeding tests to adult Schistocerca are given in Table 2.3-a.

Wheat and barley seedlings were adequately protected for two weeks in the soil treatments of 1 and 1,000 ppm of azadirachtin and aqueous extracts of neem kernels respectively; however, the complete protection lasted for one week only.

Those treated seedlings which had been partially attacked recovered fully by sprouting afresh, while in the case of the control treatments- which had been eaten to ground level the sprouting was poor. Tomato seedlings were fully protected for over three weeks. One of the reasons for the longer protection of tomatoes might be the comparative less attractiveness of tomato seedlings to Schistocerca than wheat and barley.

(b) Rice and cotton: The aqueous extract of neem kernel was tested and was applied to the cotyledonous stage cotton; and three inch high rice seedlings. The plants were, as usual, tested by feeding to adult

Schistocerca. The results are reproduced in Table 2.3-b.

None of the treatments prevented complete feeding of locusts on the rice seedlings but, from the practical point of view, even the lowest level of 10 ppm KNL proved satisfactory as the rice seedlings in the treated pots sprouted again to normal size after only a few days, while there was very little sprouting in the control pots after attack.

The cotton seedlings were not eaten for upto two weeks but, then the stems were cut in some cases though the leaves were not eaten.

Under field conditions where the locusts would have a choice of food and are likely to move away from the treated crop, the portection might last much longer. 91.

TABLE 2.3. Protection against adult histocerca of different crops with soil applications of neem formulations.

(a)Wheat, Barley, and Tomatoes Wheat Barley Tomatoes Day Day Day 2 7 14 2 7 14 2 7 14 21 PPM AZD 1 0 0 1 KNL 100 1 1 2 1,000 0 1 1

Control 0 5 5 5

(c)Rice and Cotton Rice Cotton Day Day 2 7 14 2 7 14 KNL I 1,000 1 2 2 0 0 5 100 2 3 3 1 3 5 10 3 4 4 1 4 5 Control 1 0 5 5 5 5 5 5_1

( c ) S,indle tree and Chrysanthem Week 3 (day) 1 2 3 4 5 6 FNL 1_11000 1 2 2 2 2 5 4 (Spindle) RNL 1,0W 1 1 1 2 2 3 2 (Chrysanthemum)

Average of three replications of each treatment ("1: 1).

Fed to about 150 locusts over 16 -, 18 hours. Rankin; index: same as in-Table 2.1. 92.

(c) Cabbage, sugarcane and grass: These crops were not used in a regular trial for the study of uptake and persistence of neem products but the uptake of neem in their case was observed during the course of other tests. For instance, cabbage was used for tests with Pieris

(Part III), sugarcane leaves were used in theltranslocation experiments

(page 100) and grass was used to study the effect of neem on the development of the Desert Locust hoppers. The neem products were taken up systemically by all the three species of plants and translocated within the plant system.. (a) 22,=0.111aliaLan1211aiLoasIm:1 Neem formulations were applied as kernel dust at 1,000 ppm of soil weight to one small spindle tree, one cm stem diameter and about 4 feet tall, and a chrysanthemum bush of 8-10 sprouts of 1.5 ft. height. The plants were grown in 10" diameter pots and the soil was watered after mixing of the neem seed dust.

Small branches were removed from the treated and untreated plants regularly and fed overnight to Schistocerca. The results of the feeding tests are given in Table 2.3 - c. The application of neem formulation gave adequate protection to the spindle tree for over six weeks and to chrysanthemums for over three weeks against the Desert Locust. The locusts were able to descrimineate between the treated and the untreated chrysanthemum bushes for at least six weeks: they would eat the treated leaves after the untreated ones had been consumed and the pressure of hunger mounted further.

It can be concluded that neem formulations were absorbed from the soil and translocated in several plant species including examples of monocot, dicot and wooded plants.

B. FOLIAR APPLICATION OF NEEM FORMULATIONS

A systemic chemical may be absorbed through the leaf surface and translocated within the plant. The absorption is, however, influenced by several interdependent factors such as age, surface and cuticular 93. nature of the leaf and conditions of temperature, radiation and rain.

Heavy losses of more than 50; have been recorded of leaf-applied Schradan under field conditions (Heath and Llewelly0 1953). Some of the main conditions affecting the uptake of systemic chemicals through the leaf route are considered by Benett, 1957.

Although leaves may not be considered the normal absorption areas of the plant, foliar applications have proved of considerable practical use. An insecticide may have immediate contact effect combined with a more sustained systemic use. Similarly, foliage applications are, sometimes, preferred for edible crops because of their short term effects compared with the more persistent, heavy soil or granular applications.

Against a highly mobile, sporadic and rather unprdicatable pest like the Desert Locust which may visit a particular locality for an overnight halt only, foliar application of neem may provide immediate protection • from feeding on the treated crop. Spraying operation may be undertaken even when the/swarm was alighting on the crop, while soil application has to be made in advance to allow time for translocation. To study the behaviour of foliar applications of neem formulations was, therefore, of considerable practical importance.

Foliar Application of Neem Formulation to Beans and Barley

The persistence of spray applications was studied on two crops, beans and barley. The spray was applied by means of a modified chromatographic all glass sprayer at the rate of 10 ml per plant or pot, which would corrospond to drip spray of approximately 100 gallon/acre under field conditions. The effect of the use of a wetter (Teepol) was also investigated. Both a kernel and a fruit aqueous extract; were tried. There were three bean plants of flowering stage per treatment and the experiment was repeated twice. The barley crop was grown in

plastic pots of 150 gm capacity and sprayed when about four inch tall

The results of the feeding tests are given in Table 2.4. 9L.

The water extracts of the neem fruit at 0 conc., and the kernel extract at 0.11, cone. protected both barley and bean crops for two weeks against the damage of adult Schistocerca: the lower levels of o.1 and Loll; of the two formulations gave partial protection. The use of the wetter

'Teepol' did not give a better protection and would not seem necessary inthocase of these two crops.

C. SEED-SOAKS AND SEEDLING-DIPS

1. Seed-Soaks

All external surfaces of plants are able to absorb some materials under certain conditions. Absorption of a deterrent into the seed confers the great advantage of protection for the plant during its critical early seedling stage. The relationship between seed, rate of growth and plant size would appear to be important so that the seed is large enough to absorb sufficient material to protect the resultant plant. The protection afforded to the seedlings of both large and small seeds by soaking them in neem products was, therefore, studied. eac4,4-Ma (a)Bean seeds: Five bean seeds were soaked in/ several concentrations of the neem preparations for 24 hr,and seed weight was recorded before and after soaking. On an average, the seed absorbed liquid material equal to its own weight, 1.4 gm pertseed. There were three repeats of the experiment. The protection was tested by feeding the seedlings to adult

Schistocerca. The results are given in Table 2.5.

The full protection of the seedlings lasted in general for one week, while partial protection was observed upto three weeks. The concentrations of AZD and ALC used in the experiment proved slightly less effective than ENL treatments.

(b)Barley seeds: The seeds were soaked in neem formulations for

24 hr. In tRefirst instance, the soaked seed were seeded to the soil in a wet condition and such wet seed might not pass through a mechanical 95.

TABLE 2.4. Persistence of foliar sprays of neem on bean and barley crops.

Beans (RNL) Beans (FRT) Barley (I?ItT) (Interval post-treatment) Day Day . Day (mg/L) 3 7 14 3 7 14 3 7 14

101000 0 0 0 0 0 1 0 0 1

1,000 0 0 1 0 2 3 1 2 2

1,000 0 0 1 0 1 3 1 2 3 (Teepol)

100 2 3 3 3 .4 4 4 4 4 10 4 - - 5 - - 5 - - 1 5 - - 5 - 5 5 - -

0 5 5 5 5 5 5 5 5 5 Teepol = Dilution with 0.05 Teepol solution.

Average of two repeats of three replications each (.2.- 1).

Feeding test with 150-200 locusts over 12 hours.

Feeding ratings: (same as in Table 2.1)

TABLE 2.5. Protection of bean seedlin s a ainst Schistocercafollowing seed soaks in neem products

Azadirachtin ALC Ext. ENL Ext. (Days post-seeding) Day Day Day PPM 18 25 PPM 18 25 PPM 18 25

100 0 3 1,000 1 5 10,000 0 2

10 1 100 2 5 1,000 2 3

1 5 10 4 5 100 3 4

0 E 5 5 0 5 5 0 5 5 Average of two repet112LILEttuplicates each (± 1).

Seeds soaked for 24 hours.

Germination: day 7-10 post-seeding.

Rank-index: (same as in Table 2.1) 96. drill. A second lot of soaked seed was, therefore, dried at room temp. for 24 hr before being seeded in the pots. Such seed was suitable for seeding through a mechanical drill. There were more than 50 seeds per pot, with two repeats of each treatment. The protection to the seedlings in the two cases as determined by a rank index of feeding to Schistocerca, is given Table 2.6. (Fig-0 ).

The dipping of seeds, especially in the.highest concentration of 10%1- neem kernel water extract, protected barley seedlings against damage of the Desert Locust for one week, although feeding was not prevented fully.

The damaged seedlings sprouted again and when fed to the locusts were eaten less as compared to the fresh controls. The deterrent effect of the strongest neem treatment lasted for more than three week post- soaking of seeds.

2. Seedling -Dips

The roots constitute the normal channel for absorption of water and minerals into the plant from the surrounding media, the absorption being greatest from solutions. Seedling-dips are, therefore, used experimentally for rapid intake of systemic chemicals into the plant system for protection during the early stages of the plant growth. The practice is very useful in case of crops such as rice, tomatoes, etc., which submit easily to transplanting.

Seedlings of rice, beans and tomatoes were used to study the effectiveness of neem seedling soaks. Different neem formulations were compared at several concentrations with two soaking periods of 2 and 10 hr respectively. The results are given in Tables 2.7 and 2.8.

At the higher concentrations, tomato-seedlings were protected for over three weeks; bean, and rice seedlings were protected for over two weeks from the damage of Schistocerca. The rice seedlings were s lightly nibbled presumably being smaller in size they absorbed less amount of water and also they might be more attractive to the locusts.

97.

TABLE 2. .Protection of barle seedlin s on ermination a ainst lehistocerea following soaking the seed in neem formulation.

Kernel Aqueous Extract of neem Wet seeding Dry seeding (week post-seeding) (week post-seeding) Cone.% 1 2 3 Conc.% 1 2 3

10 1 2 3 10 1 3 4

1 3 3 4 5 2 3 4

0.1 4 ii 5 2.5 3 3 5

0 5 5 5 0 5 5 5

Average of three pots of more than 50 seeds per pot.

Seeds soaked for 24 hours. Germination: 4-5 days post-seeding.

Rank-index: Same as Table 2.1.

TABLE 2.7. Protection of tomato seedlings against Schistocerca adults following dips in neem formulations.

Dipping period = 2 hrs Day

Product Conc.; I 3 7 14 21 KNL 1 1 0 0 0 1

0.1 0 0 1 3

0.01 C* - - -

ALC 0.1 0 0 C* -

A2D 0.01 0 0 0 1

Control 0 5 5 5 5

* C = seedling stem was cut but the foliage was not eaten.

Average of three repeats of five seedlings each.

Rank-index of damage: same as in Table 2.1.

Feeding test with 150-200 hungry locusts over 12 hours.

98.

TAME 2.8. Protection of rice and bean seedlings against Schistocerca following dipping them in neem formulations.

RICE BEANS

ENL Ext. FRT Ext. KNL Ext. 10 hr soak _2 ,hrsoak 10 hr soak

Day Day Day (post-dip) (post-dip) (post-dip)

Conc. 4 11 14 3 7 14 3 7 14

10 0 0 1 0 0 1 0 0 0 0 0 0

1 0 1 3 0 2 3 0 0 1 0 0 1 0 5 5 5 5 5 5 5 5 5 5 5 5 Average of three repeats of five seedlings each.

Rank-index of dama,e: same as in Table 2.1.

Peedinn test With 150-200 hunr locusts over 12 hours. Fig. 9 - Protection of barley seedlings -:grown from seeds soaked in kernel suspension, against Schistocerca after exposure to 200 hungry locusts for 12 hours. 100.

D. TRANSLOCATION AND PERSISTENCE OF NEEM FORMULATIONS

1. Translocation - lateral and vertical tranc' ort of the active

ingredient of neem in plants.

Systemic chemicals depend for their efficiency on the extent of their distribution through the plant, in both amount and direction.

Translocation implies the movement of materials in the vascular system of plants, and our understanding of this phenomenon is closely related to our knowledge of plant physiology. Studies on the uptake and translocation arebestundertaken quantitatively with the help of radio-tracers. Such

experiments were, however, not possible in this instance as the structure of the active ingredient of neem was not known. Preliminary experiments were, therefore, undertaken to study the movement of neem in plants, using as a biological assay,feeding tests with Schistocerca.

(a) U take and movement of neem throe-=h different arts....EL9,p1.2at:

The following four types of experiments were carried out using a standard rate of application of 1,000 ppm hNL in soil WO and in aqueous solution (w/v).

(i)normal soil application: neem was applied to soil as part of first watering at the time of transplanting of the bean seedlings.

(ii)root immersion: the roots of.bean seedlings were immersed

in 1,000 ppm aqueous preparation of ENL, w/v.

(iii)basal stem immersed in neem: the roots were removed and the basal stem was dipped in neem.

(iv)inverted bean plant immersed in neem: bean seedlings were immersed in neem preparation in an upside down position with half of the plant dipped in the suspension from the apical end andthe roots held up- right in the air.

The treated parts of the plants in every case, i-iv above, were removed and separated carefully from the undipped part to avoid smearing of the latter with neem. The unimmersed parts were then fed to the 101. locusts so that the protection was due to the translocated neem only.

There were three replicates of each treatment. Bean seedlings used were about six inch tall and were kept at room temperature of about 22° C.

After different intervals of time allowed for translocation, the treatments were fed to hungry adult Schistocerca for eight hr. Comparable control plants were fed along with the treated plants. The results for different immersion intervals are given in Table 2.9.

The movement in the bean plant was the fastest via roots and basal stem as complete protection in these two positions was imparted to seedlings immersed in 0.1% KNL in neem for one hr. The seedlings were about 15 cm tall, so that the rate of movement within the plant was cw approximately 157hr. The epidermis of the roots might, however, have been broken at numerous places during the uprooting of the seedlings permitting faster intake of the aqueous materials. From the soil when the roots . were obviously intact, the minimum period required for an effective translocation was 18-24 hr at 220 C in a wet sandy loam soil. The movement through the top half of an inverted bean plant was slow (though it did

to take place) as the protection impartedAthe beans was partial even after

ixQ an interval of 24 hr. There are two processes in^intact plant,

(i) absorption through root or leaf, (ii) translocation of these; (i) seems to'be the limiting factor in this instance.

(b) Uptake andmovement of neem within a single leaf: Leaves of different plant species were immersed in part in 0.0 concentration of aqueous extract of crushed kernels of neem contained in a large plastic tray. The leaves were secured to the sides of the tray in different

positions with tabs of cellotape. Half of each leaf in any particular

position vas immersed for 24 hr intheneem formulation and there were

three replications of each- position. The placement-on-side was made in

one of two ways, with the rachis or petiolar end of the leaf either

in or out of water. Six inch long pieces of sugarcane from the apical 102.

TABLE 2.9. Translocation of neem in beans studied by immersing different parts of a plant. (rank-index of feeding against Schistocerca)

Post-immersion interval (hours) Plant part 1 1 immersed in neem 4' 2 1 3 6 12 18 24 FL(MU 0.10 Roots (in soil) - - 5 5 - 3 1 Roots (immersion in 3 2 1 - - - aqueous Ext.) Basal stem 3 2 1 - - - - Foliage 5 5 4 - 4 3 2 (inverted 1 plant) Control 5 5 5 5 5 5 5

Aymag2_9f three replicates each (± 1).

Feeding rating: same as Table 2.1. 103. end were used in the experiment. After the treatment, undipped parts of the leaves were carefully cut to avoid smearing of neem onto the them, labelled and fed to Schistocerca for six hr. The results are given in Table 2.10.

Materials are transported within plants by two routes: water and salts are transpotted from the roots via stems and petioles to the apical ends of leaves through xylem vessels, while sugars etc., synthesised in the leaves are transmted downwards supposedly through the phloem.

These experiments were designed to determine the relative importance of the two routes in the transport of the active ingredient of neem, assuming that the potential rate of absorption through the leaves was similar at apical, petiolar and central regions.

When half of the leaf from the petiolar end was immersed in 0.1% 1011J for 24 hr, sufficient active material was absorbed and translocated within the other undipped half of the leaf of beans, cabbage, tomatoes and sugarcane to impart it complete protection against adult Schistocerca over a six hr feeding period. However, the protection imparted to a leaf half-dipped in similar suspension from the apical end varied from one crop to another: bean leaf was fully protected, tomatoes nearly so, sugarcane more than 50% and cabbage not at all. Similarly, when half part of a leaf placed on its side was dipped, the protection imparted to the un-immersed half of the leaf was complete if the mid-rib was dipped in the neem preparation and partial if the mid-rib was not dipped.

The results indicate that the translocation of neem took place via both xylem and phloem. Xylem route was, perhaps, more active because of better absorption through the 'open' petiolar end, while the absorption was poor through the leaf cuticle. This contention seems to be supported by the full protection of the cabbage leaf dipped from the petiolar end and complete lack of protection dipped from the apical end,

The 'open' petiolar end permitted uptake easily while the waxy cuticular 104. surface prevented absorption. However, further work is necessary by injecting the chemical on the apical end to byepass the cuticular layer.

The nature of the leaf cuticle and the distance involved in the trans— port of the material, at this stage, seemed to influence the trans— location of neem substance(s).

paired— leaf comparisio s: To study further the lateral and vertical uptake and translocation of neem products, bean seedling of,5-6 leaf stage were transplanted three to a one Hg pot. There were eight such pots and the leaves of seedlings in five of the potswehemoved leaving only two leaves of about the same size and age on each plant

(younger top,and older lower leaves were removed). Further, the two intact leaves on the stem of each seedling were arranged in one of the following three positions: i. two leaves on one side of the plant, ii. two leaves apposite to each other, iii. two leaves on opposite sides of the stem in lower and upper positions (see pictorial arrangement below). The leaves of the seedlings of the remaining three pots were left as such. One of the two leaves of each seedling in the first five pots was treated with 0.01% azadirachtin while half of the leaves of the seedlings in the other three pots with ALC 0.1% in the tvaner desribed below:

Treatment AZ.D 0.01% (one of the leaves was treated)

1. Leaf arrangement Relative position Pictorial of the treated arrangement leaf

1.Two leaves on one side bottom

2. ibid roP 3.Two apposite leaves one of them

4.Two nearly apposite leaves lower.

5. ibid upper 105.

Treatment 0.1% ALC (half of the plant treated)

6.upper half of theoplant was treated.

7.lower half of the plant was treated.

8.side half of the plant was treated.

There were three replicates in each case. Azadirachtin 0.01% and

ALC 0.1% were painted three times on both sides (once daily on three

consective days) of -f-Aejeaves with a camel hair brush and the leaves

were fed to hungry locusts on -01elith day (after 72 hr of the Ist

cation). No application was made to the stem or stalks of the leaves.

The results of tAe feeding tests are given in Table 2.11. Since there was

no feeding in the first three hr after introduction of the plants to the

locust cage, the plants were left in the.aage overnight (18 hr)before recording the damage.

It may be stated that (i) translocation of neem had taken place in

all the three directions — upwards, downwards and sidewgys for there was

no feeding on the leaves during the first three hr after the plants were

offerred to the locusts, (ii) the conferred protection decreased partially with the increase of the level of hunger of locusts as some of the leaves were nibbled after 2qpr of feeding. Foliar uptake and translocation was comparatively less active than the uptake through the roots; the. leaves were treated three times and still the protection afforded was only partial, (iii) When the lower leaf was painted with neem formulation, position 1, the protection of the upper leaf was less compared to position 2, when the upper leaf was painted indicating a more active downwards translocation. A similar trend was also observed in the case of paired positions of 4to5 and 6 to 7: treatment of lower leaveS gave slightly less protection of upper leaves than vice versa. This might also be partly due to a more active metabolism and attractive nature of the younger top leaves than older lower leaves, although seedlings on the whole were very tender. (iv) As expected, the directly painted leaf surfaces were not eaten due to the presence of free neem 106.

TABLE 2.10. Systemic movement of neem (a.i.) in yartially immersed leaves of different crows - beans cabba,e tomatoes and sugar-cane.

Leaf-end immersed in Rank-index of feeding to Schistocerca (an-immersed neem formulation part of the leaf was fed following translocation (TM 0.01%) over 24 hours) Bean leaf-let Cabbage Tomato

Apical - 0

Petiolar - 0

Side with mid-rib 0 0

Side without mid-rib 2 0

Control 5 5 Average of three replications (± 1). Feeding test was carried out with about 150 locusts over 8 hours. Feeding-index ratings: same as in Table 2.1.

TABTR 2.11. Lateral and vertical translocation of Azadirachtin and Alcohol Ext. of neem in beall_Elant following paired leaf-treatments.

Rank-index of feedint, to adult Schistoserm. feeding done after 72 hours of treatment) Treated Untreated Site of application Movement (leaf-position) (painted) (translocated) AZD 1. Side - bottom Vertical - up 0 1 0.01% 2. Side - top Vertical - down 0 0 3. Opposite Lateral - opposite 0 1 4. Opposite - lower Latero - vertical 0 2 - up 5. Opposite - upper Latero - vertical 0 1 - down 0 2 ILc 6. Lower - half Upwards - sideways 0 2 O.i % 7. Upper - half Downwards - sideways 0 1 8. Side - half Latero - vertical 0 :1 Con- - - 5 5 trol Treatment painted with camel hair brush thrice (once on three consecutive days) on to one of the paired leaves. Average of three replications (± 1). Feeding test was carried out with about 1 0 locusts over 8 hours. Feed-index ratings: same as in Table 2.1. 107.

on them, while leaves and plants of the control treatments were consumed fully.

(d) Translocation of neem in full-grown bean plants: In the

preliminary experiments described above, it was determined (i) neem was translocated both laterally and vertically, (ii) foliar uptake was slow and (iii) lower leaves were protected less than top leaves when application of neem was made to top leaves and vice versa.

To study foliar uptake and translocation of neem in grown up plants using conventional spray, bean plants of flowering stage (about 10 inch tall) of dwarf broad variety were sprayed with 0.1% KU at 10 ml per plant as follows:

i. upper half of plants was sprayed.

ii. lower half of plants was spryed.

side half of plants was sprayed.

The untreated half of the plant and the pot soil were carefully covered with polythene so that the spray did not penetrate on to them.

There were three replicates of each treatment. In the first instance, four sprays (with 0.1% KNL at 10 ml per plant) were given on consective days. The purpose was to see if translocation would occur even with a heavy dosage. In the second case, a single spray corresponding to usual field practice was made and a spray of AO 0.01% was also included for comparison. The.treated plants along with comparable controls were fed to locusts on day 4, 11, and 18 post-treatment: the results are given in Table 2.12.

It may be concluded:

(i) Foliar applications of neem provided adequate protection against adult Schistocerca to the untreated parts of bean plants.

On day 4 post spraying, the protection was partial as the top younger leaves were nibbled slightly but the plant was protected fully after one week. It took about one week for the active chemical to be abSorbed 108. and translocated within the plant to a threshold of 100% inhibition.

(ii) A few of the plants had side-sprouts growing out from the basal stem underneath the soil, these sprouts were also protected except for small nibbling of the top leaves indicating that the chemical was translocated downwards through the stem and then upwards in another direction.

2. Persistence of the Protective Effect of Neem Formulations

Neutralisation or detoxification of active materials in plants and soils is a common phenomenon. Many factore are known to influence such a breakdown. These include metabolisation by plants and soil organisms, volatilisation, leaching, transpiration, adsorption and absorption. In case of toxic chemicals, a high persistence would be undesirable for reasons of mammalian toxicity expecially in edible crops.

As far as is known, neem products are non-toxic to mammals . These have been and are/Still being used for the washing andcleansing of wounds and for oral administration against various ailments. A high persistence of neem both in soil and on foliage would appear to be tolerable and indeed adv=antageous.

In the absence of quantitative analytical methods, preliminary bioassay studies on the. persistence of neem residues were carried out by

(i) sowing a crop to neem applied soil, lifting the crop after 48 hr and transplanting to clean soil, and (ii) by repeated transplanting of bean seedling at regular intervals to a treated soil. Since Schistocerca is very sensitive to neem, hi)50 being about 0.04 ng/ cm2, a positive feeding response would indicate a very low quantity of neem in the plant.

Comparatively heavy applications of neem 1 and 0.1% by soil weight were made as the information pertaining to persistence of lower levels was already available from various other experiments. 109.

(a) Persistence of neem in grass:russocksof grass, Holcus mollis, were transplanted to soil treated with neem seed dust at 1 and 0.1%, w/w.

The grass was lifted from the treated soil after 48 hr at 220C, and the soil from the roots washed under a tap of water and the tussocks replanted to a clean soil. There were three replicates per pot, containing one Kg of soil. The persistence was tested by feeding the grass to locusts and the results are given in Table 2.13.

In the heaviest 1% (w/w) treatment of neem, the protection lasted for over three weeks, indicatingthat, once absorbed into the plant, the active ingredient was metabolised rather slowly or the metabolites were also distasteful to the locusts. The grass, however did not grow vigorously as the transplants took time to take roots, so the results under field condition might be slightly different.

(b) Repeated sowing of bean seedlinus in a neem treated soil:

The different treatments of neem were applied to the soil by weight.

The heaviest treatment 10,000 ppm was mixed in soil as seed dust while other preparations, ENL 1,000, 100 ppm; ALC 100 ppm; AZD 1Oppm, were watered to soil as suspension or emulsion. The weight of soil in each pot was one Eg'and three seedlings of beans were transplanted to each pot at regular intervals. After allowing 36 hr for translocation, the seedlings were fed to hungry locusts. After the feeding test, the seedlings were removed from the treated soil and disposed off. After 4th week, the pot soil was emptied to a tray and thoroughly mixed and returned to the respective pot. This was done to even out the distribution of neem in the soil. The pots were kept at room temperature of 220C, and to prevent loss of the chemical through leaching, a judicious watering was done at the time of each transplanting. The results of feeding tests are given in Table 2.14.

The heaviest treatment of 10,000 ppm perissted in the soil for more than 12 weeks showing that the active ingredient(s) of neem had a fairly long persistence. 110.

TABLE 2.1g. Foliar uptake and translocation of neem (a.i.) in bean plants.

Four sprays Single spray (RNL 0.1%) (ENL 0.1%; A2D 0.01%)

Portion of Direction Days post-treatment plant of 4 11 18 4 4 7 7 15 15 sprayed movement ENL KNL ENL KNL AZD ENL AZD ENL ALP Upper Downwards 0 0 0 0 0 0 0 0 0 -sideways

Lower Upwards - 0 0 1 1 0 0 0 sideways

Side Latero- 0 0 MOM Mom .11 now vertical Control 4 4 4 4 4 tf 4 4 4 Average of three re)lications

Feeding rating: same as in Table 2.1. Feeding test made with 150 locusts over 12 hour ex osure.

TABLE 2.13. Persistence of protective effect of neem (a.i.) in grass transplanted from neem treated to clean soil.

FI1T Dust Days post-transplantation

(PPM) 7 14 21 10,000 0 1 2 3

1,000 2 5 0 5 5 5

Average of two repeats of three replications each

Feeding ranks: same as Table 2.1. TABLE 2.14. Persistence of soil applied neem products - protection of bean seedlings against adult aghistoeernn through repeated sowings.

Post-treatment period (weeks)

Product PPM 2 3 4 6 8 12 ...... _ _ _.. _... _...... ,...... _ Seed dust 10,000 0 0 0 1 2 3

1,000 0 1 3 4 -

100 3

ALC 100 1 3 5

AZD 10 0 2 3 4 3 4

Control 0 5 5 5 5 5 5

Average of three replications each ) • Intervals represent the week when a fresh crop of beans was sown, lifted and fed to Schistocerca after 48 hours in the treated soil.

Feeding was done over 12 hours to about 150 hungry locusts.

Ranking Index: same as Table 2.1. 112.

E. SOME °TIM ASPECTS OF APPLICATION OF NEIN FORMULATIONS

1. Phytotoxic Aspects of Neem Applications

Systemic chemicals permeate plant cells of the tissues of plants.

The phytotoxic aspects of the application of such chemicals and of their metabolites are, therefore, of greater concern than those of the conventional pesticides. Systemic pesticides vary in their potential phytotoxicity but for any one compound the injury is related to the rate of absorption of the active material into the plant system. Optimum protection with minimum phytotoxicity calls for accurate regulation of dosage. The phytotoxic response to a given systemic treatment varies greatly within and among species of plants and is related to several factors of age and overall vigour, soil conditions, seasonal fluctuations and differences in anatomy and physiology of the plant species,

Preliminary observations were recorded on the possible phytotoxic effects of neem formulations on bean and barley plants.. After-effects such as yellowing, wilting, scorching and general decline in plant health were observed visually. Germination of seeds soaked in neem was also recorded. Root length and development of secondary root-lets were studied in bean seedlings grown from seeds. soaked in neem.

(a) Foliar spray aoplications: Spray applications to dripping, and actual dipping of foliage in neem kernel aqueous extracts did not produce visible phytotoxic symptoms in both bean and barley plants even at concentrations as high as 10%.

(b)Smearing of bean plants with neem paste: A thick smear of kernel paste on to the bean leaves produced local wilting of the smeared portion of the leaf. This could also be due to the indirect effect of excessive absorption of moisture (dessication) from the plant tissue bythe paste or interference with the photo-synthesis rather than direct chemical injury. 113.

(c) Soil application: Neem seed dust applied at 2% to barley and 10% to beans (by soil weight) did not produce visually detectable deleterious effects on the seedlings grown from seed sown in the treated soil.

(d) Seedling dips: Bean seedlings were dipped in 5% and 1% concentrations of KNL and allowed to grow in such suspensions for one week.

At the end of the experimental period, the seedlings dipped in neem t44tment were much more vigorous in growth than those dipped in water alone. This may be due to the nutritional value of the organic material in the neem treatments.

(e) Germination of seeds: Barley and bean seeds were dipped in different concentrations of neem (0.1, 1, 10% ENL) for lieriods -4to 48 hr.

Germination of barley on moist filter was observed. Beans were grown in moist sterilised sand. The results of the germination tests are given in Table 2.15.

There appears to be no significant difference in germination between the treated and control seeds.

2. Possible Growth Stimulating Effect of Neem Extracts

During the course of routine experiments, it was observed that bean seedlings from neem treated seeds usually came out of the soil bed first. A pilot experiment was undertaken to check whether the application of neem formulations hastened germination. Twolots of bean seeds were soaked in 1% ENL extract for 2 and 24 hr respectively and allowed to germinate in moist sterilised sand in plastic trays kept at 27°C.

Germination was recorded after eight days and the results are given in in Table 2.16.

The percentage germination of the treated and non-treated seeds differred only marginally being 83.3 and 76.7% in the treated lets to 73.3 and 60.0% in th!non-treated lots respectively. A marked TABLE 2.15. Percentage germination of bean and barley seeds soaked in neem formulation.

BARLEY (N = 100) BEANS (N = 30) Soak time Soak time Neem (KNL) 2 hrs 24 hrs 24 hrs 48 hrs Conc.% (%) (6/0) (%) (5') 10 . 70 75 78 86 1 78 76 85 82 0.1 73 78 82 79 0,0 71 72 84 84 115. difference was, however, noticed in the root-length of the two lots of the young seedlings. The seedlings from neem treated seeds were much longer and also had developed secondary root-lets, compared with those from control seeds. The length of the roots and the number of root-lets in the two cases were significantly different at $ level.. The experiment was not repeated and the results are of preeiminary nature only. Further work on this subjects is desirable.

(Toniand invigorating nature of neem preparations is believed in by several enthusissts of the neem.)

3. Effect of Rain on Neem Deposits

The distribution and displacement of Schistocerca in both space and time are largely governed by meteorological factors (Rainy, 1963).

In the desert, soil moisture is necessary for the development of eggs, and the sprouting of the vegetation to sustain the young hoppers.

Major swarm movements of locust take place down wind, towards and with zones of convergent surface wind flow. Apparently, there is a close association between swarm movement and rainfall. The visitation of locusts are, therefore, either preceded or followed by rain.

To investigate the effect of rain on neem deposits, three kinds of experiments were carried out under conditions of simulated rainfall.

Neem deposits on (a) filter paper, (b) on plant foliage and (c) in soil were washed under a modified 'Sandhurst Mistifier' apparatus( of the

Nematology Section). The pressure was adusted so that the spray fell in a pattern similar to the heavy tropical rain. Spray jetting from different nozzles overlapped and it was necessary to determine with graduated glass cylinders a number of points of equal distribution.

These points of equal 'rainfall' were used for placing the experimental material to be wahsed on the floor of the apparatus. The actual rainfall in each case was measured with the help of graduated glass cylinders kept alongside the material being washed. 116.

In addition to these experiments under simulated conditions,

effect of natural rain was observed on the applications of neem for

formulations during other semi-field and field experiments and is

described there.

(a) Leaching from deposits on filter papers: The different

dilutions of neem products were spread (1.5 cc of each formulation on 11 cm

Whatman No I filter paper) uniformly with a pipette. The filter papers

had been earlier impregnated with 0.1 M sucrose solution. These treated

sucrose impregnated filter papers were then placed on inverted pertri-

dishes' mountedfn plastic cups with knocked out bottoms and washed under

three inch lain. Such filter papers were fed to Schistocerca before a ftrh andi being washed. There was no feeding on any treated paper except

that of the unwashed sucrose impregnated control. Apparently, the washed sucrose filter paper had been washed until the sucrose concen-

tration was below the threshold. Accordingly, another feeding test was run after re-impregnating the washed filter papers with sucrose. There were three replicates of each treatment and the amount of feeding was ranked visually. The results are given in Table 2.17.

It would appear that rain washed away an appreciable fraction, but not all the neem active material. The ALC extract was elated the least, followed by the FlIT aqueous extract and azadirachtin respectively.

Sucrose, as expected, was eluted below the threshold and had to be replenished to induce feeding.

(b) Neem deposits on plant foliage: Three types of experiments were carried out as describeA below.

(i) Immersion of bean leaves in neem and washing them under

simulated rain: three rectangular plastic trays were filled with

BNL o.1%, FUT 1.0%, and water respectively to a level deep enough to

immerse bean leaves. The leaves were dipped with the petiole end held

outside the solution with cellotape tabs stuck to the sides of the trays,

117.

TABLE 2.16. BOrmone-like growth stimulant effect of neem on the development of roots of bean seedlings.

Germination

Soak time Mean ro9t lyngth 4. kerns) Neem (KKL) - SE Mean no. Conc.% 2 hrs 24 hrs (24 hr dip) rootlets 1 83.3 73.3 5.6 ± 0.4 3.8 ± 0.7 0 76.7 60.0 3.6 ± 0.3 0.0

No.of seeds (2 hrs) = 30 (24 hrs) = 15

TABLE 2.17. Effect of simulated rainfall (3") on neem deposits upon filter papers - feeding test with SchistocercA,

Treatment Feeding test over 18 hr exposure------(PPM) Pre-rain Post-rain Post-rain sucrose AZD 1.0 +3 ALC 100 +1

1,000 2 SUC 0.1 rl M +lf

of three replications per treatment.

1.5 ml of each conc. spread uniformly over 11 cm filter paper. no. after '+' mark indicates amount of feeding. 118. so that the uptake of neem if any was through the leaf surface. The leaves were lifted out of the treatments after different intervals of time and washed thoroughly under a tap of water, and fed to locusts after being air-dried. There were four leaves per treatment and the results of feeding tests are given in Table 2.18.

Immersing the bean leaves in neem for even a short period of five 24 minutes protected them partially (60%) while immersion overAhr gave complete protection. Presumably protection was conferred by the fraction of neem which had been absorbed into the plant tissue as the surface deposits would have been largely removed by the thorough washing.

(ii) Effects of spraying bean plants and washing them under siTtAlated_rain: Bean plants of 8-10 leaf-stage were sprayed with different concentrations of neem using 10 ml of spray material per plant.

The plants were then washed under a simulated rainfall of three inches after a post treatment dry period of 6 and 24 hr respectively.

There were four plants per treatment, three of which were washed under the shower while the fourth served as the check. The results of feeding tests with Schistocerca are given in Table 2.19.

A rainfall of thre.:inch within six hr of spraying substantially washed away spray deposits, while'after a dry period of 24 hr the effect of such rain was only marginal. This agrees with the results of experiment at (i) above, showing that sufficient neem istaken up systemically with in 24 hr to impart satisfactory protection to foliage subject to heavy rainfall.

(iii) Use of wetting agent under rainy conditions: To find out whether the use of a wetting agent would improve the retention of foliar spray deposits of neem, ENL 0.1% was sprayed on bean plants using two diluents, 'Teepol' 0.05% and water alone. Also, a series of post- treatment dry periods were tested. Otherwise the spraying, washing and feeding was carried out as in (ii) above. Observations of the damage

119.

TABLE 2.18. Protection of bean leav22,....L1t222. in neem extracts for different ifdand fed to adult °perm. RANK-INDEX OF DAMAGE Time dipped in neem formulation (pre-washing)

Minutes Hours

Conc.% 5 15 30 1 6 12 24

KNL 0.1 3 3 3 2 1 0 0

FRT 1.0 3 3 3 2 2 1 0

0.0 5 5 5 5 5 5 5 Average, of four replications per treatment ct 1). Feeding ranks: same as Table 2.1. Kruskal-Wallis test: H Ratio DF P value Treatments 14.26 2 0.001 (less than) Interval 4.21 6 0.7 (n.s.)

TABLE 2.19. Protection of bean plants sprayed with neem and washed under simulated rain (3") after different intervals (dry periodj--- fed to 4ghjatacerco

RANK-INDEX OF DAMAGE Treatment (neem spray) (Percent concentration) Dry Period A2D ALC KNL FRT FLIT Control (hours) 0.001) (0.01) (0.1) (1.0) (0.1) (0.0)

6 I 3 5 3 3 5 5 24 J 1 2 1 0 1 5

Average of three replications per treatment (1- ). Feeding ranks: same as in Table 2.1. 120. to the plants were recorded twice, 3 and 12 hr after the plants had been placed in the locust cage. The data are given in Table 2.20.

'Teepol' may not prevent washing effect except in so far as it might increase the rate of absorption and the coverage of foliage.

However, the use of Teepol 0.05 did not improve the protective effect of neem under rainy conditions of the experiment.

The locusts did not accept the treated foliage during the first three hr, even when the deposit was washed only after five minutes.

It was, therefore, possible that under o_ field conditions, when the locusts will have achoice of clean food, the effect of rain might not be so marked and the treated crops might escape locust damage.

Perhaps, the use of a sticker based on synthetic or natural glues might give better retention of neem deposits under rainy conditions.

Glues may,. however, also alter the taste of neem making it less repugnant..

Rain may wash away soil concentrations of neem and leach the active material beyond the root zone of the plants. Preliminary laboratory experiments are described to determine the effects of washing and leaching of neem fomulations applied to soil

(i) Mild leaching: Neem derivatives, AZD, ALC and KNI at 1,10 and:1 000 ppm respectively, were applied to 500 Oitof soil in small plastic pots

(with the usual holes at the botto4as part of watering just sufficient to wet the soil. Soon after the application, the 'it soil was washed by pouring water slowly at the top of the pot. The soil column in the pot was three inch high. There were four washings each with 100 cc of water

(2.5 inch of equivalent rainfall). The percolating aqueous contents were collected at the bottom in separate containers. Three different types of soils - organic, sand, and loam - were used in case of KM, treatment.

A control pot without being used for leaching of its soil was kept for each treatment as check. One bean seedling (4-6 leaf stage ) was

121.

TABLE 2.20. Effect of a wetting agent (Teepol 0.05%) on the persistence of neem spray deposits under a simulated rain of 3". (Protection of beans from Schistocerca)

Spray treatment ENL Ext. 0.1% Dry period before rain

Minutes Hours

Observation 5 30 3 12 24 48 (Post-feeding) (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) (-) (+)

3 hrs 2 2 1 1 1 1 1 1 0 0 0 0 12 hrs 4 4 3 3 2 2 2 1 1111

Average of three replicates (1.- 1 ).

(-) wetter not used. (+) Teepol 0.05io used as a diluent. Feeding_ranks: same as in Table 2.1. 122.

transplanted to the residual soil and also to the soil in the untreated pots. The roots of the seedlings were immersed in various lots of aqueous percolates. After 48 hours,a feeding test was carried out by placing the pots in a locust cage. The plants from the water cultures were lifted out and transplanted to clean moist soil in labelled plastic pots to facilitate the feeding test-

(ii) Heavy leaching: The experiment was repeated using five washings of 200 cc of water (5" of equivalent rainfall) instead of four washings of 100 cc in the first case (i) above. The results are given in Table 2.21. There was no feeding in the check pots indicating that the treatments were otherwise effective but for the washing of the soil.

It may be concluded that :

(1)washing resulted in the leaching of neem deposits but the

residual soil still retained sufficient active material to

protect bean transplants;

(2)as expected, the leaching was the heaviest in sandy soil

and least in the organic soil;

(3)the effect of rapid leaching of the chemical in sandy soil

was apparent from almost complete protection of the seedlings

lots of percolates and poor protection in P3 and in P1 and P2 P4. (4)AZD and KNL applications protected the bean transplants better than ALC at levels under test.

(5)heavy leaching resulted, as expected, in more washing away of

neem as none of the residual soil gave complete protection to

the seedlings; the low protection afforded in case of P-

treatments of the heavy leaching lot might also be due to excessive dilution of the chemical.

123.

TABLE 2.21. Leaching of neem in different soils: washing the treated soil with water and bioassaying the residual soil and per- colated aqueous contents by growing bean seedlings and feeding to S.c.11-istQqRU0..

(a) Mild Leaching: four washings each of 100 cc of water.

Neem Product ENL (1,000 PPM) A2D ALC Control (PPM) (I PPM) (10 PPM) 0.0

Soil Type Org. Org. Sand Loam Loam Loam

Feeding Time(hr) 3 24 3 24 3 24 3 24 3 24 3 24

Column no. Pot soil 0 0 0 3 0 0 0 0 0 2 1k 5 P-i 0 1 0. 1 0 0 0 0 0 3 4 5 P-2 0 4 0 1 0 2 0 2 0 4 4 5

P-3 0 3 2 2 0 2 0 4 0 4 •4 5 P-4 1 4 3 3 0 2 0 2 0 3 4 5

(b) Heavy Leaching.: five washings each of 200 cc of water.

Pot soil 1 2 1 5 2 4 1 2 2 4 ti 5

. P-I 1 5 0 1 1 4 1 5 3 5 4 5 P-2 1 5 1 5 2 3 1 3 3 5 5 5

P-3 1 4 2 5 1 5 1 5 4 5 5 5

P-4 1 5 3 5 2 4 1 3 4 4 3 5

P-5 1 5 3 5 2 5 2 3 4 5 5 5

A516.1.010. 48 hours were allOwed for translocation before feeding.

Feeding ranks: same as in Table 2.1. 124.

(6) despite heavy washing (5" equivalent rain), neem treatments

were retained in the soil after 48 hours to give protection

for the first three hours of feeding, which might prove

adequate under field conditions.

(iii) Leaching of neem in different soils under very heavy rain:

The experiment was performed in a similar manner as (i) and (ii) above except that the treated pots were washed by a simulated rain of 24" under a modified Sandhurst mystifier instead of by pouring water over the pots. The data are given in Table 2.22.

TABLE 2.22. Leaching of neem in different soils: effect of simulated rainfall of 24" soon after soil treatment. Protection of beans against Schistocerca. KNL Ext.(1,000 PPM) A2D ALC Control (1 PPM) (10 PPM) ,(o.o) Feeding time Org. Sand Loam Loam Loam Loam

5 hours 2 3 3 1 1 4 24 hours 3 5 3 3 3 5 Feeding rating: same as Table 2.1. Bean seedlings of 5-7 leaf stage were transplanted for 48 hours to the washed soil before the feeding test.

The results conform to those of test (i) and (ii) above: organic soils retained maximum neem and sandy soils the minimum; bean plants might get sufficient protection against Schistocerca even under very heavy rains.

(d) Effect of natural rain under field conditions

(i) Bean plants grown in a large peastrc tray were sprayed with

0.1% KNL aqueous preparation to dripping. After 48 hours

the tray was kept out in the rain (2.3 cm) overnight. When

fed to locust only slight nibbling on some of the top leaves

was observed.CFI. 103 Pig. 10 - Persistence of foliar application of neem after rainfall. The control plants were treated with 0.1% KNL foliar spray and exposed to 2.3 cm rainfall 48 hours later. Locusts were deterred from feeding on treated plants, but stripped the untreated control plants either side; 126.

(ii)The pots treated with neem (see semi-field experiment on

page16) were kept out in the open from 15 May to 18 June

1971 and a total of 7.5" of rain fell during the period.

The protection to the bean plants lasted for over four weeks.

(iii)Similarly, soil applications of neem were tested under field

conditions (experiment on page 145) from 27 May to 15 July

1971 and during this period a total of 7" of rain was

received; still the protection to beans lasted for over

three weeks.

4. Effect of Soil Type on the Persistence and Uptake of Neem Products

Soil type is known to be an important factor in the uptake and persistence of systemic chemicals. Some of the soil factors which influenced the availability of a.i. of neem in some such way as are:-

(i)Particle size: The fine particles have greater surface area per unit weight and thus provide greater potential loss by

adsorption.

(ii)Soil pH: Influences ion-exchange.

(iii)Organic content: The higher biological activity of organic matter helps in rapid decomposition and decay of the active

material.

The following five soil types were used:

Itsar01: 'EFFt soil-less compost for growers.

Muck: rich in peat, clay and silt.

Sandy-acidic: pH - 5.4; clay - 5.1; sand - 78.8%.

Clay-alkaline: pH - 7.3, clay - 24.7, and sand - 60.8%.

Sand: sterilized and in common use at Ashurst.

Muck, acidic and slightly alkaline soils were obtained through the courtesy of Dr Skrentny and a more detailed composition of these soils was described by Pain and Skrentny, 1969. 127.

Azadirslehtin and the alcohol extract were applied to the soil at four different levels as part of 1st watering at the time of transplanting of bean plants. The feeding tests were carried out on day 2 and 10, by placing the pots randomly in a cage of Schistocerca for 6 hours.

There were two repeats of the experiment. The results are given in

Table 2.23 (Fib. 11 ).

TABLE 2.23. Effect of soil type in the persistence and effectiveness of applications of neem. Protection of bean seedlings against Schistocercq. Azadirachtin Alcohol Extract Clay Clay alka- Sandy alka- Sandy PPM Day Org. Muck line acidic sand PPM Day Org. Muck line acidic sand

1 2 0 0 0 0 0 10 2 3 1 1 0 0

10 4 3 2 1 0 10 4 3 3 '0 0

0.5 2 1 1 1 0 0 5 2 4 3 2 1 0 10 4 3 1 2 0 10 5 4 3 1 0

0.25 2 1 1 1 1 0 2.5 2 4 4 3 1 1

10 4 3 4 2 1 10 5 4 4 3 2

0.1 2 3 2 2 1 0 1.0 2 5 4 4 3 3

10 5 4 3 4 3 10 5 5 5 Li 5 0.0 2 5 5 5 5 5 0.0 2 5 5 5 5 5 10 5 5 5 5 5 10 5 5 5 5 5 Average of two repeats of three replications each k-).

Feeding ranks: same as in Table 2.1.

Bean seedlings were exposed to 150 hungry locusts for 6 hours.

As expected, the protection of bean seedlings was the least in the organic soil, followed in order of increasing availability by muck, clay - alkaline, sandy - acidic, and sand. The results conformed to the general pattern of the uptake of pesticides (Getzin and ILosefield, 1966; Konrad tt al., 1967). High micro-biological degradation and the 'binding' Fig. 11 - Effect of soil type on the availability of neem

(alcohol extract, 5 ppm applied by dry soil

weight). The bean plants were exposed to 200

hungry locusts for 18 hours. The transplant in

the sandy soil (S) is virtually undamaged, while

the damage in descending order, for other soils

was sandy-acidic (GO, clay-alkaline (II), muck (MK) and orgaine (oa). The plant in the control treatment (0) was eaten completely. 129.

effect of the colloidal particles might have reduced the availability of the active ingredient in organic and clay soils. Irreversible sorption on clay particles and rapid hydrolysis could be a possible cause of the lower protection in the slightly alkaline-clay soil.

The results, however, are of preliminary nature and call for more work in this line. The levels of application might have to be adjusted to the appropriate soil type.

5. The Effect of Ultra Violet Light on Neem Deposits

(a) Deposits on filter papers: The three neem products Azadirachtin,

the alcoholic extract, and the aqueous kernel extract were applied

to sucrose impregnated filter papers at 1.5 cc per 11 cm filter

paper. The filter papers were dried at room temperature and

placed randomly under an U.V.lamp (Hanovia unit no.3/217, 125

watts) at a distance of 9 inches and exposed to the light for

different daily periods of 0, 0.25, 1.0, and 3 hours respectively.

The potency of the deposits was tested by feeding the filter

papers to Schistocerca. The data are given in Table 2.24.

TABTg 2.24. Effect of ultra-violet light on neem deposits upon filter- papers by feeding tests with Sehiatocerea.

Azadirachtin ALC Ext. KNL Ext. Control Days (1 mg/L) (1 mg/L) (1,000 mg/L) (0.0 ) Post- treatment (Daily Exposure - hours) ,_ _ .25 .25 1 3 0 .25 1 3 0 .25 1 3 1 - - - + + + 2 ------+ - + + + 3 - - + + - - - - - + + + + + + + 4 4. + - + + + 5 - + + + - - + - + + + + - + - 6 - + - - - - + + + + - + + -

+ = positive feeding; (-) = no feeding. 150.

It would appear that AZD and KNL deteriorated faster than ALC under the conditions of the experiment . (The results are of a preliminary nature.)

(b)'Deposits on foliage: Three bean plants - 2 to 1 leaf stage

were sprayed with a micro-sprayer with 10 cc of spray material

per plant for each treatment. The plants were exposed to u.v.

light for 5 hours daily in the same way as the filter papers at

(a) above and tested by feeding to Schistocerca for 3 hours in

like manner. The observations are given in Table 2.25.

TAME 2.25. Effect of ultra-violet light on neem foliage deposits: feeding to adult Schistocerca.

Azadirachtin ALC Ext. RIZ Ext. Control Day (1 mg/L) (100 mg/L) (1,000 mg/L) (water) Post- treatment RA R2 R1 R2 R1 R2 R1 R2

+ _ - + + 1 - - 2 3 4- 4 4. 5 - + - - 1 1 +3 4

ALC - alcohol extract; ENL - Kernel aqueous extract.

= positive feeding; higher nos. indicate higher feeding rate.

(-) = no feeding.

Bean seedlings were fed to about 150 hungry locusts for 3hrs.

Here also, the alcohol extract deteriorated less than the other treatments. It was not possible to determine whether the loss of activity was entirely due to the disintegration caused by the u.v. light alone: the systemic property was not known at the time of these experiments.

6. Shelf-life and Storage of Neem

The neem tree flowers once in a year. Therefore, the fruits have to be stored for several months before being used. Under present(iruG) farming conditions, the aqueous extracts of neem may, sometimes, be 131. prepared in bulk and the entire stock may not be used soon after preparation, necessitating the use of stale, left-over extracts.

The following preliminary observations were made on the shelf-life and storage of neem products:-

(a)Three lots of fruit and kernel of neem were stored at 5°C,

22°C, and 27°C respectively, for three months. Neem seeds were

originally procured from India in 1968 and were kept in a cold room

prior to these tests and would be , thus, over two years old. At

the end of the storage period, aqueous extracts, 1% and KNL 0.1%,

at 10 ml per plant were sprayed on beans. All the treatments gave

full protection against the locusts. Persistence of the deposits

was not studied.

(b)Extracts prepared from the three lots of seeds and kernels were

also tested by using the bio-assay technique of impregnation of the

preparation on a filter paper: no significant difference was observed

in the deterrent action of the above three lots.

(c)An aqueous extract of KNL 0.1% concentration was kept at room

temperature of about 22°C for 14 days: a bio-assay of the stale

product when compared with freshly prepared extract indicated no

significant difference in the potency of the two preparations.

Both the preparations protected the bean plants against the attack

of Schistocerca.

(d)Once a similar product (KNL 0.1%) was left standing in a

graduated glass cylinder for about two months without apparent loss

in the potency tested by spraying the product on bean plantsi even

though the product was strained through a 100 mesh sieve from time

to time to remove the putrefying organic material.

The neem seeds, thus, seemed to possess a long shelf-life which also suggested a high stability of the active ingredient. Some loss, no doubt, might occur during storage and might be .detected by chemical 132. analytic techniques, but the deterioration was not apparently rapid and might be considered negligible for practical field use.

7. General Feeding Behaviour of Schistoeerca on Neem Treated Plants The present work was planned mainly to test the effectiveness of neem formulations and was ancillary to a separate study on mechanics and physiology of feeding behaviour. The reported observations are, therefore, of a general nature made during numerous feeding tests to study the behaviour of neem deposits. The feeding behaviour of locusts was watched usually at the time of the introduction of plants into the cage and also subsequently at random intervals. The nature of plant damage was noted at the time of evaluating feeding and during occasional visits to the cages. The main points are summarized as follows:-

(a) Common feeding pattern: When the plants were introduced

to a cage, the hungry locusts alighted on or moved to the plants

indiscriminately, apparently under the olfactory stimulus of food.

They did not shun the treated plants. Once on the plant, the

locusts felt the leaf-surface with their palps and, if the

deterrent was strong enough, made no attempt at biting. They

lingered on the plant for some time exploring other surfaces of

leaves, before moving on to another plant, proceeding in this manner

until they found an untreated plant: they would start eating the

untreated leaves soon after. After consuming part of the leaf

a locust would move on to another leaf, and would be joined by

other locusts. This movement from plant to plant including the

treated ones continued until the treated plants were fully consumed.

There were usually more locusts on the untreated plants as they

tended to stay there much longer while eating than on the treated

plants: however, the treated plants were not completely without

locusts and the number on such plants depended on the density in

the cage. 133.

When the clean plants were eaten (many a time no trace was left

of them), the activity of locusts on the plants decreased and

eventually the plants were used by the locusts as perches to sit

without being eaten. The locusts would go through the usual et diurnal rhythm of rest and movement (Ellis and Ashall, 1957).

Similar observations were made with Vth stage hoppers using neem

sprayed and unsprayed grass heaped at the floor of a wind tunnel in

five lots at different locations; the floor was large enough to

simulate field conditions. The hoppers moved about from one heap

of grass to another regardless of the treatment, although the food at

a particular heap was in excess of consumption. They, however,

stayed much longer for feeding on the untreated grass. Eventually,

they mounted the sides of the tunnel for rest and became quiescent.

(b)Extreme starvation: Bean seedlings were transplanted to a soil

treated with neem kernel dust at 5,000 ppm by weight, and 15 three

day old Vth stage Schistocerca hoppers were released on the treated

plants after 48 hours of translocation. No other food was provided

to the hoppers; the hoppers did not eat the treated plants and 13

(86.6%) of them died by the 9th day. Cannabalism took place

amongst the hoppers.

Ten adult locusts of about two weeks old were similarly confined

on three bean plants sprayed with 1% kernel aqueous extract; 7 of

the locusts died after eight days without feeding on the plants.

(c)Plant damage: when either low concentrations of neem were used

for the treatment of plants or soil or when the neem treatment

became stale and old, the locusts were not put off feeding and

damage of one kind or the other occurred to the plants as follows:-

(i) The edges of leaves were bruised and dented showing attempts

at feeding but the plant was practically undamaged. 134.

(ii)The leaves were partially eaten here and there, more so

at the top.

(iii)The plants were partially eaten; the eating would start at the

apical end and progress downwards. Both buds and leaves were

eaten.

(iv)Occasionally, especially with beans, tomatoes and cotton,

the basal stem was damaged at places sufficiently or wholly

to make the plant fall on the ground or buckle under its

weight, but the foliage was not eaten.

(v)The leaves were cut off the plant and then stalks and mid-ribs were eaten, pieces of the leaves being left

scattered on the floor of the cage.

(vi)The bark of the spindle tree and the chrysanthemum were

eaten before the leaves.

(vii)Cotton seedlings were cut near the base of the stem before

being eaten and the leaves were eaten the last.

(viii)No feeding might take place during the first few hours

of the plants being offered to the locusts, but the damage

would occur overnight.

Thus, the damage varied according to the persistence of the neem residue, its distribution in the plants and also the level of hunger of locusts.

When the level of stimulation of the gustatory receptors of the palps fell below a certain threshold, the locusts were prompted to take a bite which, perhaps, brought some other possibly more sensitive sensilla in the mouth into operation, detecting the presence of the chemical 'and inhibiting feeding. This threshold centrally' determined would vary with the state of hunger (cf. Haskell and Mordue, 1970). Repeated bites caused the bruising of leaves on the edges. Partial eating of the leaves 135.

possibly indicated a further fall in the deterrent level, the insects

becoming aware of its presence after biting and chewing of the leaves.

The preferential eating of certain parts of the plants, such as bark,

upper leaves, etc., might be due to either the distribution pattern of

the chemical or attractiveness of the parts. The absence of eating

in the first few hours on the treated foliage was apparently related to

hunger; increased hunger resulted in the lowering of the inhibitory

limit or threshold value (cf. Blaney and Chapman, 1970).

F. STUDIES CONCERNING PERSISTENCE IN THE FIELD

1. Semi-Field Scale Soil Application of Neem

To test the efficacy of soil-applied neem under semi-field conditions,

two experiments were carried out based on a dosage per acre instead of

parts per million of soil. The average top eight inches of soil per

acre is estimated to weigh approximately 1,000 tons or one million Kg

(Jones, 1970), making an application of one Kg per acre equivalent to

one part per million by soil weight. Accordingly, an application of

neem fruit dust at 10 and 100 kg per acre would correspond to 10 and

100 ppm by soil weight. These two levels of application of neem were,

therefore, tested, in the first instance. On an area basis, the

application rates of 10 and 100 kg/acre were equivalent to doses of

0.115 and 1.15 gm of neem dust per pot of 9" diameter.

(a) Preliminary Experiment: (application to potted plants in the field.)

Soil of sandy-gravel type from a field lying between the old

Glass House and the Stable Block at Ashurst was used in size

9" diameter plastic pots. Neem fruit dust was applied as a

pre-sowing treatment as follows:-

Spray: the required amount of neem fruit powder - 0.115 and

1.15 gm respectively - was ground to a thick paste with pestle

and mortar, extracted with 50 cc of water and filtered through

a coarse cloth. The water extract was sprinkled over the pot soil. 136.

Dust: the treatments were applied in three ways; neem dust was bulked with fine soil to ensure uniform distribution.

Broad-cast: the neem dust was uniformly broad-cast by hand over the pot soil.

In-furrow: neem dust was placed by hand in a 4.5" deep furrow in the centre of the pot; the bean seedlings were transplanted on the furrow only.

Side-dressing: neem dust was applied in two 4.5" deep side- furrows at a distance of 2.5" from the plants, which were planted in a row in the centre of the pot.

Three bean seedlings of 6-8 leaf-stage were transplanted to each pot in a naturally moist soil and no water was applied after transplanting. However, it rained during the evening.

The treated pots were kept out in the open. The experiment was in progress from 15 May to 18 June, 1971 and a total of 7" of rain fell during the experimental period.

On the first three occasions, day 1, 2, and 3 respectively, one leaf (2-3 leaflets) from each of the three plants per treatment was plucked, labelled and fed to hungry locusts and, subsequently one plant each was fed on day 7, 17, and 31 with a feeding period of 18-24 hours in a locust cage. To test whether the soil was still active at the end of the experiment, fresh young bean plants were transplanted to.the pots and fed to locusts after being 72 hours in the soil. The results of the feeding tests are presented in Table 2.26.

Conclusions

It would appear that:- (i) spray and broad-cast dust applications proved effective in preventing Schistocerca damage to beans even at the

relatively low level of 10 kg of fruit dust per acre;

however, the uptake and translocation of neem at that 137.

TABLE 2.26. Efficacy of spray and dust soil applications of neem to beans under semi-field conditions

Rank-index of Feeding to Schistoce_rW

Day post-treatment

Treatment Egiacre New transplants (equivalent} (same soil) 1 2 3 7 17 31 34 3 hrs 24 hrs

SaraE 100 1 1 0 0 0 0 2 4 10 2 1 1 0 0 3 3

Dust

Broad-cast 100 1 1 0 0 0 0 1 4

10 3 2 1 1 1 0 3 4 In-furrow 100 5 5 5 5 2 0 1 4

10 5 5 5 5 3 3 3 4 Side 100 5 5 5 5 2 0 4 4 placement 10 5 5 5 5 5 5 5 5 Control R1 5 5 5 5 5 5 5 5

R2 5 5 5 5 5 5 5 5

Average of three plants per treatment.

E99(1121Z11119Z same as in Table 2.1. Bean leaves and plants were exposed to about 150 locusts for 18-24 hours. 138.

level was relatively poor during the first 48 hours

compared with that obtained with treatments of 100 kg per

acre, a result which would be expected because of the lower

concentration of the active ingredient in the treated soil;

(ii) in-furrow and side-dress applications were less effective and

there was no protection during the first week. Probably,

the roots did not reach the active soil due to either late

development of new roots and rootlets or leaching of neem

with rain beyond the root zone; adequate protection to.

beans was afforded by these treatments on day 17 and 31

respectively, especially in the case of 100 kg level;

(iii) the protective effect of neem lasted for over four weeks

and, curiously, the protection increased with the lapse of

time. This could be due to two reasons:

(a)with age the plants became less attractive to the

locusts and a serious aphid attack and the resultant

poor condition of the plants might have increased the

unpalatability still further;

(b)Once translocated in the.plant the protective effect of

neem application lasts for about two weeks (see page109);

in this test, as the plant growth was poor, presumably

no rapid dilution of the chemical within the plant took

place and the protection persisted despite the poor

availability of the chemical in the soil. This was

borne out by the observation that fresh bean seedlings

planted in the same soil on the 34th day were almost

completely eaten. (iv) the bean seedlings transplanted to the treated soil on the

34th day post-treatment (specially dust applications) were 139.

not eaten by Schistocerca during the first three hours of their

introduction into a locust cage (although the seedlings were

fully consumed after 24 hour exposure). This would indicate

that the residual activity of'neem in the soil after five weeks

post-treatment was adequate for protection of beans up to three

hours of exposure to locusts but was not sufficient for longer

exposure.

(v) the effect of dust applications persisted longer than similar

spray applications (on day 31 and 34 dust treatments gave more

protection than spray treatments).

(b) Confirmatory Experiment

To confirm the results of the preliminary experiment described

above, and also to test the efficacy of pre-sowing and pre-,and post-

emergence applications of neem, the above experiment was repeated

with some modifications. Soil from the same field was used in

similar pots (9" diameter), but the pots were kept in the greenhouse

in a random fashion instead of being out in the open. A tall bean

variety was substituted for the dwarf variety to study the effect of

a more vigorous plant growth. Different levels of application -

5, 10, 20, and 40 kg per acre - were tested in the case of pre-,and

post-emergence applications: otherwise the treatments were applied

as in the case of the first experiment.

(i) Pre-sowing application: After application of the different spray

and dust treatments of neem as described in the above experiment,

six seeds of broad beans were seeded to each pot in a moist soil.

Occasional watering was done to prevent the soil from drying out.

The first feeding test was carried out on day 17 post-seeding when the seedlings were about one week old. One plant from each 140.

treatment was fed to about 150 hungry locusts; two similar

feedings were done subsequently on day 24 and 40 post-treatment

(seeding) respectively. The results are given in Table 2.27.

TABLE 2.27. Efficacy of pre-sowing soil application of neem to a bean crop under semi-field conditions

Rank-index of feeding to Schistocerca

Rate- 17 24 40 Treatment equivalent . (1) (2) (4)* (Kg/acre) 24 3 24 3 21i** (hours) Spray

100 1 0 2 3 5 10 2 1 2 4 5 Soil

Broad-cast 100 0 1 2 2 4

10 2 3 4 4 4

In-furrow 100 0 1 2 1 4 10 3 3 4 4 5 Side-dress 100 1 1 2 4 5 10 3 3 3 5 5 Control R1 5 4 5 4 5 11.2. 5 3 5 4 5 Average of three replicates.

* week post-germination of seedlings.

** Hours of feeding.

Rank-index of feeding: same as in Table 2.1.

The application of neem JRT dust at 100 kg per acre gave adequate

protection to bean seedlings against the attack of Schistocerca

for one week post-germination (18 days post-seeding and treatment 141.

of soil with neem), satisfactory protection for 15 days and

some protection for as long as 40 dayslunder_the conditions of

the experiment. Even an application of 10 kg per acre was

adequate for protection up to one week, although subsequent

protection at that level was only marginal.

Dust treatments lasted longer than spray treatments (as also

in preliminary experiment). However (unlike the first experiment),

in-furrow and side-placement treatments also proved effective.

In fact, the in-furrow treatment was slightly better than others.

Apparently, the plant roots did not spread to the active soil in

the previous experiment.

Pre-.and post-emergence application In view of the effectiveness

of a dosage rate of 10 kg per acre in the first experiment, it was

decided to test a series of graded dosages; applications

equivalent of 5, 10, 20, and 40 kg per acre were made as spray

or broad-cast dust; other details of the application remained the

same. The results of feeding tests are given in Table 2.28.

(ii) Pre-emergence application: The treatments were applied at day 5

post-seeding when the seedlings had not come out of the soil as yet.

The feeding tests were carried out by lifting one plant each from

the respective pots and feeding them to adult Schistocerca in a

cage containing 120-150 locusts. Since complete feeding was not

prevented (except in one case of dust 40 kg/acre on day 12 - first

feeding), an additional observation on the amount of damage done

to the plants was made after three hours. Such information might

be useful for possible protection under field conditions as the

locusts were likely to move on to other food if they did not find

the treated food palatable during the first three hours or so.

142.

TABLE 2.28. Efficacy of pre- and post-emergence soil application of neem to beans under semi-field conditions.

nk-jndeN_of Feeding

Pre-emergenc e Post-emergence

'Day Day 12 19 26 . 3 7 15 Treat- Rate I hours ment equivalent 24 3 24 3 24 24 3 24 24* 3 i eeding (neem) (Kg/acre) Spray 40 2 2 3 3 5 1 1 4 2 3 20 3 3 3 3 5 2 2 4 3 3 10 3 3 3 3 5 3 3 5 3 7

5 4 3 5 5 5 4 3 5 4 4 Dust

40 0 1 2 3 5 1 3 4 2 3

20 1 3 3 3 5 2 3 4 3 3

10 2 3 3 , 3 5 3 3 5 3 4 5 3 4 5 4 5 4 3 5 5. 5 Control

111 5 4 5 4 5 5 4 5 4 5 112 5 3 5 4 5 5 4 5 4 5

Pre-emergence: 'neem soil treatments applied on day 5 post-seeding.

Post-emergence: neem soil treatments applied to 12-15 day old plants.

*Damage to foliage recorded after 3 and 24 hours of feeding respectively.

Feeding ranks: same as in Table 2.1. 143.

Conclusions

It may be concluded that:

(i)none of the spray treatments prevented complete feeding of

the locusts; however, an application rate of 40 kg per acre

afforded satisfactory protection for one week post-germination

(day 12 post-seeding) and, possibly, may prove effective up to

15 days under field conditions (where the locusts would have

the choice of feeding on untreated foliage). Marginal

protection lasted for three weeks.

(ii)dosage rates of 5, 10, and 20 kg per acre neem (seed dot)

were only partially protective, although the locusts were able

to differentiate between the treated and the un-treated plants

and ate the treated plants less than the control.

(iii)dust applications proved more effective than spray applica-

tions; even the lower levels of 10 and 20 kg per acre dust

gave satisfactory protection for one week post-germination;

a dosage of 40 kg/acre prevented complete feeding.

(iii) Post-emergency application: The application of neem was made to

comparatively grown up 12-15 day old plants. None of the levels

of neem under test prevented complete feeding of the seedlings;

satisfactory protection was afforded for one week and partial

protection for two weeks. There was no marked difference between

spray and dust treatments.

In general, pre-sowing treatments of neem proved more effective

than post-emergence. This would seem contrary to what was expected.

Possibly, the larger plant mass of the grown-up plants in the post-

emergence treatments diluted the available active material below

the threshold, i.e., the concentration per unit weight of leaf tissue

was less, although the total uptake per plant may well have been

greater. 144.

The better protection of the neem treatments in the first

experiment (Table 2.26), especially with 10 kg per acre rate,

might also be attributed to the smaller size. There were only

three plants per pot and being transplants roots took longer to

develop and an attack of aphids prevented vigorous growth. In

the second experiment there were 5-6 plants per pot and their growth

was also 2-3 times more vigorous than that of the plants of the

first experiment. The broad variety grows much faster and taller

than the dwarf one. However, the correct position could be

determined only by a quantitative study of the neem residues.

The spray treatments proved more effective than the dust

treatments in the case of post-emergence application. The

difference was, however, only marginal and marked during the

first week only. Perhaps the spray application being readily

available, was taken up quickly by the roots, while dust application

was absorbed slowly as most of the neem might hare stayed in the top

soil of the pot.

2. Field-scale Soil Application of Neem

A Latin-square, 3 x 3, layout was used to test the efficacy of neem

application under field conditions. Each sub-plot measured 3' x 3'; row

to row distance was one foot; plant to plant distance was 9"; there were

three rows of four plants each and a buffer strip of 1.5' width on each

side separated the sub-plots. The experiment was laid out at Ashurst

on a sandy gravel soil (in the plot of land lying at the bottom of the

field between the old Glass House and the Stable Block).

The variety of dwarf broad beans - Sutton's - was grown as the

test crop; two side rows were left out to avoid a possible border effect

and the remaining central plants constituted the six replicates. Neem was applied broad-cast or sprayed at pre-sowing or at post-emergence as described below. 145.

(a) Pre-sowing application

The neem treatments were applied as dust or spray. Each sub-plot

measured 9 sq.ft and on the basis of area 10.04 gm of neem was needed

per plot to provide a treatment equivalent of 50 kg per acre. The

neem dust was prepared by thoroughly grinding neem fruits in an

electric blender. The required quantity of dust was bulked with fine

soil and broad-cast by hand; the soil was raked to ensure uniform

distribution. For spray application, 50.04 gm of fruit dust of neem

was soaked in water overnight and was extracted with 600 cc of water

by electric blending and sieving through a muslin cloth; 200 cc of the

extract was sprayed over each plot after further diluting with about

1,000 cc of water.

The bean seeds were dibbled in after treating the soil. The

experiment was in progress from 27 May to 15 July 1971; a total of

7" of rain was received during this period.

The plots were seeded on 27 May and geniiination took place between

7 - 12 June; the first feeding test was done on 19 June - at day 23rd

post-treatment. The feeding tests were carried out by up-rooting

one plant from each plot randomly (three per treatment), transplanting

the plants to moist clean soil in labelled plastic pots and offering

them to hungry locusts in a cage. When the plants grew very large,

one shoot from each plant was fed to locusts so that the locusts

remained hungry to the end of the feeding test. The damage to plants

was recorded at 3 and 24 hours post-feeding. At the end of the

experiment, fresh bean seedlings were transplanted to the plots to

test the residual activity of neem in the plots on day 52 post-

treatment of the soil. The results are given in Table 2.29. 146.

TABLE 2.29. Efficacy of pre-sowing soil application of neem to bean crops under field conditions.

Rank-index* of Feeding to Schistocerca

Day Post-treatment

Treatment - 23 30 37 45 52 50 kg/acre 3 24 3 24 3 24 3 24 24 (equivalent) (. New trans lants

Spray 2 4 1 2 1 2 1 2 5

Dust 2 4 1 2 1 1 1 3 5

Control 4 4 3 5 3 5 3 5 5

* Average of three repeats of six replicates each.

Rank-index: same as in Table 2.1.

Kruskal-Wallis test:

H Ratio DF P value than) ( eess Treatments 11.63 2 0.01

Days 9.86 7 0.2 n.s. None of the neem treatments - dust or spray at 50 kg per acre -

gave full protection to bean seedlings under the conditions of the

experiment. However, satisfactory protection (leaves were nibbled

only at the margins) was afforded up to and beyond five weeks (37 days)

post-treatment (three weeks after germination), and partial protection

up to 45 days. Dust application was slightly better than spray.

The protection was better on day 30, 37, and even on 45 than on

day 23 at the 3 hour feeding interval: this might possibly be due

to a less attractive nature of the old plants compared to the young

seedlings, or perhaps the chemical is metabolised and transformed

to a more deterrent chemical over a period of time. A similar

effect was noticed in the case of semi-field tests also.

147.

(b) Post-emergency application

Since none of the pre-sowing treatments gave complete protection

to beans, the plan of the post-emergency experiment was modified to

include higher dosage rates equivalent of 100 and 200 kg per acre.

The application of dust or spray was made as in the case of pre-

sowing treatment (see above). Instead of seeding in, the seedlings

were transplanted and allowed to establish before the neem treatments

were applied. The practice of using seedling transplants was

adopted to save on time. Growing seedlings from seed would have

taken 3-4 weeks under field conditions. The experiment was in

progress from 25 June to 22 July 1971 and 0.5" of rain was received

at Silwood during the period. The results of the feeding tests are

given in Table 2.30.

TABLE 2.30. Efficacj of post-emergence soil application of neem to bean crop under field conditions

`Rank Index of 'Feeding Days

Treatment ' 1 10 17' 31 24 (Kg/acre) 3hr 24hr 3hr 24hr 24hr 24hr 24. A. knew transplants) Spray 200 0 0 0 0 0 0 0'

100 2 2 0 0 1 0 0

50 4 5 2 3 1 0 0

Dust 200 0 0 0 0 0 " 1 1

100 1 3 1 1 1 1 1

50 5 5 3 3 1 1 1

Control 4 5 4 5 5 5 5

Rank-index: same as Table 2.1.

Average of six replicates.

Kruskal-Wallis test: II Ratio DP P value ( less than) Treatments 25.88 6 0.001

Days 2.15 5 0.80 n•s. 148.

Neem applications at rate equivalents of 100 and 200 kg per acre protected bean plants fully against Schistocerca adults for over four weeks. The feeding tests were not run beyond this period.

However, the residual activity of the soil was tested by transplanting fresh bean plants. Even in plots treated with 50 kg per acre the soil still contained sufficient active material 24 days after treatment and the fresh bean transplants were fully protected.

On the whole, the post-emergence application was fairly effective in protecting beans from the locust attack.

The comparison between treatments before and after seedling growth is confounded. Beans made much poorer growth in the post-emergence treatments than in pre-sowing treatments. The transplants took much longer to establish and were also heavily infested with aphids at one stage. Thus, the better effectiveness of post-emergence applications might in part be due to less plant mass per unit area. Earlier, under semi-field test, pre-emergence was the better treatment and post-emergence the least effective of the three methods. The plants in that case were grown from seeds and were much more vigorous and taller than the plants in the present test.

It may be stated in conclusion that an application rate of 100 -

200 kg per acre of neem fruit dust provided adequate protection to bean crop against damage of Schistocerca for over four weeks.

Useful concentrations of neem residue seemed to last for 6-8 weeks in soil. As expected, the plant-mass had an important bearing on application rate and tall crops might need heavier applications: conditions of application, thus, shall have to be worked out for each crop before large scale use of neem.

The present recovery of azadirachtin, the active ingredient, was

0.73 gm/kg (1:1333), and thus a rate of application of 1-2 lb/acre 1149. of the pure chemical is equivalent to one ton of seeds of neem.

The results indicate that the chemical in the neem possessed high biological activity with a long persistence; 3-6 ozs of the pure, chemical per acre was adequate for the protection of dwarf beans against Schistocerca for over four weeks. 150.

DISCUSSION

Most of the literature on systemic chemicals pertains to insecticidal materials. Any discussion of systemic activity, even in the context of a feeding deterrent must, therefore, take into considera— tion the knowledge relating to systemic insecticidal action. The literature on systemic insecticides prior to 1956 is reviewed by Bennett

(1957). Other reviews include those of Ripper (1957), Reynolds (1959), Mitchell et al., (1960), Goodman (1962), and Norris (1967). The main advantages claimed for systemic over contact insecticides are as follows:

(i)Selectivity. Parasites, predators and other beneficial insects

are preserved and only the 'target' phytophagous species are

eliminated.

(ii)Longer persistence. Soil applied insecticides, especially

when in the form of granules may persist for a long time.

(iii)Uniform coverage. With conventional spray methods-, complete.

coverage of all the plant parts is difficult to achieve, particularly

in thick growing crops and trees.

(iv)Efficacy of seed treatments. Seed and seedling treatments

with systemic chemicals protect the plant during its critical young

stages.

(v)Simplicity of operations. Soil applications are easier and less

expensive to make than foliar sprays and usually do not need

sophisticated spraying equipments. The hazards, both of application

and pollution of spray drifts are minimal,

All plant surfaces are absorbent to some extent and nearly all

organic insecticides, which are predominantly lipoid soluble,

penetrate the cuticles of roots, stems, leaves and fruits, However, 151.

a true systemic compound, besides being readily absorbed, should

possess (1) sufficient water solubility to enable it to move with the

transpiration stream, and (2) sufficient stability in plant environment

to enable it and its metabolic products to exert the desired degree of residual action.

Systemic Activity of Neem Products

The systemic property of neem products has been discovered for the first time in these experiments. In the absence of analytical methods, the systemic activity of the products was studied by a bio-assay technique using adult Schistocerca as test insect. The information gathered through such tests is of necessity more qualitative than quantitative. However, applications of neem made to soil (via roots) to foliage or to seed and seedling protected all parts of several plant species for 2-4 weeks against locust attack.- This indicates that the active ingredient was readily absorbed and translocated.within the plants and persisted for fairly long periods. Neem active ingredient is, therefore, a true systemic.

The systemic activity involves (i) absorption by a plant, (ii) translocation to other parts of the plant, and (iii) storage and metabolism within the plant. These three aspects are influenced by different though inter-related physico-chemical factors (see Mitchel et al., 1960) and should normally be studied separately. In the absence of quantitative methods of assay only preliminary observations were possible on soil and foliar applications, seed treatments and on the persistence of protection.

These are discussed as follows:

Soil applications

Roots are the normal channels of absorption of water and minerals from the surrounding soil media. Such absorption is governed 152.

by several physico-chemical factors. The selective absorption of dimefox by the roots of broad beans from an aqueous solution in preference to schradan was explained on the basis of lipoid solubility by David, 1951, 1952; absorption of schradan in plants was co-related with availability of inorganic phosphorous (Caisda, et,a1., 1952), while the preferential absorption of demeton by the roots of Phaseolus vulgaris was attributed to increased intake correlated with permeability through increased respiration (Tietz, 1954). Other factors affecting uptake of chemicals by the roots were: water solubility, volatility, soil conditions, age and stage of plant, and environmental conditions of temperature, light, rain, and humidity (David, 1951; Getzin and Chapman,

1960; Reynolds and Metcalf, 1962; Burt, et al., 1965).

David (1952) found that schradan and dimefox were absorbed more from sand than from soil: Tietz (1954) observed that demeton Was taken up rapidly from a solution, slowly from sand and still more slowly from a humus-containing soil. The difference in uptake was, perhaps, related to physical factors, such as better contact between the roots and the insecticide in a solution and more adhesiveness or greater affinity of the chemical to some particles or constituents in the soil. Orgahic matter and clay contents of soil have been found to be important factors correlated with reduced absorption and faster decline of schradan, demeton, phosdrin, and aldicarb (Casida et al., 1952.; Way and Scopes, 1965; Pain and Skrentny, 1969). This loss of activity of the chemical might be due to increased adsorption or greater micro-biological degradation, or even thermal decomposition.

An initial 4-8% organic matter (content of the soil) may favour the persistence of some organo-phosphorous insecticides (Way etal., 1969). 153.

Sandy and acid conditions may make certain insecticides more toxic and

persistent than clay and alkaline conditions (Griffiths, et al., 1967).

The neem derivatives were readily absorbed and translocated

through the roots of several plant species, namely, beans, barley, wheat, rice, grass, cotton, chrysanthemum, and the spindle tree. Older, pod— bearing bean plants were also fully protected, against locust attack by soil application of neem. There was no evidence of any serious impediments to the root absorption and subsequent translocation of neem products under the conditions of these experiments. Further work is necessary to establish the role of the individual factors mentioned above.

At a constant level of application of neem, the protection against Schistocerca, given to bean plants grown in the treated soils was less in organic and clay—alkaline soil than in sandy soils. However, the protective effect persisted over a longer period in organic than sandy soils. The clay and organic soil particles probably had a binding effect on the active chemical\which was released slowly over a longer period for uptake by the roots. As expected, the absorption by the roots of the active chemical was the most rapid in solution, less so in sand and least in soil, because bean plants grown in three different media, i.e., aqueous solution, sand treated with neem, and loam soil treated with neem, aquired protection against Schistocerca, first in solution, then in sand and last of all in loam soil, over a given period of exposure.

Foliar applications

The absorption and translocation of systemic chemicals in plants are complex phenomena, influenced by many factors. In uptake by roots, the major factor consists of movement of the substance to the roots, for

151k.

this water solubility is important. Once in contact with roots,

compounds of many kinds are actively taken up as discussed above. But

in foliar uptake the outstanding problem is the movement across the • cuticle. This consists of an outer waxy layer which may be very thin

or very thick, presenting a serious barrier to compounds of low lipid

solubility. Underlying the wax layer is a complex of cutin, pectins,

and cellulose. In many leaves cutin is present in flakes cemented

together by hydrophilic substances, probably pectins, providing limited

pathways for absorption of hydrophilic substances which may be quite

important. Stomatal openings are probably of minor importance, for the surface tension of most liquids is too high to permit penetration of such. small apertures. Thus, the penetration of the lipoid cuticle is almost certainly the principal barrier, compared with which penetration'of cell membranes is unlikely to present a major problem.

In the present investigations, despite the limitations imposed by the simple bio-assay technique, information of a practical nature has been, collected on the foliar absorption and translocation of neem products.

The main points are as follows:

(i)It has been shown that the active ingredient of neem is absorbed

and transloeated in leaves of several crops. The treatment of upper,

lower, and side parts of bean plants protected unsprayed parts.

The active ingredient, thus, was transloeated both laterally and

vertically.

This was further confirmed by making applications to paired

leaves; application to an upper leaf protected a lower leaf and

vice versa. Nearly apposite leaves were also protected.

(ii)The application of neem to the top half of a bean plant also

resulted in the protection of side shoots growing from the basal stem. 155.

The chemical travelled downwards to the basal stem and then upwards

in the side sprouts.

(iii)When side half-portions of the leaves of beans and tomatoes

were immersed in neem (KNL 1,000 ppm.- without the rachis end being

dipped in the chemical), the other untreated half of the leaves

became protected, indicating that the chemical was translocated

across the mid-rib, whether through the mid-rib or via the apical

portion is not clear. However, the mid-rib is usually eaten while

the remaining leaf is still distasteful to locusts which shows that

retention of the chemical in the mid-rib is poor.

(iv)Effective translocation of the chemical from one part to another

of the bean plant took 4-7 days. As expected, absorption and

translocation of the active material via leaves was slow compared

with uptake and translocation via the roots.

(v)Three inches of simulated rainfall 24 hours after application

of foliar sprays to beans did not affect the protection given against

Schistocerca; a similar artificial rainfall after 6 hours washed

away part of the chemical and protection was only partial.

Apparently, a sufficient amount of the chemical was absorbed in the

leaves in 24 hours to confer protection.

(vi)When only half of the apical side was immersed in 1,000 ppm

KNL for 24 hours, the untreated parts of cabbage leaves were eaten

completely by Schistocerca. Similarly, the comparatively waxy

leaves of sugar-cane absorbed the deterrent more slowly than leaves

of beans and tomatoes. The leaf cuticle, thus, acted as a barrier

in the case of cabbage and sugar-cane. The chemical was either

not absorbed due to its hydrophilic nature or was retained by the waxy contents of the cuticle. However, when leaves were immersed in a 156.

similar way from the basal end, the untreated apical parts of

leaves were not eaten by locusts - even when the leaves were of

cabbage or sugar-cane.

(vi) As expected, the distance to be travelled was a factor in

translocation. When a half portion of a leaflet of beans was

dipped in neem, the other half was protected, but where one of the

leaflets was dipped over the same period of time the other leaflet

(across the rachis) was not fully protected.

Seed and seedling soaks

The use of seed and seedlings soaks with systemic chemicals to protect seedlings through the critical period of growth at the young stage is too well established to need to be supported by references from the literature. Obviously, the protection is related to seed and plant size; larger seeds are capable of absorbing larger quantities of a given solution which may afford longer protection to the germinating seedlings.

Seed and seedling soaks in neem protected cotton, beans, barley, and rice seedlings for one to three weeks; the protection was related to the size of the seed. For example, the protection of bean seedlings lasted for two weeks, compared with one week for barley and rice.

Seedling soaks of tomato seedlings (roots only) protected the transplants up to three weeks as against one week for rice. This difference might be either due to better absorption and utilisation of the chemical by tomatoes, or to a greater palatibility of rice to Schistocerca.

Persistence of neem applications

The persistence of chemicals in plants can vary from 2-3 days, as in the case of phosdrin (Casida et al., 1954) to several years, as in 157.

the case of R-61999 in pines (Norris and Coppal, 1961). Obviously, the persistence depends on whether the metabolic process increases or decreases the toxic action of the resultant metabolites. A detailed study was not possible because the chemiStry of the active ingredient is unknown. The following factual information is, however, relevant:

(i)Grass planted in soil treated with 1,000 ppm of kernel dust

for 1 8 hours and then transferred into clean soil, retained full

protection against Schistocerca for a further two weeks. Once

absorbed, the chemical (or possibly deterrent metabolites) persisted

for two weeks.

(ii)Soil application of neem seed dust at rates of 100, 1,000,

and 10,000 ppm, w/w, protected bean transplants for 1, 3, and 6

weeks respectively. Partial protection, given by the highest level

of 10,000 ppm which persisted for over 12 weeks, suggests that a ten-

fold increase of the chemical increased the protection three-fold

during the first three weeks and only two-fold during the next three

weeks. From these results it can be deduced that at higher concen-

trations detoxification rates are disproportionately faster; i.e.,

rate of detoxification varies with concentration in a non-linear way.

The possible metabolites, even if toxic, were themselves detoxified

rapidly. Incidently, this result also indicates that frequent small

doses should be much more economical than one large single dose.

(iii)Foliar applications of 0.1% ENT, to run off gave complete

protection for over two weeks.

(iv)A rate equivalent of 100 kg seed dust per acre under field

conditions protected beans for over three weeks, while partial

protection lasted for eight weeks. 158.

(v)The period of protection afforded by the use of systemic

insecticides is inversely related to the rate of growth (Reynolds,

et al., 1957) and the persistence may also be shortened by excessive

leaching (Gardner, 1961). Heavy leaching reduced the availability of

neem in the soil but did not wash out the chemical completely; the

residual soil retained enough activity to protect beans for more than a week after 24 inches of simulated rain.

The rate of growth and the plant mass per unit area seemed to

affect the protection given by neem applications; tall and rapidly

growing variety of beans needed higher doses than a dwarf variety.

It thus may be deduced that the application rate should be related

to the crop size.

(vi)Selectivity and persistence can be improved by way of application

as well as formulation of insecticides (Ripper, et al., 1951;

Metcalf, 1956; Way and Scope, 1968). Soil applications of phorate

were non-toxic to bees, while similar foliage sprays were toxic;

in-furrow placements of phorate and menazon proved less toxic to

certain not-so-active soil organisms; and the persistence was

prolonged.

Broad-cast dust applications of neem proved slightly better than

those sprayed on soil at equivalent rates; dust formulations may

have released the active ingredient slowly over a longer period and

were, perhaps, also less affected.by leaching. In-furrow, side-

dress, pre-sowing, pre-,and- post-emergence applications did not

differ significantly in the protection afforded to beans.

The active ingredient of neem seems to meet all the basic requirements of a good deterrent It is distasteful to Schistocerca at 159,

2 a very low concentration, ED50 0.04 ng/cm . It is apparently non—toxic

to mammals (and possibly to parasites and predators; more information is

required here). It is fairly persistent and easily available (there are

25 million neem trees in India and according to one estimate, 200,000 tons

of seed goes waste every year). Above all, it is systemic. It might,

therefore, prove to be an excellent deterrent against the Desert Locust.

Despite the lack of an analytical method for determining the

active ingredients, sufficient preliminary information has been gathered

to warrant the use of neem for further development under field conditions.

Basic information on soil and foliar application, persistence in plants

and soil, together with effects of rain, soil type and plant species,

has been collected. Preliminary field trials indicate that the chemical

may be equally or even more effective under these conditions than was

indicated by experiments with potted -plants.

Of course, a good deal more work is needed, notably experiments

on species other than the Desert Locust. Preliminary results for several

species are given in the final section of this Thesis.

Certainly, a quantitative method of analysis is necessary to understand the fundamentals of the systemic behaviour of the chemical:

absorption, translocation, persistence and metabolism. Its very

interesting property of lateral and downwards movement, possibly in phloem,

needs to be investigated further. PAaT III. EFFECTS OF NEEM APPLICATIONS AGAINST

SEVERAL INSECT SPECIES. 161.

INTRODUCTION

The active principle of neem fruit extracts can be absorbed by

the roots and translocated in bean plants was shown by Gill and Lewis,

1971. Further experiments on uptake, translocation and persistence of

soil and foliar applications of neem were reported in Part II.

Neem products were translocated in several plant species including

wheat, barley, rice, sugar-cane, tomatoes, cotton, chrysanthemum, and

small spindle tree, as evidenced by biological assays using Schistocerca.

Foliar sprays applied to either upper, lower or sides of potted bean

plants protected the unsprayed foliage from locust attack indicating

that translocation had taken place apparently through both xylem and

phloem vessels. The persistence, in general, lasted for 2-3 weeks, although soil application at 10,000 ppm seed dust protected bean seedling from the attack of the desert locust for eight weeks. Even when the soil was washed by a simulated rain of 20", sufficient protective activity was retained to prevent damage of the desert locust to bean transplants.

Neem products, thus, show great potential as systemic anti-feeding compounds.

The deterrent and insecticidal properties of neem have been known in India for a long time (see also under 'General Introduction'). The

effects of neem applications were, therefore, studied against several insect and non-insect species to determine the spectrum of neem activity. 162.

MATERIALS AND METHODS

1. Test Species

The test species were taken from standard cultures maintained at

Imperial College. Exceptions were the white fly, Trialeurodes vaporariorum and the aphis, Myzus persicae, from natural field

population. The nematode, Pratylenchus spp. was taken from a field soil culture at Ashurst. The fungus, BoArtis fabae, was from the culture of Dr Wheeler (Botany Department). The field mouse, Ludemus

Lylvatieus, was trapped in the laboratory where it apparently strayed in search of food from the adjoining fields. The mosquito eggs were obtained from Mr P.G.Shute of the Malaria Reference Laboratory, Epsom,

Surrey. Further details are given under the sections devoted to the test species.

2. Neem Products

The preliminary experiments were carried out with neem seed aqueous extracts. The fruits were originally obtained from India and were stored at 4°C. Azadirachtin, the pure chemical was included in the final test, wherever possible, as only a limited quantity of the compound was available.

The following abbreviations are used for neem products:-

AL) - Azadirachtin; ALC - Alcohol extract;

ENL - Aqueous extract of neem kernels; and

FRT - Aqueous extract of the fruit or drupe of neem.

(See under "Part I, Methods and Materials".)

3. Experimental Conditions

The experiments were carried out at 27°C and 65% R.H. in a constant temperature room at Ashurst Lodge, unless otherwise stated; the details appear alongside the results. 163.

4. Statistical Treatment

Since a large number of test species (fourteen) were studied in a limited period of time, some of the information is of qualitative rather than quantitative nature. However, depending upon the quality, quantity and the experimental design used, the data were submitted to the following statistical treatments:

(i)Standard errors were calculated to indicate variability.

(ii)Sample means were compared using 't' tests to determine the

significance of the difference and the respective probability

values are given.

The amount of feeding (or damage) was assessed by a visual ranking system: fl = no feeding; 1 = 1-10%; 2 = 11-30%; 3 = 31-50%;

4 = 51-80%; and 5 = 81-100%.

The ranks or scores were devised with the practical use of the product in view. Thus, a ranking of 0 and 1 would indicate adequate, 2 or 3 a marginal, and 4 or 5 a poor protection of the treated crop.

Since the results were self-evident and there was very little variation amongst the replicates, a statistical treatment of the data was not likely to elicit extra useful information and was not carried out.

(Also see under Part II, where this contention has been actually tested by analysing the data of examples of such tables.) 164.

EXPEUIMENTAL RESULTS

The experiments were carried out according to the availability

of the test species. These are, however, not described in a

chronological order but are given according to the precedence of the

significance of the results and the volume of data. The species

belonging to the same order are, however, grouped together..

The Cabbage Butterfly, Pieris rassicae Linn.) (LepidoRtera: Pieridae

The cabbage butterfly, Pieris brassicae is a serious pest of many

Cruciferous crops. Although. Lepidopterous larvae are easily amenable

to insecticidal control, repeated applications are required to suppress reinfestation. Besides being expensive, repetitive spraying is likely to build up hazardous residues. An alternate method of control with relatively persistent non-toxic material, such as neem, is, therefore, worthy of investigation.

A culture of Pieris was available with the College Tnsectory. The larvae were bred on cabbage leaves at 20°C and were utilised in these experiments. Both foliar and soil applications of neem were tested.

Foliar applications

Experiment 1. Cabbage leaves were dipped in different concentrations of neem products (see Table 3.1). After six hours, the leaves were presented for feeding to over 200 larvae, 4th and 5th stage, in a cage of 3.8 cu.ft capacity, alongside the daily feed of cabbage leaves.

Being placed at the top, the leaves were readily attacked by the larvae.

The damage was assessed by using a visual ranking system. The results of four repeats of two replicates (leaves) each per treatment are given in Table 3.1. 165.

TABLE 3.1. Inhibition of feedinr, of Piers larvae on cabbage leaves dipped in neem formulations.

Mean Damage Rating (24 hr post-feeding) t 0.001 0.01 I 0.1 1.0 (% cone.) Azadirachtin 5 4 1 -

Alcohol Ext. - 2 0 -

Kernel Ext. - 4 - 0

Control 5 5 5 5

Visual ranking code: 0 = no damage; 1 = 1-10%; 2 = 11-30%; 3 = 31-50%; 4 = 51-80%, 5 = 81 - 100%.

Average of four repeats of two leaves each.

Azadirachtin at 0.01 and 0.001% (100 and 10 mg/L) failed to inhibit feeding of the late stage larvae of Pieri; however, considerable, though not complete inhibition of feeding was obtained at 0.1% levelAzd: 0.1% alcohol extract and 1.0% kernel extract inhibited the feeding of the larvae completely.

These results suggest that Azadirachtin was much more active against Schistocerca, against which species it is about 1,000 times more potent than the aqueous extract, and that some additional factor(s) repugnant to Fieris were present in the neem seed.

Experiment 2. A noticeably higher larval mortality was observed in the breeding cage used for feeding larvae on neem treated cabbage leaves, and this aroused suspicion of a possible toxic effect. To test this possibility and to study the deterrent action further, a range of extracts in 0.05% Teepol were sprayed on potted cabbage plants at the 3-4 leaf stage with a modified chromatographic all glass sprayer 166.

(see under Part Il,Methods and Materials) using 10 cc of spray

material per plant. The plants covered with glass chimneys, were

then offered for feeding to three different batches of Pieris larvae,

one after the other, in the manner described below. The open end

of the chimney was secured with a piece of muslin to prevent the

escape of the larvae. The concentrations of neem used, amount of feeding (rating values), mortality percentage and weight of the larvae

'after introduction', are given in Table 3.2.

TABLE 3.2. Inhibition of feeding, mortalit and weirht of Pieris larvae fed on cabbage plants sprayed with neem preparations.

Treatment (conc.%)

Day post- AZD ALC KILL FRT Control spraying 0.01 0.1 1.0 1.0 0.0

(a) First batch

Feeding 2 1 0 0 0 2

TI 8 2 1 1 1 3

ti 8 3 2 2 2 4

Mort.% 8 100 100 100 100 10

(b) Second batch

Feeding 15

Mort.% 15 100 100 100 100 0

Feeding 21 4 3 3 4 3

Mort.% 21 40 80 80 60 10

Weight 21 12.6 11.5 11.5 11.2 26.6 (mg/Larva)

Rankina code. same as Table 3.1.

First larval batch released on sprayed foliage after 24 hours; second, after seven days; and third, on 15th day. There were ten larvae per plant. 167.

(a)The first batch of ten third stage larvae was released on each

plant 24 hours after spraying. No feeding was observed on the

plants sprayed with 0.1% ALC, 1.O liNL, and 1.0% FIT, during the

first 24 hours: 72 hours after introduction to treated foliage,

some larvae looked sick. All the larvae on the treated plants

died within one week. The control larvae were healthy and grew

in size. The feeding on the treated plants was markedly less than

on the controls.

(b)A second batch of 3-day old larvae was released to the plant

remnants from 8-15th day post-spraying. Since the larvae were

very young, only a small amount of spot feeding took place,but

sprayed foliage was eaten less than the control. The growth of

the larvae was retarded and these also died within one week. The

size of the larvae after this period was about half of those in

the control treatment.

(c)A third batch of ten 6-7 day old larvae was fed on the still uneaten

parts of the plants from 15-21st day post-spraying; fresh plants

were used for the control. The larvae on the treated foliage

failed to grow and after seven days the treated larvae weighed less

than half the control's weight. A high larval mortality of up to

80% was observed in larvae fed on 1% KNL and FRT.

SOIL APPLICATION

Experiment 3.

The three neem products Azadirachtin, alcohol extract, and kernel aqueous extract, were applied to soil as part of watering to 3-4 leaf stage potted cabbage plants in a range of 10 to 10,000 ppm to dry weight of soil. The plants were kept at 20°C for 72 hours before being fed to Pieris larvae. Each treatment had three replicates. 168.

Ten 3rd stage larvae were released on each plant (placed under a glass

chimney) and were allowed to feed for 72 hours. The larvae were then

removed from the plants and reared on unsprayed fresh cabbage leaves.

The following observations were made:-

(a)The inhibition of feeding of Pieris larvae at 24 and 72 hours after

introduction to food was recorded as given in Table 3.3.

(b)The development of the larvae removed from the treated plants

after 72 hours and transferred to clean food was observed for

after-effects of consumption of neem contaminated food. The

results are given in Table 3.4.

(c)The plant remnants were fed to Schistocerca to confirm translocation

of neem.

TABLE 3.3. Inhibition of feedinj of Pieris larvae by soil application of neem to cabbage plants.

Treatment (PPM)

Interval Kernel Ext. ALC Ext AZD Control Post- 4 2 2 introduction 10 103 10 10 10 0.0 (hours) (feeding ranks)

Batch I 24 1 1 2 1 1 3

72 2 2 3 2 2 Li

Ranking Code: As in Table 3.1. TABLE 3.4. Development of Pieris larvae fed for 72 hours on foliage sprayed with neem and then transferred to clean food.

Days Average larval Neem Average days Level weight (mg/L) Product 21 to pupation (PPM) 4 10 pay210 (of.-Di".) AZD 10 28 59 86 64 21 2 ALC 10 33 48 89 70 15 4 KNL 10 35 77 100 56 - 103 33 52 100 74 - 2 10 25 36 82 84 12 Control 0 3 17 21 144 10 Average of 30 larvae _key treatment. Fig. 12 - Partial protection given by foliar application of kernel suspension of neem against Pieris. (1 1.0c/o; 2 = 001/0; 3 = control).

i.reirAtaiir-!f

Fig. 13 - Partial protection given by soil application of neem derivatives against Picris. After 48 hours of soil application, each plant was exposed to 10 V stage larvae for 24 hours. The treatments were: 1. azadirachtin 10 ppm; 2. alcohol extract 4 100 ppm; 3. TL 10 ppm; 4. KNL 103 ppm; 5. KNL 100 ppm and 6. control. 170.

CONCLUSIONS

(1)None of the neem formulations when applied to soil and taken up

systemically through the roots prevented complete feeding of Pieris.

However, a significant degree of protection (90%) was afforded during

the first 24 hr post-introduction. About half of the treated foliage

was eaten over the next 48 hours (72 hr post-introduction); in

comparison 80% foliage of the control was eaten.

(2)The survival rate of larvae fed on the treated foliage for 72 hours

and subsequently bred on unsprayed cabbage leaves was 14%, 11%, and

18% for AM (lOppm), ALC (100 ppm),and KNL (1,000 ppm) respectively,

compared with 79% in control. All the larvae fed on plants treated

with 1,000 and 10,000 ppm died before pupation.

(3)The average larval weight (70 mg) of the treated larvae on day 10

post-transfer to clean food was about half of the control larvae,

despite elimination of weaker larvae through death.

(4)Amongst the surviving larvae, the larval period was prolonged,

average days to pupation being 18 for treated and 10 for control.

(5)Similarly, the emergency of adults from the pupae of the treated

larvae was delayed for periods up to 55 days, compared with pupation

periods of 10-15 days in the control (at room temperature of 22°C).

(6)The plant remnants were fed to 150 adult Schistocerca on day 4 and

21 post-treatment of soil with neem. The locusts did not eat the

treated plants even after 72 hr post-introduction to the cage, while

the unsprayed remnants were eaten completely overnight. This would

indicate a comparative specificity of neem to Schistocerca and that

the after-effects on the larval growth of Pieris were presumably due

to presence of neem in the food. 171.

Experiment 4.

Oviposition behaviour of adult Pieris on plants treated with neem and subsequent larval feeding and development.

To test the reaction of Pieris adults to neem bearing plant surfaces, three potted cabbage plants (each with three leaves) were treated with neem formulations as follows:

(i)Sprayed with 1% ENL with 10 ml/plant.

(ii)Pot soil treated with FRT dust at 04%, Lo/o.

(iii)No application was made to serve as control.

After 48 hours of application the plants were placed for two days in a cage full of cabbage whites. The butterflies did not discriminate between the plants and laid eggs profusely on all the three plants.

Feeding and development of the hatched larvae.

The eggs of Pieris on the treated and control plants (as described above) hatched after 7-10 days. The larvae fed least of all on the plants treated with neem via soil application. The larvae did not disperse and died in clusters 3-5 days after hatching. They remained small in size, less than 5mm long. The larvae on the sprayed ti leaves (the deposit of neem was already 11 days old at the time of their hatching) fed more than those on soil treated plants (the leaf area eaten by the latter was more than twice of the former) and grew to be approximately 1 cm long; but all these larvae also died within 10 days of hatching. The growth was much retarded compared with that of control larvae (length of larvae: 1 cm in spray treatment in comparison with more than 2 cm in control). The control larvae also consumed almost all the plant foliage and then started to migrate.

The treated, damaged plants recovered subsequently by putting forth new growth while the control plant withered and died. The application ofneem thus gave some protection against the attack of the cabbage butterfly. 172.

In summary, these experiments have brought out two main points:

(i)The application of neem derivatives at concentrations which

inhibited the feeding of Pieris larvae affected their growth

and eventually caused their death. The retardation in growth

was not due to starvation alone as the larvae fed initially on

contaminated foliage failed to regain growth when fed

subsequently on unsprayed cabbage leaves. Despite clean food,

the larval weight was reduced and larval and pupal periods were

prolonged. In some treatments up to 800 or more of the larvae

died before pupation; the larvae seem to have difficulty in

moulting. Neem intakes thus retarded growth and affected

larval growth. Since the effect of the chemical persisted for

a considerable time after its intake was stopped (even affected

pupal period, delaying it up to 55 days), the chemical appears to

have more than a simple toxic effect; since moulting seems to be

affected, the chemical may be hormonal in nature.

(ii)The application of foliar sprays of 0.01%, ALC; 1.0%, KNL; and

1.0%, FRT, inhibited the feeding of Pieris larvae completely

during the first 24 hours post-treatment but application of

0.01% AZD failed to do so. Against Schistocerca, AZD applica-

tions were about 1,000 times more potent than similar KNIA

treatments; the pure chemical AZD was highly specific to

Schistocerca, or some other substance(s) more repugnant to

Pieris were present in the neem seed.

As all the freshly emerged larvae on sprayed leaves died before dispersal and the damage to foliage was small, further development may lead to a satisfactory method of control of Pieris with neem products. 173.

The House Fly, Musca domestica Linn. (piptef.5:7--nugfTrgr-----

The insects were obtained from a standard laboratory culture and were kept at 27°C. Experiment 1. Non-deterrent action of neem to the adults of the House Fly

Neem derivatives, a kernel water extract, the alcohol extract and azadirachtin were mixed into a IM solution of sucrose at 100 ug/L.

Balls of cotton wool, soaked in these neem-containing food media, were offered for feeding to batches of 30 adult flies in replicates of ton, each kept under a glass chimney. The flies fed normally on all the treatments: there was no mortality during the first 72 hours.

The cotton balls were then removed to starve the flies: all the flies died over the next three days. Neem did not, therefore, prevent the adult flies from feeding at these concentrations.

Experiment 2. Non-chemosterilant effect of neem

About 200 pupae of Musca were placed in a cage kept at 27°C.

The emerging flies were fed for three days on (1) a mixture of 10, neem kernel dust and sucrose thoroughly ground to a powder and (ii) powdered sucrose alone. A container of water was placed in each cage and replenished from time to time. There was no unusual mortality in either of the cages and the flies fed on the two lots of food to the same extent. The left-over food was also carefully examined under a binocular for a possible selective feeding of sucrose alone. Apparently, neem did not deter the feeding of adult flies.

A high protein food was then (after three days of emergence) placed in each cage to provide for ovarial egg development. After day seven post-emergence, the flies were offered milk soaked balls 174. of cotton wool wrapped in muslin for oviposition. The petri-dishes containing the cotton balls were examined for the presence of eggs after 8 hours. The eggs were laid in both the cases abundantly.

Groups of 100 eggs each were collected from the cotton wool balls with a fine paint brush on to a wet piece of black satin cloth, laid on the top of a standard larval food medium in a container.

There were four replications of each of the two lots of eggs. After three days, the cloth pieces were removed from the containers and hatch percentage was worked out by examining the egg shells under a stereo-microscope: the larvae on hatching crawled into the medium.

After 10 days, the contents of the plastic containers were emptied into a large dish of water and the floating pupae were recovered, and their numbers were recorded to calculate percentage pupation. The pupae were kept at 27°C until hatched and a count was made of the emerging adults.' The mean percentage for hatching, pupation, and emergence and the respective standard errors are given in Table 3.4.

TABLE 3.4. Mean percentage hatch, pupation and emergence of M.domestica fed on a standard and a neem-based diet.

No.of eggs under test Percentage + St Error

Adult food Hatching Pupation Emergence

10/0 neem + 541 84.1 + 2.3 81.5 + 3.6 90.4 + 7.2 standard diet 382 82.4 + 2.3 89.8 + 2.7 standard diet 95.5 + 1.5

The feeding of adult house flies on the two kinds of diet did not affect oviposition, hatching, pupation and emergence of Musca significantly. There was, therefore, no evidence of a sterilizing effect of neem on the house fly.

175.

Experiment 3. Development of larvae on food medium mixed with neem

The effect of mixing neem in the larval food medium of Musca was studied in a preliminary experiment. The required quantities of

the neem kernel dust in a range of 1 to 10% were mixed in a standard larval diet. The mixtures were shaken well to ensure uniformity, moistened with water and then put into transparent plastic cups.

There were four replicates of each treatment. 50 two days old larvae were introduced into each replicate and the mouth of the container secured with a piece of muslin cloth. The containers were kept at 27°C. The contents were examined from time to time and finally emptied into a dish of water after four weeks to recover the pupal shells and the dead adults. The datalwe presented in Table 3.5.

TABLE 3.5. Effect of neem-mixed food on the development of ======N.domestica larvae Level of neem Mean no.of Mean no.of in food pupae per batch adults emerged (% cone.) of 50 larvae per batch of 50 larvae (+ SE)

10 0 0

5 0 .0

2.5 0 0

1.0 1.0 + 0.4 0.5 ± + Control 39.5 3.5 25.3 - 2.9

Levels of neem above 2.5% prevented larval development completely and no pupae were formed. At 1% level only two adults were recovered from a total of 200 larvae. There was a marked reduction in growth rate as reflected in the size of the larvae, some of which were visible through the container walls. The larvae in the 5 and 10% treatments died within one week and those in 2.5% and 1% grew at a slow rate. The larval duration was prolonged up to

25 days (1% level) as against 7 days in the control. 176.

Neem would thus appear to inhibit or reduce larval growth

of Musca.

The simplest explanation of these results is that neem dust

at the above concentrations inhibits larval feeding, at least partially,

and the larvae die from starvation. The possibility of other toxi-

cological effects, e.g., interference with the hormonal control of

growth, cannot be inferred nor yet entirely ruled out, and further work would be of interest.

C. The Stable Fly (StomoAys calcitrans (Muni)) (Diptera: Muscidae)

The larvae of M.domestica failed to develop on a diet containing neem (see under 'Musca'). It was of interest to study,

the development of another species of fly on a similarly treated diet.

A standard culture of Stomoxys calcitrans was available at AShurst

Lodge and was utilised for these experiments.

Effect of Neem on the Larval Development

Experiment 1. Neem kernel dust in the range of 0.1 - 10%, w/w, was mixed in a standard diet. Fifteen, 3-4 day old larvae of Stomoxys were introduced to each of the three replicates of the respective treatments.

For observations, the contents of the small plastic containers were

emptied onto a black sheet of hard paper and. the whitish larvae counted by shifting around the food medium. The mean numbers of

the surviving larvae, after different intervals of time, are given

in Table 3.6. 177.

TABU'. 3.6. Larval development of S.calcitrans on a diet containing neem kernel dust

Neem content Mean no.of larvae of diet (Day post-treatment) (conc. %) 2 7 10 P Value (± S.E. 0

1.3 0

9.6 9.0 5.3 ± 1.1 0.01

15.0 13.6 12.0 t 0.7

15.0 13.3 10.6 ± 0.4

Average of three replications of 15 larvae each.

The application of neem at levels of 5 and 10% (rather high) resulted in the complete mortality of the larvae within 48 hours.

There was a highly significant reduction in the number of larvae at 1% level (p less than 0.01). However, at 0.1% conc., there was a small but not significant increase in the number of surviving larvae over the control (which may be due to the nutritive value of the neem).

Experiment 2. It was observed that the larvae in neem treatments, in Experiment 1 above, were smaller in size than those in the control medium. In the repeat experiment observations were therefore made on the larval weight as well as mortality. Three levels of application of seed kernel dust, 1, 2, and 4% by weight, were tested with three replications each; 10 one week old larvae were used per replicate,

30 per treatment. For observation, the larvae were taken out from the food medium, washed in water, dried on a black filter paper, weighed, and returned to the respective containers. The data for the surviving larvae, the pupae and the mean larval weight at different intervals is given in Table 3.7. 178.

TABLE 31.2. Effect of neem on the development of Stomoxys - larval weight, mortality and percentage maturity.

Neem content Original Day 4 Day 10 Day 17 Maturity of diet no.of of (conc. %) L Wt L Wt L , Wt P L Wt P adults (%) 4.00 30 1.44 21 5.41 16 11.26 0 7 9.62 6.6

2.00 30 1.18 28 6.49 19 12.22 0 9 11.95 20.0

1.00 30 1.36 29 8.53 20 12.23 0 9 19.45 33.3

0.00 30 1.26 29 14.59 22 21.69 2 7 21.58 80.0

L = no. of larvae; wt = weight in mg/larva; P no. of pupae.

The mixing of neem in the diet affected the larval growth

at all the levels (1, 2, and 4%) under test. The difference between

the means of the control and the least effective level of treatment

(1.0%) was highly significant (p less than 0.01). The larval

duration was prolonged, pupation delayed, and fewer adults emerged.

Experiment 3. Development of ens incubated on filter papers moistened with neem products.

Batches of eggs of Stomoxys were placed on 9 cm filter papers

moistened with 3 cc of the respective dilution of neem extracts and

allowed to incubate at 27°C. In this preliminary test, 0.010

Azadirachtin, 1.0% ENL, and 1.0% FRT were used. A speck of moist

food was placed in the centre of the filter paper to attract and to

collect the hatched larvae. The larval counts were made after 48

hours post-treatment. The unhatched eggs were kept under observation

for one week and no further hatching took place. The results are

given in Table 3.8.

The percentage hatch of Stomoxys was markedly affected by

azadirachtin. Some of the eggs treated with azadirachtin exhibited

longitudinal fissures partially exposing the larvae, but most of the

eggs did not hatch at all. 179.

TABLE 3.8. Incubation of eggs of Stomoxys on filter paper moistened with neem products.

AZD 0.01% ENL 1.0% FAT 1.0% Control

No eggs 175 178 235 188

No.of larvae 12 120 195 165

% Hatch • 6.9 67.4 83.0 87.8

D. The Mosquito, Anopheles stephensi (Diptera: Culicidae)

Anopheles stephensi is an important vector of malaria in

India. An alternate method of its control would help to minimise

the serious pollution risk of the continual use of persistent

insecticides, such as DDT.

Experiment 1.

In a preliminary test, a water extract of neem, prepared by grinding the kernels and sieving through 100 mesh, was added at 1.6

(w/v), to a culture of over two thousand fourth-stage mosquito larvae, maintained in a round plastic dish; other similar trays contained the control insects. The treated larvae started to die after 12 hours and were all dead in 72 hours.

Experiment 2.

Subsequently, three products (a) azadirachtin 0.01%, (b) an alcohol extract 0.01% and (e) water extracts of the neem kernels ranging from 0.01 to 5.0% (of total breeding medium) were tested against third-fourth stage larvae. 100 larvae were introduced to

100 cc of each of the dilutions of the neem products in small circular plastic bowls, 4" x 1.5" deep. To avoid excessive evaporation and the escape of the emerging adults, the bowls had lids perforated

180.

with muslin covered holes. The contents were daily topped up with

water to make up the small evaporative loss. Regular counts were

made of the population of larvae and pupae until the emergence of

adults was completed. A standard diet of crushed sweet biscuits

was used for feeding; there were two repeats of three replicates

each, kept at 27°C. The results are given in Table 3.9.

TABLE 3.9. Effect of neem derivatives on the larval development of A.stephensi.

Mean no.of adults and pupae

Day post-treatment

Neem content Larval Day 3 Day 6 Day 10 of medium mort.% (cone. %) (24 hr) Pupae Pupae Adult Pupae Adult

KNL Ext.

5.0 100

1.0 100

0.1 0 32 2 0 3

0.01 0 0 71

ALC Ext.

0.01 31

0.01 100

Control 0 68

Initial no.of 3rd-4th stage larvae/treatment was 100 ± 5%.

All the neem treatments, except KNL 0.01% resulted in a

significant, reduction in the number of larvae reaching maturity.

The levels, KNL 1 and 5%, killed the larval stages completely in 24

hours while most of the other concentrations proved effective in one

week; concentrations of 0.1% KNL also killed pupae. 181.

Experiment 3.

The sieved neem suspension used in the above experiments

contained suspended organic matter which emitted a strong smell on

decomposition, To exclude the suspended organic material, the water

extract was filtered and used in this test. Also, careful larval

counts were made instead of the pupal counts; 100 - 5 larvae of 2-3 _

stage were used per replication. The results are presented in

Table 3.10.

TABLE 3.10. Larval mortality of A.stenhensi reared on food media containing neem products.

Mean larval mortality (%)

Day post-treatment

Neem content No.of of diet 60 Day 1 3 - SE 10 adults

KNL EXT. 1.0 55 94 100 0.1 10 28 55 1.54 85 0.01 0 5 22 0.61 30 ALC EXT.

0.01

AZD ,

0.001

0.0001

Control

Initial number of larvae (2nd-3rd stage) was 100 ± 5. 182.

The neem products RNL 1.0%, ALC 0.1% and AZD 0.01% killed all the larvae in one week; other treatments were effective in ten days. The difference between the control and the least effective dilution of AZD 0.0001% was significant at 1% level (P being less than 0.01). Although no data on weight of the larvae was recorded, a noticeable retardation of growth, reflected in the smaller size of the larvae, was observed; the pupation was also delayed. On day 7 adult mosquitoes emerged in the control treatment. The filtered extract proved more effective than sieved water suspension of neem;

0.01% KNL suppressed complete emergence of adults, while the ordinary suspension at that level proved ineffective. However, at 1% sieved suspension killed all the larvae in 24 hours but it took more than three, days with the filtered extract.

Ex,)eriment 4.

When batches of 150 eggs each were incubated in a culture media containing iNL 1.0% and 2.5%, ALC 1.0%, and AZD 0.01% respectively in separate containers, the eggs hatched normally but all the young larvae died within 48 hours.

E. The White Fly, Trialeurodes vaporariorum .(Westwood) (Homoptera: Aleyrodidae)

There was a very heavy attack of the white fly, T.vaporariorum on the standing field crops during May 1971 at Ashurst Lodge. The brussels sprout plants (left-overs from the previous season) harboured many thousands of flies, which would arise in swarms on shaking the plants. The opportunity was taken to test the effect of neem treatments upon this naturally occurring population. 183.

Experiment 1.

Three plants of brussels sprouts in the field were sprayed with

1% NIT (neem aqueous fruit extract), using a lady hand sprayer, at

60-80 gallons per acre. Three other plants, sprayed with water only,

constituted the control.

Preliminary observations, made by shaking the plants and

assessing the size of the ensuing swarm of-flies and by roughly

estimating the number of adult flies sitting on individual leaves,

showed no obvious differences between the sprayed and the unsprayed

leaves. Another spray with 1.0% FRT was, thereforel applied to•the

same three plants on the fourth day and water was again sprayed on

the control; the sprayed plants were covered with muslin sleeves to

prevent reinfestation from the adjoining plants. After one week

(day 10 post first spray), six leaves from each plant were removed and the number of eggs and larvae in standard areas (4 sq cm) were counted under a binocular; the mean values are given in Table 3.11.

TABLE 3.11. Mean no.of eggs and larvae of T.vaporariorum on brussels sprout ylants sprayed with neem (Day 10 post-treatment)

Mean - SE DF P Value

Eggs YET 1.0 82.1 - 14.8 31

Control 98.7 ± 15.7 31 n.s.

Larvae 1E/ 1.0% 7.1 ± 1.4 31

Control 44.2 - 7.3 31 0. of

The difference in larval numbers was highly significant in the case of neem treated plants. The adult flies were still alive on both the sprayed and the unsprayed plants; they continued to lay eggs on both kinds of leaves and the difference between egg counts was not significant.

184.

EIEMLEDILI• To confirm the results of the above experiment, three lots of

six leaves each were removed from the unsprayed brussels sprouts

plants in the field and the three batches respectively treated as follows: (i) petiole end of leaf immersed in 1.0% FRT for 48 hours; (ii) leaves sprayed with 1.0% kRT (both made in 0.05% Teepol); (iii)

sprayed with Teepol solution only. The leaves were planted in a

moist sand bath to prevent drying. Regular counts were made of eggs

and leaves and the data for day 8 post-treatment are given in Table 3.12.

TABLE 3.12. Number of eggs and larvae of Trialeurodes on brussels srout leaves treated with neem.

Mean - SE

No.of Day 8 unhatched Post- eggs Original no. treatment DF P Value

/RT 1.0% 110.3 t• 14.5 5.5 -±• 0.7 10 (petiole dipped) ±1T 1.0% 113.7 ±• 18.3 7.2 ±1•.8 10 0.01 (spray) Control 123.8 ±• 33.1 24.50 ±4.8 10 No.of larvae

FRT 1.0% 42.0 ±• 10.2 9.8 ± 1.6 10 0.01 (petiole dipped)

FRT 1.0% 36.2 ± 8.8 14.8 ±• 1.1 10 0.01 (spray) Control I 39.3 ± 6.6 56.7 ±11.7 10

Average of six leaves per treatment. The counts were made over a standard area of 4 sq.cm. constituting about 1/10 of total leaf surface. 185.

The treatments led to reduction in larval numbers (being

10, 15, and 57 on petiole dipped, sprayed and control leaves -

Table 3.12). The size of the larvae on treated leaves was much

smaller and they looked dessicated. Those which died finally withered away to a brownish mass.

Another conspicuous feature was the presence of many 'semi- hatched' larvae on the treated leaves; the larvae did not fully

emerge out of the egg shells and were lying dead in a 'half-in and half-out' position. This observation suggests that the active ingredient of neem penetrated the egg shells, affecting the developing

embryos; this phenomenon would no doubt repay further study.

The mean differences between the numbers of both eggs and larvae on the treated and untreated leaves after 8 days were significant at 1.0% level.

Experiment 3. White fly on Cotton* A serious infestation of T.vaporariorum was observed again during July 1971 on cotton plants grown in the glass house, one cotton plant each - 8-10 leaf stage - was (i) sprayed to dripping with 1.0%

FRT; (ii) 1.0% FRT dust w/w, was applied to soil, (iii) the control plant was sprayed with water alone. The plants were kept at room temperature of 22°C (approximate).

Portions of leaves (2 cm sq) at intervals, were cut and

examined under a stereo-microscope. The larvae on the sprayed leaves appeared emaciated and many died within one week (seen as yellowish dried mass on the leaves). The sprayed leaves had fewer empty puparia and less honey dew than the controls. On the plant subjected to soil treatment, there was a marked retardation of larval growth at first. The leaves were full of young larvae which did not

grow. However, the 'withering' effect (larvae in the process of

* This species was identified by the British Museum (Natural History), London.

186.

drying up) was less marked during the first week, as compared

with the withering of larvae on sprayed leaves. Eventually, however,

there was no significant difference between them. This was perhaps

due to a slower uptake and ingestion of the chemical from the soil

treated cotton plant. In the control treatment, many more larvae

grew into adults, as observed from the number of the empty puparia

on the leaves. The number of larvae and adults (either newly

emerged and still sitting on the leaves or dead and stuck in the honey dew) and the presence or absence of eggs over a unit area of

2 cm sq on day 14 post-treatment, are given in Table 3.13. The distribution of leaves, numbers 1 to 6, was from the bottom to the apical end of the plant - the 6th leaf being the youngest.

TABLE 3.13. Effect of the application of neem on Trialeurodes infesting cotton plants. 1 Observations on Day 14 post-treatment

Treatment - FRT 1.0;x: sample area - 2 cm. sq. Larvae Adults Eggs Honey-dew Sample from Con- Con- Con- Con- leaf no. Soil Spray trol Soil Spray trol Soil Spray trol Soil Spray trol (1%) (1%) (0.0) (1%) (1%) (1%) (1%) (0.0)(1%) (1%) (04

1 2 1 10 2 0 1 . 0 0 + L L M 2 2 5 15 0 0 0 0 + L L M 3 1 5 6 1 0 0 0 + L L 4 7 8 50 1 1 5 0 0 + L L H 3 13 1 5 4 2 1 + + + .11 M H 6 8 7 10 0 2 1 + + + L Iii 11

Total 24 29 106 i 5 5 11 +2 +2 +6

Mean 4.0 4.8 17.7 -

SE 1.2 2.4 6.6j - 1

L = Low; M = Medium; H = High. 187.

The effect of neem on the population of Trialeurodes was

clearly reflected in the numbers of larvae (and even adults) in the

treated and untreated plants; there was an associated reduction in

the production of honey dew. In fact, the comparative absence of the

honey dew was the main difference which would strike a casual observer;

otherwise, the adult flies were present equally on the leaves of both

the categories of plants. The leaves of the control plants glistened with honey dew on which fungus had started to grow.

The absence of eggs in the older leaves of, the treated plants needs further investigation; the adult flies did not seem to discriminate between the treated and untreated leaves in the matter of alighting on them and the older leaves of treated plants were greener than the older leaves of the control plants; yet there was less oviposition on them. The sense organs on the ovipositor might possibly be able to detect neem.

Neem formulations, thus, affected the larval growth of kialstualea, egg and embryonic development, and possibly oviposition,

o)but this conclusion is tentative and requires confirmation. The effect on the advanced stages of larvae was less marked(and some reached maturity the adults were not affected.

F. The Desert Locust, Schistocerca gregaria Forsk. (Orthoptera: Acrididae)

In the numerous experiments reported under parts I and II, adult

Schistocerca populations were fed on neem treated food and no unusual mortality or other ill-effects were observed amongst the treated locusts. only They presumably only fed on food containing trace quantities: Dipping adult locusts and V instar hoppers in 1% ENL (neem kernel water extract) and then rearing them on clean food also did not result in any mortality. 188.

A preliminary experiment was, therefore, carried out to test the

effect of feeding locust hoppers on foliage sprayed with neem.

Experiment 1. Development of locust hoppers on neem treated grass.

It was determined that grass sprayed with concentrations of

5 ug/L AZD (azadirachtin) and 5 mg/L KNL (kernel water extract of neem), at 10 cc. of the respective dilution per 50 gms of green grass deterred the feeding of locust hoppers to approximately 50%.

Batches, each of 35 (28 late IV and 7 early V) Schistocerca hoppers were fed, until fledging, on grass treated with (i) AZD 5 ug/L, (ii)

KNL 5 mg/L, (iii) sprayed with water alone, and (iv) no food was given to the hoppers. The food was replaced daily and each batch was weighed periodically. The hoppers were bred in circular polythene cages with muslin windows. The cages were kept at 27°C1cleaned.daily, and dead insects, if any, were removed. The details of the observa..- tions are given in Table 3.14.

The application of the neem products killed a proportion of the locust hoppers, the mortality being 77.1, 41.4, and 34.3% in KNL and

AZD and 'clean food' respectively. A high mortality of 91.4% was recorded in the case of totally starved hoppers. There was no large difference in the mean weight of the surviving larvae, being 1.64,

1.78, 1.48, and 1.4 mg/Larva on day 14 for AZD, KNL, food,and no food categories of hoppers respectively. In fact, the average weight was slightly greater in neem treatments, which might be due to the death of the weaklings and the survival of the heavier and more vigorous animals. The hoppers fledged at about the same time (day 14), indicating no marked delay in the maturity of the larvae.

As expected, there was a high rate of cannabalism in the trio-foodt treatment, which was included in the experiment for comparison of weight loss due to starvation rather than the mortality. 189.

awever, an interesting fact came to light that about 10% of the

locust hoppers survived total lack of food. Apparently, short of

a complete failure of hatching, the locust progeny could survive

in the field under the worst food conditions and give rise to an

adult population.

The information is of preliminary nature only and further

evidence would be necessary to confirm the findings. The effect of

starvation is not separate from that of neem. Injecting known

quantities of neem products into locusts could yield data both on

acute and chronic toxicity. Replacing 'no-food' treatment with

partial feeding with a known starvation period could provide more

useful information on weight loss and effects of starvation. The

feeding of young larval stages on neeM treated food may result in

complete mortality of the hoppers as was the case with some other

test species, D.cinagatus, for instance.

Experiment 2. Treatment of locust egg-pods in soil with neem.

The eggs of the white fly, T.vaporariorum laid on the foliage

sprayed with neem did not hatch fully (see under Trialeurodes).

To test if neem application had some similar effect on Schistocerca

eggs, a preliminary experiment was carried out. Six sand filled

cylindrical aluminium tubes, in common- use as oviposition sites for locusts, were obtained from the Centre for Overseas Pest Research,

London. Each of the tubes contained 2-3 egg pods. A small longitudinal hole was made in the moist sand at the centre of the top of the tube running to about half the length of the tube and a solution (1 or 2 cc) of 0.01% An and 1% ENL each was introduced at the

end of the hole with a pipette, so that it could spread around and reach the eggs; water was used for the control treatment. The holes 190.

TABLE 3.14. Development of Schistocerca hoppers (IV & V) on grass sprayed with neem products at ED50 level.

• Day post-treatment 0 3 7 10 14 17 20 Azadirachtin (5 ug/L)

No.of IV 28 17 2 •. 2 AMID •••

tl V 7 13 25 23 16 8

" Adult 7 15 17

Total 35 30 27 25 23 23 17 Mortality - 14.3 22.9 28.6 34.3 34.3 51.4

wt/ (mg) 0.61 0.76 1.14 1.46 1.64 Kernel Ext. (5 mg/L) No.of IV 28 16 1 0 0 0 V 7 11 16 15 8 1 Adult 6 12 8 Total 35 27 21 15 14 13 8

% Mortality - 22.9 40.0 57.2 60.0 62.9 77.1

wt/L (mg) 0.62 0.79 1.24 1.59 1.79 'Clean' grass No.of IV 28 17 3 1 1 V 7 18 30 30 18 6

Adult - - 11 • 21 23

• Total 35 35 33 31 30 27 23 • Mortality 5.7 11.4 14.3 22.9 34.3

wt/L (mg) 0.59 0.79 1.11 1.41 1.48 - 'No Food' 1 0 IV 0 28 14 2 V 7 10 10 8 6 4 Adult - - - 1 3 . , Total 35 24 12 9 6 5 % Mortality - 31.4 68.7 74.3 82.9 85.7 91.4 wt/L (mg) 0.65 0.75 1.03 1.23 1.4

• 191.

were closed by pressing the sand. The eggs in the tubes were

incubated at 30°C. There was no significant difference in the

hatch percentage of the treated and control egg-pods. Apparently,

the infusion of neem in the tubes had no marked effect on the egg hatch

at the concentration under test. The eggs were laid on 29 May 1971

and hatched on 11 June 1971; there was no delay in their incubation

period.

G. The Nematode, Prat lenchus spp. (Tylenchida: Tylenchidae)

Barley seedlings were grown on moist filter paper. Ten day

old seedlings were dipped in neem kernel water extract 0.5 and 1.0% for two hours. The treated seedlings were transferred to a naturally infested soil in plastic cups and grown further at 25°C with 24 hours artificial light. After 20 days in soil, the roots were stained in acid fuchsion and cleared in lactophenol. The roots were pressed between two glass sheets and the number of nematodes counted under a stereo-microscope. There were four replicates of each treatment.

The data are given in Table 3.14.

TABLE 3.15. Prevention of invasion of barley roots from Pratylenchus spp. through neem root-dips.

No.of nematodes

Level of Reduction of neem (IcNL) incidence (conc.%) R3 R4 Mean - SE (4A) 1.0 5 3 r6.50 + 1.71 52.5 0.5 6 14 10.25 i 1.93 25.5 n.s.

0.0 14 IS 13.75 ± 2.10 -

The infection of the nematode was thus significantly reduced (the mean difference was significant at 5% level between 1.0% treatments and control). 192.

H. Preliminary Experiments with Neem Formulations against Certain Pest Species

These very preliminary experiments were carried out to explore further the spectrum of activity of neem products. The results suggest that neem formulations affected the behaviour of some of the test animals and that more critical laboratory and field experiments would be worth while.

1. The Diamond Back Moth, Plutella xylcstella (Curtis) (Lepidoptera: Plutellidae)

(i)One cabbage leaf each was painted with 1, 0.1, 0.01% conc. of

ENL, ALC, AZD with 2 ml of the respective preparation. All the

formulations were prepared in 0.05% Teepol. After 24 hours five

fourth stage larvae of Plutella were released on the leaves. To

prevent wilting, the leaf stalks were put in moist sand. None of

the treatments prevented feeding of the larvae completely. However,

there was a marked difference in the extent of damage after 48 hours,

visually ranked in the descending order of 1, 1, 2, and 3 for KNL,

ALC, AZD, and the Control respectively.

(ii)Neem formulations - AZD 0.01%, ALC 0.1%, and ENL 1.0% in Teepol

0.05% - were applied to potted cabbage plants with a camel hair-brush

at 6-8 ml/plant. Soil application was made at 1.0%, w/w, of neem

fruit dust by mixing the dust to the top soil of the other pots and

watering down. Nine one-day old larvae of Plutella were released

on each plant 48 hours after the application of neem. Observations

were made on the number of surviving larvae and the length of the

tunnel per larva. Foliar applications of 1.0% ENL prevented both

entry and tunnelling by the larvae into cabbage leaves; 0.01% ALC

and 0.01% AZD resulted in complete larval mortality one week after 193.

application and the tunnelling was significantly less than the

control plants. The soil application also killed all the larvae

and reduced tunnelling in comparison with untreated plants. Further

work is suggested.

2. The Cotton Stainer, ilysdercus cinn;ulatus (ilemiptera: rrhocoridae)

(i)The insects were bred at 27°C on a standard diet of cotton seeds.

Ten gm of cotton seed were moistened with 5 cc of an aqueous

preparation of 10 ppm azadirachtin and another batch with a 1,000 ppm

neem kernel water extract; water was used for the control treatment.

Each batch of seeds was put in a small glass dish and a wet piece of

cotton wool was placed alongside to provide water. The small glass

dish was then placed in a bigger plastic dish having a bed of

sterilized peat. Fifteen Dysdercus larvae of V stage were fed in

each dish and the number developing to adults was recorded at

rftular intervals.

Although fewer adults emerged from larvae fed on neem treated

seeds the reduction was not significant. A delayed emergence of

adults in azadirachtin was, however, noteworthy. The adults from

the experiment were reared further on clean food and they matured,

mated and oviposited normal viable eggs.

(ii) The larvae fed on the neem treated seeds in the above

experiment were of V instar. The experiment was repeated using

II stage larvae; 50 larvae per treatment were used. All the tiny

larvae fed on seeds treated with AZD 10 ppm and ENL 1,000 ppm died

within the week, while those in the control treatment developed

normally. 194.

3. The Green Peach Aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae)

As the neem products were taken up systemically by plants apparently through both the xylem and phloem vessels, it was of interest to study their effect on the feeding behaviour of aphids.

(i)Bean plants were sprayed to dripping (100 gallons/acre) with

1.0% FRT and also grown in soil mixed with as high as 1.0% FRT, w/w.

The treated and the control plants were then exposed in a choice

experiment to a large population of winged aphids in a single cage.

One week after exposure to adult aphids the plants were examined for

aphid larvae. All the plants were found infested with aphid larvae.

Thus, the adult aphids were not deterred from laying on the treated

plants and the young aphids on the treated plants did not suffer from

serious ill effects in the first week of life.

(ii)Bean plants in field experiments undertaken for other purposes

(Part II) became naturally infested with aphids; this indicates these

aphids are not deterred by concentrations of neem which are effective

against Schistocerca.

The possible growth disturbing property of neem was not known

at that time and the effect of neem application on the rate of growth

of individuals and their rate of multiplication was not investigated,

but would appear, desirable.

4. The Wire Worm, Affriotes spp. ••••••.•IMPII (Coleoptera: Elateridae)

The 'wire worm' group of larvae of the genus Agriotes are serious pests of pastures, cereals and root crops. With the restrictions placed on the use of organo—chlorine insecticides, there isji, need for other methods of control. 195.

A technique for the screening of candidate repellents has been described by Griffith (1969). Filter paper discs are soaked in a standard nutrient solution, dried and the candidate repellent applied to one end of the nutrient impregnated filter paper. The treated discs are then offered to individual larvae of the 'biting phase' of wire worms.

A preliminary investigation by dipping filter paper discs in up to 10% KNL showed no significant difference in the number of bites by the worms on the treated and control side of the papers. The presence of bites, however, does not mean that the test papers were acceptable as food; a modified test technique using some quantitative assessment of food intake might be more revealing. The effects of neem intake on the larval development may also be investigated. 196.

DISCUSSION

The traditional beliefs in the insecticidal properties of neem have been put to test by several earlier research workers. An application of macerated juices of neem leaves, Azadirachta indica, to lucerne resulted in 25% mortality of the larvae of a weevil (unspecified species)

(Chopra, 1928). A compost of neem leaves applied to wheat at the rate of seven tons per acre reduced the incidence of termites from 8% in the. control to 0.7% in the treated field (Hussain, 1929). An extract of 32 gms of Melia azedarach seeds with 100 cc of alcohol, sprayed as an aqueous solution on cabbage, killed 98% of the population of the aphid species,

Brevicoryne brassicae (Astrakhantzev, et al., 1937).

Neem cake (residual cake left after the extraction of oil from the neem seeds) was mixed with wheat grains at 5% by weight and the seed stored in small glass jars for one year; the damage from C. oryzae to the treated seed was 2.9 - 9.4% in comparison with 50% in the control (Pruthi,

1937). Fry (1938) found that mixing.of neem seed dust in stored grains was an effective method to prevent the damage of Ephestia cautella;

Erishnamurti and Rao (1950) showed that putting of neem leaves in storage receptacles minimised the damage of different stored grain insects, but the practice could not be depended upon for full protection of grains.

The deterrent effect of neem on the feeding of locusts has been observed by a number of workers (Hussain and Mathur, 1936; Volkonsky,

1937 a, b; Roonval, 1938, 1953; Bhatia, 1940; Hussain et al., 1946;

Chauvin, 1946).

Some Recent Research Work in India

In preliminary laboratory tests, Cherian and Gopalamenon (1944) found neem applications to be an effective feeding deterrent against: 197.

Eupterote mollifera, Achoea janata, Plusia peponis, Atteva fabricella, pilachna spp., Aphis Lpssypii, Aphis malvas, Urentius echinas, Saissetia nigra. A high mortality of the test insects, i.e., 70-98%, was noted in many cases over a period of 2-4 days. The formulations were sprayed as oil emulsions, or water extracts of seed at rates from 0.01 to 1.0%, upon a variety of plants to run off.

According to Trehan (1946), Neem (A.indica) formulations possessed appreciable contact toxicity to Lipaphis erysimi, and extracts of the allied species of neem, M. azedarach had promising contact insecticidal property against the lar:vae of Pieris brassicae and L. erysimi, and adults of Aulacophora foveicollis and Bavada pieta.

Further testing of the extracts of the drupes Pf M. azedarach in alcohol, petroleum ether, or water, was carried out against the larvae of P. brassicae by Atwal and Pajni (1964). The larvae, kept in glass jars, were fed on cabbage leaves sprayed with a 'flit' sprayer. The food was replaced every 24 hours. A mortality of 78.3, 4.26, and 3.8% was observed after 96 hours to sprays containing 10% alcohol, 10% petroleum ether or 3.9% aqueous extracts, respectively. Dusting the leaves with

100% drupe dust killed 60% of the larvae. Prevention of feeding on the treated leaves was also observed.

The hairy caterpillar, Amsacta moorei, did not feed on crops sprayed with formulations containing 1-10 gm neem kernel paste to one litre of water; some cutting of shoots and petioles was noticed but the caterpillars did not eat the cut parts of the plants, (Patel et al., 1968).

Babu and Beri (1969) noted a similar deterrent action of neem sprays against the larvae of Euproctis lunata.

The mixing of kernel dust in stored grain at 1-2% by wt, protected them from the damage of certain Coleopterous insect pests, 198.

namely, Trogoderma granarium, Lhimplrtha dominica, Sitophilus uzzae,

Calloso bruchus maculatus, for 9 to 21 months (Jotwani and Sircar, 1965,

1967).

The spraying of crops with 0.1% neem kernel suspension at 0.1% at 100 gallons per acre is recommended by Pradhan et al., (1962); in their laboratory trials foliar sprays of 0.001% suspension deterred feeding of

Schistocerca on the treated plants. The protection under field conditions lasted for two weeks. However, the solvent extracts of neem seed and its pure products, i.e., nimbidin-T and nimbix (Siddiqui, 1942; Siddiqui and

Mitra, 1945) were found to be less effective as. a feeding deterrent to lucusts than the aqueous extracts of seed kernels (Pradhan et al., 1963).

The activity of neem was further investigated by a number of students at the Indian Agricultural Research Institute, as part of their theses; Sinha (1960), Deshpande (1967), Mane (1968), and Babu (1969).

The insect species to which neem applications proved deterrent and prevented d.mage included:-

Lepidoptera - Euproctis lunata, Lephygma eximua, Prodenia litura,

Agrotis xypsilon, Utetheisa pulchella, Diacrisea

obliques.

Coleoptera - Rhizopertha dominica, Trogoderma granarium,

Callosobruchus maculatus, Sitophilus oryzae.

Orthoptera - Schistocerca gregaria, Locusta migratoria migratoriodes,

Acrida exaltata.

The present status of the work has been reviewed by Pradhan and Jotwani, 1968. Neer is stated to be an excellent feeding deterrent against locusts, a good protectant against stored grain pests, and mildly insecticidal to some other phytophagous insects.

199.

The experimental results discussed above bring out the following points:-

(i)Neem formulations, notably seed dusts and simple aqueous extracts

(prepared by grinding the seeds and 'sieving the aqueous mixture through

a muslin cloth), affected many insect species. Mortalities of 25 -

100% over a period of 2'-96 hours with concentrations of up to 10%

sprayed to run off over a variety of plants were recorded by several

research workers. Mixing of seed dusts with stored grains up to

20%, w/w, protected them substantially from the damage of several

Coleopterous and Lepidopterous pests for periods of 9-21 months.

The active chemical(s) was a very good deterrent against Schistocerca;

high volume sprays of aqueous kernel suspension at a concentration of

0.001% deterred locusts from feeding on treated plants under

laboratory conditions.

The mortality of test insects was attributed to starvation,

though some workers noticed 'mild contact insecticidal' property.

(ii)The majority of test insect species belonged to Lepidoptera and

Coleoptera. Probably, because neem was considered mainly an anti-

feedont, more chewing and biting species of insects were tested, in

general. However, some examples of Hemiptera, including aphids

brassicae, A. ossypii, A. malvas), the painted bug, Bagrada

and the Coccid, 1„ liams, have also been reported in these . experiments. There are no test species reported from Hymenoptera

or Diptera.

Contribution of Present Investigations

In the present investigations, the larvae of several species, including Diptera, failed to develop to maturity when reared on a diet containing neem products. Larvae of Musca and Stomoxvs did not develop 200.

on food mediums mixed with 2-4% seed dust by weight; a reduction of larval size and loss in larval weight was observed. Anopheles larvae reared in medium contaminated with neem products at concentrations of

0.001 to 0.1% died before pupation. At higher concentrations a complete mortality was observed within 48 hours. /bung Dysdercus larvae fed on cotton seeds soaked in neem preparation also died within 48 hours.

Similarly larvae of Pieris Trialearodes and Plutella fed on foliage sprayed with concentrations of 0.0t to 1.0% died within a week of confinement on treated leaves.

The simplest explanation of this failure to develop is that it was due to starvation caused by the feeding deterrent action of neem

extracts. But there are also certain indications of another, more indirect action. Larvae of Pieris fed on neem treated foliage for 48 hours and then transferred to clean food did not recover fully. They either failed to develop further or died during moulting. In some instances, malformed pupae were produced and in others emergence of adult butterflies was delayed for as long as two months compared with a normal pupation period of 10-14 days. These are not effects which can be explained by starvation. Eggs of Trialearodes on cabbage leaves treated with 1% kernel suspension spray did not hatch properly. Many of the larvae died in their egg shell in a semi-hatched position, with the larvae lying in partially emerged condition in their shells. When eggs of Stomoxys were incubated on filter papers moistened with 0.01% aqueous azadirachtin, egg-hatch was completely suppressed. Adult insects, however, did not seem to suffer serious ill effects.

The physiological reasons for the death of larvae are not clear.

Some interference in the growth regulating mechanism seems implicit; the 201.

effects are maximal to young stages. The larvae seem also to have some

difficulty in moulting indicating possible activity of neem on the

ecdysone-sensitive system.

A hormone-like growth disrupting property of neem might also, to some extent, explain the'mild contact insecticidal' effects of neem sprays observed by some earlier workers. Apparently, the feeding of the contaminated foliage was not prevented completely (otherwise the results could be attributed to starvation rather than insecticidal activity).

The mortality observations were recorded usually over 48-96 hours, during which period, hormonal .effects following ingestion of sprayed vegetation might have started to operate, resulting in partial mortality of test insects. When observations were made over longer periods, t.g., stored grain pests, almost complete mortality of the test population was recorded over periods longer than nine months.

Some adults appear to be less sensitive to the toxic effects of neem than larvae.

In the present experiments, adult Musca accepted a diet of sucrose containing 10% of neem seed dust without suffering ill effects; the insects bred normally and laid viable eggs. Adult Schistocerca were fed regularly on food treated. with neem products and nomortality was observed. In preliminary experiments against M. Ealtaaa, the aphids were not inhibited from feeding on artificial medium containing 0.1% neem kernel susp. or 0.01% azadirachtin. The aphids also infested the plants treated with neem, both under laboratory and field conditions, and could apparently feed on contaminated cell sap.

Further evidence of hormonal activity of neem products is indicated by the American experiments on extracts of Melia azedarach, an allied species of neem. 202.

Experiments on Extracts of M. azedarach

'The Chinaberry tree, Melia azedarach Linn., was introduced to the United States from Asia. It, grows abundantly and rapidly along roadways in certain localities and is, apparently, undesirable to insects since few, if any, species seem to feed on it.' These observations and that substances repellent and toxic to some insects are present in chinaberry are made in the reports of Mclndoo (1945) and

Jacobson (1958). Accordingly, McMillian and Starks (1966) bio-assayed leaf extracts of chinaberry in studies of host plant resistence to corn earworm, Neliothis zea (Boddie) and the fall army-worm, Spodoptera frugiperda (J.E.Smith). They found chinaberry extracts no more phago- stimulatory than sucrose, but noted some larval mortality of test insects.

In a follow up study (McMillian et al., 1969), the effects of chloroform extracts of chinaberry were studied, mixed at doses of 0.03 to 30.0 mg equivalent/gm in an artificial diet or applied with a chromatographic sprayer to corn seedlings grown in the greenhouse. All larvae of Heliothus and Spodoptera died after feeding on a diet containing 30 mg equivalents of chinaberry extract/g diet. Also, all corn earworm larvae and 90(10 of the fall army-worm larvae died after feeding on a diet containing 10 mg equivalents; though the remaining 10 of the fall army-worm larvae pupated, no adults emerged. Pupal mortality in control tests was negligible. The incorporation of the 3 mg equivalents caused considerable- mortality in corn earworm larvae, but little mortality in fall army-worm larvae compared with the check. Also days to pupation and emergence of both insects increased, and adults fed the intermediate concentration often emerged deformed.

When fall army-worm larvae were fed on corn seedlings treated with dilutions of a chinaberry extract, a visual rating of 8-day leaf 203.

feeding by larvae indicated no statistically significant differences

at the 5% level of confidence. The larval weight difference was,

however, significant at 5% level, being 4.3 mg/larva in 5.0 gm

equivalent/plant to 7.83 in the check. No mortality of larvae was observed in this treatment.

In these results there is, however, no mention of the systemic activity of the extracts. The aqueous extracts proved comparatively ineffective in preventing feeding of test larvae on the sprayed plants, while solvent extracts were phytotoxic. In the circumstances, the information obtained would not look very promising from the practical application point of view, though it added Melia to the list of plants possessing growth retarding property. But the present results on

A. indica add substantially to the knowledge of the possible mode of action of neem extracts and suggest they may have a wider field of potential practical value.

Activity of Neem against Nematodes

The control of plant parasitic nematodes with organic amendments has been reviewed by Singh and Sitaramaiah, 1970, It has been shown that (1) application of leaves of neem to pot cultures at

5-10%, w/w, reduced the incidence of root-knot on tomatoes and okra by

50% and afforded satisfactory control of the disease; (ii) oilcake of neem applied at 1,600 lb/acre resulted in consistent and significant control of root-knots; once application was adequate; (iii) neem cake gave the best additional cash return. Applications of neem cake also reduced the population of fungi and stylet bearing nematodes around the roots of some fruit trees (Mobin and Khan, 19gq).

The above observations support the present finding of 50% reduction in the incidence of Pratylenchus spp. on barley seedlings dipped 204.

in 1.0% aqueous suspension of neem kernels for 2 hours and then grown for 20 days in a naturally infected field. .However, the reference to the effects of neem applications in the former experiments was incidental only, as the main purpose of the research was to study the effect of

'organic content' of soil on nematodes rather than that of neem applica tion. This is evident from the large quantity (1,600 lbs) of neem leaves used per acre (and not neem seeds), and neem cake constituted one of the treatments in tests of several oil cakes. The present results are, therefore, significant in suggesting a possible use of neem formulations against certain nematodes in crop protection. Moreover, the reduction of nematode incidence took place as a result of dipping of seedlings in neem solution, so the effect must have been systemic.

This is interesting for seedling dips and seed soaks are a much more practical proposition than the use of oil cakes and leaf compost of neem.

The effect of neem on nematodes has been studied further by

Singh (1971). (The research was undertaken at the Imperial College at the author's suggestion as part of an M.Sc. Thesis.) The motility of nematode species of Ditylenchus depsaci and Pratylenchus spp. was affected in a 1,000 ppm aqueous solution of neem; the nematodes did not move and infectivity in the presence of neem was nil. There was 57% mortality among nematodes kept in 10% suspension of neem kernel for 24 hours. The loss of motility was irreversible as many nematodes trans-- ferred to clean media failed to regain normal mode of movement. This observation suggests that the physiological effect of neem on test nematodes species may include effects other than starvation only.

Miscellaneous Possible Effects of Nees on Fungi,' Bacteria, Mites and Certain Mammalian Pests.

The comparative resistence of neem timber to wood—rot has been mentioned previously. Reduction in fungal population was observed by 205.

Mobin and Khan (1964) by the application of neem oil cakes around the

roots of fruit trees. In very preliminary experiments in the present

series, reduction in the infectivity of Botrytis favae by 66.6 with

systemic application of neem at 5,000 ppm to soil weight was observed

on young bean plants; there was an irregularity of growth of Fusarium

on agar medium treated with neem formulations.

Antiseptic properties of neem are mentioned in the Indian

Pharmacopoeia. An infusion of neem leaves is used for washing of wounds and cooked neem leaves are used as a poultice. Randahawa (1968) reported control of bacterial disease of citrus canker through the application of neem. Neem products may, therefore, be tested further for the control of certain bacterial and fungal plant diseases. These organisms, however, differ widely in susceptibility to toxicants.

A decoction of neem leaves is used for the cure of the skin disease of mange in dogs caused by an arachnidan parasite. The use of neem proved effective against Tetranychus telarius (Cherian and

Gopalamenon, 1944).

The mouse, Apodemus, discriminated between neem treated and non-treated food pellets and did not accept the treated food during the first 24 hours. Rats may, therefore, be put off the treated food under a situation involving choice of food. Cattle, such as buffaloes and cows, do not normally eat neem leaves and cakes.

These observations have been included as some of them might prove useful under peasant farming conditions, e.g., stray cattle may be put off feeding by border application of unfenced crops, and soaking of rat burrows with neem soaks may force the rats to abandon nests at some critical stage, such as a breeding season.

In summary, the present investigations have extended the spectrum of activity of the active chemicals of neem to include new insect species, notably the Dipterous larvae of Stomoxyq, Musca and 206.

Anopheles, and the white fly, Trialearodes. These have also shown that neem products possessed a possible hormone-like growth disrupting activity against Pieris larvae. The neem chemical(s) penetrated the egg-shells of Stomoxys to affect the embryonic development.

It may be, therefore, of interest to test the effects of regular sprays of neem on the pest complex of field crops, such as cotton, rice and sugar cane, because such a practice may disrupt the development of larval stages of several pests. Such studies should preferably be preceded by investigator l of the effects of neem on individual pest species under controlled conditions. CONCLUDING REMARKS

AND

SUIDIARY 208.

CONCLUDING Rai/IRKS

The application of suitable insecticides to crops constitutes the main method of pest management. Such a use of toxic chemicals is, however, fraught with the dangers of environmental pollution and upsetting of the natural balance. To exercise selective control, feeding deterrents may be employed as alternatives to pesticides. Against certain highly gregarious pest species, e.g., Schistocerca, the use of rejectants, in conjunction with insecticides, is likely to afford greater protection of crops. The application of insecticides would kill locusts while the use of rejectants would prevent their feeding.

In the work presented in this thesis, out of the 28 compounds tested for rejectant action against Schistocerca, 22 chemicals deterred feeding to a varying degree. It has been pointed out that to be effective an anti-feedant has to be persistent and systemic, otherwise selective feeding on the new plant growth will occur. Three of the effective chemicals - TD-5032, 2,4, 6 - trichlorophenoxy acetic acid, and Phosphon - are known to possess limited systemic activity. However, at the same time, having plant hormone activity, they would retard plant growth and could be used under certain conditions only. Moreover, they are too expensive and not sufficiently active as deterrents to be used for general prophylactic spraying of crops. The neem derivatives, which were more than a thousand times more potent as deterrents than the other chemicals, were therefore the only candidates to be studied further.

Two 'Newt Properties of Neem

Further studies of neem products revealed two new aspects of

their activity. 209.

(i)The systemic activity of neem derivatives is reported for the

first time in this Thesis. Soil applied neem formulations were

readily taken up by the roots of several plant species including

woody ones and translocated throughout the aerial parts, to

inhibit feeding of Schistocerca. Foliar sprays applied to upper,

lower, or one-side of potted bean plants protected the unsprayed

parts against locust attack, indicating translocation presumably

through both xylem and phloem vessels. The active chemical proved,

stable under different environmental conditions of rain and soil,

and persisted, in general, for 2-3 weeks, applied as foliar•

applications of high volume at 0.1‘A concentration to beans, or as

soil applications of 50-100 kg equivalent rate per acre; higher

applications protected bean plants up to 8 weeks against Schistocerca.

(ii)The neem products possessed growth disrupting property.

Pieris larvae fed for 48 hours on foliage treated with neem products

were subsequently reared on clean food; they failed to develop to

maturity and most of them died while moulting. Malformation in

pupae and prolongation of pupal period up to 50 days as against a

normal period of two weeks was observed. Eggs of Stomoxys incubated

on filter papers moistened with azadirachtin did not hatch properly;

the young larvae died without fully emerging out of the egg-shells.

The same effect was observed in the case of Trilearodes eggs sprayed

with neem formulations.

As a result of the above findings, further development of neem products in pest control should receive a new impetus and the use of crude extracts of neem may eventually be adopted on a large scale by 'the village farmers in Asia and Africa. For example, the deterrent action of neem 210.

preparations against Schistocerca has been known for a long time, but little practical use has been made of this knowledge in actual protection of crops, so far. The lack of interest may be attributed to two reasons. Firstly, enough information was not available on the behaviour of neem applications on plants and in soil; the information was frag- mentary and sometimes contradictory in nature. Secondly, spraying required water which is not easily available in semi-arid areas. Now much more information is available on the persistence and action of neem application and systemic soil applications should obviate the difficulties of finding water for spraying purposes. Since locusts usually follow rains, adequate soil moisture would generally be available before invasion for the translocation of the active chemical within the plants.

Neem was considered to be mainly a feeding deterrent and the mortality of the test species was presumed due to starvation. Although a high 'contact' mortality of test species was claimed in some of the experiments, the performance of neem applications was not generally so spectacular. The partial prevention of feeding of the treated crops would not carry much conviction with the farmers. However, the finding of the growth disrupting activity of neem brings in a new factor for the evaluation of neem applications. Further investigations of this property might make the use of neem more worth while.

It has been shown that the larvae of Musca and Anouheles, two important vectors in Tndia of cholera and malaria, respectively, failed to reach maturity in food mediums containing neem extracts. There might be some link between these findings and the general belief that the villages with preponderance of neem trees have lower incidence of cholera and malaria. The berries of neem ripen and fall to the ground during 211. the monsoon period, and are likely to be washed away by torrential rains into ponds and also over rubbish heaps, which are located in pits in low lying areas. The stagnant waters of ponds and the rubbish heap constitute the main breeding grounds of the flies and the mosquitoes. The neem products might, thus, affect the multiplication of these insect vectors resulting in the reduction of the incidence of the diseases. However, the hypothesis, though a valuable lead to follow, needs experimental proof through further research.

Chemical Constituents of Neem

Because of its medicinal properties, interest in the chemical investigation of the active principle of neem in India dates back to

1880-90; these efforts seem to have culminated in the isolation of three products from neem oil reported by SiddiquL, 1942. The total

'bitters', Nimbidin-T constituted 2% of the oil. Further purification yielded two crystalline products Nimbin (Ci8113808), and Nimbinin

(C7111003)n and an amorphous Nimbidin (C-68.5, H-7.8, and S-7%).

Other chemical, pharmacological and toxicological data on these and other products isolated from neem oil are reviewed by Mitra, 1963.

The products were, however, found less effective than kernel aqueous extracts for inhibiting feeding of the locusts (Pradhan et al., 1963).

The isolation of another product from the seed of M. azedarach was reported by Lavie et al., 1967. Meliantriol, a triterpenoii alcohol, had a marked deterrent effect against locusts.

Azadirachtin (C H 0 c) a new type of highly oxidised 35 44 lo 9 triterpenoid compound (Butterworth and Morgan, 1968 and 1971), appears to be the most potent of the products in preventing feeding of.

Schistocerca. However, on the available evidence, azadirachtin is 212.

either relatively specific to Schistocerca, or other active chemical

compounds are present in the neem seed. For instance, the ED values 50 of inhibition of feeding of Schistocerca for the crude aqueous extract 2 and azadirachtin are 19.0 and 0.036 ng/cm 'relative potency of 530.

Against Pieris, a spray application of 1.0% crude extract inhibited

feeding of larvae completely, while 0.1% azadirachtin inhibited feeding

partially only. In fact, Butterworth and Morgan (1971) have reported

azadirachtin as relatively ineffective against Pieris.

The possibility of the presence of other compounds is also

suggested by the results of the experiments of MacMillian et al., 1969)

and Atwal and Pajni (1964), who have reported that alcohol extracts of

M. azedarach were more effective than aqueous extracts; While Pradhan

et (1963) found the aqueous extracts of A. indica more potent than

alcohol extracts. Both extracts, however, possessed similar

properties of inhibiting feeding of locusts and affecting the behaviour

of other insects. Thus, the extracts mny contain two separate,

possibly related chemicals, one mainly water soluble and the other

alcohol soluble. It will be of interest if the alcohol and aqueous

extracts of Melia also possessed systemic property.

This wider appreciation of the potentials of neem products is

likely to stimulate further investigation of the chemical structure of

azadirachtin and other possible active compounds which may be isolated from Neem and Melia, and also lead to studies of analogues-which may be

yet more potent as has been the case with Pyrethrins.

There are some apparent inconsistencies in the reported results

regarding the performance of the neem extracts. For example,, the

protective effect against Schistocerca of 0.1% kernel water extract 213.

sprayed to run off upon several crops, including maize, lasted for about 15 days under field conditions (Pradhan et al., 1962), a result which is consistent with those obtained in the present work. In contrast, Venkatesh et al., (1970) reported the effect of some concentration of sprays to last for three days only. Normal feeding of V stage hoppers of Schistocerca was resumed on the sprayed foliage after one week. In the latter case, the tests were carried out under the dry conditions of the Rajasthan State and the waxy nature of the leaves of Pennisetum might have caused most of the spray material to bounce off the leaves; the contamination of the soil with the spilled spray was, perhaps, not adequate for systemic protection, which also needs a reasonable level of soil moisture for adequate translocation. In the latter case, the criterion of faecal pellets voided by the two sets of locusts as an index of feeding was used; this may not be a sufficiently sensitive technique under certain conditions (c.f. Ellis and Ashall, 1957). :There is, thus, a need to standardise the testing techniques for feeding deterrents. One improvement which may be suggested is that a pure chemical, such as azadirachtin should be included in the tests to provide a reference for the comparison of unpure extracts which may vary from one laboratory to another.

TOaiClt~ of Neem to Mam als

As far as is known, neem products are non-toxic to human beings.

Neem preparations have been, and are still extensively, consumed for medicinal use in India. Pregnant women are reported to consume neem oil. Neem preparations are also administered to infants against worms, and scalp applications are made to contol vermin. 214.

In studies with sodium nimbidinate (a pure chemical from neem oil) Bhide et al., (1958) reported an oral acute ID of 1,000 mg/kg 50 in rats and 750 mg/kg in mice; feeding of 50 mg/kg over 10 days did not produce chronic toxicity. Administration of 1,000 mg intra- musdular.and 7,000 mg orally to human males produced no local or general side effects. however, more studies under controlled conditions would be necessary to investigate further the toxicological aspects of neem intakes before a wide-spread use of neem products can be recommended.

Possibility of Control of 'Internal Borers' of Crops

As has been suggested elsewhere, that feeding deterrents may . be of little use because the pest insects will move away to adjacent untreated crops, but there are some examples of possible control where movement away is unlikely, to be important. Applications of neem may prove potentially useful for the control of internal borers of crops.

For example, neem applications may affect the borers in three ways:

(i) preventing entry into the plant, (ii) affecting development inside the plant, resulting in failure to reach maturity, and (iii) delaying the entry into the plant and so facilitating control by conventional insecticide.

According to previous experience, the application of systemic chemicals has not been a great success against the tissue borers of crops, and actual field trials are needed to justify the above contaions. Some evidence of the effectiveness of neem applications as a general treatment is available from the results of the experiments of fertiliser use of oil cakes (Sinha, 1960; Sinha and Gulati, 196$).

In applications of neem cakes (residue left after extraction of. oil) to sugar-cane, rice, and cotton crops, it is reported that many field 215.

workers had observed an 'improvement in quality' attributed to the

'supposed' pesticidal properties of neem, in addition to increased yields. The fertiliser effects of neem in the these experiments were compared with those of other oil cakes and a standard chemical fertiliser.. Apparently, the improvement in quality in the case of neem treatments was not due to the addition of plant nutrients but was due to some peculiar property of neem. The difference was also highly marked to attract attention and commend mention in the research reports, despite lack of a valid explanation.

One of the main reasons for deterioration of quality of crop yields is the attack by insect pests. In crops like sugar-cane, rice and cotton, several pest species, including internal borers, leaf- hoppers, white flies, are known to affect seriously the quality and quantity of yield. The failure of the larval stages of several insects on media containing neem has been shown in the present findings.

Systemic uptake of neem has also been demonstrated.in several plant species, including, rice, cotton, andsugar-cane.

It is, therefore, suggested that the improvement in quality in these general 'fertiliser' experiments was probably caused by •- a reduction in.pest incidence, including internal borers. This explanation is further supported by the observations of Randhawa (1968), who reported control of leaf-miner of citrus with neem.

Hate of Application

In the present experiments, soil application of neem seed dust to potted beans was detected by tests with Schistocerca at a rate of one kg equivalent per acre but a dose of 100 kg per acre was necessary for adequate protection under field conditions. Higher doses may be required for tall crops. 216.

The present recovery of azadirachtin from neem seed is 0.75

gin/kg ti : 1333). The usual application rate for pesticides of 1-2 lbs per acre of active ingredient will come to 1-1 ton of seed of neem.

Researches, therefore, may not be put off by the seemingly high rates in terms of seed, which may be necessary in some cases. In view of the prospective usefulness of the chemical, industry is likely to develop isolation techniques and possibly other competitive analogues.

The application of neem oil cakes might also yield equally useful results.

Foliar application of 1% seed and 0.1% kernel suspension at about 100 gallons per acre protected bean plants against the attack of

Schistocerca for three weeks. The active factor was translocated

Idthin the plants in all directions, including downwards, in laboratory experiments. In the ease of tall crops with larger leaf area, the uptake via the foliar route may be more economical in terms of dose per acre. The shell or husk of the fruit contains a mucilaginous substance and the kernel has oill making the water extract a stable emulsion. Thus a suspension of ground kernels provides a good formulation which can be prepared locally by farmers.

A 'Back-yard' Pesticide

The growth retarding activity of neem and its systemic uptake in plants may be helpful in reducing pest incidence substantially through regular prophylactic spraying of crops. The systemic property would also help to make a fuller use of the remarkable anti-feeding property of neem, against predicted attacks by Schistocerca.

Neem products may, therefore, provide a readily available, inexpensive form of crop protection to the peasant farmers without the 217.

need for the use of sophisticated methods of application. The neem

tree improves soil fertility (Radswanski, 1969), and provides shade,

timber, and fuel wood, besides preventing soil erosion and bringing

about a general improvement of the environment. Thus, the tree,

which has been described in medical terms as a 'village dispensary'

may prove to be a 'plant-health dispensary', as well. According to

one estimate, there are 25 million neem trees in India and 200,000 tons

of seed goes waste every year. Apparently, there is no problem of

supplies.

However, much more research work is necessary to understand and develop the potentials of neem fully. Quantitative methods of analysis are required to study in detail the systemic behaviour and other activities of the chemical. The nature of the growth disturbing,

possibly 'hormone-like' effects needs to be determined further.

Information on the chemical constitution of the active ingredient and further isolation work on other constituents is another long term prospect. Toxicological studies are called for before extensive use can be recommended. Laboratory and field trials should be carried out to know more about the spectrum of activity against different pest species, and uptake and persistence in different plants. The present findings may, perhaps, serve as a valuable lead to a whole new system of pest management. 218.

SUMMARY

1. A technique for the biological assay of anti-feeding compounds was developed using simple feeding tests. Known quantities of the test chemicals were applied uniformly to sucrose impregnated filter papers.

Treated and control papers were then fed to 150-200 adult Schistocerca for a specified period in a food choice experiment. The amount of each filter paper eaten by the locusts was determined gravimetrically, and from the results, an index of inhibition of feeding relative to standard sucrose papers was calculated. The results obtained by this method were more consistent than those of feeding tests carried out by confining small batches of insects to single treated filter papers in individual cages.

An alternative bio-assay method for the rapid evaluation of acceptable and non-acceptable chemicals to Schistocerca by stimulating the taste receptors on mouth-parts, notably the palps, with chemical films mounted on a wire-loop has also been described.

2. A primary evaluation of 28 potential deterrents, including neem derivatives, alkaloids, saponins, organotins, triazenes, plant growth regulants, carbamates, and miscellaneous insect repellents was carried out using the 'multiple-choice' method described above, of feeding treated sugared filter paper discs to hungry adult Schistocerca. Each chemical was tested at four dose levels, i.e., 158, 15.8, 1.58, and 0.158 ug/cm2, applied to paper discs sugared with 0.1M sucrose solution, with three replications per treatment. 2 Compared at 15.8 ug/cm dosage, ten chemicals (azadirachtin, alcohol extract of neem, aqueous extract of neem, gramine, veratrine, 219.

Brestan, Tinicide, TD-5032, carbaryl, and mercuric chloride) inhibited feeding (by Schistocerca) to the extent of 76 to 100 inhibition, seven chemicals (hyoscyamine, lobeline, diosgenin, stannous chloride, phosphon, phenoxy acetic acid, indalane) to the extent of 26.75% and five chemicals (hrdnine, lupinine, digitonin, copper stearate, copper resinate) to the extent of 1-25p. The remaining six chemicals (B-nine dimethyl amino-succinamic acid, CL-24,055, MGK repellent 874, Metadelphene,

Rutgers 6-12, dimethyl phthalate) stimulated feeding.

3. A secondary evaluation using the technique of feeding treated sugared (0.1M) multiple papers to Schistocerca gave the following" ED50 values of inhibition of the ten most active anti-feedants, in units of 2 ug/cm : azadirachtin 3.6 x 10-5 alcohol extract of neem 1.0 x 10-3, -2 kernel extract of neem 1.9 x 10 veratrine 0.22, carbaryl 0.28, gramine 1.58, TD-5032 4.74,,Du-ter 6.0, Brestan 6.6/4 and phenoxy acetic acid 23.6. Neem formulations were, thus, more than a thousand times more potent than the next best commercial chemical.

A number of extracts of different parts of the neem fruit were prepared, all of which inhibited feeding of Schistocerca, although the preparations based on kernels were more potent than the others. There was, thus, no advantage in using formulations based on kernels only, and the use of extracts of neem seeds is recommended.

-4 2 5. When a constant concentration, 0.8 x 10 ug/cm of azadirachtin was applied to filter papers treated with different concentrations of sucrose, i.e., 0.06, 0.12, 0.25, 0.5, and 1.0M, the amounts of filter papers eaten by adult Schistocerca were 81, 96, 136, 245, and 1,352 mg, respectively, compared with 201 mg of control papers treated with 0.1M sucrose only. The efficacy of a given dosage, therefore, decreased on 220.

the concentration of phagostimulant present. As an analogy, dosages

of deterrents might have to be adjusted to the palatibility of crops to

a given species.

6. Adult Schistocerca supplied daily with filter papers treated

with a constant level of neem became habituated to it and began eating.

However, the inhibitory effect of the neem treatment was restored when

the locusts were taken off the treated food and fed on green grass for

four days and then re-introduced to the treated food. The habituation

was, therefore, temporary.

7. Systemic anti-feeding activity was shown for soil applied neem

products absorbed through the roots of young seedlings. The uptake and

translocation of the active ingredient of neem through soil application was also confirmed for old bean plants, several different varieties of

beans, and the following other plant species: wheat, barley, rice, grass,

sugar-cane, cotton, cabbage, brussels sprouts, tomatoes, chrysanthemums,

and the spindle tree. The protection against starved locusts in general lasted for 2-3 weeks at levels of 1,000 ppm of kernel ext., w/w., applied

to the soil.

8. In paired-leaf comparisons, if one of the paired leaves of beans in upper, lower, or nearly apposite position was treated with neem, the other untreated leaf was protected, indicating translocation from one part of the plant to the other. Similarly, when only half of a leaf was immersed in a neem preparation (0.1% kernel ext.), the other half was protected. As expected, the translocation was faster when the 'open' rachis-end was dipped in a suspension than when the apical end vas dipped.

In the case of the cabbage leaves, when the apical half-side was immersed, the other half undipped portion was not protected. In this 221.

instance, the waxy surface of the leaf might have prevented penetration, and subsequent translocation.

Similarly, foliar sprays of neem applied to upper, lower, or one-side of bean plants protected the unsprayed parts from locust attack, indicating lateral andvertical translocation through both phloem and xylem vessels.

9. Seedlings of several plant species, i.e., beans, barley, rice, cotton, were protected from Schistocerca when seeds were soaked for 24 hours in neem formulations before sowing. Seedling-dips of these crops also protected the young transplants against Schistocerca for 2-3 weeks.

The young bean seedlings grown from seeds dipped in 1.0% kernel suspension for 24 hours had significantly longer roots and developed more secondary rootlets than the controls. The reason for this effect is unknown.

10. Foliar sprays of 0.1% kernel ext. and 1.0 seed extract at 10 ml/ plant protected beans from Schistocerca for over 2 weeks under laboratory and semi-field conditions.

11. Soil applications of azadirachtin, alcohol extract, and kernel suspension at the rates of 10, 100, and 1,000 ppm, w/w, to potted bean plants protected them from Schistocerca for over three weeks.

A heavy soil application of 1.0% seed dust by weight of soil protected tussocks of grass against locusts fully for six weeks and partially for 12 weeks.

12. Under field conditions, application of seed dust of neem at rate equivalent of 50 kg/acre made before seeding to dwarf broad beans, in a sandy loam gravel soil, protected the seedlings fully against Schistocerca for one week and partially for three weeks, post-germination.

The seeds took 15-18 days to germinate. 222.

A post-germination application of neem seed dust at a rate of 100 kg/cm protected the bean plants fully for three weeks.

13. Different methods of soil application of neem, i.e., (i) dust-

broadcast; (ii) side-band placement of dust in a furrow at 2.5" on both sides of the plant row; (iii) in-furrow placement of dust at 3" below the seeds, and (iv) spraying of the seed bed, were tested under semi-field conditions. The protection afforded to beans lasted for 10-12 days at

40 kg seed dust equivalent/acre. Broad-cast application of dust gave better results than the other treatments.

14. No phytotoxic effects were observed after foliar applications of concentrations up to 10(i'L of neem seed aqueous preparations to crops of beans, barley, and rice. Likewise, soil applications of 20 neem seed dust, w/w, did not produce phytotoxic effects.

15. Effects of rain on foliar and soil applications of neem were studied under simulated rainfall conditions. Foliar applications of

1.0; seed suspension to beans were washed under three inches of rain- equivalent, 6 hours following application. In feeding tests with

Schistocerca, the washed plants were fully eaten, showing that the neem deposits on the plants had been substantially removed by the rain. But the same amount of rainfall 24 hours after spraying did not diminish the protective effect of the neem spray.

Soil applications of neem at 1,000 ppm, w/w, subject to the leaching action of even 20" of rain equivalent, immediately following application, were retained in the soil in sufficient quantities to protect from Schistocerca beans transplanted to the soil after the leaching action of the rainfall. As expected, the losses of the active chemicals due to leaching were greater in sandy soils than organic, and clay soils, as indicated by the degree of protection given to the beans. 223.

16. Soil type affected the availability and persistence of neem derivatives. At a constant level of application of neem, the protection given to beans from hungry Schistocerca was less in alkaline clay and organic.. soils than in sandy soils. However, the protective effect diminished more rapidly in sandy than in organic soils, indicating that the availability of the chemical declined faster in sandy than in organic soils.

17. Experiments showed that Pieris larvae were much less sensitive to the deterrent action of neem extracts.

A spray of 1.0% kernel extract of neem, or 1.0% alcohol extract to cabbage plants inhibited the feeding of IV and V stage Pieris larvae completely during the first 24 hours after application, but sprays of

0.1% kernel ext., 0.1% alcohol ext., and 0.01% azadirachtin, and also a soil application of 1.0% seed dust, w/w, inhibited feeding of Pieris partially only. The damage was, however, markedly less during the first

2-3 days of application.

18. The larvae of several species (Pieris, Muscat Stomoxys,

Anopheles, Trialearodes) fed on diets (or in medium) containing neem

(up to 4% by weight in the diet mediums, or deposits of foliar sprays of up to 1.0%) failed to develop to maturity. The reasons for this failure could be simple starvation due to deterrent action of neem and/or some other indirect growth disrupting properties.

19. When the larvae of Pieris, fed initially for 48 hours on foliage contaminated with neem were subsequently reared on untreated cabbage leaves, they failed to regain normal growth. The pupae were malformed and the pupal period was, in some cases, prolonged up to 55 days in comparison with the normal period of 15 days. The results indicated that the intake of neem affected the physiology of the larvae in some way which interfered with the growth control mechanisms. 224.

Eggs of Trialeurodes laid on leaves of brussels sprouts sprayed with 1.0% seed suspension to the run off point either did not hatch, or the larvae died partially hatched in the egg-shells. Similarly, eggs of

Stomoxys incubated on filter papers moistened with 0.01/0 azadirachtin did not hatch. The observations suggested that the active ingredient of neem penetrated the egg-shells and affected the developing embryos.

The above results suggest that neem products may possess hormone- like growth disrupting properties. Since in many cases, moulting behaviour of the larvae appeared to be affected, the chemicals might be interfering with the normal effects ofecdysone.

20. Very preliminary experiments showed that neem preparations affected a species of nematodes, Pratylenchus, and a species of fungus,

Botrytas.

21. In a final discussion it is argued that crude neem extracts may provide a readily available, inexpensive 'grown in the backyard' form of crop protection for the peasant farmers in Asia and Africa. 225.

ACKNOWLEDGEMINFS

I wish to express my thanks, first of all, to my

Supervisor, Dr C.T.Lewis, for his helpful advice and constructive

criticism. I am indebted to Dr G.Murdie for help and advice on

statistical matters, and Mrs C.M.Black for typing the manuscript.

Thanks are also due to Drs D.Campion and J.Pollock for their

interest in this work. The receipt of samples of azadirachtin from Dr Morgan of ICeele University and of certain other compounds from Mr Griffiths of Rothamsted Experimental Station is gratefully acknowledged.

My special thanks go to Professor T.R.E.Southwood and

Dr P.T.Haskell for research facilities.

I ath grateful to the F.A.O. of the United Nations for the award of a Research Fellowship,and to the Government of India for permission to take up the Fellowship.. 226.

BIBLIOGRAPHY

1.Ahmd, M.K., Newsom, L.D., Roussel, 3.S., and Emerson., R.B., 1954. (Systemic chemicals - uptake and persistence.) J. econ. Ent., ;ID 684-91.

2.Ahmed, M., 1970. (Persistence of soil applied systemic aphicides.) Ph.D. Thesis, London University, June, 1970.

3.Akeson, W.R., Mangletz, O.R., Corz, H.F., and Hoskins, F.A., 1967. (Bioassay of stimulants and antifeedants.) J. econ. Ent., 60: 1082.

4.Al-Azawi, A.F., Norris, D.M., and Casida, J.E., 1961. J. coon. Ent., 54: 124-29.

5.Annonymous, 1965. (Environmental Pollution Panel, 1965.) President's Science Advisory Committee Report of November, 1965.

6.Ascher, K.R.S., 1964. (Antifeeding - a novel approach.) Proc Ist Int Conc. Parasito•, Rome.

7. ibid 1965. (Organo-tin antifeeding.) Proc. 2nd.W1d. ELIu. Pl. Protection, Naples, May, 1965.

8. ibid , 1971. (Pest control by chemosterilants and antifeedants.) Rev. Pest Control, 51.: 140-55. 9.Ascher, K.R.S., and Avdat, N., and Kamhi, J., 1970. (Organotin antifeeding.) Int. Pest Contro1412: 11-13,33. 10, Ascher, K.R.S., and Meisner, J., 1969. (Pennsalt TD- 5032 has systemic deterrent action.) Pft Hrankh. Pft Path PlfSchutz, 2k; 564-75.

11.Ascher, K.R.S., and Moscowitz, J., 1969. (Pennsalt TD- 5032, antifeeding properties.) Int. Pest Control, 11: 17-20.

12.Ascher, K.R.S., and Nissim, S., 1964. (Organotin antifeeding.) Wld. Rev. Pest Control, 3: 188, 211. 227.

13.Ascher, K.R.S., and Nissim, S., 1965. (Quantitative aspects of antifeeding.) Int. Pest Control, 2.: 21-24. 14.Ascher, K.R.S., and Rones, G., 1964. (Fungicide has residual effect on larvae feeding.) Int. Pest Control, 6: 6-8. 15.Asquith, P.E., 1966. (And ten other graduate students.) Selective insect control - a study prepared by graduate stdents at the Harvard Business School, MIR Management Reports, Boston, Mass; 1966, pp 81. 16.Astrakhantzev, P.1., Belagin, N.D., Bogolyubov, N.V., and Borozdina, K.L., 1937. (Chemotoxicological investigations of Melia azedarach, pp 455-57, R.A.E., 1937, p. 159. 17.Augustin, M.A., Fisk, F.W., Davidson, R.H., Lapidus, J.B., and Clearly, Ra., 1964. (Best plant selection by Mexican bean beetle.) Ann. ent. Soc. America, 127. 18.Babu, T.H., 1969. (Efficiency of neem seed solvent extracts against some insect pests.) M. Sc. Thesis, Indian agric. Res. Inst., New Delhi, 1969. 19.Babu, T.H., and Berl, Y.P., 1969. (Neem seed. solvent extracts against Euproctis lunata.) Andhra agric. J., 16: 107-11. 20.Bardner, R., 1961. (Some factors affecting systemic chemicals.) Ann. Rep. Rothem. Expt. Stn., 1960, 150-51 pp. 21.Beck, S.D., 1965. (Resistence of plants to insects.) Ann. Rev. Ent., 10: 207.

22.Benett, S.M., 1957. (Behaviour of systemic insecticides applied to plants.) Ann. Rev. Ent., 2: 279-96.

2 28 .

23.Bhasin, G.D., Roonwal, MIL., and Singh, B., 1956. (A list of insect pests of forest plants in India and adjacent countries.) Indian Forest Bull., Dehra Dun, 171: 6-9. 24.Bhide, N.K., Mehta, D.J., and Lewis, R.A., 1958. (Toxicological aspects of neem.) Indian J. med. Sci., 12: 141-46. 25.Blaney, W.M., and Chapman, R.D., 1970.* (Functions of maxillary palps in Acrididae.) Ent. ±a2.. 12: 363-76. 26.Brandish, D., 1906. (Neem timber - properties of.) 'Indian Trees', pp. 139: Archibald Constable Co., London. 27.Bullen, F.T., 1969. (Damage potential of Schistocerca.) Anti-Locust Memoir 10, 17. 44, Anti-Locust Research Centre,London. 28.Burt, P.E., Bordner, R., and Etheridge, P., 1965. (Influence of volatility and solubility on movement of systemic insecticides.) Ann. appl. Biol., 56: 411. 29.Busvine, J.R., 1971. 'A Critical Review of Techniques for Testing Insecticides', The Commonwealth Institute of Entomology, London, pp. 345. 30.Butler, C.G., Finney, D,J.I and Schiele, P., 1943. (Repellency of lime-sulphur to bees.) Ann. appl. Biol., .311: 143-50. 31.Butterworth, J.N., and Morgan, E.D., 1968. (Isolation of a chemical that suppresses feeding in locusts.) Chem. Comm., 23: 4. 32 ibid , 1971. (Investigation of the locust feeding inhibition of the seeds of nim.) J. Insect Physiol., 12: 969-77. 229.

33.Carlisle, D.B., Ellis, P.E., and Betts, E., 1965. (Influence of aromatic shrubs on sexual maturation of Schistocerca.) J. Insect Physiol., 11: 1541-58. 34.Casida, J.E., Chapman, R.K., and -Alen, T.C., 1952. (Uptake and availability of Schr.radan in plants.) J. econ. Ent., 568-78.

35.Casida, J.E., Chapman, R.I., Stahmann, M.A., and Allen, T.C., 1954. (Persistence of phosdrim.) • J. econ. Ent., Az 64-71. 36.Cela, G., 1968. CA 6830, tin fungicide; data sheet on Decafentin - cited by Ascher,

1971. 37.Chao, S.T., 1950. (Seed dips with Sch::.radan.) Nature, London., 166: 909-10. 38.Chapman, R.F., 1966. (Mouthparts of Xenocheila zarudnyi.) J. Zool., 148: 277-88.

39.Chauvin, R., 1946. (Deterrent chemicals from Melia azedarach for locusts.) c.r. Acad. Sci. Paris., 222: 412-14. 40.Chauvin, R., 1952. (Elder pith discs for testing feeding stimulants for Colorado beetle.) Ann. Epiphyt., .2: 33. 41.Cherian, M.C., and 0opalamenon, E.R., 1944. (Neem oil emulsions for the control of insect pests.) Madras agric. J., m: 10-13. 42.Chopra, R., 1928. (Neem formulation as contact poison against larvae of weevil on lucern.) au. Dept. agria. Punjab, 1925-26, pp. 67-125, Lahore, 1928. 43.Chopra, R.M., 1958. (Therapeutic properties of neem.) 'Indigenous drugs, of India', 2nd. Edn., pp. 260, Dhur, Calcutta. 230.

44.Crafts, A.S., 1961.

'Translocation in Plants', Holt, Rinehar, & Winston, New York, pp. 182. 45.Dadd, R.H., 1963. (Feeding behaviour and nutrition in grasshoppers and locusts.) 'Advances in Insect Physiology', 1: 47-107, Academic Press. 46.David, W.A.L., 1951. (Uptake of systemic chemical, SchTradan.) Ann. aul. Biol., : 508-24. 47. ibid , 1952. (Uptake of Schradan.) Ann. appl. Biol., 22: 203-10. 48.David, W.A.L., and Gardiner, B.O.C., 1951. (Sodium nuoroacetate (1_ too toxic as a systemic.) Ann. appl. Biol., 39: 91-110. 49. ibid 1955. (Seed and seedling soaks with systemics.) Ann. appl. Biol., AI: 594-614. 50.Deshpande, A.D., 1967. (Neem seed as a protectant against storage pests.) M.Sc. Thesis, Indian agric. Res. Inst., New Delhi. 51.Dethier, V.G., 1947. 'Chemical Insect Attractants and Repellents', McGrawhill, New York, pp 289. 52. ibid 1947,b. (Chemical stimulation of ovipositor of Hymenopterous parasites.) J. exntl. Zool., 112: 199-208. 53. ibid 1950. (Central summation from chemo-receptors.) Fed. Proc., 31-32. 54. ibid 1953. In 'Insect Physiology' (ed. Roeder, LB.), chapter on 'Contact Chemoreception', Wileys, London & 231.

55.Dethier, V.G., 1954. (Mode of feeding: orientation, biting, swallowing.) Evolution, 8: 33. 56. ibid 1956. (Problems associated with repellency.) Ann. Rev. Entomol., 1: 181.

57. ibid , 1966. In 'Insect Behaviour' (Ed. Southwood, T.R.E.), Ch. on 'Feeding Behaviour', Royal Ento: Society, London, Symposium no. 3, pp. 46-58. 58. ibid 1967. In 'Handbook of Physiology', See. 6, 1: 79 (Ed Code, C.F.). Amer. Physiol. Soc., Washington, D.C.

59. ibid , 1970. (Some general considerations of insect responses to the chemicals in food plants.) In 'Control of Insect Behaviour by Natural Products', (Eds. Wood, D.L., Sikprstein, R.M., and Nakajima, M.), Academic Press, New York. 60.Dethier, V.G., and Chadwick, L.E., 1947. (Rejection thresholds, alcohols: blowflies.) J. Gen. Physiol., 30: 247-53. 61.Dethier, V.G., Browne, L.B., and Smith, C.N., 1960. (Proposed the term feeding deterrent.) J. econ. Ent., OL 134. 62.Ellis, P.E., and Ashall, C., 1957. (Field studies on diurnal behaviour of Schistocerca.) Anti-Locust Bull. no 25, pp. 94; Anti-Locust Research Centre, London.

63.Ellis, P.E., Carlisle, D.B., and Osborne, D.J., 1965. (Desert Locusts: sexusl maturation delayed by feeding on

senescent vegetation.) Science, 1111: 546-47. 232.

64.Findlay, J.B.R., 1968. (The use of anti-feeding compounds as protectants against damage by pests.) M.Sc. Thesis, University of,Pretoria, S. Africa, May, 1968. 65.Finlayson, D.G., and MacCarthy, H.R., 1965. (The movement and persistence of insecticides in plant tissues.)

Residue Rev., 2: '114-52. 66.Finney, D.J., 1964. 'Probit Analysis', Cambridge Univ. Press, pp. 218. 67.Fraenkel, G.W., and Gunn, D.L., 1940. 'The Orientation of Animals', Oxford University Press. 68.Frazer, A.C., 1965. (Proposed the term 'rejectant') Pept. of Res. Committee on Toxic Chemicals; Agri. Res. Council, London, 1964. 69.Frings, H., and Frings, M., 1949. (Loci of contact chemoreceptors.) Amer. Midland Naturalist, 41: 602-58. 70.Georghiou„ G.P., and Metcalf, R.L., 1962. (Phenylcarbamates feeding deterrent to salt marsh caterpillar.) J. econ. Ent., 51: 125. 71.Getzin, L.W., and Chapman, R. K., 1960. (The fate of phorate in soils.) J. econ. Ent., 151: 47. 72.Getzin, L.W., and Rosefield, I., 1966. (Persistence of diazinon and zinophos in soils.) J. econ. Ent., a: 512. 73.Gilbert, B.L., Barker, G.E., and Norris, D.M., 1967. (Uptake and persistence of systemic chemicals.) J. Insect Physiol., 12: 1453-59. 74.Gilbert, B.L., and Norris, D.M., 1968. (Systemic uptake.) J. Insect Physiol., 14: 1063-8. 75.Gill, J.S., 1971. (Possible uses of neem in pest management.) FAO Desert Locust Tech. Series, Rome (under print). 233.

76.Gill, J.S., and Lewis, C.T., 1971. (Systemic action of neem.) Nature, London, 232: 402. 77.Goodhue, D., 1963. (Feeding stimulants required by polyphagous insect, Schistocerca.) Nature, London, !: 403-6. 78.Goodman, R.N., 1962. (Impact of anti-biotics upon plant disease control.) Advan. Pest Control Res., 5.: 4-46.

79.Griffiths, D.C., 1969. (Laboratory tests of deterrents against Agriotes spp.) Proc. ja Br. Insectic. Fungic. Conf., 1969, 398-405. 80.Griffiths, D.C., Raw, F., and Lofty, J.H., 1967. (Effects on soil fauna of insecticides.) Ann. appl. Biol., 60: 479. 81.Guerra, A.A., and Shaver, T.N., 1968. (Laboratory evaluation of feeding stimulants.) J.econ. Rat., 61: 1398. 82.Halimie, 1970. (Factors affecting uptake of selected systemic chemicals.) Ph. D. Thesis, Univ. of Wales, Bangor, July, 1970.

83.Haskell, P.T., 1970. (The hungry locust.) Science, 6: 61-67. 84.Haskell P.T., and Nordue(Luntz), A.J., 1970. (Role of mouthpart receptors in feeding behaviour.) Ent. saa. & appl., 12: 363-76. 85.Haskell, and Schoonhoven, L.M., 1969. (Function of mouthpart receptors in feeding.) Rat. exH. & appl., 12: 423-40. 86.Hansberry, f.A., 1966. ' Lecture on the Pesticide Symposium, 12th Annual Western Agri. Chem. Assoc. NW Conf., January, 1966. 234.

87.Hansberry, R.A., 1968. (Allocation of USDA funds for research.) Bull. ent. Soc. Am., 14: 229-35. 88.Unnson, F.E., 1970. (Sensory responses of phytophagous Lepidoptera.) In 'Control of Insect Behaviour by Natural Products', (Ed: Wood, D.L, Silverstein, R.M., and Nakajima, M.), Academic Press, Newprk & London, pp. 81-88. 89.Hardee, D.D., and David, T.B., 1966. (Systemic Chemicals - uptake.) J. econ. Ent .211: 1267-70. 90.Harley, K.L.S., and Thornsteinson, A.J., 1967. (Influence of plant chemicals on feeding.) Can. J. fool., 505-19. 91.Heath, D.F., and Llewellyn, N.Y., 1953. Proc. Isotope Techniques Conf., 1: 445-51. (Oxford, July, 1951; published 1953.) 92.Hill, L., and Goldworthy, G.J., 1968. (Growth in larvae of Locusta.) J. Insect amaka, 14: 1085-98. 93.Hodson, E.S., 1951. (Threshold of Laceophilus to salts and alcohols.) Physiol. Tool., 24: 131-140., 94.Hurd-Karrer, A.M., and Poos, F.W., 1936. (Selenium tests against aphids and spiders.) Science, New York, 84: 252. 95.Hussain, M.A., 1928. (Annual Report of the Entomologist to Punjab Government, 1927-28.) Rept. Dept. Agric,. Punjab, Lahore, 1929, pp. 55-79. 96.Hussain, M.A., Mathur, C.B., and Roonwal„ M.L., 1946. (Food and feeding habits of Schistocerca.) Indian J. Ent., New Delhi, 8: 141.-65. 97.Jacobson, M., and Crosby, D.G., 1971. 'Naturally Occurring Insecticides', Dekker, Ne0ork, pp. 585.

235.

98.Jermy, T., 1966. (Evaluation of feeding inhibitors against chewing insects.)

.922. & aPP1.1 1. 99.Jermy, T., and Matolcsy, G., 1967. (Anti-feeding effect of some systemic compounds.) Acta phytopath. Acad. Sci. Ihmg.., 2: 219-24. 100.Johnson, N.E., and Redske, J.H., 1965. (Systemic pesticides in wooded plants.) Bull ent., Soc. Am., 11: 190-95. 101.Johnson, N.E., and Zingg, J.G., 1967. (Translocation of four systemic insecticides.) J. econ. Ent., 60: 575-78. 102.Jones, F.G.W., 1970. (Nematodes take an annual toll.)

Farmer & Stock breeder, 7 April, 1970, p.39,41, 43. 103.Jotwani l M.G., and Sircar, P., 1963. (Neem seed as a protectant against stored grain pests.) Indian J. Ent., j: 160-4. 104. ibid , 1967. (Neem seed as a protectant against stored grain pests.) Indian J. Ent., 29: 21-24. 105.Kanjilal, U.L., 1901. 'Forest Flora of the School Circle, Superintendent, Government Printing Press, Calcutta, India. 106.Kennedy, J.S., 1951. Trans. Intern. Congr. Entomol., 9th Meeting, Amsterdam, 1951, 2: 106-10 (printed 1953). 107. ibid , 1956. Proc. Intern. Congr. Entomol., 10th Meeting, Montreal, Que., 1956, 2: 397-404. 108.Kennedy, J.S., and Moorhouse, J.E., 1969. (Locust behaviour to

grass odours.) Ent. ex-A. & appl., 12: 487-503. 236.

109.Kennedy, J.S., and Stroyan, H.L.G., 1959. Ann. Rev. Entomol., 4: 139-60.

110.Konrak, J.G., Armstrong, D.E., and Chesters, G., 1967. AMR. J., 59: 591. 111.Erishnamurti, B., and Rao, D.S., 1950. (Control of stored grain pests.) Bull. agric. Coll. Res. Inst., Mysore, 14: 1-93. 112.Kruskal, W.A., 1952. (A non-parametric test for the several sample problem.) Ann. Math. Statist., 21. 525-40.

113.Kruskal, W.H., and Wallis, W.A., 1952. (Use of ranks in one-c (etton -variance analysis.) J. Amer. Statist. Ass., Li.: 583-621. 114.Lavie„ D., Jain, LB., and Shpan-Gabrielith, S.F., 1967. (A locust phagorepellent from two Melia species.) Chem. Comm.,18: 910-11. 115.Lindquist, D.A., Bull, D.L., and Ridgway, R.L., 1965. (Systemic activity of bidrin.) J. econ. Ent., 2§: 200-3. 116.Ma, W. C., 1969. (Ecdystrone inhibition to Pieiris.) Ent. exnt. & appl., 12: 584-90

117.MacMillan, H.F., 1935. 'Tropical Planting and Gardening', 4th edn., pp. 234,

MacMillan & Co., London. 118.Mane, S.D., 1968. (Neem spray as a repellent against some foliage feeding insects.) M. Sc. Thesis, Indian Res. Inst., New Delhi.

119.Mangunath, B.L., 1948. (Ed.) 'The Wealth of India',vol.II, Council Sci. Indus. Res.,

New Delhi, pp. 140-42.

120.Marfurt, T.A., Toscani, H.A., 1968. (Organotin anti-feeding.) Delta del Parana, 6: 45-8. 237.

121.Martin, J.T., 1966. 'Chemical and physical nature of the plant cuticles in relation to the deposition and penetration of pesticides - radio isotopes in the detection of pesticide residues, International Atomic Energy Agency, Vienna, 59-66 pp. 122.Matterson, J.W., Taft, J.M., and Rainwater, C.R., 1963. (Systemic tests: plants.)

J. econ. ,m•ammaaEnt., 56: 189. •••• 123.Maxwell, F.A., Jenkins, J.N., Keller, J.C., and Parrot, W.I., 1963. (Feeding stimulant tests with boll weevils.) J. econ. Ent., 56: 449. 124.Metcalf, R.L., 1956. 2nd Intern. Plant Prot. Conf. Fernhurst, 23: 129-42.

125.Metcalf, R.L., March, R.B., Fukuto, T.R., and Maxon, M.G., 1954. J. econ. Ent., !ID 1045-55. 126.McCuig, R.B., 1968. 'Insectide Index', Anti-locust Research Centre, London. 127.McGovran, E.R., Cassil, C.C., and Mayer, E.L., 1940. (Stomach poison tests on leaves: Epilachna.) J. econ. Ent., 211. 525. 128.McMillian, W.W., Bowman, M.C., Burton, R.L., Starks, K.J., and Wiseman, R.R., 1969. (Extract of Chinaberry leaf as feeding deterrent and growth retardant.) J. econ. Ent., 62: 708-10. 129.Mitchell, J.W., Smale, B.C., and Metcalf, R.L., 1960. (Absorption and translocation of regulators, etc.) AAvan. Pest Control Res., 3: 359-436.

130.Mitra, C.R., 1963. 'Teem', Indian Central Oilseeds Committee,

Hyderabad, India, 190 pp. 238.

131.Mobin, M., and Than, A.M., 1969. (Effect of organic amendments on the population of rhizosphere fungi and nematodes.) First. All India Nematology Symposium, 1969, New Delhi, India.

132.Mulkern, G. B., 1967. (Food selection by grasshoppers.)

Ann. Rev. Ent., ••••...12: •••••••./ONg• a/M/OmMO NV* 59-78. 133.Norris, D.M., 1965. (Systemics in trees.) Proc. Midwest Chap. Intern. Shade Trees Conf., Chicago, Ill., 20, 33-36. 134. ibid , 1967. (Systemic insecticides in trees.) Ann. Rev. Ent., 12: 127-48. 135.Norris, D.M., and Coppel, H.C., 1961. (Translocation and stability of Chapman R - 6199 in Pinnus strobus.) eon.Fht., 54: 159-61. 136.Onsager, J.A., and Rusk, 1967. (Translocation of diazinon.) J. econ. Ent., 60: 586-88.

137.O'Brien, R.D., 1967. 'Insecticides - action and metabolism', Academic Press, London & New York, 332 pp. 138. Pain, B.F., and Skrentny, R.F., 1969. (persistence and effectiveness of thionazin.)

J. sci. Fd. uric., 20: 485-88. 139.Painter, R.H., 1959. (Melia azedarach not attacked by pests - quoted by Thorsteinson, 1960.) Ann. Rev. Ent., 5.: 193.

140.Patel, H.K., Patel, V.C., Chari, M.D., Patel, J.C., and Patel, J.R., 1968. (Neem deterrent to hairy caterpillars.) Madras agric. J., 22: 509-10. 141.Perera, H.C., 1941. (Neem as indigenous drug in the treatment of livestock.) Indian Vet. J., j: 261-65. 239.

142.Perte, S.L., 1942. (Neem leaves and extracts for control of carpet beetles. )

Indian I. But., /m 94.

143.Pivar, G.A., Dimitrijevie, M., Valencic, L., and Nikolic, M., 1968. 'The use of Dimecron with other pesticides in the protection of sugar beet', Brochure printed by A.G. Matos, Samobor, 1968,

7 PP.

144.Pivar, G.A., Valencic, L., and Vukcevic, R., 1965.

Agrohemija, Beograd, 4: 249-56.

145.Pradhan, S., and Jotwani, M.G., 1968. (Neem as insect deterrent.) Chem. /a India, 12: 756-60. 146.Pradhan, S., Jotwani, M.G., and Rai, BeR., 1962. (Neem seed

deterrent to locusts.) Indian Fm0-., 12: 7,11.

147. ibid , 1963. (The repellent properties of some neem products.) Bull. xte Res. Lab. Jammu, 1: 149-51.

148.Pruthi, H.S., 1937. (Neem as an insecticide -stored grain pests.) Sci. au. agric. Res. Inst. New Delhi, 1935-36, pp. 123-37. 149.Radswanski, S.A., 1969. (Improvement of red acid sands by the neem tree.)

J. Appl. Ecol., 6: 507-11.

150.Rainey, R.C., 1963. 'Meteorology and the migration of Desert Locust - application of synoptic meteorology in locust control', Anti-Locust

Memoir no 7, 115 pp. (also issued by WMO, no 8, TP. 64.)

151.Randhawa, M.S., 1968. 'The flowering trees', National Book Trust, India, New Delhi,

pp. 205'. 152. Reynolds, H.T., 1959

Advan. Pest Control Res., 2; 135. 240.

153.Reynolds, H.T., Fukuto, T.R., Metcalf, R.L., and March, R.]3., 1957. J. econ. Ent., 50: 527-39. 154.Reynolds, H.T., and Metcalf, R.L., 1962. J. econ. Ent., 55: 2-5. 155.Richardson, C.H., and Sciferle, E.F., 1938. J. uric. Res., 24: 1119.

156.Ripper, W.E., 1957. Advan. Pest Control Res., 1: 305-45. 157.Ripper, W.E., Greenslade, R.N., and Hartley, G.S., 1951. J. econ. But., 44: 481-502. 158.Rodger, A., 1936. 'A handbook of forest products of Burma', Superintendent, Govt. Printing and Stationery, Burma, Rangoon, pp. 101.

159.Roonwal, ILL., 1953. (Food preference of Schistocerca.) J. Zool. Soc. India, 5: 44-58. 160.Shama Sastry, R., 1929. 'Treatise on Polity', Kautilya's Arthashatra), Mysore. 161.Siegel, S., 1956. 'Non-parametric Statistics - for the behavioral sciences', McGrawhill, London, 312 pp. 162.Siddiqui, S., 1942. (Isolation of three new bitter principles from neem oil.) Curr. Sci., 11: 278-79.

163.Simons, J.N., Silverstein, R.M. and Bellas, T., 1968. (D valuation of feeding stimulants.) J. econ. Ent., 61: 1448. 164.Singh, A., 1971. (Effect of Azadirachta indica products on, nematodes.) M. Sc. Thesis, London University, Aug. 1971. 165.Singh, Gurdas, 1951. (Control of Schistocerca.) Indian bal., 2.: 10-13. 241.

166.Singh, R.S., and Sitaramaiah, K., 1970. (Control of plant parasitic nematodes with organic soil amendments.) Pans., 16: 286-97. 167.Sinha, N.P., 1960. (Use of neem seedcake.) M.Sc. Thesis, Indian agric. Res. Inst., New Delhi. 168.Sinha, N.P., and Gulati, K.C., 1968. (Studies on the use of neemcake.) J. Inst. Chem. India, 22.1: 199-202. 169.Sinoir, Y.G., 1968. (Factors in feeding of locusts.) Ent. exp. &Ecia., 11: 195-210. 170.Skrentny, R.F., 1970. (Translocation and fate of soil applied systemic pesticides.) Ph. D. Thesis, London University, 1970. 171.Smith, G.N., Watson, B.S., and Fischer, P.S., 1967. (Investigations on Dursban insecticide.) J. agric. Fd. Chem., 15: 127-132. 172.Soo Hool C.F., and Fraenkel, G., 1964. Ann. ent. Soc. Am., 22L 788-90.

173.Tahori, A.S., Zeidler, G., and Varley-, A.H., 1965. (Plant growth regulants possess anti-feeding property.) J. Sci. Fd. 2gEic., 16: 570. 174.Teitz, H., 1954. (Factors affecting uptake of systemic chemicals.) Hofchen-Briefe, 1-55. 175.Thomas, J.G., 1966. (Sense organs of mouthparts of Schistocerca.) J. Zool.,•148: 420-48. 176.Thomas, B.A., 1969. (anti-feeding of Fentin acetate.) Res. Notes Forest Ser. USDA, SE -118, 3pp.

177.Thomas, W.D.E., and Bennet, S.H., 1954. (movement of Schradan in apple tree.) Ann. apyl. Biol., 41: 501-19. 242.

178.Thorsteinson, A.J., 1960. (Host selection in phytophagous insects.) Ann. Rev. Ent., 2: 193-218. 179.Thorsteinson, A.J., and Nayar, 1963. (Plant phospholipids as feeding stimulants for grass hoppers.) Can. J. Zool., 41: 931-35. 180.Trotter,11., 1940. 'Manual of Indian Forest Utilisation', Oxford Univ. Press, pp. 269-98. 181.Troup, R.S., 1909. 'Indian Woods and Their Uses', Indian Forest Memoirs, Economic Product Series, 1: 187. 182.Venkatesh, Sikka, H.L., and Singh, Brajendra, 1970. (Efficacy of neem kernels as deterrent to Schistocerca.) FAO UNDP (SF) TS/ 6-(k), Locust Project Progress Report, pp. 1-10. 183.Volkonsky, M., 1937, a. (Protection from locusts.) c.r. Soc. Biol., Paris, 22D 417-18. 184. ibid , 1937, b. (Action of extracts of Melia azedarach.) Archs Inst. Pasteraur Alger., 427-32. 185.Wada, K., and Munakata, K., 1968. J. agric. Fd. Chem., 16: 471-4. 186.Waite, R., and Boyd, J., 1953. (Iheeding Of carbohydrates of grassers.} J. Sci. Fd. Agric., 4: 197-203. 187.Warden, C.D., 1921. 'Forest Resources of Baroda State', Bull. no 2, 2nd Fdn., pp. 10, 19. 25; Baroda Printing Works, Baroda (India).

188.Way, M.J., Murdie, G., and Galley, D.J., 1969. (Integration of chemical and biological control of aphids.) Ann. an 1. Biol., 459-75. 189.Way, M.J., and Needham, P.W., 1957. (Factors in systemic uptake of chemicals.) Plant Path., 6: 96.

2113.

190.Way, N.J., and Scopes, N.E.A., 1965. (Side effects of some soil applied systemic insecticides.) Ann. 9121. Biol., .22: 340.

191. ibid 2 1968. (Effects on soil fauna of some soil applied systemic insecticides.)

Ann. anol• Biol9 62: 199-214. 192.Williams, L.H., 1954. (Feeding. habits and food preferences of Acrididae.) Trans. 221:. Ent. Soc., London, 212: 423-54. 193.Woefenbarger, D.A., Lowry, W.L., Scales, A.L., and Parencia, C.R., 1968. ( Control of cotton pests with Organotins.) J. econ. Ent., 61: 235-8. 194.Wood D.L., Silverstein, R.M., and Nakajima, M., 1970. (Editors) 'Control of Insect Behabiour by Natural Products', Academic Press, New York. 195.Woodbury, E.N., 1943. (Repellent testa far leaf-feeders.) Publ. Amer. Ass. Advano. Sci., no 20, pp 174. 196.Wright D.P. (Jr.), 1963. Anti-feeding compounds for insect control.) Advances in Chemistry, ser. 41, Washington, Av. Chem. Soc., 56-63 pp. 197. ibid , 1967.. 'Anti-feedants' in Pest control - biological, physical and selective chemical methods', (Eds. Kilgore and Douff), Academic Press, Yew York, pp. 287-93. 198.Yamamoto, R.T., and Fraenkel, G., 1959. (Tests of attractants for plant feeding insects.) Nature, London, 184: 206. 199.Yu. S.J., and Morrison, F.O., 1969. (Systemic uptake.) J. econ. Ent., 62: 1296- 1303. 200.Zeid, M.M.I., and Cutkomp, L.K,, 1951. (Schradan in plants.) J. econ. Ent., 44: 898-905. 244.

ADDENDUM

Atwal, A.S., and Pajni, H.R., 1964. Indian J. Ent., 26: 221-27. Chauvin, R., 1951. Bull. Off. nat. Anti-acrid. Paris 1: 15-18. Dadd, R.I., 1960. J. Insect Physiol. 301-16. Fraenkel, O., 1969. Proc. 2nd Int. Sym., Wageningen, June, 1969. Fry, J.S., 1938. Gold Coast Farmer, 6: 190 (quoted by Sinha, 1960). Ilamamura, Y., and Naito, K., 1961. Nature, Loud. Ds) 879-80. Ilsiao, T.H., and Fraenkel, G., 1968. Ann. ent. Soc. Amer., 61: 44-54. Hussain, M.A., and Mathur, C.B., 1936.

Indian J. aerie. Sci., Delhi, 6: 263-67. Kennedy, J.S., 1965.

Ann. apple Biol., 56: 317-22. Mc Indoo, N.E., 1945. 'Plants of possible insecticidal value, a review of literature, up to 1941', USDA Handb., E-661, p. 286. McMillian, W.W., and Starks, K.J., 1966. Ann. Entomol. Soc. Amer., 2.: 516-19. Munakata, Katsura, 1970. 'Insect anti-feedants in plants' in Control of Insect Behaviour by Natural Products, Academic Press, New York. Nayar,J.K., and Fraenkel, G., 1962. Ann. Entomoi. Soc. Amer., LI 174-78. 245.

Nayar, J.K., and Thorsteinson, 1963.

Canad. j. '&01., 41:: 923-29. Rees, C.J.C., 1966. Ph. D. Thesis, Oxford (quoted from Fraenkel, 1969). Trehan, K.N., 1956. 'Final Report of the Scheme for Research on Insecticides

of Vegetable Origin, October 1954 to 1960', Controller of Printing and Stationery, Punjab, Chandigarh, India. Thorpe, W.f., 1963. 'Learning and Instinct in Animals', 2nd Edition, Methuen, London. Thorsteinson, A.J., 1958.

Ent. via. appl., 1: 23-27. APPENDICES 247.

APPENDIX 1. Deterrent effect of candidate antitEjltliEL z milla evaluation with Schistocerca.

% Concentration applied 1.0 0.1 0.01 0.001 Control Dose/cm2 (ug) 158 15.8 1.58 0.158 (Sucrose 0.11,1) Repli- (mgs of filter paper eaten) cates 1. Azadirachtin 1 - 0 0 0 283 2 - 0 0 0 301 3 - 0 0 0 276 Total - 0 0 0 860

2. Alcohol Ext.of Neem 1 - 0 0 0 361 2 - 0 0 0 253 3 - 0 0 0 322 Total - 0 0 0 936

3. Kernel Ext.of Neem 1 - 0 0 11 406 2 - 0 0 23 389 3 - 0 0 20 343 Total - 0 0 54 1138

4. Gramine 1 0 29 30 - 368 2 0 21 56 - 468 3 0 39 36 - 275 Total 0 89 122 - 1111 248.

APPENDIX 1 (Continued)

% Concentration applied 1.0 0.1 0.01 0.001 Control (Sucrose 0.1M) (mgs of filter paper eaten)

5. Hordenine 1 399 430 563 515 413 2 267 364 514 585 563 3 422 501 641 496 569 Total 1088 1295 1788 1596 1545

6. ElataaMiat 1 203 183 285 232 283 2 178 198 230 301 303 3 263 202 255 320 373 Total 644 583 770 853 959

7. Lobeline 1 - 307 551 527 413 2 339 485 683 563 3 338 '368 570 569 Total 984 1404 1780 1558

8. Lupinine 1 258 360 402 531 2 307 431 361 467 3 301 347 430 412 Total 866 1138 1192 1410 249.

APPENDIX 1 (Continued)

% Concentration applied 1.0 0.1 0.01 0.001 Control (Sucrose 0.1M)

9. Veratrine 1 0 4 21 33 216 2 0 3 29 28 182

3 0 2 18 30 174 Total 0 9 68 91 572

10. Digitonin 1 - 258 326 307 350 2 - 275 248 412 330

3 - 288 306 298 398. Total - 821 880 1017 1078

11. Diosgenin 1 509 415 340 350 2 473 378 465 330

3 605 572 435 398 Total 1587 1365 1240 1078

12. Brestan - 93 111 255 315 2 - 81 98 279 354

3. - 72 169 268 460 Total - 246 378 802 1129 250.

APPENDIX 1 (Continued)

Concentration applied 1.0 0.1 0.01 0.001 Control (Sucrose 0.1M) 13. Du-ter

1 112 182 405 488 2 128 77 386 500

3 168 189 445 457 Total 408 448 1236 1445

14. TD -3035 1 31 75 141 132 2 29 48 139 189

3 -- 31 66 173 167 Total 91 189 453 488

15. Stannous chloride 1 51 83 153 85 2 93 55 164 104

3 70 ' 105 156 99 Total 214 243 473 288

16. Phosphon 1 0 127 243 242 2 0 186 222 370

3 0 193 274 134 Total 0 506 739 746

17. B -nine 1 236 199 157 137 2 241 248 204 208 3 229 225 210 167 Total 706 672 571 512 2 51.

APPENDIX 1 (Continued)

% Concentration applied 1.0 0.1 0.01 0.001 Control (Sucrose 0.1M) 18. Phenoxy acetic acid

1 51 211 293 433 2 39 164 306 508 3 46 158 249 375 . Total 136 533 848 1316

19. Carbaryl 1 - 36 68 195 204 2 - 38 59 134 198

3 - 29 98 157 305 Total - 103 225 486 707

20. Cl.. 24,055 1 . 24 90 133 79 2 31 99 90 61

3 43 86 125 74 Total 98 275 348 214

21. Copper stearate 1 184 158 153 201 208 2 129 135 182 172 167

3 156 191 162 198 213 Total 470 484 497 571. 588 252.

APPENDIX 1 (Continued)

% Concentration applied 1.0 0.1 0.01 0.001 Control (Sucrose _ 0.1M) 22. Copper resinate 1 296 374 498 - 394 2 308 353 396 - 512

3 327 327 356 - 389 Total 931 1054 1250 - 1295

23. Mercuric chloride 1 1 37 183 289 2 1 61 198 203 3 0 57 182 199 Total 2 155 563 691

24. Indalane 1 61 86 89 56

2 53 88 112 32 3 72 142 83 73 Total 186 316 284 282

25. MGK repellent 1 85 125 119 83 2 - 108 101 106 67

3 - 94 98 130 68 Total - 287 324 355 218

253.

APPENDIX 1 (Continued)

Concentration applied 1.0 0.1 0.01 0.001 Control (Sucrose - 0.1M)

26. Meta-delphene 1 - 111 124 ' 114 83 2 - 100 77 86 67 3 - 150 99 90 68 Total - 362 300 290 218

27. Rutgers 6-12 1 - 76 104 145 78 2 - 109 138 70 96 3 - 83 87 59 93 Total 268 329 274 267

28. Di-methyl phathalate 1 79 65 86 - 79 2 60 97 75 - 68 3 76 107 113 - 86 Total 215 269 274 - 233 254.

APPENDIX 2: An example of the Kruskal-Wallis one-way analysis of variance by ranks. Table 2.18. Protection of bean leaves dipped in neem against attack of Schistocerca

Rank-index of damage Dipping interval Minutes Hours

Neem treatment 15 30 1 6 12 24 (% cone.) ENL 0.1 3 3 2 1 0 0 FRT 1.0 2 2 1 0 Control ... N = 21 (7 x 3); ranks 1 to 21.

Ties: N Total Av.rank 3 6 2 2 9 4.5 3 21 7.0 6 69 11.5 7 126 18.0 21 231

Correction for ties: t _ 2 2 6 , 2 T - 24 24 210 336 (24 = 1 6 24 + 210 + 336) _ 0.94. (21)3 - 21

1 2 3 4 5 6 7 Total A 11.5 11.5 11.5 7 4.5 2 2 50 B 11.5 11.5 11.5 7 7 4.5 2 55 C 18 18 18 18 18 18 18 126 41 41 41 32 29.5 24.5 22 231 255.

12 + 222 H (between the treatments - 21 x 22 * (412 + ) - 66/0.94

= 14.26 with 2DF (P at 0.001 = 13.82).

H (within the treatments) = 12 x 1 (502 552 + 1262) - 66/0.94 21 x 22 7 4.21 with 6DF (13 at .05 = 3.96 n.s.). 256. Appendix 3. B. L. Mangunath (Ed.) 19)48. The Wealth of India- Raw Materials, vol. I. pp. 1)10-1421 The Council of Scientific and Industrial Research, New

AZADIRACIiTA A. Juss. MELIA CEA E This is a genus of tall, evergreen trees, comprising appear from March-May and the fruits ripen during June-August. They are greenish yellow when ripe, two 'species. A. indica, native to India, is now widely distributed throughout the Endo-Malayan and usually contain only one seed. region and is also found in tropical Africa. A. ink- The tree grows wild in the dry forests of the gnyclia Merril is indigenous to the Philippines. Deccan and in the open scrub forests of the dry zone of Burma. It is cultivated all over India and A. indica A. Juss. Syn. tlielia azadirachta Linn. Burma, but thrives best in the drier climate of the NEEM DIEE, MAnoos.A TREE north-western parts, where normal rainfall varies -D. E. P., V, 211 ; C. P., 780 ; - Pl. Br. Ind., I, 54 ; from 18-45 inches and maximum shade temperature P1.XXII,L• . . may be as high as 120°F. It grows on al/ kinds of SAN'S ; soils but does well on black cotton soil. It is well azad-daraloht-hi2;di ; suited for afforestation in dry situations and is a HIND. & BENG ; MAR- & j common avenue tree. • TEL., TAM . & MAL .—Vepa ; KAN Almost every part of the tree is bitter and has This is a medium-sized tree, with a clear hole of found application in indigenous medicine. The seed ..10-25', and a girth of 6-8'. Although described as . oil (margosa oil) is acrid, yellow, bitter in taste, evergreen, in drier areas it becomes deciduous. and has a disagreeable garlic-like odour. It is used Its bark is moderately thick with longitudinal in skin diseases such as scrofula, indolent ulcers, or oblique furrows on the outer surface. It is dark- and sores, and ringworm. It is also applied in grey outside and reddish inside. The flowers cases of rheumatism as a liniment. It is reputed to possess anthelmintic and insecticidal properties. The bitter principles of mem Oil have been investigated by several workers (Watson •&-, Chatterjee, J. Soc. chem. Ind., Lord., .1923, 42, 387T ; Rangaswami & Seshadri, Indian J. Pharm., 1940, 2, 206). Siddique and Mitra (J. sci. industr. Res., 1945, 4, 5) have recently obtained the bitter principles by extraction with alcohol (yield, 2%). Nim- bidin (m.p., 90-100° ; 1.2-1.6%), the principal component, is highly bitter and contains sulphur. On hydrolysis, it gives nimbidinic acid • which is.equally hitter and retains sulphur. Besides nimbidin, two bitter compounds, free from sulphur, (m.p., 192°, and 205'), have been obtained in very small quantities. Nimbidin and sodium nimbidinate are almost non-toxic (m.l.d. for frogs, 0.25 mg. per g. body wt.). Nhnbidin preparations are .reported to be free from the unpleasant smell of the oil and to be efficacious in a variety of skin diseases, sceptic sores, and ulcers due to burns, and useful in bleeding gums and pyorrhoea. • The bark is a good bitter tonic, astringent and antiperiodic.. It is said to contain a resinous bitter principle and is usually prescribed in the form of a tincture or an infusion (Birdwood, 104). It is also regarded as beneficial in malarial fever. But, preliminary experiments carried out recently, at the School of Tropical Medicine, Calcutta, have shown that a tincture (1 : 10) made from the dried- and 1. AZADIRACHTA INDICA Powdered bark has no action on monkey malaria 257.

A2ADIRACIITA inS AVgArlin am r main

or malaria in human beings (Mukerjee, Private greasy lather. been. oil could be mixed with other communication ; cf. Central Indigenous Drugs oils and fats for the manufdeture of washing soap Comm., .3rd Rep., 1916, 185 ; and Koman, (llatta, Basu and Na,ndy, Dep. Indu,str., Bengal, 1910, 18). • • • Ball. No. 47, 1936). Considerable quantities of the The bark also is considered useful in skin diseases oil aroused for the preparation of cheap washing (Komar, loc. cit.). The leaves arc bitter and have a .soap. . Medicated soaps with the odour of neem • faint, but characteristic - unpleasant smell. Dried -are found in the market. • in the shade, they are commonly placed in books, - The oil -cake is regarded es a useful fertilizer (mo- paper and clothes to protect them from moths, etc. -isture, 6-S• 7; organic matter, 84-89 ; inorganic mat- The odour produced by the burning of powdered lea- ter, 1. 69-5.07 ; salts, 3-9 ; P2 05, 0 ' 68-1 • 4%). ves is said to be fatal to insects. The leaves are appli- The gum which exudes from. the bark occurs in ed to boils in the form of a poultice, and a decoction is recommended in ulcers and eczema. The dry flow- the form. of clear bright amber coloured tears or ers are considered. tonic and stomachic. The berries 'fragments. It is found mixed with guui ghatti. It contains : water , 13-15 ; ash,-3 ; galaetans, 12 ; are regarded as purgative, emollient and anthelmin- pentosans, 26% ; some albumins and oxidase tic. But, Otitis and Mhaskar (Indian J. med. Res., (Wehmer, II, 662 ; and Koolhaus, 1923, 11, 364) have shown that the juice of the leaves Chem.. Abstr., 1940, 34, 6118). and the oil do not have any anthelmintic proper- • ties. Fresh tender twigs are used to clean teeth, par, A sweet liquid, neem toddy is occasionally ticularly in pyorrhoea. The presence of the tree obtained as an exudation from the upper parts of around villages and on road-sides is considered some trees. It bas•the unpleasant odour of neem beneficial. and contains : sugars, 6.5, and albuminous and • gummy matter, about 6.5% (Chesil, Indian For., The seeds are reported to contain up to 45% of 1913, 39, 261). oil. According to Rao and Seshadri (Proc. Indian : Acad. Sci., 1912, 15, 161) the oil has the following - The wood is moderately heavy, medium-textur- constants : d/31°, 0 • 9129 ; ni3/31°, 1 • 4658 ; sap, ed, and narrowly interlocked.-grained (sp. gr., val., 195.6 ; iod. val., 69.2 ; acid val., 11.2. The 0 ' 68 ; air-dry Wt., 44 lb. per c. ft). The sapwood is fatty acids are chiefly oleic (about, 53%), stearic greyish-white. The heartwood is red when first ..(about 18%), and palmitic (about 14%), together exposed, and later turns reddish-brown. It is • with small amounts of Enoleie and arachidic acids. not very lustrous, and is slightly aromatic. Ana- tomically it is featured by distinct growth rings, The margosic acid ' of earlier workers was shown by medium-sized vessels occluded by reddish-brown Roy and Du tt (J. Soc. chem. Ind., Lond., 1029, 48, 333T ; see also Child and Ramanathan, ibid., 1036, gum, pa.renchyrna cells with deposits 'of guru, 55, 124T) to be a mixture of the usual fatty acids. and fine rays. The wood has often been mistaken The glycerides of neent oil have been investigated for that of Melia azedarach Linn., but can be easily distinguished as the latter is one of the few ring by Ililditch and Murti (ibid., 1039, 58, 310T) and found to follow the usual rules cf glyceride structure porous woods of India (Pearson and Brown, I, 253), in seeds and fats, Investigations on the odorous The timber seasons well, and is durable even in constituents of the oil which are considered to be exposed situations. It is not attacked by white .'sulphur compounds have so far been inconclusive, ants, perhaps clue to its bitter character. !:(vide Marti, Rangaswami and Seshadri, loc. cit.). It is not difficult to saw. It can be worked both by In India, the oil is usually prepared in ghavis -hand and by machine, and is suitable for carving, (wooden oil mills) for local use, either as medicine but it does not take polish well. The timber is used in housebuilding and for making boards, .or as an illuminant. 110\s-ever, while burning, panels, toys and ploughs (Pearson and Brown, it smokes badly. If seeds are heated before crushing, the odour becomes intolerable. The oil loc. cit.). gives soap of unattractive brown colour which still •retains some smell. This could be considerably decreased by a second boiling, and by salting out the soup. The soap produces a profuse though slightly

258.

Appendix 4. Uses of Neem: in 'Neem by C.R. Mitra, Indian Central

Oilseeds Committee, Hyderabad, India.

Domestic uses The twigs are largely used as tooth cleaners and for general month hygiene. The oil is employed for burning in open lamps. The villagers sometimes apply the oil to the hair in order to kill vermin. The oil is also reported to be taken internally by certain women in pregnancy.4

"The leaves- are largely used to protect clothes, books, papers, etc. from the ravages of insects, but they are inferior to camphor for this purpose and require to be renewed frequently.

"The leaves and twigs are used as cattlefeed and for manure. The oileake is largely employed for manurial purposes." Recently, the dried ncem fruit pulp has been found to be an effective manure for jowar crop."

The tree is held sacred by the Hindus and its parts arc used in many of their rituals and ceremonies. It is believed that a few drops of heavenly nector fell on neem; hence on New Year's Day, the Hindus eat its leaves and take a bath with water in which a few neem leaves are boiled, in the hope of acquiring freedom from disease. It is believed that Sri Chaitanya, the father of Vaishnava cult, was born under a neem tree and hence his nickname `Nimar. A festival called Chatastizapana (installation of the sacred pot) is well-knoWn in many parts of India; on certain occasions the villagers collect together and instal at a public place, a pot filled with water on which they put five branches of neem and a 'coconut. This is covered with flowers and worshipped. Sacrifices (bail) are made before it. This festival is considered to avert ill- luck and disease. •

Amongst the superstitious people, it is customary to chew a necm leaf as an emblem of grief on returning from funerals. The tree is held in high esteem by orthodox people and is worshipped along with pi pal.

The neem. bark exudes a clean, bright, amber-coloured gum which is collected in small tears and fragments. At one time, it used to be known as East India. gum and was a commercial commodity. It is considerably estee- med medicinally as a stimulant. It makes a weak mucilage and is of little value. Local si]k dyers use the gum in every preparation of their colours.

The bark yields a fibre, commonly employed in the local manufacture of ropes, but it is of little economic importance.

Uses of neem ica indigenous system of medicine

The earliest authentic record of information relating to the knewledge of chemistry, metallurgy and medicine of the ancient and medieval India is found in. the Arthashast:a (Treatise on Polity)~7 of Kautilya which gives a magnificent account of the political, serial, industrial, civil and military organisations of the 4th Century B.C. 259.

The very name of the tree, nimba, seems to have been derived from a remote knowledge of its medicinal properties,. nimba being synonymous with Arishla meaning relieving sickness ; Pidoi91:irda, another name of the plant being equivalent to destroyer of leprosy."

Nana bark is retrarded as a bitter tonic, astringent and useful. in fevet.5,,, thirst, nausea, vomiting as well as in skin diseases, The active part is the inner layer of the bark. A decoction is prepared by boiling two ounces of the bruised inner layer of the bark with a few cloves or a small piece of cinnamon in 30 ounces of water for 15 minutes and straining. When cold, the decoction is administered in 2 oz. doses, thrice daily in cases of fever."

The bitter leaves are used as a pot herb, being made into ;oup and curry with other vegetables. The slightly aromatic and bitter taste which they impart to curries is Mi nch relished by some people.

The leaves are, extensive/y used as an old and popular, remedy for skin diseases. For internal administration, the fresh juice of the leaves is given with salt in cases of intestinal worms and with honey in skin diseases and jaundice. The leaves are also reported to be efficacious in the treatment of prurigo, boils and urticaria.2 As an external application to ulcers and skin diseases, neem leaves are used in a variety of forms such as poultice, wash, ointment and liniment. A poultice made of equal parts of neem leaves and sesamum seeds is recommended by Chaltradutta" for unhealthy ulcerations. Neer leaves bruised and moistened with tepid ‘vater are used as a stimulating poultice for chronic ulcers."

The fruits are described as a purgative and emollient and useful in intestinal worms, urinary diseases and piles. The oil obtained from the seeds is employed in skin diseases and ulcers.

As is customary in ayurvcdic medicines, the different parts of the neem tree like other herbal medicines arc seldom prescribed alone but enter into the composition of numerous complex preparations. Sonic of the well-known ayurvedic preparations incorporating various parts of neem in them and Which are extensively in vogue even. to the present clay arc reproduced"° • in the following paragraphs.

Kautilya or Chanakya, known as Indian Machiavelli, recorded" extraction of oils from linseed (Linton usitathsinum), seeds of nimba (neem), sesamum (Sesanzum indica/1z), (Balagites roxburgliii), etc. in his Arthashas- Ira.

The record shows that neon oil was being utilised even in hoc days when hardly a caul-A) et' maj.h. oilseeds were in common use in India. While ses-mium oil appeals to he the oldest known edible oil, non-edible neem oil is perhaps the oldest known medicinal 260.

Soap was possibly introduced into India by the Mohammedans though, earlier, the Hindus had made use for a long time, of alkaline lyes obtained from the ashes of plants. They had also a substitute for soap in several berries. The" crude soaps were made in the beginning from sarfikakdiara (trona or natron), common salt, vegetable oil (scsamum) and !Iniirol suet.

Uses of nee& in 4yurveria. Almost every product a this invaluable tree has been largely employed for medicinal purposes. The parts used and their reported phYsiologiczd actions are recorded as follows: •

Root bark, stem bark and young : Tonic and antiperiodic. Seeds, oil and leaves : Antiseptic, insecticide and local stimulant. • Flowers : Stimulant tonic and stomachic. Gum : Demulcent tonic. Toddy : Refrigerant, nutrient and alterative tonic.

The bark, leaves and fruits have been used in ayurvedic medicines from a very remote period, and are mentioned in the ancient Sanskrit writings, vie., those of Shushruta.