- 1 -

Development of Controlled-Release Pesticide

Formulations for use Against Plant Parasitic

Nematodes.

Alan John Birtle B.Sc.(Hons).

A Thesis Submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of

Membership of Imperial College.

Department of Pure and Applied Biology

Imperial College Silwood Park,

Ascot,

Berkshire SL5 7PY. September 1985 - 2 -

*

To

Mum and Dad

and

My late brother Keith who "fell asleep" aged 21 months. - 3 -

ACKNOWLEDGEMENTS

Foremostly, I wish to thank my supervisor, Dr. D.J. Wright, and his wife,

Tanya, for their assistance, encouragement and friendship throughout my study and for the helpful criticism and typing of this thesis.

I wish also to thank my advisors, Dr. A.A.F. Evans and Dr. G.N.J. le Patourel.

I am very grateful for the technical assistance provided by Dr. P.K. Ramdas of Hindustan Insecticides during my formulation work and Dr. R.H. Bromilow of Rothamsted Experimental Station for the use of their g.l.c and H.P.L.C.

My sincere thanks to Mrs. Jackie Pugh (Stores), Mrs. Rosemary Prince

(librarian) and Mr. Tom Rogers (gardener) for their assistance and friend­ ship during my stay at Silwood.

I am also indebted to Mr. J.T. Buckingham of Alder Farm, Prickwillow, for his help and co-operation during my field trials, and to many of my friends and colleagues at Ashurst Lodge who assisted with the harvest.

My thanks also to Mr. David Prince for providing me with gainful employment and Mrs. Diana Dolby for providing a roof over my head during the writing of this manuscript.

I also acknowledge the financial support provided by the Ministry of

Agricultural, Fisheries and Food for this project.

Finally, my love and thanks to my family for their support and under­ standing during my academic pursuits. - 4 -

TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENTS 3

TABLE OF CONTENTS 4

ABSTRACT 6

INTRODUCTION 8

Controlled-release formulations of pesticides. 8

The beet cyst nematode, Heterodera schachtii (Schmidt). 29

Nematicidal activity of oxamyl (Vydate). 44

MATERIALS AND METHODS 47

Chemicals. 47

Nematode culture. 48

Effects of oxamyl on life cycle stages of H.schachtii. 48

Effect of long-term exposure to oxamyl on nematode 51 migration, infectivity and lipid levels.

Assessment of two methods of nematode inoculation to plants. 52

Preparation of controlled-release granules. 54

High Performance Liquid Chromatography (HPLC). 56

Selection of experimental formulations for field trials. 56

Sugar beet trials, 1982-1984. 57

RESULTS 67

Effects of oxamyl on life cycle stages of H.schachtii. 67

Effect of long-term exposure to oxamyl on nematode 77 migration, infectivity and lipid levels.

Assessment of two methods of nematode inoculation to plants. 81

Relative release rates of oxamyl from experimental 81 granular formulations.

Selection of experimental formulations for 1984 field 91 trial.

Release rate characteristics of experimental oxamyl 91 formulations used in 1983 microplot trial.

Sugar beet trials: 1982-1984. 96 - 5 -

PAGE

4. DISCUSSION 126

4.1 Laboratory studies. 126

4.2 Formulation studies. 137

4.3 Field studies. '144

5. CONCLUSIONS 161

REFERENCES 165

APPENDICES 188 - 6 -

ABSTRACT

Controlled-release formulations of non-fumigant nematicides have the potential for extending the period of nematode control for the same amount of active ingredient or for achieving equivalent control with less pesticide than is present in a conventional fast-release formulation.

In the first part of this work, the effects of the oximecarbamate nematicide, oxamyl, on hatchingsmotility, migration, invasion and development of the beet-cyst nematode, Heterodera schachtii (Schmidt), were examined under laboratory conditions to determine the most vulnerable stage in the life cycle and to give an indication of the minimum pesticide concentration necessary for control. The effect of prolonged exposure to oxamyl on the subsequent infectivity of second stage juveniles of H.schachtii was also investigated.

Initial field work involved testing several existing formulations of oxamyl including several experimental slow-release granules, and another oximecarbamate nematicide, aldicarb, both to reassess their potential and to help determine the most useful methods of appraisal for the development of new formulations. To supplement field trial data, nematode-infested microplots at Silwood Park were prepared as these enabled much greater initial soil nematode populations to be used under less variable conditions than are normally present in the field.

In the second part of this study, clay-based granular formulations of oxamyl were developed using plastic coatings of cellulose acetate, cellulose triacetate or urea-formaldehyde resin containing different - 7 -

combinations of surface-active agents and plasticizers. Six formulations, whose release rates approximated most closely to that of an "ideal" controlled-release granule, were selected for field evaluation on a peat fen soil in East Anglia against H.schachtii on sugar beet. Residual concentrations of oxamyl in the soil were monitored during the growing season and the efficacy of each formulation in controlling invasion, development and reproduction of the nematode was determined together with the final yield of sugar beet.

It is suggested that controlled-release granular nematicides would be particularly useful with long-term annual crops where conventional treatment can prevent major yield losses but cannot prevent late-season nematode reproduction. - 8 -

1. INTRODUCTION

1.1 Controlled-release formulations of pesticides

Farmers worldwide continue to over-apply pesticides either through use of excessive dosage rates or by increasingly more frequent applications to obtain desired levels of control. One of the principal factors contributing to such over-application is that the majority of compounds used today only offer short periods of residual activity and in some cases 60-90% of the chemical never reaches the target organism

(Kydonieus et al., 1976). For example, a pesticide may be removed from the soil water by a variety of processes including: chemical and microbial degradation, evaporation, leaching and adsorption, and, because of these processes, application rates in the field often need to be an order of magnitude greater than those suggested by laboratory tests (Bunt, 1975).

Most conventional formulations of pesticides, including granules, release the active ingredient (a.i.) relatively rapidly and the initial pesticide concentration reaching the target organism is often far greater than that required for adequate -control. Thus, a major proportion of the pesticide applied enters the environment without contributing to pest control and may have potentially deleterious effects on non-target organisms and also contaminate ground water. The objectives for a controlled-release formulation are therefore to provide the necessary soil concentration of pesticide to achieve a more appropriate timing and duration of control. The requirements for such a formulation may include: immediate release for short-term pest problems;

release of a.i. at a steady rate to balance losses; an induction period - 9 - with no release followed by a burst to achieve the necessary concentration and then a steady release; release in which the concentration increases with time (Marrs and Seaman, 1978). In theory, at least, release rates could be programmed either for the entire crop season or for the period at which a particular pest infestation occurs.

Cramer (1976 a, b) has proposed the term "minimum pest inhibitory concentration" (MIC) which is the minimum concentration of pesticide necessary to immediately inhibit the pest population. In an ideal pesticide formulation, there would be a conventional fast-release component to attain the MIC and immediately inhibit the pest species, and a bound component which would not be degraded by the environment.

The latter would be released at the same rate as the a.i. was degraded or otherwise removed. In this way, an effective level of pesticide would be maintained at the MIC and the amount of bound component would determine the duration of control. The theoretical release curves of conventional and controlled-release formulations are shown in Figs 1.1a, b, and c.

The initial application, whether conventional or controlled-release, must include sufficient unbound, excess pesticide to satisfy the adsorptive capacity of the soil. Where the release rate is too slow, and there is insufficient unbound pesticide to saturate adsorptive sites, or where losses from leaching and decomposition are similar to the rate of release, the apparent MIC will not be attained at once (Fig. 1.2).

Possible solutions to this problem would be to enclose the controlled-release granule within a fast-release coating or to apply a mixture of conventional and controlled-release formulations. In this way, the required concentration of free a.i. should be achieved - 10 -

Fig. 1.1 a) Conventional release

Approximate weeks persistence b) Controlled-release (short-term control) Application level _ (eg.2 .OKg a.i./ha) Bound Required period of control Component } MIC Fast-release component 0 4 8 12 16 20 Approximate weeks persistence

c) Controlled-release (long-term control)

(After Cramer, 1976b) - 11

Fig. 1.2 Controlled-release with insufficient unbound pesticide.

Approximate weeks persistence

"I" Estimated 4 weeks to MIC

Fig. 1.3 Fast and controlled-release combination

Approximate weeks persistence - 12 -

immediately (Fig. 1.3).

In practice, the requirements for controlled-release are difficult to

achieve but even partial attainment of this type of release pattern has

several important advantages. A controlled-release formulation can

either maintain equivalent control to a conventional formulation with

far less a.i. or maintain control longer with an equivalent amount of

pesticide (Figs. 1.1b and c). In both instances, the environmental

impact of pesticide treatment is likely to be reduced. For example,

controlled-release formulations should be: 1. Less toxic to non-target

organisms; 2. Less persistent after the required control period, since

pesticides of shorter residual-life may be used; 3. Less mobile and there­

fore less likely to cause ground waterpollution; 4. Of minimum novelty

hazard, since existing well-tested chemicals may be used.

Such formulations may also increase the stability of otherwise

impractical unstable compounds, or may be suitable for use on or near a wider variety of crops including those which show phytotoxicity to

conventional formulations.3

Controlled-release formulations do not necessarily have to be more

expensive than the conventional counterpart as savings in a.i. may

offset the increased cost^of formulation. Futhermore, the greater

efficacy of such formulations may extend their usage to crop systems where conventional formulations have not proved to be cost effective

3 An alternative method for prolonging pesticidal activity would be to use equal amounts of conventional and delayed-release granules. This method could achieve a greater concentration of a.i. with time in order, for example, to protect a more extensive root system (Fig. 1.4a, b, c). - 13 -

Fig. 1.4 a) Conventional release

b) Delayed release

Approximate weeks persistence

c) Combined effect - 14 -

1.1.1 Methods of controlled-release.

There are various methods by which some degree of controlled-release of the a.i. can be achieved from a formulated product (Bakan, 1976;

Theeuwes, 1977; Cardarelli, 1975, 1976; Kydonieus, 1980; Lewis, 1981).

The physical characteristics of the a.i. itself are important. For example, the water solubility of the chemical will control its movement in plants and soil (McFarlane and Pedley, 1978)), while its partition coefficient (log K ,) will determine the extent to which the chemical is adsorbed onto organic matter in the soil (Bromilow, 1973; McFarlane and

Pedley, 1978).

The choice of formulation will also influence the release rate

(Furmidge, 1972). Granules offer the greatest opportunity to modify the way in which the a.i. is released (Turnipseed, 1963; Furmidge et al.,

1966, 1968). Thus, the behaviour of granular formulations will depend, for example, on the size of the granules and the distribution and concentration of a.i. (Stokes et al., 1970; Furmidge, 1972).

Controlled-release formulations and their test and registration procedures have been reviewed by Cardarelli and Walker (1978). These authors list 114 pesticides and other agents (eg. fertilizers and pheromones) which have been tested, together with 100 materials suitable for use as binders, matrices and wall membranes (including 15 categories of polymer) and 87 additives.

There are basically four types of binding materials: 15 -

1. Elastomers, such as natural rubber, which releases the a.i. by

diffusion-dissolution and leaching processes (Cardarelli,

1976).

2. Natural and synthetic polymers such as polyvinyl chloride where

release of the a.i.is through leaching. In particular, the use

of biodegradable polymers as formulating agents offers a number

of advantages, especially for the controlled-release of

systemic pesticides. Such polymeric materials include lignins,

starches, cellulose derivatives, clays, vegetable gums, waxes,

proteins and chitin (Hall, 1977; Wilkins, 1978)

3. Encapsulation, where the pest control agent is surrounded by a

polymeric wall which permits loss through diffusion, permeation

or degradation. The field of microencapsulation is rapidly

expanding (Scher, 1977) and can be described as techniques to

apply uniformly thin, polymeric coatings to small particles of

solids, droplets of pure liquids, or solutions and dispersions.

These coatings can be produced by a number of processes,

including: air suspension; coacervation - phase separation;

electrostatic deposition; interfacial polymerization;

multiorifice - centrifugal extrusion; pan coating; and spray

drying and congealing, (Bakan, 1976; Somerville, 1977).

Microcapsules can vary from a few tenths of a micron to a few

thousand microns in diameter, and capsule release mechanisms

are usually by coating fracture, coating degradation or,

especially important for controlled-release products, by

diffusion. They possess many advantages when compared with

conventional products including: increased residual activity;

greater stability during storage; lower mammalian and - 16 -

phyto - toxicity; reduced environmental contamination; reduced

evaporation and leaching; masking of repellant properties, and

more specifically, decreased contact without reduced stomach

activity (thus improving selectivity against leaf-chewing

insects). The release rate can easily be altered by varying

the capsule size, wall thickness or permeability (Phillips,

1974; Scher, 1977; Simkin and Galun, 1983).

4. Chemical combination of agent and polymer, where the a.i.

slowly diffuses through the polymer or breaks off the polymeric

backbone; since each pesticide molecule released exposes

another pesticide - polymer linkage beneath, breakdown extends

over a period of time. They are of two basic types: 1) The a.i.

is physically dissolved or dispersed in a polymer matrix.

Release occurs by diffusion, or by the chemical or biological

degradation of the matrix. This method has general

applicability to a wide variety of pesticides (Allan et al.,

1971; Harris et al, 1973; Nichols, 1975; Harris, 1976). 2) The

a.i. is either chemically linked as pendent side chains to a

polymer substrate or incorporated as part of the polymeric

backbone. In either case, the a.i. is released by the

sequential chemical or biological degradation of.the pesticide

- polymer bonds providing sustained release, the rate of which

is determined by the nature of the bond, chemical

characteristics of the pesticide and polymer, and the

dimensions and structure of the resultant molecular

combination. This method depends on the possession of

appropriate functional groups on the pesticide and is thus less

universal than (1) in its usage (Allan et al., 1971; Feld et

al., 1975;Harris, 1976; Wilkins, 1976). - 17 -

A large variety of polymers have been used for controlled-release

formulations (Schacht et al., 1981). Besides their relatively low costs,

these polymers are known to degrade slowly when applied to the soil

(Katz and Fassbender, 1966), thus releasing the a.i. at a controlled

rate on contact with water. New classes of water soluble polymeric

substances (Beasley, 1970) have increased the potential of this method.

Other, less used, methods of producing a controlled-release formulation

include adsorption and pumping systems. For example, attempts have been made to increase the half-life of pesticides by adsorbing them onto

diatomaceous earth and polymeric foams, although this approach has not

been adopted commercially (Baker and Lonsdale, 1973). Similarly,

although the use of osmotic pumps (Baker and Lonsdale, 1973; Theeuwes,

1977, 1980) is widely used in the pharmaceutical industry where cost is

not a constraint, they have yet to be expoloited for pesticide delivery.

1.1.2 Usage of controlled-release formulations.

Conntrolled-release formulations have long been used for various purposes

(Table 1.1) and many of these applications have been comprehensively

reviewed by Cardarelli (1975) and Batterby (1978).

Types of existing controlled-release formulations are compared on the

basis of a number of criteria including reliability of performance, lack

of residues after termination of the effective release period, cost of

materials and production and adaptability to a wide range of farming

techniques. 18 -

Table 1.1 Usage of controlled release formulations.

Use Reference

Anti-fouling Engel hart et al_. (1974); Montemarano et al. (1975)

Fertilizers Lunt et al_. (1961); Allen (1977); Shartna (1979); Michaud (1982).

Herbicides Allan et aJL (1972, 1973, 1975); Thompson (1974); Janes (1975); Feld et al_. (1975); Wilkins (1976); Janes and Mansdorf (1977); Wilkins (1978).

Molluscides Stephenson (1972); Cardarelli (1974); Shiff (1974, 1975); Cardarelli et al_. (1981).

Insect Pheromones Glass et al_. (1970); McKibbon et al. (1971); Cunningham and Steiner (1972); Beroza et al. (1974);

Hardee et^ a\_. (1975); Brooks and Swenson (1976); Johnson et al_. (1976); Campion et al. (1978); Kydonieus and Beroza (1982); Kydonieus et al. (1982)

Anthelmintics Guerrero et al. (1980)

Insecticides McFarlane (1970); Allan etal_. (1973, 1974); Coppedge et al_. (1975b); Kydonieus et_ al. (1976); Knapp (1977); Wilkins (1978); Thompson et al. (1981)

Fungicides Hall (1975); Hartill (1980); Cooke et a U (1981, 1982).

Juvenile hormone Scheafer and Wilder (1972); Dunn and Strong (1973) mimics

Insect repellents Simkin and Galun (1983)

Baits Andrewes et_ al_. (1975). - 19 -

However, despite their variety of uses, controlled-release formulations

are far more widely used for pharmaceuticals than for agrochemical

products. This is due in part to economics, but also to the more stable

environment to which pharmaceuticals are exposed. It is hoped that this

degree of success will be extended to the pesticide industry in the near

future.

1.1.2a Insecticides

Various controlled-release formulations of organophosphate and carbamate

insecticides have been tested in the field.

The first commercially available formulation contained dichlorovos

(Anon, 1964) and this was regarded as the beginning of a new era of

insect control in medical and veterinary entomology (Knapp, 1977).

Formulations of dichlorovos in polyvinyl chloride (PVC) [Vapona^], were

reported to control a number of public health pests (Smittle and Burden,

1965; Harvey and Ely, 1968; Bailey et al., 1971; Wright, 1971) and

agricultural pests (Green et al., 1966; McFarlane, 1970). Dichlorovos

has also been formulated using montan wax and hydrogenated cottonseed

oil or phthalic acid esters,- and found to be effective in the control of

adult mosquitoes and flies (Miles et al., 1962). The use of such

formulations with a controlled-release vapour action had the particular

advantage of preventing contamination of food handling areas as would

occur with dusts and sprays (Quisumbing and Kydonieus, 1977).

Plastic formulations of insecticides have also been developed for less

volatile compounds. For example, pellets of PVC and polyamide containing

temephos, naled and malathion (Whitlow and Evans, 1968) and foamed PVC - 20 - formulations of temephos (Miles and Woehst, 1969), were observed to extend the period of mosquito larval control. Other controlled-release insecticide formulations, including chlorpyriphos in polyethylene and

PVC pellets (Parker, 1970; Wilkinson et a!., 1971; Miller et al., 1973 a, b) and temephos in elastomeric and plastic formulations (Quick et al., 1981), have also been reported to give control of mosquito larvae

% for up to several years.

Controlled-release systems for the control of livestock pests have also been based on dichlorovos, with the aim of avoiding repeated applications. Such devices include: impregnated ear-tags and leg bands for use against insect pests on cattle (Harvey and Brethour, 1970;

Miller et al., 1981) and horses (Knapp, 1962; Dorsey, 1966); impregnated collars for flea control on dogs and cats (Kibble, 1968; Fox et al.,

1969); and systemic implants in bolus form for use against subcutaneous pests (Miller et al., 1981).

Controlled-release polymer formulations of organophosphate and carbamate insecticides have also been tested in the field for use against soil insects and demonstrated to extend their effective systemic life.

Kydonieus et al. (1976) describe several experiments using the multi-layered plastic Hereon dispenser with the organophosphates diazinon and phorate. They found that longer periods of protection were obtained on corn against the banded cucumber beetle, Diabrotica balteata and the southern corn rootworm, D.undecimpunctata, when compared with standard applications. The carbamates, carbofuran and methomyl have been combined with polyamide and a carbofuran - polyamide formulation was found to suppress the mahogany shootborer, Hypsiphyla grandella, in

Spanish cedar plantations for up to a year (Allan et al., 1973, 1974). - 21

Applications of carbofuran in lignin were also found to give long-term control of shoot-boring insects in seedling trees (Wilkins, 1978), and

Thompson et al. (1981) demonstrated extended control of pine tip moths using polymer combinations of carbofuran and disulphoton.

Results obtained by Gauthier (1977) showed that encapsulated formulations of diazinon, chlorpyriphos and acephate gave longer and better control of insect pests on vegetable crops when compared with conventional formulations. Furthermore, improved persistence and control of the cotton boll weevil, Anthonomus grandis, has been achieved using encapsulated formulations of methyl parathion (Ivy, 1972; Pass and

Dorough, 1973; Simkin and Galun,. 1983); such encapsulated formulations offer not only increased persistence but also improved handler safety, reducing the risks of highly volatile pesticides (Speight, 1980; Shasha,

1980; Riley, 1983).

In a simulated controlled-release experiment, Ridgeway et al. (1969) found multiple applications of aldicarb to be more effective against boll weevils than a single application, even though the same total amount of a.i.was used in both cases. Further work by Stokes et al.

(1970) demonstrated that compacted carbon granular formulations of aldicarb prolonged the release of incorporated a.i. in the laboratory, and that by varying the base material, types and percentage of binder and mesh size, different rates of aldicarb release could be achieved.

This was also reported by Coppedge et al. (1974) using similar formulations of aldicarb in greenhouse and field tests; they found such products were less phytotoxic but more effective against cotton pests than the conventional corn cob formulation. - 22 -

Work in preparing granular formulations of aldicarb with various plastics and additives was conducted by Stokes et al. (1973). Such formulations were reported to have slower release rates in water immersion tests and extended period of systemic activity against boll weevils. While, in greenhouse tests, formulations of aldicarb, dimethoate and methomyl with slower release rates were found to be less damaging to plants and active for longer against the cotton aphid, Aphis gossypii, than a fast-release standard formulation (Stokes et al., 1973;

Coppedge, 1975 b).

Finally, coating pirimicarb granules with an increasing number of layers of resin or wax was found to adjust the release rate of a.i. (Seaman and

Warrington, 1972).

1.1.2b Nematicides.

Plant parasitic nematodes are now well known as potential limiting factors in crop production (Feldmesser et al., 1976).

Table 1.2 lists a selection of nematicides currently available worldwide. They include many soil fumigants which are often highly phytotoxic as well as facing increasingly severe governmental restrictions. The remaining compounds are organophosphates and carbamates which are also used as insecticides and these may be used on and around established plants. The use of the latter compounds as nematicides is often of secondary importance to their role as insecticides and their method of application often makes them less efficient therefore in the former role (Hague and Pain, 1973; Cardarelli and Feldmesser, 1975). - 23 -

Table 1.2 Nematicides available on world markets.

Fumigants Trade name Formulation

Methyl bromide Dowfume MC-2 Gas

1,3-Dichloropropene Telone Liquid

Dichloropropene- D-D Liquid dichloropropane

Ethylene dibromide Dowfume W-85 Liquid

c h lo ro -j- Dibromopropane ' Nemagon Emulsifiable and non-emulsifiable liquid Metham-sodium Vapam Liquid

Dazomet Basamid Dust (prill)

Methyl isocyanate Di-Trapex Liquid

Chloropicrin Dowfume MC-2 Liquid

Organophosphates

Thionazin Nemafos Granular or emulsifiable liquid

Ethoprophos Mocap Granular or emulsifiable liquid

Fenamiphos Nemacur Granular or emulsifiable liquid

Fensulfothion Dasanit Granular

Terbufos Counter Granular

Isazophos Miral Granular - 24 -

Table 1.2 contd

Carbamates Trade name Formulation

Aldicarb Temik Granular

Oxamyl Vydate Granular or emulsifiable liquid

Carbofuran Furadan Granular and flowable

Cloethocarb Lance Granular

t Use restricted to Hawaii on pineapples. - 25 -

Plant parasitic nematodes are difficult to kill in the field because of the large bulk of soil which must be treated, a formidable and difficult barrier to penetrate. The efficiency of chemical treatments is hindered further by chemical and biological degradation, leaching and dissipation of toxicant to the atmosphere. In addition, many nematodes can persist due to physiological and physical factors such as resistant stages,

including in many cases the eggs, and because they are often protected from the soil environment by layers of plant tissue (Feldmesser et al.,

1976). All these factors help to reduce the likelihood of nematicides reaching their targets, making it necessary to use large amounts to attain some degree of control. However, because nematicides can be costly and phytotoxic, they should ideally be used sparingly.

For the above reasons, it is extremely difficult to find and develop cost-effective nematicides which result in subsequent crop yield

increases (current economic considerations prevent too much concern over field nematode populations). The small number of currently registered compounds reflects these difficulties. Cardarelli and Feldmesser (1975) have suggested that the answer to these problems may be found in two areas: 1) The continued search for new, more efficient, cheaper, and more specific nematicidal moieties. 2) The development of new techniques that may enable us to use available nematicides more efficiently.

Control!ed-release technology in the form of nematicides incorporated in granules, coatings, fibres, encapsulations, polymers and controlled porosity matrices may provide some of the answers for (2) although none are yet available commercially. Such formulations of nematicides could conceivably provide season long protection against nematode damage to crops with a minimal number of applications through improved residual - 26 - activity.

As mentioned previously, currently available compounds with nematicidal activity can be divided into two groups: those with fumigant action, and the non-fumigant organophosphates and carbamates.

Soil fumigants diffuse through the soil to kill nematodes and their eggs and are thought to involve a number of potential target sites (Moje,

1960; Castro, 1964; Wade and Castro, 1973; Castro and Belser, 1978).

Unfortunately large amounts (up to 1000 Kg a.i. ha ) must be used and because of their long residual activity and mobility some fumigants are now causing ground water contamination. Also, because with the exception of DBCP (now virtually banned) fumigants are highly toxic to plants, they are restricted to use in soils prior to planting.

The non-fumigant compounds, despite their failure to kill nematodes directly have several advantages when compared with fumigants which sometimes outweighs their greater basic cost. They are generally much less phytotoxic, relatively easier to apply - requiring no special equipment, are effective in controlling nematodes at much lower dosage rates and have less persistent residues (Van Berkum and Hoestra, 1979).

Formulations which could maintain such toxicant levels in the soil over an extended period might therefore control damage to plants and decrease final nematode populations, enabling closer rotation of susceptible crops.

The concept of a lethal dose as a cumulative product of concentration and time is said to be applicable to nematodes (Hague, 1975; Munneke and

Van Gundy, 1979). Therefore, even sublethal concentrations may - 27 -

eventually reduce nematode populations, either by cumulative build-up or by weakening them sufficiently to make them more susceptible to attack by other soil organisms (Munneke and Van Gundy, 1979).

As previously discussed (Section 1.1), controlled-release nematicides would be useful where crop residue problems are not a serious danger that is with long-term crops like potatoes or sugar beet. Also those nematicides now considered to be phytotoxic may prove less so when applied as a controlled-release treatment. Reduced costs associated with lowered rates of pesticide application and savings in labour and energy due to the reduced need for frequent treatments, may open the way for the practical treatment of nematode-infested crops of low cash value for which there are at present almost no economically feasible control measures (Feldmesser et al., 1976) and introduce nematicide registration for minor crops (Elson, 1982).

Laboratory studies have been made with formulations of DBCP and diazinon which show controlled-release patterns (Feldmesser, 1974; Feldmesser ert al., 1976), and a DBCP-starch xanthide granule was found to control

Meloidogyne incognita on tomato at a rate that was ineffective using emulsifiable concentrates of this nematicide (Feldmesser and Shasha,

1977). However, formulations of vinylite VYHD resin-coated granules of aldicarb that showed controlled-release characteristics in the laboratory were not successful in the field, failing to give an improved control of H.schachtii on sugar beet when compared with standard coal and gypsum formulations (Batterby et al., 1980). More recently, the use of nematicidal amines in various polymers has been reported (Feldmesser et al., 1979, 1981). - 28 -

The best evidence for the usefulness of controlled-release nematicides is perhaps by analogy with recent work on the multiple application of nematicides in irrigation systems in California and Arizona. Liquid formulations of fenamiphos and oxamyl applied at a low rate (1.1 Kg a.i. ha *) at monthly intervals (April to November) through closed, low pressure irrigation systems were found to give good control of the citrus nematode, Tylenchulus semipenetrans (Van Gundy et al., 1982).

Oxamyl, in particular, reduced nematode numbers and increased root growth, fruit size and yield after the first year. Similarly, Apt (1981) found that by applying oxamyl or fenamiphos at monthly intervals by drip irrigation during the first 16 months of pineapple growth at 1.7 Kg a.i. ha”*, Rotylenchus reniformis populations could be reduced by 91-93% and yield increased by over 80%. While Hylin et al. (1982) stated that four monthly applications of 50 mg 1"* of oxamyl via a drip irrigation system resulted in nearly complete eradication of nematode populations in a heavily infested Hawaiian soil. Similarly, Park et al. (1982) showed an economic yield response after drip irrigating citrus and vine crops with

1.2 Kg a.i. ha * of oxamyl per month, and Garabedian and Van Gundy

(1983) found that whereas a single application of nematicide was ineffective at controlling M.incognita on tomato, multiple application through a low pressure drip irrigation system at-0.33 (for the novel nematicide, avermectin) and 3.36 Kg a.i. ha"* (for oxamyl and carbofuran) effected complete control.

It is obvious from the above work that oxamyl applied incrementally via an irrigation system is more efficient than when applied more conventionally and this compound has now been registered for use in drip and flood irrigation systems in California (Park et al., 1982). - 29 -

Another form of simulated controlled delivery is the multiple application of oxamyl as foliar sprays or in combination with soil treatments to non-irrigated crops. For example, control of the burrowing nematode, Radopholus similis on banana was achieved by up to four applications of oxamyl at 1.2 - 2.4 Kg a.i. per plant (Park et al., 1982). While McKenry and Buzo (1984) obtained yield increases on vines treated with monthly or bi-monthly applications of oxamyl at low dosage rates, and several nematode species on pineapple in South Africa have been controlled through the use of oxamyl at 2.5 Kg a.i. ha"* plus three sprays at three-monthly intervals (Anon, 1983). More effective control through multiple applications of oxamyl has also been reported for H.sacchari (Garabedian and Hague, 1981), H.schachtii (G?iffin, 1975;

Potter and Marks, 1976b), Globodera rostochiensis (Brodie, 1983),

M.incognita and Pratylenchus scribneri (Radewald et al., 1970) and

P.penetrans (Thompson and Willis, 1976).

1.2 The beet cyst-nematode, Heterodera schachtii (Schmidt)

The frequent cropping of land with sugar beet may lead to a condition known as "beet-sickness" where the plants are stunted, wilt readily, and have a small tap root wi-th an excessive development of fibrous lateral roots, all of which results in a greatly decreased yield. This condition is caused by the beet cyst-nematode or beet eelworm, H.schachtii

(Schmidt), a major pest in Europe and the United States.

In Britain, H.schachtii is firmly established in the peat soils of the

East Anglian fens and is slowly becoming more widespread elsewhere

(Southey et al., 1982; Whiteway et al., 1982). In a recent survey by the

Ministry of Agriculture, beet cyst-nematode infestations were found to - 30 -

occur in up to 20% of randomly selected beet fields in the Fens and in

5% in other beet-growing areas (Cooke, 1983).

The areas likely to be particularly affected by "beet-sickness" are

those where beet and other host crops (members of the Cruciferae and

Chenopodiaceae) are grown in the same rotation. For example, the rapid

expansion in oilseed rape cultivation (an efficient host crop for

H. schachtii) into traditional beet-growing areas in the U.K. and its

inclusion in normal five year rotation systems will almost certainly

lead to an increase both in beet cyst-nematode infestation levels and

the acreage infested (Evans, 1984).

In 1977, it was estimated that 1,000,000 ha of arable land in the U.K.

was influenced by sugar beet production with approximately 200,000 ha of

sugar beet being grown each year, rising to 250,000 ha by 1980 (Rieley

et al., 1977); while in England and Wales the area of oilseed rape was

over 160,000 ha by 1982 (Evans, 1984). This increasing area of potential

host crops suggests that H.schachtii is likely to become an economically

more important nematode pest.

-As more land becomes infested the rate of spread is likely to increase.

In the United States, the percentage of infested fields in the Imperial

Valley, California was stable at 20% from 1964 - 1978 but from 1978 -

1981 this increased to an estimated 40%; an increase which probably

resulted from increased economic pressure to re-plant sugar beet

(Caswell and Thomason, 1984).

I. 2.1 Life cycle of H.schachtii. - 31 -

The entire life cycle of the beet cyst nematode occurs below the soil surface. This nematode has a sessile, lemon-shaped female and a motile vermiform male, both preceded by four juvenile stages. Fig. 1.5 shows the basic outline of the life-cycle.

The second stage juvenile (J2) is the resistant, dormant and infective stage remaining inside the egg within the cyst formed by the tanning of the female cuticle. Cysts are usually found in the top 30 cm of the soil, most containing 200 - 300 eggs (Jones and Jones, 1984) although the egg content of large cysts may exceed 800 (Goffart, 1952). Hatching of the J2 can occur spontaneously in water but is influenced by the presence of exudates from the roots of developing host seedlings; these diffusates increasing the rate of hatching rather than the total hatch.

This is in contrast to some other cyst-nematode species where, for example, G.rostochiensis hatches mainly in response to the presence of host root diffusates (Shepherd and Clarke, 1971).

After emergence from eggs and cysts, the J2 is attracted towards the roots of the host (Peacock, 1961; Viglierchio, 1961). Invasion occurs just behind the root tip or at the site of a new lateral root. Following invasion, the J2 starts to feed on a group of cells in the pericycle or endodermis. Its saliva rapidly induces the formation of an enlarged multinucleate syncytial transfer cell on which the nematode feeds throughout the rest of its development.

The sex of the juveniles becomes apparent in the third stage and from this point, the development of males and females is divergent. The J4 male re-elongates within the cuticle of the J3 stage and adult males eventually leave the root. In contrast, the J4 female remains saccate; - 32 - Fig. 1.5 Life cycle of Heterodera schachtii

Egg contains fully developed J2.

Hatches in ___^ ^ Juvenile moults or more freely i once within egg. to host root dii

Nematode cyst full of eggs as occurs in soil (dormant stage).

Immature adult ? (white, darkening to brown) projects from root.

^ Few eggs i passed out in mucus sac.

estis

2 inside

J4 ? cuticle

Adult free in soil. - 33 - the adult female swells greatly, ruptures the root cortex and protrudes from the root exposing the vulval region with head and neck remaining inserted in the root. Females are then fertilized by vermiform males (1 mm in length) which can survive in the soil for as long as 10 days. Up to one third of the eggs produced may be laid into the gelatinous egg-sac found adhering to newly formed cysts (Whitehead, 1973). The mature female (1 mm in length) which is initially white, darkens to a brown colour after death due to a tanning process in the cuticle.

The cysts remain in the soil after the host crop has been harvested and in the interval between host crops, a proportion of the eggs hatch annually and others may be destroyed by natural enemies and disease.

However, after a heavy infestation, some viable eggs may be found in the soil for up to ten years (Jones and Dunning, 1972).

The eggs of H.schachtii, unlike those of the potato cyst nematode, are unable to survive desiccation (Ellenby, 1968). Apertures at the head and vulval regions of the cyst aid water ingress and emergence of hatched J2

(Jones and Jones, 1984).

The number of generations of H.schachtii per annum is governed by the soil temperature, soil moisture and, where these are not limiting, the duration of the sugar beet season (Jones, 1950). In laboratory studies,

H.schachtii was found to reproduce most rapidly at 21-27 °C (Kampfe,

1955; Wallace, 1955; Raski and Johnson, 1959; Thomason and Fife, 1962) with an optimum of 25 °C. Thus, in the U.K., development is slow in the spring when soil temperatures are low and the life cycle is completed in approximately ten weeks, whereas in the summer, when soil temperatures are higher, the life cycle may take only six weeks. As soil temperatures - 34 -

fall in the autumn, nematode development slows and almost ceases in

November (Jones and Dunning, 1972).

In the U.K., sugar beet is generally grown from March to October or

November which allows for the completion of two and sometimes a partial

third generation of H.schachtii (Duggan, 1959). In contrast, in the

Imperial Valley of Southern California, where sugar beet is a winter

crop, three to five generations are possible (Jones, 1950; Thomason and o Fife, 1962) due to an optimum soil temperatures at 15 cm of 25 C for

much of the season (Cooke and Thomason, 1979). The number of generations

per season is of practical importance as there is an inverse

relationship between nematode populations in the soil prior to planting

and final yield (Olthof et al., 1974).

The rate of increase of H.schachtii under a host crop can vary from ten

to fifty fold. This is because invasion of J2 is density dependent. At

low initial pre-plant densities, a large proportion of J2 can develop r within the roots and multiplication rates are high, whereas at high

initial densities, sugar beet seedlings are unable to support large

populations and only a few J2 develop (Whitehead, 1973; Cooke and

- Thomason, 1979; Fichtner et al., 1983). Eventually an equilibrium

density is reached; microplot studies have put this at around 140 eggs g"*

soil (Jones, 1956; Greco et al., 1982).

As a consequence of H.schachtii populations increasing, to devise

effective pest management stratagems to minimise crop loss in infested

fields we have to have an understanding of the relationship between

nematode density at sowing and damage to the crop in terms of yield

loss. Jones (1956) found a linear inverse relationship between the - 35 - pre-planting density of H.schachtii and sugar yield. Seinhorst (1965) used this information to produce the first equation to relate the two mathematically. From this and earlier work on infested fields (Jones,

1945), a "tolerance limit" for H.schachtii on sugar beet of 10 - 20 eggs g “*soil was produced (the initial population level below which damage was not measurable).

In the United Kingdom, 10 eggs per g soil is recognised as the level above which it is unwise to grow sugar beet in the peat soils of the fens (Jones, 1945), while Steudel and Thielemann (1970) suggest 20 eggs per g soil as the level above which significant yield losses will occur in

Europe. However, it has always been recognised that low population densities are capable of causing appreciable losses particularly on mineral soils, and work by Cooke (1983) showed that as few as 2.8 and

6.2 eggs g~* soil were capable of causing yield losses of 5 and 10 tonnes ha”* respectively.-Similar levels, of 3 - 8 eggs g“* soil, were considered to give a rather large chance of damage to sugar beet in

Holland (Heijbroek, 1973). In Italy, Greco et al. (1982) found in practice that no yield losses occurred in soil infested with fewer than

8 eggs g -1 soil although they still derived a tolerance limit of 2 - 4 eggs g ”* using the Seinhorst equation (Seinhorst, 1965).

Tolerance limits were also found to vary with temperature. Thus, although 10 eggs g~* soil may be a suitable value in Britain where the average monthly temperature rarely reaches optimum for nematode development, this is not the case, for example, in Southern California.

Thus, Cooke and Thomason (1979) found that under irrigation - controlled field conditions in the Imperial Valley, a tolerance limit of only 1 egg g"1 soil was more applicable. Under the latter conditions, sugar yield - 36 - was reduced from 38 - 92% by 1.7 - 14.4 eggs g“* soil respectively

(Kontaxis et al., 1975; Olthof, 1978) and in contrast with British soils, only 1.7 and 2.5 eggs g“* soil caused yield losses of 5 and 10 tonnes ha~* (Cooke and Thomason, 1979).

The above observations may help agriculturalists integrate cultural and chemical control methods by predicting crop losses based on the initial nematode population density, before the crop is sown.

With the rising costs of nematicides and reduced profit margins it is equally important to know at what point treatment with a nematicide would be cost effective. This is known as the "economic threshold" and can be defined as the initial population level at which the value of the increased yield from an effective nematicide treatment becomes greater than the cost of treatment. The economic feasibility of chemical control depends on the value of yield increases and prevailing sugar prices.

Cooke and Thomason (1979) produced an equation to calculate this threshold level, taking into account such factors as the cost of treatment, the price of sugar beet, the potential yield and the tolerance limit. They estimated that although damage could be expected to occur with an initial population of 1 egg g _l soil, not until a population of 1.4 eggs g “l soil was present would there be a sufficient loss in yield to make chemical treatment economical.

However, many sugar beet growers often use pesticides as insurance treatments which do not always produce yield increases of sufficient value to offset costs of treatment (Mumford, 1979; 1981). This view is supported by Cooke et al. (1979) and Maughan et al. (1984), who found that at several field sites in East Anglia there was no response to - 37 - granular pesticides on the establishment and yield of sugar beet. These authors have therefore suggested that many growers use pesticides unecessarily and have advised greater selectivity in their use on the sugar beet crop.

1.2.2 Field control of H.schachtii.

Between 1977 and 1982, the U.K. national root yield loss caused by

H.schachtii was estimated as approximately 10000 tonnes of roots per annum on mineral soils and, assuming a similar yield loss relationship for all soil types, 30000 tonnes on organic soils (Cooke, 1984). At present, damage by the beet-cyst nematode is controlled by a four to six course rotation or a shorter rotation in combination with fumigant or granular nematicides.

1. Crop rotation

Studies have shown that a decrease in H.schachtii populations ranging from 33 - 78% eggs per annum occurs in the absence of a host crop

(Jones, 1956; Den Ouden, 1956; Jones, 1959; Moriarty, 1961; Shepherd,

1962; Moriarty, 1963; Southey, 1965; Greco et.al., 1982); the wide variation being due to a number of factors, including soil type and condition and the age and biotype of the cyst populations. The generally accepted average figure for decline is 50% per annum; the rate of decline appearing to be independent of the population level (Jones,

1956).

For many years, crop rotation was enforced by a clause in the grower's contract with the British Sugar Corporation (now British Sugar pic) and, - 38 -

in infested areas, by the Beet Eelworm Parliamentary Orders of 1960 and

1962. The latter stipulated that in a "scheduled area" comprising mainly

the organic soils in Eastern England and in all other areas known to be

infested, host crops of H.schachtii (i.e. most brassicas as well as

sugar beet and related crops) should not be grown more than one year in

three and that in fields known to be infested (whether inside or outside

the scheduled area), one year in four. The grower's contract stipulated

that sugar beet should not be grown in any field which had had a host

crop of H.schachtii in either of the two previous years. Due to the

above measures, very little damage occurred and the spread of the beet

cyst-nematode was more or less contained, although some growers,

especially on the peat soils of the fens, were seriously limited in the

frequency with which they could grow beet.

The situation has now changed due to the introduction of the Beet Cyst

Nematode Order of 1977. The Ministry of Agriculture has decided that the

cost of administering the old Beet Eelworm Order was no longer justified

and believed that the growers themselves should be held responsible for

adopting a suitable rotation system. However, the 1977 Order in revoking

-all existing statuatory restrictions does still enable controls of

cropping to be re-introduced should nematode infestations increase

(Dunning and Dyke, 1977). More recently, and because no apparent

increase in infestations had occurred following the above changes,

British Sugar removed the above mentioned restrictions on cropping prior

to sugar beet (from 1983 onwards).

In the Imperial Valley of Southern California, where the threat of

nematode damage is much greater as discussed previously, infested fields

cannot be replanted to sugar beet for at least three years and annual - 39 - surveys, conducted since 1960, monitor infestations in all planted fields by sampling soil adhering to sugar beet delivered to the factory

(Thomason et al., 1984).

2. Resistant varieties

The potential value to the sugar beet industry of sugar beet varieties resistant to H.schachtii is well documented (Winslow, 1954; Viglierchio,

1960; Steele, 1975).

Research to breed sugar beets which are resistant or tolerant to

H.schachtii has long been in progress and although resistant, interspecific crosses between resistant wild-type and commercial species of beet have been produced (Savitsky, 1975; Cooke, 1982) we are still some way from having a commercial nematode-resistant beet variety.

Even if resistance to H.schachtii were to be developed, this may not be the solution. For example, when resistant varieties were introduced for the soybean cyst-nematode, Heterodera glycines, physiological strains developed which were able to multiply on the resistant strains of soybean (Ross and Brim, 1957; Ross, 1962; Price et al., 1978). Since

H.glycines and H.schachtii are closely related (Steele, 1965; Potter and

Fox, 1965; Miller, 1975), it seems likely that physiological strains of

H.schachtii could also develop (Griffin, 1981) which would be able to overcome any plant resistance.

3. Chemical control

Chemical control of cyst-nematodes is necessary when other control - 40 - measures prove inadequate. The longevity and wide host ranges of

H.schachtii make control solely by crop rotation difficult or

impractical in modern farming. Similarly, the lack of effective biological control measures and the lack of resistance to nematode

increase in commercial cultivars can make use of nematicides essential.

A cyst-nematode is considered to be fully controlled by a nematicide when a susceptible crop can profitably be grown in infested soil without

increasing the number of nematodes in the soil; partial control is achieved when multiplication is lessened. As female cyst-nematodes produce many eggs, nearly all juveniles must be killed or immobilised if partial control is to be achieved.

Rapid progress in the control of cyst-nematodes followed the manufacture of the non-fumigant granular nematicides. Of these, some organophosphate compounds can be effective, for example, fenamiphos, but generally the most active compounds belong to the oximecarbamate group.

At present, carbamates with known nematicidal as well as insecticidal activity (aldicarb, oxamyl and carbofuran) are used on approximately 35

- 45% of the sugar beet crop in the U.K. to control nematode and arthropod pests (Cooke, 1982; Maughan et al., 1984). These compounds have low phytotoxicity and may be applied to the crop at sowing or around established plants. They are relatively easy to apply requiring no special equipment, are effective in controlling some nematode populations at low dosage rates and have a short residual activity (Van

Berkum and Hoestra, 1979). Unfortunately these materials have little fumigant action and are only effective in the soil in the immediate vicinity of the granule. However, they are systemic being taken up by - 41 the plant roots and translocated to all parts of the plant thus deterring aerial as well as root pests. Overall, they have been shown to be far superior to soil fumigants as demonstrated by Jorgenson (1969) who found that plots treated with aldicarb consistently produced higher sugar beet yields than plots treated with conventional soil fumigants.

Aldicarb and oxamyl at rates of 0.5 - 1.0 Kg a.i. ha"* applied in the seed-furrow were first used on a wide scale on the British sugar beet crop in 1974 - 1975 (Cooke, 1975a). At these low rates, they are economically effective in controlling ectoparisitic nematodes

(Trichodorus, Paratrichodorus, and Longidorus species) which causes a disease of sugar beet known as Docking disorder; a disease particularly prevalent on sandy soils (Dunning and Winder, 1968; Cooke et a!., 1974;

Cooke, 1975a; Maughan et al., 1984). As insecticides they are effective in controlling soil arthropod pests which decrease seedling establishment (Dunning and Heijbroek, 1981; Brown, 1983) and aphids which transmit yellowing viruses (Cooke, 1975b; Maughan, 1977; Maughan et al., 1984).

For treatment of cyst-nematodes, much higher rates are required than for ectoparasitic species. In a wide range of arable soils, 5.0 Kg a.i. h_a-l of aldicarb or oxamyl incorporated in the seed-bed before the crop is sown will prevent serious crop losses in sugar beet, potatoes and peas, and except for sugar beet, will reduce nematode increase by about 90%

(WHitehead, 1977). Applied broadcast, these granular nematicides are used commercially at the above rate to control cyst-nematodes on potatoes in the U.K. but because of the occurrence of 2-3 generations per year, controlling H.schachtii populations has proved less effective

(Muller, 1979). - 42 -

Thus, at the present recommended field rates for cyst-nematode control, aldicarb or oxamyl may increase yields in soils heavily infested with

H.schachtii but they are unable to control nematode reproduction (Cooke,

1982). Much higher rates are needed for effective population control to increase persistence but this is likely to be uneconomic and may possibly result in phytotoxicity.

Soil fumigants have limited uses in beet production, mostly because they are phytotoxic and must escape from the soil surface before crops are grown. They are also strongly adsorbed to organic soils thus rendering them less effective and they can present problems in application.

Nevertheless, fumigants are used to control H.schachtii populations in the Netherlands and California at a cost of approximately £250 ha“* .

Treatment costs have usually prevented their use in the U.K. (Cooke,

1982).

Thus, although the large rates of nematicide required for chemical control of H.schachtii are too expensive to be recommended for routine use at present, nematicide usage is likely to increase in the future with the development of new compounds, formulations and modes of application (Cooke, 1978).

4. Current pest status of H.schachtii in the U.K.

It remains to be seen whether the removal of enforced rotational control will alter the pest status of H.schachtii through changes in cropping practices; so far (to 1982), surveys have shown no changes in cropping policy (Cooke, 1982). - 43 -

Proposed increases in sugar beet growing in this country, due, for example, to the possible use of this crop for fuel alcohol production

(Doney and Theurer, 1980; Doney, 1980), may result in an increase in beet cyst eelworm infestations. However, the single greatest threat to maintaining control of H.schachtii comes from the increased cropping of the alternative host crop, oilseed rape in beet growing areas (Evans,

1984; see Section 1.2).

Oilseed rape is a particularly efficient host of H.schachtii and with two pest generations a year, as on sugar beet, it can cause a quick build up of this nematode (Anon., 1980), as well as provide an overwintering site for the aphid vector of virus yellows, Myzus persicae. With as yet no nematicide cleared for use on oilseed rape

(Evans, 1984), it would appear likely that H.schachtii will become a more economically important pest in the near future.

5. The need for an improved nematicide formulation to control

H.schachtii on sugar beet

The most damaging effects of attack by H.schachtii are on young beet seedlings. In the U.K., populations of H.schachtii are controlled during the early part of the growing season by standard commercial formulations of aldicarb, carbofuran or oxamyl. Steudel and Thielemann (1967) have estimated that 90% of final yield loss could be prevented by applying aldicarb (5 Kg a.i. ha"*) over the rows immediately after sowing.

However, ideally a nematicide should also prevent multiplication of

H.schachtii. Despite early control, the soil population at harvest may have increased to a level similar to, or even above, that found in untreated control plots (Steudel and Thielemann, 1967; Jorgenson, 1969; - 44 -

Heijbroek, 1973; Thielemann and Steudel, 1973; Dunning and Winder,

1974). This is because the a.i. usually declines rapidly in the soil

(Bromilow, 1973; Leistra et al., 1976; Bromilow et al., 1980) and rapid nematode reproduction by late-hatching or second generation J2 can then occur on the extensive root systems formed during the initial period of nematode control (Batterby et al., 1980; Cooke, 1982).

Whitehead et al. (1979) concluded that small amounts of aldicarb or oxamyl were often as effective as the recommended commercial rates at preventing early damage to seedlings and minimizing multiplication of

H.schachtii. However, because of the short 2 - 3 week half-lives of these compounds in U.K. soils, doses of up to 19.2 Kg a.i. ha-l must be used to delay invasion of sugar beet roots long enough to prevent later root injury and rapid nematode population increase. This rate of nematicide application is both uneconomic and likely to cause severe phytotoxicity problems if applied as a single dose.

Thus, in the U.K. and many other countries, a potential usage for controlled-release nematicides is the of H.schachtii. The advantages of such formulations would be twofold: they would give economic control of nematode damage to the sugar beet crop to which it was applied and, by reducing final nematode populations through extended control, allow more frequent cropping with sugar beet in favourable growing areas.

I.3 Nematicidal activity of oxamyl (Vydate)

The oximecarbamate, oxamyl[S-methyl-N,N-dimethyl-N-(methylcarbamoylloxy)

-1-thiooximimidate] (Fig. 1.6) is a contact-type moderately residual nematicide and insecticide developed by E.I. du Pont de Nemours and Co. - 45 - and marketed under the name of Vydate 106.

Fig. 1.6 Structure of oxamyl (a) and its oxime (b)

a) (CH )?N.C0.C(SCH ) = N0.C0.NHCH O L O j

b) (CH ) N.C0.C(SCH ) =N0H O u j

Oxamyl is fully systemic in action, moving both upward (acropetally) in the xylem and, to a lesser degree, downward (basipetally) in the phloem

(Radewald et al., 1970; Taylor and Alphey, 1973; Potter and Marks,

1976a, b; Gowen, 1977; Peterson et al., 1978; Atilano and Van Gundy,

1979; Wright and Womack, 1981).

The primary degradation product of oxamyl, the oxime (Fig. 1.6) has no nematicidal activity at field rates (Wright et al., 1980; Evans and

Wright, 1982; McGarvey et al., 1984). The effects of non-fumigant nematicides on various stages of the nematode life cycle have been examined in a number of plant parasitic species (McLeod and Khair, 1975;

Hough and Thomason, 1975; Wright et al., 1980; Evans and Wright, 1982).

Oxamyl, like other carbamate and organophosphate nematicides, controls nematode populations by sublethal rather than lethal effects and its action is known to be reversible (Wright, 1981). It is believed that all of these compounds inhibit the enzyme acetylcholinesterase (AChE) in the nematode nervous system (Spurr, 1966; Spurr and Chancey, 1967; Evans,

1973; Wright and Awan, 1976; Hogger et al., 1978; Wright, 1981). In nematodes, acetylcholine is thought to be the excitatory transmitter at - 46 - neuromuscular junctions and inhibition of AChE would thereby result in an accumulation of excess transmitter and subsequent impairment of neuromuscular and sensory activity thus disrupting the life cycle

(Wright, 1981). It is known, for example, that hatching, invasion, feeding behaviour, orientation behaviour and development of plant parasitic nematodes can be impaired at low concentrations of oxamyl

(Wright et al., 1980; Evans and Wright., 1982).

In the present work, oxamyl has been selected as a suitable candidate for development in a controlled-release formulation. Oxamyl is particularly appropriate for this role as it is known to have a relatively short half-life in the soil (Bromilow et al., 1980; Gerstl,

1984) with a minimum recommended interval between applications and harvest of only 14 - 21 days (Jones and Jones, 1984). As part of this study, and in order to determine the MIC required, experiments on the effects of oxamyl on the eggs and infective J2 of H.schachtii and on the development of this species within the sugar beet root have been used to determine which stages of the life cycle are most susceptible to chemical control. - 47 -

2. MATERIALS AND METHODS

2.1 Chemicals

Analytical samples of oxamyl and its oxime (>99% pure) were supplied by

Du Pont de Nemours and Co., Wilmington, Delaware, USA. Stock solutions of pesticide were prepared in re-distilled acetone and stored at -20°C.

Granular formulations of oxamyl: Vydate 10G, DPX 4702 and DPX 5578-2, together with Florex 30/60 LVM blank granules were supplied by Du Pont de Nemours and Co. Experimental formulations of oxamyl were also prepared by I.C.I. Ltd., Plant Protection Division, Jealott's Hill,

Bracknell, Berks. Commercial granular formulations of aldicarb: Temik lOG-f and Temik lOG-bc, were donated by Union Carbide U.K. Ltd. Vinylite

VYHD-coated aldicarb granules were prepared by Dr S. Batterby (Batterby,

1978).

GPR grade cellulose acetate, polyethylene glycol-6000, urea and the plasticizers - dimethyl phthalate, dibutyl phthalate and triphenyl phosphate were supplied by British Drug Houses Ltd., Poole, Dorset.

Sodium sulphate (anhydrous) and FI ori s i1 (60-100 mesh) for column chromatography were also from BDH Ltd. Florisil was activated by heating at 120 °C for 16 h before use.

Cellulose triacetate (Grade SP1) was donated by Hercules Powder Co.,

Ltd., London, and the Aerosol 0T-B (Cyanamid) was donated by Dow

Chemical Co., Ltd.

Sebacic acid dimethyl (90-95%) and dibutyl (Grade II, 95%) esters were - 48 - obtained from Sigma Chemical Co., Ltd., London and the formaldehyde solution (40% w/v) was supplied by May and Baker Ltd., London.

2.2 Nematode culture

Cysts of Heterodera schachtii (Schmidt) used for laboratory experiments were obtained from sugar beet plants, Beta vulgaris L. cv. Vymoto, grown in sandy-loam soil (50:50) in the greenhouse. Plants were watered weekly with a 0.05% (w/v) Phostrogen solution (Phostrogen Ltd., Corwen, Clwyd, pH 4.7). Cysts were extracted from soil using a Fenwick can (Shepherd,

1970).

Hatched second stage juveniles (J2) of H.schachtii were obtained by placing cysts on a 180 p nylon sieve in a solid watch glass containing sugar beet root diffusate obtained from three to ten-week-old seedlings grown in acid-washed silver sand. Nematodes which hatched over the first

24 h at 2 0 ° C were discarded; only those which hatched between 24-48 h were used in experiments.

2.3 Effects of oxamyl on life cycle stages of H.schachtii

2.3.1 Hatching.

Ten similar sized cysts of H.schachtii were placed on a 180 p nylon sieve in a watch glass containing oxamyl in Phostrogen solution (2 cm? ), oxamyl in sugar beet root diffusate (2 cm ) or control solutions. After

o 7 days at 15 C, the number of hatched juveniles was recorded and the cysts placed in fresh Phostrogen solution daily for 3 days and finally in sugar beet root diffusate for 4 weeks. The number of hatched - 49 - juveniles was recorded at each stage. At the end of the experiment, the cysts were broken open and the viability of the remaining unhatched eggs was determined using New blue R stain (Shepherd, 1962). Each treatment was replicated four times.

2.3.2 Nematode activity.

Ten H.schachtii J2 were placed in a watch glass containing either oxamyl or its oxime in Phostrogen solution or in Phostrogen solution alone (2 cm*). The activity of the juveniles was recorded after 1, 2, 4, 8, 12 and 24 h at 15 °C using two methods of assessment:

1) A 0-4 scale, where 0 = no movement during a 10 sec. observation period, 1 = little movement with spasms in anterior and posterior regions, 2 = movement with spasms, 3 = normal undulatory movement but considerably slower, and 4 = normal undulatory movement. The juveniles 9 were then transferred to fresh Phostrogen solution and their activity recorded after a further 24 h.

2) The mean number of body undulations per min. for five nematodes; an undulation being taken to represent one complete cycle or movement at the anterior end (Peters, 1928). Preliminary observations indicated a wide variation in the amplitude of the undulations between the various treatments, a conclusion also drawn by Nelmes (1970). Thus, to supplement the above observations and to obtain a direct comparison between treatments, the percentage of juveniles actively undulating during each observation interval was calculated (Wallace, 1962; Nelmes,

1970). - 50 -

The percentage of juveniles exhibiting abnormal stylet movement was also determined; stylet movement being defined as axial extrusion and retraction.

Each treatment was replicated five times

2.3.3 Migration.

The experimental procedure was essentially that of McLeod and Khair

(1975). Thirty H.schachtii J2 were added to a watch glass containing oxamyl or control solutions (1 cm?). After 8 h pre-incubation, the juveniles were placed on top of a sand column (1.5 x 0.5 cm; particle size 600 jj) in a polythene tube sealed at the bottom with nylon mesh (50 p) and standing in a specimen tube containing oxamyl or control solution. After 16 h at 15 °C, the number of juveniles which had migrated through the column into the outer solution was recorded. Each treatment was replicated five times.

2.3.4 Infectivity.

Approximately 150 H.schachtii J2 were incubated in oxamyl or control solutions (2 cm *) for 24 h before inoculation onto a dry silver sand:loam mixture (70:30; 7.5 cm?) containing a freshly transplanted four-week-old sugar beet seedling. After 48 h at 15 °C, the seedling was carefully removed, its roots washed and replanted in a clean sand:loam mixture and left for a further 10 days. The roots were then stained in methyl cotton blue (0.1% w/v) in boiling lactophenol (Hooper, 1970) for

3 min. and nematodes within the roots counted. Each treatment was replicated five times. - 51 -

2.3.5 Development.

Four-week-old sugar beet seedlings were inoculated with approximately

150 H.schachtii J2 in Phostrogen solution (2 cm*) and left for 48 h at o 15 C to allow for invasion of the roots to occur. The seedlings were then carefully removed, their roots washed, and seedlings transferred into fresh sand:loam mixture (70:30). Oxamyl in Phostrogen solution or

Phostrogen solution alone (2 cm*) was then added to the soil. After 14 o days at 15 C, during which the plants were watered with oxamyl or control solutions respectively, the roots were stained (Section 2.3.4) and the number of vermiform and saccate juveniles determined. All treatments were replicated five times.

2.4 Effect of long-term exposure to oxamyl on nematode migration,

infectivity and lipid levels

More than 5,000 H.schachtii J2 were incubated at 15 °C in a screw-capped

Erlenmeyer flask (50 cm*) containing oxamyl in Phostrogen solution with benzyl penicillin (100 units cm*) and nystatin (10 units cm3 ), or

Phostrogen solution with antibiotics alone (total volume 10 cm3). The incubation medium was changed weekly.

Batches of juveniles were removed after 7, 14, 28 and 56 days incubation, washed three times in distilled water and left for 24 h in

Phostrogen solution. The juveniles were then tested for their ability to migrate through a sand column over 24 h at 1 5 ° C (Section 2.3.3) or their relative infectivity assessed (Section 2.3.4) after the respective incubation periods. - 52 -

Nematode neutral lipid reserves were estimated after 7, 14, 28 and 56 days incubation using the Oil Red 0 staining procedure as described by

Storey (1984). The relative degree of staining of twenty nematodes was assessed visually.

2.5 Assessment of two methods of nematode inoculation to plants

1. Horizontal inoculation technique.

A newly germinated sugar beet seedling was transplantedinto a sandisoil

(70:30) mixture in one half of a split pot, divided down the centre by a polystyrene strip (Fig. 2.1a). After 14 days at 15° C, the pot was opened and the polystyrene strip removed to reveal an extensive root system (Fig. 2.1b). The split pot was held horizontally and approximately 150 H.schachtii J2 were inoculated onto the exposed surface in Phostrogen solution (1 cm?), the exposed surface was then covered with a moist cotton wool milk filter and the pot left in a horizontal position at 15 °C for 48 h. The cotton wool pad was then removed and thepot resealed with its other half filled with a sand:loam mixture (70:30) to allow extensive new root growth. Thepots were kept at 15 °C for a further 14 days, and the number of nematodes within the roots determined as previously described (Section 2.3.4).

2. Vertical inoculation technique.

A freshly transplanted, three-week-old sugar beet seedling in a sand:soil (70:30) mixture was inoculated with approximately 150

H.schachtii J2 in Phostrogen solution (1 cm*) by applying the nematodes - 53 -

Fig. 2.1 Horizontal Inoculation Technique

a) Seedling grown in split pot

Polystyrene strip

b) Pot opened and nematodes inoculated onto exposed root surface

Pot resealed after 48 h at 15° C. - 54 -

to the soil surface. After 14 days at 1 5 °C, the number of nematodes in the roots was determined (Section 2.3.4).

2.6 Preparation of controlled-release granules

Experimental granules were formulated using Florex 30/60 LVM blank granules as the base material. The final product had a size range of 20

- 30 mesh and contained 5.4% (w/w) active ingredient (a.i.). Pilot samples (25 g) of each type of formulation were prepared and their release characteristics determined.

2.6.1 Extraction of oxamyl and dye from Vydate 10G granules.

Vydate granules (625 g) containing 10% (w/w) oxamyl, were divided equally into four 1000 cm3 round-bottomed flasks fitted to a Griffin flask shaker. Analytical grade acetone (200 cm3 ) was added to each flask and shaken for 2 h. The solvent was then decanted into a collecting vessel. This was repeated a further six times using acetone (100 cm3 ), shaking for 1 h intervals. Finally, the granules were suction-filtered using a Buchner flask. The acetone extract containing the oxamyl and an intense blue dye (incorporated with a.i. in commercial formulations) was evaporated down to 250 cm3 on a rotary film evaporator and the oxamyl content determined by gas-liquid chromatography (Section 2.9.8).

2.6.2 Cellulose acetate and triacetate formulations.

Oxamyl with cellulose acetate (10% w/v) or triacetate (15% w/v) and any other adjuvant were applied in acetone to blank granules and thoroughly mixed. The solvent was evaporated under a jet of air. - 55 -

2.6.3 Urea-formaldehyde formulations.

Oxamyl in acetone was first absorbed onto the blank granules. After evaporation of the solvent, the granules were sprayed with a urea-formaldehyde pre-polymer solution (prepared heating a urea - formaldehyde solution at 90 °C for 3 h while maintaining the pH between

5.5 and 6.5), followed by mixing with orthophosphoric acid to produce the polymer. The granules were blended thoroughly until dry.

2.6.4 Polyethylene glycol formulations.

Polyethylene glycol-6000 in hot acetone was sprayed onto granules impregnated with oxamyl. The mixture was blended until the coating was hardened.

Other formulations with different coatings were prepared employing a combination of the above methods.

Formulated granules contained approximately 2.5% (w/w) of polymer except those formulations with urea-formaldehyde resin which contained approximately 15% (w/w), or where otherwise stated.

2.6.5 Measurement of oxamyl release rates.

The relative release rates of oxamyl from different granular formulations were determined, by placing the granules (1 g) on a glass column (150 x 18 mm) of acid-washed sand, and eluting with distilled water at a flow rate of 1 c m 3 per min.. The column retention time for - 56 - oxamyl under these conditions was about 10 min. The eluate was collected in glass tubes which were changed at 5 min. intervals.

The oxamyl content of the eluate was shown by HPLC analysis (Section

2.7) to be directly proportional to the concentration of blue dye released from the granules. For routine analysis, therefore, the concentration of dye was measured spectrophotometrically.

2.7 High Performance Liquid Chromotography (HPLC)

Oxamyl in aqueous solution was analysed by HPLC using a Gilson pump

(model 302) and manometric module (model 803), fitted with a Spherisorb

10 ODS column. The carrier, a solution of water and methanol (70:30), was passed through the column at a rate of 1.5 cm3 per min. Oxamyl solution was introduced into the column via an injection loop and absorption measured at 230 nm using a UV monitor (Cecil CE212).

2.8 Selection of experimental formulations for field trials

A theoretical a.i. release curve was calculated for an ideal formulation where the toxicant is released at a constant, zero-order rate for the desired period of control (set at 100 min. for the purpose of the laboratory experiments; Section 2.6.5). The release rates chosen were

3.00, 2.25, 1.75, 1.50, 1.25 and 1.00% (w/w) oxamyl minT1 under the laboratory conditions described. To assess each formulation, the total area under each of the six selected release curves was given a maximum

"efficiency index" of 100. The percentage of oxamyl released from each experimental formulation was plotted and an efficiency index calculated for each standard release rate. - 57 -

The formulations with the greatest efficiency index value compared with

each of the six selected ideal release rates were selected for field

evaluation.

The percentage a.i. of the formulations selected for field trials was

determined by a gas chromatographic method (Bromilow, 1976) following extraction of oxamyl from the granules with acetone.

2.9 Sugar beet trials, 1982-1984

2.9.1 Location.

Field trials were conducted in 1982 and 1984 on peat fen soils (Table

2.1) at Alder Farm, Prickwillow, Cambridgeshire. Sugar beet had been grown in the same fields three years before each trial. A normal crop-rotation programme, using non-host crops (potatoes and cereals), was in force during the intervening years.

Outdoor pot trials were also conducted in 1982 at Silwood Park, Ascot, using naturally infested peat fen (Prickwillow) and sandy-clay (Broom's

Barn) soils (Table 2.1).

In 1983 and 1984 microplots of sandy-loam soil at Silwood Park were used

(Table 2.1).

2.9.2 Experimental design.

1. 1982 Field trial Table 2.1 Soil analysis of field sites.

% (vt/vj) composition

Sand Silt Clay Organic matter pH

Site Year Soil (2000-60m ) (60-2m ) (< 2m ) (loss on ignition) (in water)

Prickwillow 1982 Peat fen 67 28 5 44.7 7.5 Silwood 1982 Sandy-clay 55 17 28 2.7 7.3 Silwood 1983 Sandy-1oam 64 21 15 5.2 6.2 Prickwi1 low 1984 Peat fen 53 24 23 45.3 7.2 i Silwood 1984 Sandy-1oam 66 19 15 5.0 6.4 - 58 -

Plots 12.2 m long and 5 rows of beet wide (50 cm between rows) were arranged in a fully randomized block with five replicates per treatment.

The treatments were:

A. Untreated.

B. Vydate 10G (10% a.i. w/w, gypsum-based granules, Du Pont).

C. Vydate DPX 4702 (9% a.i. w/w, experimental controlled-release

granules, Du Pont).

D. Vydate 10G Incremental.

All nematicide treatments were applied at 5.6 Kg a.i. ha"*.

The oxamyl granules were broadcast by hand using a "pepper-pot" and the plots rotivated to a depth of 15 cm using standard farm equipment.

Seed-bed preparation and drilling were carried out by the farmer four days after pesticide application. In treatment D, 2.8 Kg a.i. ha~* was broadcast at week 0 and an equal amount applied at week 4 when the granules were mixed into the soil between the rows of sugar beet seedlings using a hand fork.

All plots were sprayed with the post-emergence herbicide, Clout

(disodium methanearsonate) by the farmer eight weeks after application of oxamyl. The seedlings were thinned to a stand of 20 cm the following week.

2. 1984 Field trial

Plots 10 m long and 5 rows of beet wide were arranged in a fully - 59 - randomised block with five replicates per treatment. The treatments were:

A. Untreated.

B. Vydate 10G.

C. CA + DMP (0.5% w/w) + PEG (1% w/w) coating (5.4% a.i. w/w; 3.00%

oxamyl min"*).

D. CA + PEG (1% w/w) coating (5.4% a.i. w/w; 2.25% oxamyl min-*).

E. CA + PEG (0.5% w/w) coating (5.4% a.i. w/w; 1.75% oxamyl min-*).

F. CA + DBP (0.5% w/w) + PEG (0.5% w/w) coating (5.4% a.i. w/w; 1.50%

oxamyl min"*).

G. CA + DBP (0.5% w/w) + PEG (0.25% w/w)coating (5.4% a.i. w/w; 1.25%

oxamyl min“l).

H. CA + DBP (1% w/w) coating (5.4% a.i. w/w; 1.00% oxamyl min_l).

CA = Cellulose acetate.

DBP = Dibutyl phthalate.

DMP = Dimethyl phthalate.

PEG = Polyethylene glycol.

All treatments were applied at 5.6 Kg a.i. ha"* as described for the -

1982 trial. Drilling was carried out by the farmer seven days after nematicide application.

Field plots were sprayed with two post-emergence herbicides at standard rates; an application of "Betanal E" (phenmedipham) during late May was followed by a "Goltix/Actipron" (metamitron) mixture in mid June

(Actipron is an adjuvant oil used to increase contact activity). - 60 -

3. 1982 Pot trial

The treatments were:

A. Untreated.

B. Vydate 10G.

C. Vydate DPX 4702.

D. Vydate 10G Incremental (applied at weeks 0 and 4).

E. Temik lOG-f (10% a.i. w/w; gypsum-based aldicarb granules, Union

Carbide).

i F. Vinylite VYHD (11.5% a.i. w/w; Vinylite VYHD-coated montmorillonite

SYK aldicarb granules).

The nematicides were pre-mixed with soil to give a rate equivalent to

5.6 Kg a.i. ha“* , and the soil transferred to plastic pots to a depth of

15 cm. The pots were arranged in four fully randomized blocks with one replicate per block, and buried in a field site at Silwood Park to soil level. Seven inch pots were used with peat fen soil (1.5 Kg, 2050 cm3 soil); eight inch pots were used for sandy-clay soil (4.0 Kg, 2750 cm3 soil). Sugar beet seeds (cv. Vytomo) were sown to give three seedlings per pot.

4. 1983 Microplot trial, Silwood Park

Each treatment consisted of a row of sugar beet 3.05 m long with 50 cm between rows. Treatment rows were separated by guard rows and arranged in a fully randomized block design with five replicates per treatment.

The treatments were: - 61

A. Untreated.

B. Vydate 10G.

C. Experimental fast-release oxamyl granule (5% a.i. w/w; I.C.I.).

D. Experimental medium-release oxamyl granule (5% a.i. w/w; I.C.I.).

E. Experimental slow-release oxamyl granule (5% a.i. w/w; I.C.I.).

F. Vydate DPX 5578-2 (10% a.i. w/w, experimental controlled-release

granule, Du Pont).

G. Vydate DPX 5578-2 : Vydate 10G (3:1).

H. Vydate DPX 4702 : Vydate 10G (3:1).

Pesticide granules were broadcast in a 50 cm band, at a rate equivalent to 5.6 Kg a.i. ha"* and mixed to a depth of 15 cm using a garden fork.

Pelleted sugar beet seed (cv. Sharpes Klein Monobeet) was sown by hand at 7 cm intervals, two days after granule application. Sugar beet seedlings were thinned to a stand of 21 cm, 8 weeks after germination.

Fertilizer (Fison's Growmore 7:7:7) was applied after 0 and 10 weeks at

70 g m 2 broadcast. Weeding was carried out by hand.

5. 1984 Microplot trial, Silwood Park

Each treatment consisted of a plot 3.05 m long and 5 rows of beet wide with 50 cm between rows within a treatment and 66 cm between treatments.

Only the centre three rows of each plot were assessed. The field plot was divided into four blocks with one replicate per block arranged randomly.

The treatments were:

A. Untreated.

B. Vydate 10G. - 62 -

C. Temik lOG-bc (10% a.i. w/w; coal-based aldicarb granules,Union

Carbide).

D. CA + DBP (0.5% w/w)+ PEG (0.25% w/w) coating (5.4% a.i. w/w; 1.25%

oxamyl min *).

Pesticide granules were applied in the furrow at 7.6 g a.i. 100 m-1 row

(1500 g a.i. ha-l) using a calibrated Horstine Farmery Spot Applicator.

Mono pelleted sugar beet seed (cv. Regina) was sown at 7 cm intervals using a Combi HS-300 Seed Applicator, and thinned to a stand of 21 cm between 4 and 12 weeks after germination.

Fertilizer (Fison's Growmore 7:7:7) was applied before sowing and after

12 weeks at 70 g m 2 . Six weeks after germination of beet seedlings, all plots were sprayed with one application of a post-emergence herbicide mixture; "Betanal E" (phenmedipham) and "Nortron" (ethofumesate) 7 and 5

1 ha * respectively.

2.9.3 Soil sampling.

Soil samples were taken for initial and final nematode populations and for pesticide residue analysis; these were stored at 5 °C and -20 °C respectively.

For estimation of H.schachtii populations, the following procedures were used:

1. 1982 and 1984 field trials - fifteen soil cores (18 x 2.5 cm) per

plot were taken.

2. 1982 pot trials - bulk samples were taken before potting and at - 63 -

harvest.

3. 1983 microplot trials - six soil cores per plot were taken.

4. 1984 microplot trials - nine cores per plot were taken.

For pesticide residue analysis, ten soil cores per plot were taken from the central 10 m and 9 m of the middle three rows of beet during the

1982 and 1984 field trials respectively.

2.9.4 Sampling of plants.

Plant samples were taken at random from treatment plots throughout the growing season to determine seedling or root weights, and the number of nematodes per root system.

2.9.5 Harvesting of sugar beet.

The following areas in each plot were harvested 28 weeks after pesticide application:

1. 1982 field trial - middle 10 m of the central 3 rows of beet.

2. 1984 field trial - middle 9 m of the central 3 rows of beet.

3. 1983 microplot trial - central 10 beet of each treatment row.

4. 1984 microplot trial - central 10 beet of the middle 3 rows of beet.

Each beet was lifted and "topped" (approximately 1 mm above the lowest leaf scar) by hand. Loose soil was knocked off the beet before counting them into sacks. The sacks from each plot were weighed on the field using a spring-loaded balance. - 64 -

2.9.6 Estimation of H.schachtii in roots.

The method used was essentially that of Marks and McKenna (1981). For small root systems, the normal procedure of staining washed roots in boiling methyl cotton blue/lactophenol (0.1% w/v) for 3 min, followed by clearing in plain lactophenol for several hours was used (Hooper, 1970).

For larger root systems, the main tap root was "shaved" of all lateral rootlets; the latter were wrapped in muslin cloth and stained as above.

Roots were then washed free of lactophenol on a 250 p (60 mesh) sieve, and transferred to a liquidizer (Moulinex) in 200 cm3 of distilled water. Five successive periods of homogenization were usually sufficient to finely macerate the roots. The homogenate was poured through a 178 p

(85 mesh) sieve into a 53 p (300 mesh) sieve and thoroughly washed with a jet of water. The contents of the latter sieve were transferred to a graduated boiling tube and made up to 25 or 50 cm3 with distilled water, depending on the amount of plant tissue. Five or ten drops of methyl cotton blue/lactoglycerol (0.1 % w/v) stain were added to the suspension to prevent excessive colour loss from stained nematodes. Three aliquots

(5 cm ) were taken after thorough mixing, and dispensed into plastic counting dishes. The number of nematodes and their stage of development was recorded.

2.9.7 Estimation of H.schachtii~ in soil.

Soil samples were dried overnight in a ventilated cabinet at 25 °C, and cysts extracted from 200 g (dry wt.) of soil using a Fenwick can.

Lemon-shaped cysts were crushed in an all-glass homogenizer, washed into a calibrated boiling tube and the volume made up to 20 cm3 . The tube was vibrated for 60 sec. on a Fison's vortex mixer to separate eggs from - 65 -

ruptured cysts, an aliquot (2 cm3) was transferred to a perspex counting well and the number of eggs (and J2) counted. Further aliquots (2 cm3) were observed after agitation of the boiling tube for each sample and the mean count calculated as eggs per gram of dried soil.

2.9.8 Oxamyl residue analysis

The method used was essentially that described by Bromilow (1976).

1. Extraction procedures

Anhydrous sodium sulphate (25 g), acetone (75 cm3) and dichloromethane

(75 cm3) were added to 50 g of thoroughly mixed soil in a round-bottomed flask (500-cm3), and shaken mechanically for 4 h. An aliquot (75 cm3) of the supernatant was removed and filtered through Whatman No 1 paper into a round-bottomed flask (250-cm3). The filter paper was washed with 15 cm3 of acetone. The supernatant was evaporated to the point of dryness on a rotary film evaporator.

2. Column clean-up

A chromatographic column (glass; 300 x 18 mm i.d.), with integral sintered glass support (porosity 0), and a PTFE Rotaflo stopcock, was prepared with Florisil (18 g, 60-100 mesh) in diethyl etheriacetone

(3:1, eluting solvent). Anthydrous sodium sulphate (20 mm) was added on top of the Florisil and the solvent level reduced to the top of the column bed.

The soil extract was re-dissolved in small volumes of the eluting - 66 -

solvent (total volume 20 cm3) and added gradually to the top of the column while maintaining the solvent level just above the column bed.

After adding the extract, further eluting solvent (100 cm3 ) was allowed to percolate through the column at a rate of approximately 1 drop per second and the eluate discarded. The column was then eluted with acetone

(75 c m 3) and the eluate collected in a round-bottomed flask (100-crn3), evaporated to dryness on a rotary film evaporator and the residue quantitatively transferred (2 cm3 ) into a glass vial in ethyl acetate and stored at -20 °C prior to analysis.

3. Gas-liquid chromatography

A stainless-steel column (0.9 m x 3 mm o.d.), packed with chromosorb W

(80-100 mesh) coated with a mixture of Carbowax 20M (0.5% w/w) and SE-30

(5% w/w), was used in a Pye Series 104 gas chromatograph fitted with a venting valve and a United Analysts flame-photometric detector operating in the sulphur mode (394 nm filter). Solutions were taken into a 10 pi syringe in the following order: ethyl acetate (0 .2 pi), oxamyl sample

(2.0 pi), and 0.1 M trimethyl phenyl ammonium hydroxide in methanol (0.5 pi). The mixture was then injected and the column immediately vented for

30 sec.

Calibration solutions were injected containing 1-80 ng oxamyl. Mean recoveries from fortified samples (0.05-0.40 mg a.i. Kg”* soil) were also determined to calculate the efficiency of extraction. - 67 -

3. RESULTS

3.1 Effects of oxamyl on life cycle stages of H.schachtii

3.1.1 Hatching.

The percentage reduction in hatch during the period of nematicide treatment and the percentage total reduction in hatch are given in Table

3.1.

When exposed to oxamyl and sugar beet root diffusate or to oxamyl alone, hatch was significantly reduced by all treatments compared with the control (P < 0.01; Table 3.1) but resumed on transferring the cysts to diffusate alone (Appendix 1). Figs. 3.1a and 3.1b show the cumulative percentage hatch during the test period for both types of treatment.

_3 Total hatch was significantly reduced at 10 pg a.i. cm with oxamyl _ 3 alone (P < 0.05) and at 2.5 and 10.0 |jg a.i. cm with oxamyl plus root difusate (P < 0.05). Staining of unhatched eggs with New Blue R gave an overall mortality rate of 2 %; there was no significant difference between any of the treatments (P > 0.05).

3.1.2 Nematode activity.

Oxamyl rapidly inhibited nematode activity at concentrations greater _ 3 3 than 0.5 jjg a.i. cm (Tables 3.2 and 3.3). At 2.0 pg a.i. cm" , only

58% and 2% of juveniles (J2) were actively undulating after 1 and 2 h exposure respectively (Table 3.3). In contrast, the oxime of oxamyl had no visible paralytic effect at 25.0 pg a.i. cm" (Tables 3.2 and 3.3) Table 3.1 Effect of oxamyl on hatching of H.schachtii J2.

concn 3 % initial reduction , % overal1 reduction Treatment (pg cm" ) of hatch (0-7 daysr of hatch X

1.0 68.4** 18.8

2.5 89.5*** 5.6 Oxamyl i n 5.0 89.5*** 13.7 water 10.0 9 4 ^ 7*** 33.8*

1.0 62.3** 12.7

Oxamyl i n 2.5 89.5*** 41.5*

root 5.0 98.7*** 2 2 .0 • diffusate 10.0 98.5*** 54.3*

+ (% hatch in control for 0-7 days) - (% hatch in oxamyl for 0-7 days) v (% hatch in control for 0-7 days) i (% total hatch in control) - (% total hatch in oxamyl) ^ (% total hatch in control)

*, **s *** Significantly reduced from untreated at P < 0.05, 0.01, 0.001 respectively. - 69 -

Fig. 3.1 Effect of oxamyl on hatching of H.schachtii. Figures show

the cumulative percentage hatch of cysts during treatment

with oxamyl alone (a) or oxamyl in root diffusate (b)

followed by thorough washing and transfer to root diffusate,

at 15° C: A = control; 0 = 1.0 pg cm“^;4=2.5 pg cnf^;

0 = 5.0 pg cnf^; • = 10.0 pg cnf^. i.31 a) Fig.3.1

Cumulative % hatch. Cumulative % hatch. 40 20 30 50 60 70r 10 r 0 7 0 ) xml n ot diffusate. root in Oxamyl b) -

xml n water. in Oxamyl - 70 -

- 71

Table 3.2 Effect of oxamyl and its oxime on the activity of H.scnachtii J2 : 0-4 scale.

Mean activity ^ Following removal from pesticide Treatment Exposure to pesticide (h)______for (uq cm"3 ) 1 2 4 8 12 24 24 h

Untreated 4.0 4.0 4.0 4.0 3.9 3.9 3.6

Oxime: 25.0 4.0 4.0 4.0 4.0 t 4.0 * 3.9 1 3.6

Oxamyl: 0.1 4.0 4.0 + 3.9 * 3.9 } 3.8 3.6 3.4 0.2 4.0 t 4.0 t 3.9 3.8 3.5 3.1 3.4 0.5 4.0 t 4.0 3.5 2 .8 2.4 2.0 3.2 0.75 3.9 t 3.7 2.6 2 .0 1.2 1.0 3.4 1.0 3.8 3.0 2.1 1.5 0.9 0.6 2.9 2.0 2.6 1.3 1.0 0.9 0.5 0.3 3.0

"I* Means of five replicates, 10 J2 per replicate.

^ Hyperactivity observed. - 72 -

Table 3.3 Effect of oxamyl and its oxime on the activity of H.schachtii J2: Undulations per minute.

Mean undulations min”*.

Exposure to pesticide (h)

Treatment 1 2 4 8 12 24 (pg cm-3)

Untreated 5.4±0.1 6 .0±0 . 1 6 .0±0.1 7.4±0.2 7.0±0.1 6.3±0. 1 (100)(1 0 0) (1 0 0) (100)(1 0 0) (100) Oxime :25.0 6 .1 ±0 . 2 5.4±0.1 6.4±0. 1 9.0±0.1 8 .0±0 . 2 8.4±0.2 (100)(100)(100)(100) (1 0 0) (100) Oxamyl: 0.1 6 .1 ±0 . 1 7.0±0.2 8 .0±0 . 1 8.0±0 . 1 7.2±0.2 4.8±0.3 (100)(100)(1 0 0)(100) (1 0 0) (100) 0 .2 8.0±0 .2 7.4±0.2 8 .1 ±0.1 7.1±0.1 5.6±0.2 3.0±0.2 (100)(100)(1 0 0)(100) ( 96) ( 90) 0.5 8.6±0.3 6.6±0 . 1 4.6±0.1 1 .7±0. 1 0.9±0.1 0.4±0. 1 (100) (100)(1 0 0) ( 80) ( 50) ( 34) 0.75 7.2±0.2 4.5±0.1 1.3±0. 2 0.2 ±0 . 1 0 0 (100)(100) ( 60) ( 2 2 ) (0) (0) 1.0 6.4±0.2 3.0±0.3 0.5±0.2 0 0 0 (100) ( 74 ) ( 2 2 ) (0) (0)(0) 2 .0 1.4±0.2 0.1 ±0.1 0 0 0 0 ( 58) (2 )(0)(0) (0)(0)

Figures in parentheses are percentage juveniles actively undulating (rated 3 or 4 on scale).

Means of five replicates; 5 J2 per replicate. - 73 - but did cause hyperactivity between 8 and 24 h exposure (Table 3.2).

The majority of oxamyl-treated J2 recovered some of their former activity following 24 h in distilled water; recovery was little influenced by the original concentration of pesticide (Table 3.2).

The 24 h E C ^ value was estimated by Probit analysis to be 0.53 pg a.i. cm-3 using the 0-4 activity scale data and 0.19 and 0.42 pg a.i. cm- 3 when undulations per minute and percentage actively undulating were used, respectively.

The percentage of J2 exhibiting abnormal stylet movements increased during exposure to oxamyl (Fig. 3.2). The response of J2 to 0.5 pg a.i. cm"3 was only marked after 24 h, whereas 2.0 pg a.i. cm- 3 produced an immediate response. During the recovery period in distilled water the stylet activity of treated J2 ceased.

3.1.3 Migration.

The number of J2 migrating through a sand column in 16 h was significantly reduced by concentrations of 0.5, 0.75, 1.0 and 2.0 pg a.i. cm -3 (P < 0.01, 0.001, 0.001 and 0.001 respectively; Table 3.4). _ 3 The EC was estimated by Probit analysis to be 0.53 pg a.i. cm . 50

3.1.4 Infectivity.

\ -3 Soil application of oxamyl at 0.2 pg a.i. cm significantly reduced root invasion by J2 (P < 0.05; Table 3.5), whereas 0.5vand 1.0 pg a.i. _ 3 oxamyl cm almost completely prevented invasion (P < 0.001). The EC^ - 74 -

Fig. 3.2 Effect of oxamyl on abnormal stylet movement in H.schachtii.

Figures give the percentage of juveniles actively extruding

and retracting their stylet during exposure to oxamyl at 15° C.

All values are corrected for control movement using Abbott's

formula (Abbott, 1925):

A = 0 .2 M9 cm-3; 0 = 0.5 M9 cm-3; ♦ = 0.75 pg cm”3 ; II no o • O = 1.0 wg cm”3; • M9 cm”3 ; A = 25 pg oxime cm - 75 -

Fig. 3.2 - 76 -

Table 3.4 Effect of oxamyl on the ability of H.schachtii J2 to migrate through a sand column.

Treatment Number of J2 migrating through (pg cm"3 ) column within 16 h (meant S.E.)

Untreated 28.8 ± 1.5

Oxamyl : 0.1 27.2 ± 1.6 0.2 25.4 ± 1.3 0.5 18.4 ± 2.3** 0.75 9.4 ± 1 .4 *** 1.0 5.6 ± 1A*** 2.0 0.8 ± 0.4***

Means of five replicates, 30 J2 per replicate.

* Significantly less than untreated at P< 0.01, 0.001 respectively.

Table 3.5 Effect of soil application of oxamyl on infectivity of H.schachtii J2.

Treatment Number of juveniles in roots _o (pg cm ) ______(mean ± S.E.)______

Untreated 28.6 ± 4.1

Oxamyl : 0.05 24.6 ± 5.5 0.1 20.2 ± 2.7 0 .2 16.0 ± 3.4* 0.5 4.6 ± 0.g*** 1.0 1.2 ± 0.6***

Means of five replicates

5 Significantly less than untreated at P< 0.05, 0.001 respectively. 77 -

. -3 was estimated by Probit analysis to be 0.20 pg a.i. cm .

3.1.5 Development.

Soil application of oxamyl at 1.0 and 4.0 pg a.i. cm" significantly reduced nematode development within the root (P < 0.05, 0.01 respectively; Table 3.6). Probit analysis gave an estimated EC__ value 50 of 1.38 pg a.i. cm“ .

3.2 Effect of long-term exposure to oxamyl on nematode migration,

infectivity and lipid levels.

The effects of prolonged exposure to oxamyl on migration, infectivity and lipid reserves are shown in Tables 3.7, 3.8 and 3.9 respectively.

Incubation of J2 in 0.1 - 2.0 pg oxamyl cm" 3 appeared to have no harmful effects on their subsequent ability to migrate through a sand column when compared with control nematodes of a similar age. In fact, incubation in oxamyl for up to 56 days generally increased the number of

J2 migrating through a sand column in 24 h although none of the treatments were significantly different from the control (P > 0.05;

Table 3.7). Similarly, while the mean number of J2 invading roots was greater after the majority of the oxamyl treatments these were only significantly different from the controls for nematodes incubated in 0.5 _ 3 pg oxamyl c m ” for 28 and 56 days (P < 0.05; Table 3.8).

Neutral lipid reserves of J2 showed an obvious decline during prolonged incubation in oxamyl or control solutions, but lipid levels were noticeably greater in J2 exposed to more than 0.1 pg oxamyl cm- 3 for 28 - 78 -

Table 3.6 Effect of soil application of oxamyl on the development of H.schachtii J2 in sugar beet roots.

Treatment % juveniles saccate (M9 cm~3) after 14 days

Untreated 82.7

Oxamyl : 0.25 65.4 0.5 63.6 0.75 60.1 1.0 50.6* 4.0 17.6**

Means of five replicates * ** Significantly less then untreated at P< 0.05, 0.01 respectively. - 79 -

Table 3.7 Effect of long term exposure to oxamyl on the ability of H.schachtii J2 to migrate through a sand column.

No. J2 migrating through column within 24 h (mean±S.E.) Exposure to pesticide (days) Treatment (pg cm"3) 7 14 28 56

Untreated 15.5±2.3 9.5±1.7 7.5±2.5 7.8±3.0

Oxamyl: 0.1 12.0±1.6 13.0±4.1 8.2±1.4 4.5±1.2 0.2 15.2±1.2 10.2+1.5 11.8±2.9 t 0.5 17.8±1.9 13.8±2.3 11.5±1.0 11.8±2.9 2 .0 16.2±2.5 13.0±2.3 9.5±1.4 r

^ Fungal contamination of incubation medium .

Means of four replicates, 20 J2 per replicate.

Table 3.8 Effect of long term exposure to oxamyl on infectivity of H.schachtii J2.

No. juveniles in roots, (mean ± S .E.)

Exposure to pesticide (days) Treatment (pg cm"3) 7 14 28 56

Untreated 15.5±1.3 11.5±2.4 2 .5±1.0 0.5±0.3

Oxamyl: 0.1 16.5±2.6 3.0±1.3 1.2±0.5 0.8±0.5 0.2 24.8±2.6 10.2±3. 6 9.5±3.3 t 0.5 28.0±7.8 13.0±3.7 1 0.8±2 .0* 2 .5±0.9* 2.0 24.0+8.0 15.2+5.9 10.8±5.3 t

^Fungal contamination of incubation medium. Means of four replicates. ★ Significantly greater than untreated at P< 0.05. - 80 -

Table 3.9 Effect of long-term exposure to oxamyl on neutral lipid reserves of H.schachtii J2.

Lipid level 1

Exposure to pesticide (days) Treatment (pg cm"3) 0 7 14 28 56

Untreated 4.0 3.5 3.0 1.7 0.7

Oxamyl: 0.1 - 3.3 2.5 1.3 1.0

0 .2 - 3.7 2.7 2.3 \

0.5 - 4.0 3.3 2.3 1.7

2 .0 - 3.7 3.5 2.0 \

1 Scale 0-4 (none to full intestinal lipid content).

^ Fungal contamination of incubation medium.

Means of 20 J2. - 81 - days compared with untreated J2 (Table 3.9).

3.3 Assessment of two methods of nematode inoculation to plants

The horizontal technique of inoculating plants (Section 2.5) gave a significant increase in the number of J2 in the roots (P < 0.001) and gave a better distribution of nematodes throughout the root system than with the vertical technique (Table 3.10).

3.4 Relative release rates of oxamyl from experimental granular

formulations

3.4.1 Effect of polymer coating.

The rate of release of oxamyl from granules coated with cellulose acetate (CA), cellulose triacetate (CTA), urea-formaldehyde resin (UFR) or polyethylene glycol (PEG) alone into water are given in Fig. 3.3 and

Appendix 2, together with data for Vydate 10G.

The Vydate 10G granules released all the a.i. within a short period under laboratory conditions-(Section 2.6.5). Granules prepared using CA,

CTA and, in particular, UFR considerably reduced the release rate of oxamyl compared with Vydate 10G, while granules coated with PEG displayed similar fast release characteristics to Vydate 10G. The CA and

CTA-coated granules released approximately 60% of the a.i. (w/w) over the standard elution period of 100 min. with just over 0.2 % a.i. per min. being eluted after this time. Granules coated with UFR released only 16% of incorporated a.i. (w/w) over 100 min. with virtually nothing being eluted after this time, whereas the PEG coated granules released - 82 -

Table 3.10 Assessment of two methods of inoculating sugar beet seedlings with H.schachtii J2.

No. of juveniles i n roots f Method (mean ± S.E.) % invasion

Horizontal 117 ± 11 *** 77.7 (range 70.7-84.7)

Vertical 22 ± 2 14.8 ( range 13.5-16.1)

Means of 23 and 8 replicates for horizontal and vertical respectively.

• k ' k ' k Significantly greater at P< 0.001. - 83 - over 94% of available a.i. (w/w) in 55 min.

3.4.2 Effect of plasticizers.

The rate of release of oxamyl from granules prepared with CA and CTA- containing plasticizers are given in Figs. 3.4 and 3.6 and Appendices 3 and 5.

Incorporation of the plasticizers dimethyl and dibutyl phthalate and dimethyl and dibutyl sebacate into the polymer coating increased the removal of a.i. to 70% (w/w) over 100 min. with between 0.2 and 0.25% a.i. per min.being eluted after this time. Formulations prepared using

CA released a greater proportion of a.i. (w/w) than the corresponding

CTA formulations.

Increasing the amount of plasticizer in the polymer coating resulted in a reduced initial rate of release and an improved release rate after 100 min.(0.3% a.i. per min.). Incorporation of another plasticizer, triphenyl phosphate into the polymer coating reduced the release rate compared with polymer alone. Only 44% of the a.i. (w/w) was released over 100 min. and less than 0.1 % a.i. (w/w) per min. thereafter.

3.4.3 Effect of polyethylene glycol.

The rate of release of oxamyl from granules prepared with CA and CTA plus polyethylene glycol (PEG) are given in Figs. 3.5 and 3.6 and

Appendices 4 and 5.

Granules containing PEG at a concentration of 1% (w/w) released between - 84 -

85 and 95% of the available a.i. (w/w) over 100 min. and had a very rapid initial rate of release. Reducing the concentration of PEG in the coating decreased the release rate while the addition of a plasticizer had the effect of improving the release rate after 100 min. elution to

0.15% a.i. per min.

A second coating of CA to a granule prepared with CA and PEG reduced the initial release rate, releasing 65% of the a.i. (w/w) over 100 min. with a release rate of 0.25% a.i. per min. after 100 min. (Fig. 3.5; Appendix

4).

3.4.4 Effect of wetting agent.

Granules prepared using the wetting agent, Aerosol 0T-B incorporated into the CA coating released up to 60% of the a.i. (w/w) during the first 100 min. and had a release rate of between 0.15 and 0.2% a.i. per min. after this time. Varying the concentration of wetting agent had little effect.

3.4.5 Effect of urea-formaldehyde resin.

The rate of release of oxamyl from granules prepared with CA and CTA and coated with UFR are given in Fig 3.7 and Appendix 6 .

A coating of UFR considerably reduced the release rate and the total amount of a.i. which leached from the granules. In the majority of cases, less than 14% of a.i. (w/w) was eluted from granules with less than 0.05% a.i. per min. being released after 70 min. of the elution period. Formulations which also contained PEG released up to 22% a.i. - 85 -

Fig. 3 3 Effect of polymer coating on release rate of oxamyl from

experimental formulations: A= cumulative release; PEG at

16% w/w. A = "ideal" zero-order release.

Fig. 3 4 Effect of plasticizers on release rate of oxamyl from

experimental formulations prepared with cellulose acetate:

▲ = cumulative release. All plasticizers at 0.5% w/w, unless

otherwise stated.

Fig. 3 5 Effect of polyethylene glycol and Aerosol OT-B on release rate

of oxamyl from experimental formulations prepared with cellulose

acetate: A = cumulative release. All plasticizers at 0.5% w/w.

Fig. 3 6 Effect of plasticizers and polyethylene glycol on release rate

of oxamyl from experimental formulations prepared with cellulose

triacetate: A= cumulative release. All plasticizers at 0.5% w/w.

Fig. 3 7 Effect of urea-formaldehyde resin on release rate of oxamyl

from experimental formulations prepared with cellulose acetate

and triacetate: A= cumulative release. All plasticizers at

0.5% w/w. Fig.3.3 % oxamyl released % oxamyl released 100 100 100 i 80 20 40 60 80 20 40 60 20 “ 40 10 80“ 30" 50- 90 - 60- 70“ 0 0 0 J - “

2 4 60 40 20 0 ells ctt (CA)acetate Cellulose ie f lution o i t u el of Time 8 - 86 - Cluoetictt (CTA)triacetate Cellulose 1 . ) n i m ( Fig. 3.4

% oxamyl released 100 100 80 80 - 40 20 60 20 40 " 60 " 0 0

- i

A+Dbtl hhlt (DBP) phthalateCA Dibutyl+ A+Tihnl hsht (TPP) phosphate CA+Triphenyl ie f (min). n o i t u l e of Time - 87 - 60 A+DS (1%)CADBS+ A+Dmty sbct (DMS) sebacate CADimethyl+ i.3.5 Fig. 3 % oxamyl released 100 80 40 60 20 0 ie f (min)-. n o i t u l e of Time 8 - 88 - A+O- (0.25%).OT-BCA + Fig.3.6

% oxamyl released 100 100 80 20 40 60 80 0 CTA + DMP+CTA - 89 ieo lto (min)elutionof Time - CTADBP + Fig. 3.7 Fig.

% oxamyl released 0 0 1 100 100 40 80 0 2 60 0 2 40 60 80 80 40 20 60 0 0 0 J “ “ - J - - - - - " . - - “ -

CTA + DMP + UFR+DMP+ CTA CA + DMP + UFRDMP+CA+ CA + UFRCA+ - 90 l . ) n i m ( n o i t elu f o e m i T - CTAUFR + T M E (1%)UFR +PEG CTADMP+ + A+DP+PG (1%)UFR +PEGCA DMP+ + ▲ T 40 n 60 - 91

(w/w) over 100 min. with virtually nothing being eluted after this time.

3.5 Selection of experimental formulations for 1984 field trial

Fig. 3.8 shows the theoretical a.i. release curves calculated for an ideal formulation, where the pesticide is released at constant rates of

3.00, 2.25, 1.75, 1.50, 1.25 and 1.00 % a.i. per min. for the desired period of control (set at 100 min. for the purposes of this experiment).

The total area under each standard curve corresponds to quantitative removal of a.i. from the formulation and this was given a maximum efficiency index of 100. The percentage of oxamyl released from the experimental granules during the elution period was similarly plotted

(Fig. 3.8), and the percentage area corresponding to that of a standard release curve was calculated for each of the release rates and for each experimental formulation.

These values were assigned to the formulations as "efficiency indices" for the respective standard release rates or "index groups".

Appendix 7 gives the efficiency indices for all the experimental formulations tested and for Vydate 10G. From these indices the choice of formulation to represent each of the six selected ideal release rates was made. Table 3.11 shows the order of choice obtained; out of these, six different formulations were selected for field trials (Table 3.12).

3.6 Release rate characteristics of experimental oxamyl formulations

used in 1983 microplot trial. - 92 -

Fig. 3.8 Theoretical a.i. release curves where thepesticide

is released at a constant rate. 1 = 3.00% min’*;

2 = 2.25% min’1; 3 = 1.75% min"1; 4 = 1.50% min"1;

5 = 1.25% min"1; 6 = 1.00% min"1.

♦ = release curve for formulation No. 13. i. 3.8 Fig.

Release Rate (% oxamyl min. 5- -5 0 - 0 4 10 20 - 0 3 3-5- 1-5- 2-5- - 0 - - Standard ees Curves. Release ie mi .) in (m Time CJ vo I - 94 -

Table 3.11 Formulation selection for 1984 field trial.

Index groups

(Relative release rate: % oxamyl min”1)

1 2 3. 4 5 6 Choice (3.00) (2.25) (1.75) (1.50) (1.25) (1 .0 0)

1 st 14 (6 6) 13 (64) 13 17 (60) 15 12 T

(60) (58) (58)

2 nd 24 (65) 24 (63) 17 15 (58) 17 23

3rd 13 (64) 14 12 (57) 9 24 (59) 15 ll (5?) 10 (57) 16 (60) 24 18 17 25 25

Figures in parentheses are the corresponding efficiency indices.

^ Formulation No.12 was not available for field trial. - 95 -

Table 3.12 Formulations selected for 1984 field trial.

Release Index rate Formulation group (% oxamyl min } number Adjuvants used in the formulation

1 3.00 14 CA + DMP (0.5%) + PEG (1.0%) 2 2.25 13 CA + PEG (1.0%) 3 1.75 15 CA + PEG (0.5%) 4 1.50 16 CA + DBP (0.5%) + PEG (0.5%) 5 1.25 17 CA + DBP (0.5%) + PEG (0.25%) 6 1.00 9 CA + DBP (1.0%)

All percentages are w/w. For abbreviations see Section 2. N.B. At the concentrations used, none of the adjuvants alone were toxic to H.schachtii J2. - 96 -

The rate of release of oxamyl from the three experimental formulations

used are given in Fig. 3.9 and Appendix 8.

The granules with the fastest release rate lost over 95% of incorporated

a.i. (w/w) after 30 min. elution, whereas granules with a medium release

rate required 60 min. to achieve a comparable loss. The formulation with

the slowest release rate lost 77% of incorporated a.i. over 100 min.

elution, with 0.7 - 0.8% a.i. per min. being released after 60 min.

3.7 Sugar beet trials: 1982-1984

Full statistical analyses of data are given in the relevant appendices.

3.7.1 1982 Field trial.

Results from the field trial at Prickwillow are presented in Figs 3.10 -

3.12, Tables 3.13 - 3.14 and Appendices 9 and 16 - 21.

None of the treatments showed any signs of phytotoxicity on the young

beet seedlings and germination was good (Fig. 3.10).

At 8 weeks after pesticide application, all oxamyl treatments

significantly (P < 0.05) reduced invasion of roots (Fig. 3.10). However,

by week 17, the number of nematodes per root system was greater in the

treated plots compared with the control although the differences were

not significant (P > 0.05), while the number of nematodes per g.root

weight was similar to the controls (Fig. 3.11). At week 17, root weights

were not significantly different between treatments (P > 0.05; Fig.

3.11). - 97 -

Fig. 3.9 Release rate characteristics of the experimental oxamyl

formulations used in the 1983 microplot trial:

A = cumulative release - 98 -

Fig. 3.9

100 Slow

80 '

60 -

40 “

20 “

o J 0 - 99 -

Fig. 3.10 FIELD TRIAL 1982: WEEK 8.

a) Plant weight(g). b) Nematodes per root.

Treatments

Fig. 3.11 FIELD TRIAL 1982: WEEK 17.

a) Root weight (g). b) Nematodes per root. c) Nematodes per g. root. L.S.D. L.S.D. L.S.D. P = 0.05 P = 0.0' P = 0.05

Treatments 100 -

Fig. 3.12 FIELD TRIAL 1982: WEEK 28.

a) Harvest root weight (g).

Untd. Vy. DPX Vy. 10G 4702 Inc.

Treatments - 101 -

Table 3.13 1982 Field trial: Control of H.schachtii on sugar beet using various formulations of oxamyl (Vydate).

Mean eggs g” soil (± S.E.).

Initial Final ^ x multiplication Treatment Population Population rate 6 kg a.i. ha"*) (Pi) (pf) (Pf/Pi)

Untreated 2.5 ± 0.6 20.2 ± 6.3 8.1

Vydate 106 2.4 ± 0.5 10.5 ± 4.5 4.4

Vydate DPX 4702 3.2 ± 0.5 9.5 ± 4.0 3.0*

Vydate 10G Incremental 3.3 ± 0.9 10.9 ± 3.9 3.3*

t Week 28.

* Significantly less than untreated at P<0.05. - 102 -

Table 3.14 1982 Field trial: Oxamyl residues in peat fen soil (pg g~* soil) during growing season.

Mean ± S.E.

Vydate Week ^ Vydate 10G DPX 4702 Incremental

2 3.08 ± 0.05 2.70 ± 0.46 2.03 ± 0.13

4 1.27 ± 0.46 1.18 ± 0.23 0.57 ± 0.26

8 0.41 ± 0.06 1.02 ± 0.27 2.04 ± 0.60*

17 0.04 ± <0.01 0.03 ± 0.01 0.07 ± <0.01*

^ After pesticide application. ★ Significantly greater than Vydate 10G at P<0.05.

Recovery of oxamyl from fortified soil was found to be 79% efficient and all residue data was corrected accordingly. - 103 -

The proportion of nematodes which had developed from a vermiform to a saccate stage by week 17 was significantly reduced by oxamyl (P < 0.05;

Fig 3.11); the experimental controlled-release formulation, Vydate DPX

4702, appeared to be the least effective at inhibiting nematode development.

There was no significant (P > 0.05) increase in yield between oxamyl-treated and control plots at harvest. In fact, control yields were somewhat greater than the Vydate 10G, DPX 4702 and Vydate 10G incremental treatments (Fig 3.12). However, these pesticide formulations did significantly reduce the final H.schachtii population at harvest compared with that in control plots (P < 0.05; Table 3.13).

Oxamyl residue analysis showed that pesticide concentrations in the soil decreased significantly (P < 0.05) throughout the growing season.

Oxamyl levels in Vydate 10G incremental treatments were significantly greater than in Vydate lOG-treated plots from 8 weeks after pesticide application (P < 0.05; Table 3.14).

Full meteorological data is given in Appendix 15.

3.7.2 1984 Field trial.

Results from the field trial at Prickwillow are presented in Figs. 3.13-

3.16; Tables 3.15 and 3.16, and Appendices 10 and 22-28.

None of the pesticide treatments produced symptoms of phytotoxicity and germination was good (Appendix 10). Fig. 3.13 FIELD TRIAL 1984: WEEK B. Fig. 3.14 FIELD TRIAL 1984: WEEK 12.

;' a) Root weight (g). L.S.D·I a) Root weight (g). L.S.D·I ~ P = ,0.05 P = 0.05

0·2 20

0' "" O~'~----~~--~-L--~~L---~~--~~~--~~--~~~~ b) Nematodes per root. LeSeD·r b) Nematodes per root. L.S.D. P = 0.05 P = 0.05 I

r.J % saccate ~ % saccate ...., o 60 ~

20

o I v, I v· £ I V £ I V <;, ,v , I V <, Ie c, Ie < 0' K C r::z=:J ,...... K <

c) Nematodes per g. root. c) Nematodes per g. root. L. s. D.I L.S.D·I P = 0.05 6 P = 0.05 200 5 4

3 100 2

1

0~'~----~~--~~4---~~----L-~--~--L---~~ ____ L-~ __ ~ OLI~--~~--L-L-~~---L~--~~~~~--~~~ Untd. Vy. . 3·00 2·25 1·75 1·50 1·25 1·00 Untd. Vy. 3·00 2·25 1·75 1·50 1·25 1·00 10G % oxamyl min-.' lOG % oxamyl min~l Treatments Treatments Fig. 3.15 FIELD TRIAL 1984: WEEK 16.

Fjg. 3.16 FIELD TRIAL 1984: WEEK 28.

a) Harvest root weight (g).

L.S.DJ

b) Nematodes per root. L.S, D. P = 0 05 6 0 0 I

□ % saccate i 4 0 0 0 U 1 1 1 200

Treatments 1 telv .2 a _ b . Jzz _zz a

Treatments - 106 -

Table 3.15 1984 Field trial: Control of H.schachtii on sugar beet using various formulations of oxamyl (Vydate).

Mean eggs g soil (± S.E.).

Initial Final f x multiplication Treatment Population Population rate (5.6 kg a.i. ha”*) (Pi) (Pf) (Pf/Pi)

Untreated 10.5 ± 1.0 111.8 ± 20.1 10.6

Vydate 10G 12.3 ± 1.3 112.9 ± 26.1 9.2

Experimental 3.00 12.8 ± 2.2 75.5 ± 13.4 5.9 Formulations^1 2.25 11.4 ± 3.0 70.5 ± 9.2 6.2 1.75 12.7 ± 2.1 80.3 ± 10.1 6.3 1.50 14.6 ± 3.1 89.3 ± 9.3 6.1 1.25 14.7 ± 2.1 92.9 ± 21.3 6.3 1.00 11.1 ± 2.1 102.4 ± 18.3 9.2

+ Week 28.

^ Release rate: % oxamyl min”*. - 107

Table 3.16 1984 Field trial: Oxamyl residues in peat fen soil (pg g"1 soil) during growing season.

Mean ± S.E.

Experimental formulations ^

Week t Vydate 10G • 2.25 1.50 1.00

1 2.56 ± 0.27 2.34 ± 0.72 2.12 ± 0.24 2.52 ± 0.72

2 4.03 ± 0.70 2.70 ± 1.00 4.22 ± 1.17 2.94 ± 0.84

4 1.25 ± 0.44 1.82 ± 0.33 1.98 ± 0.19 1.25 ± 0.15

8 0.41 ± 0.07 0.60 ± 0.04 0.46 ± 0.10 0.49 ± 0.07

12 0.14 ± 0.01 0.18 ± 0.02 0.24 ± 0.01* 0.24 ± 0.02*

t After pesticide application. t -1 + Release rate: % oxamyl min .

Significantly greater than Vydate 10G at P<0.05.

Recovery of oxamyl from fortified soil was found to be 82% efficient and all residue data was corrected accordingly. - 108 -

At week 8 after pesticide application, all oxamyl treatments significantly reduced invasion of seedling roots by J2 and development of J2 within the roots was also significantly inhibited in five of the experimental controlled-release treatments (P < 0.05; Fig. 3.13). While the mean root weight was lowest in the control plots, this was not significantly different from the pesticide-treated plots (P > 0.05).

By week 12, root weights were significantly greater (P < 0.05) in four of the experimental treatments compared with the control; these formulations released between 1.00 and 1.75% a.i. min"*. In addition, treatments with formulations releasing 1.25 and 1.50% a.i. min“* had significantly greater root weights than the 3.00% a.i. min"* treatment

(P < 0.05; Fig. 3.14). Nematode invasion was significantly (P < 0.05) reduced in all oxamyl-treated plots and the 1.25 and 1.75% treatments had the lowest percentage development of J2 within the roots (Fig.

3.14).

Only the 1.50 and 1.75% treatments produced significantly (P < 0.05) greater root weights than controls 16 weeks after pesticide application; these same formulations also gave substantially greater root weights than any other oxamyl treatment (Fig. 3.15). However, nematode invasion was still significantly (P < 0.05) reduced by all oxamyl treatments; the experimental controlled-release formulations releasing 1.25 and 1.50% a.i. min-1 being significantly (P < 0.05) better at preventing invasion than either Vydate 10G or the experimental formulation with the fastest release rate (3.00%). When nematode invasion at week 16 was expressed per unit of root weight, all the oxamyl treatments had significantly (P

< 0.05) lower values compared with control roots. The four slowest release experimental formulations (1.00 - 1.75%) gave nematode densities - 109 - significantly lower than the fastest release experimental formulation

(3.00%) at 16 weeks (P < 0.05; Fig. 3.15). The 1.50% treatment significantly inhibited development of J2 within the roots compared with the control and together with the 1.25% the former inhibited development to a significantly greater extent than Vydate 10G (P < 0.05; Fig. 3.15).

At harvest, root weights were not significantly different between treatments although the greatest root weights were found in the 1.50 and

2.25% treatments (P > 0.05; Fig. 3.16).

The reproductive rate for H.schachtii was lowest in those plots treated with experimental oxamyl formulations (Table 3.15).

Residual concentrations of oxamyl in the soil decreased significantly (P

< 0.05) throughout the growing season for the four formulations monitored. Residue levels in both the 1.00 and 1.50% treatments were significantly greater than in the Vydate lOG-treated plots 12 weeks after pesticide application (P < 0.05; Table 3.16).

Full meteorological data is given in Appendix 15.

3.7.3 1982 Pot trial.

Results from the pot trial at Silwood Park are presented in Figs.

3.17-3.19; Table 3.17, and Appendices 11, 12 and 29-36.

At 4 weeks after pesticide application, seedling weights were significantly greater than controls in sandy-clay soil treated with

Vydate DPX 4702, Vydate 10G added incrementally and the experimental Fig. 3.18 POT TRIAL 1982: WEEK 8. Fig. 3.17 POT TRIAL 1982: WEEK 4. Peat fen S a n d y -c la y L.S.D. L.S.D.T a) Root weight (g). P = 0.05 P = 0.05-L T l .s .d . I p = 0.05

L.S.D.T l .s .d . p = o.or P = 0.05

E2 % saccate b) Nematodes per root. L.S.D. L.S.D. P = 0.05 P = 0.05

- t

Untd. Vy. DPX Vy. Tem. Aid. 10G 4702 Inc. 10G VYHD

Treatments

10G 4702 Inc. 10G VYHD 10G 4702 Inc. 10G VYHD

Treatments - I l l

Fig. 3.19 POT TRIAL 1982: WEEK 12.

Peat fen _ Sandy-clay

a) Root weight(g). L.S.D. L.S.D. P = 0 .0 5 P = 0 .0 5

L.S.D. TL.S.D. P = 0 .0 5 J"P = 0 .0 5

E2 % saccate

I t

V/ VI i 0 VI

Treatment - 112 -

Table 3.17 1982 Pot trials: Control of H.schachtii on sugar beet using various formulations of oxamyl (Vydate) and aldicarb (Temik).

Mean eggs g~* soil (± S.E.)

Initial Final t x multipl Treatment Population Population rate (5.6 kg a.i. ha *) (Pi)

Peat fen soil

Untreated 2.8 ± 0.3 7.0 ± 1.1 2.5 Vydate 10G 2.8 ± 0.3 4.6 ± 1.5 1.6 Vydate DPX 4702 2.8 ± 0.3 5.3 ± 1.3 1.9 Vydate. 10G 2.8 ± 0.3 4.5 ± 1.3 1.6 Incremental Temik 10G 2.8 ± 0.3 3.4 ± 1.2 1.2 Aldicarb VYHD 2.8 ± 0.3 5.3 ± 2.1 1.9

Sandy-clay soil

Untreated 13.3 + 0.6 6.9 + 1.9 0.5 Vydate 10G 13.3 + 0.6 9.0 + 0.6 0.7 Vydate DPX 4702 13.3 + 0.6 8.2 + 2.0 0.6 Vydate 10G 13.3 + 0.6 10.1 + 2.5 0.8 Incremental Temik 10G 13.3 + 0.6 13.8 + 1.9 1 .0* Aldicarb VYHD 13.3 + 0.6 4.2 + 0.6 0.3

t Week 12. * Significantly greater than untreated at P<0.05. - 113 - formulation of aldicarb, Vinylite VYHD (P < 0.05; Fig. 3.17). There was no significant difference (P > 0.05) between any treatment in peat fen soil. No treatment showed any signs of phytotoxicity on the young beet seedlings. All nematicide treatments significantly (P < 0.05) reduced invasion of H.schachtii J2 in both soil types and only the two aldicarb treatments in sandy-clay soil failed to significantly inhibit development of J2 within roots (P > 0.05; Fig. 3.17).

At week 8 after pesticide application, root weights were significantly greater than controls in peat fen soil treated with Vydate 10G and the aldicarb VYHD formulation; the latter were also significantly greater than with Temik 10G (P < 0.05; Fig. 3.18). In sandy-clay soil, root weights were only significantly (P < 0.05; greater than controls in the aldicarb VYHD treatment; there was no significant difference between any of the experimental controlled-release formulations and the commercial formulations at week 8 (P > 0.05; Fig. 3.18).

In peat fen soil at week 8 , nematode invasion in both of the aldicarb treatments was significantly (P < 0.05) reduced compared with the control. When invasion was expressed as nematodes per unit root weight, all treatments except Vydate 10G incremental had significantly (P-<

0.05) lower nematode densities in the roots compared with controls.

Development of J2 in roots was significantly inhibited by all nematicide treatments (P < 0.05; Fig. 3.18).

In sandy-clay soil at week 8 , nematode invasion was significantly (P <

0.05) reduced by the Temik 10G, Vydate 10G and Vydate DPX 4702 treatments compared with the control. All of the pesticide treatments significantly reduced the number of nematodes per unit root weight - 114 - compared with the control (P < 0.05); nematode density in the Temik 106,

Vydate 10G and Vydate DPX 4702 treatments was also significantly lower when compared with the Vydate 10G incremental treatment (Fig. 3.18).

Subsequent development of J2 within the roots was not significantly (P >

0.05) reduced by Vydate 10G added incrementally, Vydate DPX 4702 or aldicarb VYHD, but development was significantly reduced by Vydate 10G

and Temik 10G in this soil type (P < 0.05; Fig. 3.18).

In both soil types 12 weeks after pesticide application, Vydate DPX 4702

significantly increased root weights compared with the controls; Vydate

10G gave significantly greater root weights than controls in peat fen

soil (P < 0.05; Fig. 3.19).

Nematode invasion per root was significantly reduced at week 12 by both

formulations of aldicarb in peat fen soil and by Temik 10G and Vydate

10G added incrementally in sandy-clay soil; invasion in the last

treatment was also significantly lower compared with Vydate 10G (P <

Q.05; Fig. 3.19). However, all pesticide treatments except Vydate 10G

incremental in peat fen soil and all treatments except Vydate 10G in

sandy-clay soil had significantly fewer nematodes per unit root weight

compared with controls (P < 0.05; Fig. 3.-19). Development of J2 within

roots was significantly inhibited by all pesticide treatments in the

peat fen soil (P < 0.05) but by none in the sandy-clay soil (P > 0.05;

Fig. 3.19).

There was no significant reduction in the final population of

H.schachtii in any of the treated pots after 12 weeks (P > 0.05).

However, there was a significant increase caused by the Temik 10G

treatment in sandy-clay soil (P < 0.05; Table 3.17). - 115 -

Full meteorological data is given in Appendix 15.

3.7.4 1983 Microplot trial.

Results from the microplot trial at Silwood Park are presented in Figs.

3.20-3.23; Table 3.18, and Appendices 13 and 37-41.

At week 6 after pesticide application, seedling weights were

significantly (P < 0.05) increased in plots treated with a combination of Vydate DPX 5578-2 and Vydate 10G compared with the control. No

treatment affected germination or showed any symptoms of phytotoxicity

(Fig. 3.20). Nematode invasion was significantly reduced compared with control roots by Vydate DPX 5578-2 and the Vydate DPX 4702/10G combination; the latter treatment also significantly reduced invasion

compared with the Vydate 10G treatment (P < 0.05; Fig. 3.20). Root

nematode density was significantly decreased by all the pesticide

treatments compared with controls, and the Vydate DPX 4702/10G-treated

roots had significantly fewer nematodes per unit root weight than the

Vydate 10G treatment (P < 0.05; Fig. 3.20). Only the Vydate 10G and

Vydate DPX 5578-2 treatments had significantly fewer developed stages within the roots than controls (P < 0.05).

By week 10, the Vydate DPX 5578-2 and Vydate DPX 5578-2/10G-treated

plots had significantly greater root weights compared with controls,

and, together with the DPX 4702/10G and the fast-release experimental

formulation, had root weights significantly greater than in Vydate

lOG-treated plots (P < 0.05; Fig. 3.21). However, none of the pesticide

treatments significantly affected nematode invasion per root (P > 0.05) Fig. 3.20 MICROPLOT TRIAL 1 9 8 3 : W E E K 6. Fig.3.21 MICROPLOT TRIAL 1983: WEEK 10. Plant weight (g). L.S.D. a) Root weight (g). L.S.D. a) T P = 0.051

.D. 0.05 116

c) Nematodes per g. root. S.D. = 0.05

10G ------reT "rate------5578^ /10G /10G Treatments Treatments Fig. 3.22 MICROPLOT TRIAL 1983: WEEK 16.

a) Root weight(g). Fig. 3.23 MICROPLOT TRIAL 1983: WEEK 28. L.S.D. a) Harvest root weight (g). P = 0.05 L.S.D. P = 0.05

Treatments

Treatments - 118 -

Table 3.18 1983 microplot trial: Control of H.schachtii on sugar beet using various formulations of oxamyl (Vydate).

Mean eggs g"1 soil (± S.E.)

Initial Final -f x multipl Treatment Population Population rate (5.6 kg a.i. ha~*) (Pi) (Pf) (Pf/P

Untreated 8.6 ± 0.6 29.7 ± 5.0 3.5

Vydate 10G 8.0 ± 0.7 26.7 ± 9.7 3.3

Experimental formulationsFast 7.7 ± 0.8 32.5 ± 3.1 4.2 Medium 7.9 ± 0.7 27.6 ± 5.5 3.5 Slow 8.2 ± 0.9 26.9 ± 6.8 3.3

Vydate DPX 5578-2 8.1 ± 0.9 27.8 ± 6.3 3.4

DPX 5578-2/10G 8.1 ± 0.9 24.6 ± 5.6 3.0

DPX 4702/10G 8.2 ± 0.6 23.5 ± 6.5 2.9

t Week 28.

* Slow to fast release rate. - 119 -

and only the fast and slow-release experimental formulations and DPX

5578-2 had significantly fewer nematodes per unit root weight compared with controls (P < 0.05; Fig. 3.21). Development of J2 within the root was lowest in three of the controlled-release treatments although none were found to be significantly different from the control (P > 0.05;

Fig. 3.21).

After 16 weeks, none of the pesticide treatments had significantly (P >

0.05) greater root weights than the control, although the number of new cysts maturing on the roots was significantly reduced by all oxamyl treatments (P < 0.05; Fig. 3.22).

By harvest, the Vydate DPX 4702/10G combined treatment gave both the greatest mean root weight and the lowest reproductive rate for

H.schachtii of all the pesticide treatments, but these were not significantly different from that in the control plots (P > 0.05; Fig.

3.23; Table 3.18).

Full meteorological data is given in Appendix 15.

3.7.5 1984 Microplot trial.

Results from the microplot trial at Silwood Park are presented in Figs.

3.24-3.28; Table 3.19, and Appendices 14 and 42-48.

No treatment showed signs of causing phytotoxicity to the growing seedling, but germination was poor (Appendix 14).

At week 7 after pesticide application, plots treated with an - 120 -

Fig. 3.24 MICROPLOT TRIAL 1984: WEEK 7.

a) Root weight (g ). c) Nematodes per g. root. L.S.D. L.S.D. P = 0.05 P = 0.05

Untd. Tem. Vy. Exp. Untd. Tem. Vy. Exp. Untd. Tem. Vy. Exp. 10G 10G form. 10G 10G form. 10G 10G form.

Treatments

Fig. 3.25 MICROPLOT TRIAL 1984:WEEK11.

a) Root weight Cg). c) Nematodes per g. root. L.S.D. L.S.D. P = 0.05 P = 0.05

Untd. Tem. Vy. Exp. Untd. Tem. Vy. Exp. 10G 10G form. 10G 10G form.

Treatm ents Fig. 3.26 MICROPLOT TRIAL 1984: WEEK 15.

a) Root weight (g). b) Nematodes per root. c) Nematodes per g. root. L.S.D. L.S.D. L.S.D. P = 0.05 P = 0.05L P =0.05 200r

1 6 0 -

120 -

80 -

4 0 -

O - Untd. Tem. Vy. Exp. 10G 10G form.

Treatments

Fig. 3. 27 MICROPLOT TRIAL 1984: WEEK 21.

a) c) Nematodes per g. root. L.S.D. P = 0.05 400r-

3 0 0 -

2 0 0 -

1 0 0 -

0 - Untd. Tem. Vy. Exp. 10G 10G form.

Treatm ents - 122 -

Fig. 3.28 MICROPLOT TRIAL 1984: WEEK 28.

a) Harvest root weight (g).

Treatments - 123 -

Table 3.19 1984 Microplot trial: Control of H.schachtii on sugar beet using various formulations of oxamyl (Vydate) and aldicarb (Temik).

Mean eggs g' 1 soil (± S.E.)

Initial Final j- x multiplication Treatment Population Population rate (5.6 kg a.i. ha”*) (pi) (pf) (Pf/Pi)

Untreated 13.1 ± 0.7 49.8 ± 1.5 3.8

Temik 10G 10.2 ± 0.8 31.6 ± 1.8 3.1

Vydate 10G 11.5 ± 1.5 28.3 ± 2.0 2.5

Experimental ^ 11.6 ± 1.1 26.0 ± 6.0 2.2* formulation

t Week 28.

Release rate: 1.25% oxamyl min”*.

* Significantly less than untreated at P<0.05. - 124 -

experimental controlled-release oxamyl formulation had significantly (P

< 0.05) greater root weights than controls and were also substantially greater than Vydate 10G or Temik 10G plots although these differences were not significant (P > 0.05; Fig. 3.24). Both Temik 10G and the controlled-release treatment appeared to have a greater effect than

Vydate 10G on nematode invasion and had significantly fewer nematodes per root than controls (P < 0.05; Fig. 3.24). All pesticide treatments significantly (P < 0.05) reduced the proportion of nematodes developing to a saccate stage.

By week 11, there was little difference in the root weights between the pesticide treatments, and only Vydate 10G and Temik 10G gave root weights significantly greater than controls (P < 0.05; Fig. 3.25). The mean root weight for the controlled-release treatment was greatly reduced by one very poor plot and would otherwise have been similar to the Temik 10G treatment weight. Nematode invasion was significantly reduced by the Temik 10G and controlled-release treatment and nematode density within the roots was significantly less compared with control roots in all pesticide-treated plots (P < 0.05; Fig. 3.25). The lowest percentage development of J2 was recorded in the controlled-release plots although there was no significant'difference between treatments (P

> 0.05; Fig. 3.25).

All pesticide treatments had significantly greater root weights and significantly (P < 0.05) fewer nematodes per root than controls at both

15 and 21 weeks after application. However, while nematode invasion in the controlled-release treatments was the lowest recorded; being particularly so compared with Vydate 10G, none of the treatments were significantly different (P > 0.05). Subsequent development of J2 did not - 125 -

appear to be affected by any of the pesticide treatments (P > 0.05;

Figs. 3.26 and 3.27).

At harvest, only the Temik lOG-treated plots had significantly greater mean root weights than controls (P < 0.05; Fig. 3.28) although the controlled-release treatment did significantly reduce the final population of H.schachtii compared with control plots (P < 0.05; Table

3.19).

Full meteorological data is given in Appendix 15. 126 -

4. DISCUSSION

4.1 Laboratory studies

The objective of the laboratory bioassays in the present study was to

determine the minimum concentration of nematicide (MIC) required to

control H.schachtii. It was shown that this species can be incapacitated

by relatively low concentrations of oxamyl; the EC^q values varying

appreciably according to the stage in the life cycle examined (Table

4.1). This agrees with numerous other reports using oxamyl and similar

nematicides on H.schachtii (Table 4.2) and related species (eg. McLeod

and Khair, 1975; Wright, 1981).

The results indicate that it is the infective stage of the life cycle which is the most vulnerable to disruption by oxamyl (Table 3.5); a concentration of 0.2 |ug a.i. cm“3 significantly reducing nematode

invasion of host seedlings and this rate was taken as the MIC for oxamyl against H.schachtii under laboratory conditions. At this concentration general activity of the nematode was hardly affected

(Tables 3.2-3.4) which suggests that oxamyl was acting principally on nematode sensory behaviour.

As mentioned in Section 1.3, impairment of nematode neuromuscular transmission by inhibition of AChE is the usually accepted mode of action for carbamate and organophosphorus pesticides, as is the case in insects and vertebrates (Corbett, 1974). However, AChE activity has also been detected in neurons associated with the amphids and other sensory organs in nematodes (McLaren, 1972; Pertel et al., 1976) and Wright et ai. (1980) have suggested that oxamyl and other similar compounds may - 127 -

Table 4.1 Effect of oxamyl on life cycle stages of H.schachtii

EC50 or lowest concentration tested which significantly _ 0 Life cycle stage inhibited process (pg a.i. cm” )

Hatching - Initial 1.0 - Total 2.5 with diffusate 10.0 without diffusate

Activity EC5q: 0.19-0.53

Migration EC50I 0.53

Infectivity EC5Q: 0.20

Development EC50: 1.38

t For full details see SEction 3. 128 -

Table 4.2 Effect of various carbamates and organophosphates on life cycle stages of H.schachtii.

Cone, reported to inhibit process o Life cycle stage Pesticide . (pg a.i. cm ) Reference

Hatching aldicarb 1.0 Steudel (1972) aldicarb 4.8 Hough and Thomason (1975) aldicarb 1.0 Steele and Hodges (1975) aldicarb 5.0 Steele (1983) carbofuran 5.0 Steele (1983) fenamiphos 4.8 Greco and Thomason (1980) fenamiphos 1.0 Steele (1983) isazophos 4.5 Greco et a 1.(1984)

Activity aldicarb 10.0 Steudel (1972) aldicarb 2.0 Batterby (1979)

Migration aldicarb 1.0 Hough and Thomason (1975) fenamiphos 0.5 Greco and Thomason (1980) fenamiphos 10.0 (cf ) Greco and Thomason (1980)

Infectivity oxamyl (foliar) 500 Griffin (1975) aldicarb 1.0 Hough and Thomason (1975) fenamiphos (foliar) 500 Griffin (1975) fenamiphos 1.0 Greco and Thomason (1980) Development oxamyl (foliar) 500 Griffin (1975) aldicarb 6.0 Steele and Hodges (1975) fenamiphos (foliar) 500 Griffin (1975)

Contd 129 -

Table 4.2 contd...

Cone, reported to inhibit process Life cycle stage Pesticide (pg a.i cm Reference

Sex attraction aldicarb 0.01 Hough and Thomason (1975) 130 - also disrupt the sensory behaviour of nematodes thereby impairing such processes as feeding, orientation and invasion. It would appear, therefore, that at the MIC for oxamyl against H.schachtii it is the relatively complex behavioural sequences necessary for successful invasion that are disrupted.

Sensory mechanisms are also involved in reproduction in amphimictic nematode species (Duggal, 1978); H.schachtii males, for example, having been shown to be attracted by secretions from the body surface of the female (Green, 1966) and disorientation of males by pesticides would result in lower rates of population increase (Hough and Thomason, 1975).

Thus, it is possible that the effects of oxamyl on H.schachtii in the field, where its concentration in the soil water may not be greatly in excess of the MIC, could be predominantly behavioural, affecting in particular invasion of host roots. Although a combination of other inhibitory effects may also be involved depending on the toxicant concentrations in the soil.

For example, newly emerged H.schachtii J2 were shown to be more sensitive to oxamyl than unhatched juveniles (Table 4.1) which is in agreement with reports by other authors (Table 4.2). Inhibition of hatching was found to be completely reversible on removal of cysts from exposure to low concentrations of oxamyl as reported previously for this and several other non-fumigant nematicides (Hague and Pain, 1973; Hough and Thomason, 1975; Wright et al., 1980; Evans and Wright, 1982; Steele,

1983) although at higher concentrations (10 pg a.i. cm"3 ) the effects of oxamyl on hatching were more permanent (Fig. 3.1a). However, when cysts of H.schachtii were exposed to low concentrations of oxamyl (2.5 pg a.i. 131

cm-3) in combination with sugar beet root diffusate (to stimulate field

situations when application and sowing are simultaneous, or when

nematicide is applied sometime before sowing) the effects of oxamyl were

less reversible (Table 3.1; Fig. 3.1b).

Irreversible effects on hatching after treatment with nematicide plus

hatching agent have also been reported by Osbourne (1973) with

G. rostochiensis and by Hough and Thomason (1975) and Steele (1983) with

H. schachtii. This enhanced inhibition of hatching by nematicide in the

presence of root diffusate may be due to the increased uptake of

toxicant by the eggs. Certainly, hatching stimulants are known to

increase the permeability of the egg shell to solutes (Dropkin et al.,

1958). Therefore, in a nematode species which lacks a specific hatching

factor, Meloidogyne javanica, it would appear that negligible amounts of

pesticide actually penetrate the nematode egg shell (McCleod and Khair,

1975). In this species, when eggs were immersed in nematicide

concentrations that inhibited hatching, the juveniles within the egg

showed normal activity although once outside the egg they showed signs of paralysis. It would seem likely in these circumstances that the toxicant acts within the egg by disrupting the complex behavioural sequences required for hatching to occur (Wright, 1981).

For example, low concentrations of oxamyl (Fig. 3.2; Bunt, 1975) and aldicarb (Nelmes, 1970) have been shown to stimulate stylet movement in unhatched nematode juveniles which normally exhibit a high degree of activity during hatching (Doncaster and Shepherd, 1967). This vigorous stylet thrusting may aid eclosion and account for the stimulation of hatching at low concentrations of carbamates experienced by many workers in the laboratory (Steele and Hodges, 1975; Hough and Thomason, 1975; 132 -

Kampfe, 1975; Steele, 1983) and in the field (Kaul and Sethi, 1984).

However, the precise orientation necessary to cut a continuous slit in the egg shell (Doncaster and Shepherd, 1967) is also likely to be affected by nematicides and this may explain the apparently greater sensitivity of the eggs of Heterodera species compared with Meloidogyne species (Hough and Thomason, 1975) which do not exhibit co-ordinated movements prior to hatching.

None of the oxamyl treatments caused excessive mortality of unhatched juveniles which while in agreement with other work on this compound

(Miller, 1970) is in contrast to studies on H.schachtii cysts treated with aldicarb, where an increasing proportion of dead juveniles was found within eggs with increasing concentration of nematicide (Steudel,

1972).

In the present study, hatch is expressed as a percentage of the original egg content of fully embryonated cysts, as recommended by Shepherd

(1959). This overcomes the problems associated with variable numbers of eggs within cysts. Excessive numbers of eggs may mean that although a nematicide is effective in reducing hatch, large numbers of juveniles still emerge. Alternatively, low numbers of eggs, as would occur in older cysts, could result in relatively few juveniles emerging in the controls making comparisons between treatments difficult. Certainly, even within cysts of the same age from the same population , the number of eggs in Heterodera cysts can be very variable (Den Onden, 1963). As

Curtis (1965) and Clarke and Shepherd (1966) state, there is a lack of conformity in the presentation of such data in the literature. If emergence was always expressed cumulatively as a percentage of the original cyst contents (see Fig. 3.1), meaningful comparisons could be 133 - made between different nematode species and nematicidal compounds.

Observations on the effects of oxamyl on the nematode stages within the host root (Table 3.6) support the theory that carbamate nematicides, although taken up by plant roots, act against plant parasitic nematodes mainly in the soil phase of the life cycle (Hague and Pain, 1973;

Boparai and Hague, 1974). Once in the root, nematode development is less affected by pesticides applied either to the foliage or to the soil

(Tables 3.6 and 4.2). The latter observations may be due to the fact that all the species examined become sedentary endoparasites shortly after invasion and these stages might be expected to be less vulnerable to the sublethal effects of nematicides. It would be of interest, therefore, to study the action of these nematicides on plant nematodes which remain mobile within the roots or aerial parts of plants.

At low concentrations of oxamyl, complete recovery of juvenile activity occured with H.schachtii. This reversibility, which also occurred in the hatching test, appears consistent with the theory that carbamates impair nematode mobility by inhibition of AChE activity; an enzyme which is known to be reversibly inhibited by a number of such pesticides fYu et^ al., 1972). In contrast, high (albeit uneconomic) concentrations of nematicide can be lethal (Evans, 1973; Nelmes et al., 1973; McLeod and

Khair, 1975). For example, McGarvey et al. (1984) found that 4000 pg oxamyl cm" 3 f 0r 40 min. was sufficient to prevent recovery of

M.incognita juveniles, although a similar concentration was unable to prevent some recovery of Ditylenchus dipsaci after three weeks exposure to oxamyl (Bunt, 1975). 134 -

This and other work (Wright et al., 1980; Evans and Wright, 1982) suggests that H.schachtii and G.rostochiensis J2 are of approximately equal sensitivity to oxamyl but about five times more sensitive than

M.incognita J2. While caution must be exercised in comparing different experiments, similar methods of assessment were used in each and perhaps more significantly, in the present work the same E C ™ values were bu obtained for oxamyl with H.schachtii J2 using two different methods.

Such variations in susceptibility to nematicides have been explained for other species in terms of differences in the rates of uptake, metabolism and elimination of the toxic compounds (LePatourel and Wright, 1976;

Batterby et al., 1977). The effectiveness of a particular compound can also vary between developmental stages and Evans and Wright (1982) have suggested that the greater sensitivity of J2 compared to adult male

G.rostochiensis is due, at least in part, to the relatively greater surface area to volume ratio of the former. The amount of AChE present in nematodes is not believed to be of prime importance in determining nematicide susceptibility (Dickson et al., 1971).

The inevitable conclusion from these observations is that it is necessary to assess the efficacy of each compound against every economically important species in order to achieve optimal nematode control.

Inhibition of movement or the ability of an infective juvenile to find a host plant would not necessarily be expected to kill a plant parasitic nematode since some species are able to survive long periods of starvation (Golden and Shafer, 1960; Slack et al., 1972; Evans and - 135 -

Perry, 1976). However, while it is assumed that a paralysed nematode will eventually use up its food reserves, lose its infectivity and die

(Hague, 1979), there is little information on the duration of control required for this to occur under laboratory or field conditions. Such information is particularly vital for the selection of controlled-release formulations of nematicides.

In plant parasitic nematodes, energy reserves are principally in the form of neutral lipids (triglycerides) and a direct relationship between nematode neutral lipid levels, activity and infectivity has been found for G.rostochiensis (Storey, 1984). In the present work, long-term exposure of H.schachtii J2 to oxamyl demonstrated that at concentrations of toxicant which induced total paralysis, neutral lipid reserves were conserved and following transfer to a pesticide-free environment these nematodes had an equal or greater infectivity than untreated J2 of the same age.

Similarly, exposure of G.rostochiensis J2 to 1.0 pg cm”^ oxamyl for 35 days was also found to reduce the rate of decline of neutral lipids and these nematodes were found to be at least as infective as controls (D.J.

Wright, personal communication). M.incognita J2 treated with aldicarb have also been reported to conserve their lipid food reserves and to be capable of delayed invasion of host roots (Nelmes et al., 1973).

In contrast, H.schachtii J2 incubated in 0.1 pg cm"3 oxamyl exhibited a hyperactive response and may have utilized their lipid reserves more quickly than untreated juveniles (Table 3.9). However, while the infectivity of such nematodes appeared to be less than that of control nematodes or of nematodes exposed to greater concentrations of oxamyl - 136 - after 14 and 28 days incubation, none of the differences were significant (Table 3.8).

If such hyperactivity does occur in the field in response to low levels of nematicide, this could explain why Hague and Pain (1973) and Hague

(1979) found that G.rostochiensis J2 recovered from the soil up to 8 weeks after pesticide treatment tended to have lower lipid reserves than untreated J2.

From these observations, it appears that soil concentrations of nematicide which are sufficient to immobilize infective juveniles may in fact be less useful than the maintenance of a much lov/er concentration of compound; the latter being sufficient to disorientate the nematodes but at the same time enhancing lipid utilization. It appears, therefore, that whether inhibition of invasion of a host plant under field conditions is temporary or permanent may depend largely on the degree of persistence of toxic levels of nematicide above the MIC.

Finally, poor invasion by untreated nematodes in infectivity experiments

(Table 3.5) makes it difficult to evaluate the efficacy of a compound and many experimental errors and false statements are likely to be made when working at low densities. Many authors have experienced this problem of low infectivity with a variety of nematode species: 5-10% for

H.avenae (Davies and Fisher, 1976), 10% for G.rostochiensis (Evans and

Wright, 1982), 10-15% for M.incognita (Wright et al., 1980), 15-20% for

H.schachtii (Greco and Thomason, 1980) and 2% for Pratylenchus penetrans

(Oostenbrink, 1958).

The commonly used method for inoculating seedlings is to apply to the 137 soil surface and allow the nematodes to migrate downwards through the

soil to the roots. An experiment to compare this method (vertical) with one in which the nematode inoculum was applied to the full length of the

root system (horizontal; Fig. 2.1) showed that whereas the former gave

10-20% invasion the latter method gave 70-85% (Table 3.10). Observations on stained root systems, showed a high proportion of H.schachtii J2 in a few roots around the hypocotyl with the "vertical" method while with the

"horizontal" method the nematodes were well distributed throughout the root system. In the latter method, the number of root tips (invasion sites) accessible to the J2 is far greater and competition for invasion sites would be much less thereby improving the chances for sucessful invasion by each individual J2.

4.2 Formulation studies

Oxamyl, being highly soluble in water, is rapidly released from commercial Vydate 10G granules; this suggests that the effectiveness of the formualtion could be considerably improved if the release curve were to approximate more to a zero-order form (Fig. 3.3).

Granules of oxamyl prepared using several polymers did greatly reduce the rate of a.i. release (Fig. 3.3). However, the rate of release is not the only criterion in assessing the efficiency of a formulation. The initial rate of release and the total amount of a.i. which can be removed while still maintaining an effective concentration of toxicant in the soil water are equally important. A theoretical minimum inhibitory concentration (TMIC), as defined for the laboratory elution tests, has been estimated as the concentration of oxamyl resulting from a release rate of 0.2-0.25% a.i. min'1 . This would approximate to a - 138 -

_3 concentration of 0.4-0.5 |jg a.i. cm in the field; a value twice the laboratory MIC of 0.2 fjg in order to allow for adsorptive and degradative processes occuring in the soil.

In the case of granules containing urea-formaldehyde (UFR; Figs. 3.3 and

3.7), only 14-16% of the total oxamyl content could be released and the rate of toxicant release rapidly fell below the TMIC. This was probably due to strong binding between oxamyl molecules and acidic sites on the UFR pre-polymer (see Schacht et al., 1981). Alternatively, the coating may have been too hydrophobic, preventing the penetration of water necessary for escape of the nematicide.

In contrast to UFR, the CA and CTA formulations were able to maintain the TMIC for a greater length of time than the commercial formulation although a large percentage of the a.i. was still unavailable for release (Fig. 3.3). Stokes et al. (1973) reported that release of aldicarb from CTA granules was much slower than from CA formulations, although little difference was observed in this study.

In an attempt to achieve a more complete removal of oxamyl from the granules, in order to improve efficiency and to minimize the-decrease in release rate with time, various plasticizers were incorporated into the polymer during preparation. The addition of the plasticizers, dimethyl and dibutyl sebacate were found to enhance the removal of a.i. from the granules and such formulations were able to maintain a more effective rate toxicant release for a longer period when compared with polymer alone (Figs. 3.4 and 3.6). The release rate with plasticizers also approximated much more to a zero-order process, releasing over 70% of available a.i. 139 -

Similar observations have been made for polymer-plasticizer combinations

with aldicarb, dichlorvos and chlorpyrifos (Miles et al., 1962; Nelson

et al., 1970; Stokes et al., 1973) but only Stokes et al. (1973)

concluded that the amount of plastizer present was of any importance.

In the present study, early observations had shown that oxamyl granules

coated with PEG alone released up to 95% of their a.i. content (Fig.

3.3). In a further series of tests, therefore, PEG was used as a

surface-active agent to aid the quantitative removal of toxicant from

the granules. Although the initial rate of release was rather rapid when

PEG was added to CA at 1% (w/w) of granule, this was reduced at 0.25%

PEG (w/w) and together with a suitable plasticizer such a formulation

released 80% of the incorporated oxamyl, sufficient to maintain the

TMIC in the soil for long-term control (Fig. 3.5).

A possible explanation for the observed effects of PEG may be that as it

is fairly water soluble its inclusion is likely to open up the polymer

matrix as it dissolves and so speed up the release of a.i. from the

impregnated absorbent granules. The addition of any water soluble material or surfactant is likely to have a similar effect.

Attempts to reduce the excessive initial release rate of a.i. by

applying a second coating of CA were successful but in doing so also

reduced the total amount of a.i. which could be utilized.

To check that complete wetting of the granules occured during elution, a

non-ionic wetting agent was also used as an adjuvant in the polymer

coating. In fact, this did not change the release rate when compared with polymer coating alone indicating that variable wetting was not a - 140 - factor in the elution experiments (Fig. 3.5).

From the elution results (Figs. 3.3 - 3.7), the release rate

characteristics of the polymer-coated oxamyl granules are roughly

proportional to J t (Fig. 4.1) or intermediate between zero-order and/t;

>/t-order release is characterised by an initial sharp rise. However, as

Kydonieus (1980) points out, zero-order‘is not necessarily better than

J t . An initial release of a.i. to satisfy the adsorptive capacity of the

soil followed by a slower, more sustained release to replace a.i. which

is lost, appears more desirable. Systems with a J t release rate are also generally less costly to produce than zero-order systems (Kydonieus,

1980).

The oxamyl release rates from pilot experimental formulations were assessed using a simple water elution test (Section 2.6.5). Leaching methods have also been used by other workers (Furmidge et al., 1966,

1968; Stokes et al., 1970, 1973; Coppedge et al., 1975b; Batterby,

1978). Although this appraisal technique is not ideal, it is difficult to see what other method would be practical for selecting a short list of formulations with different release characteristics which could

realistically be field tested. At least continuous elution, as opposed to water immersion, has the advantage that no concentration gradient is allowed to develop which may influence the release rate. During the search for a rapid method of oxamyl analysis for the elution experiments, it was found that the colorimetric method reported by

Singhal et al. (1977) for oxamyl was due to a misinterpreted observation. The colour reaction with carbon disulphide was not in fact given by the oxime of oxamyl as suggested by the authors but was due to the dye added by the manufacturers in oxamyl granules. Fig. 4.1 Representation of zero order andorderzeroof Representation Fig. 4.1

Cumulative % a.i. released Time ofelutionTime 141 --- ► T / \ order release rates.release order 142 -

Simple elution tests would appear to be of most use when the release of a.i. from the granules is mainly by the process of leaching. If a different mechanism is operating, for example, biodegradation, hydrolysis, oxidation, water vapour distillation or a temperature related effect, more complex experiments are needed (Seaman and

Warrington, 1972). However, the underlying problem associated with all of these methods of assessment is in predicting what relationship laboratory tests bear to actual field conditions.

In the soil, many factors influence the release rate of a.i. from a pesticide granule. Apart from its physical and chemical composition, the performance of a granular formulation in the field will also be influenced by its immediate environment, such as soil composition, the duration and intensity of rainfall and temperature (Furmidge, 1972;

Coppedge et al., 1975a). Comparisons between data from laboratory experiments and those expected in the field must, therefore, be treated with caution.

nevertheless, Stokes et al. (1970, 1973) demonstrated that the characteristics of release from granular formulations of aldicarb observed in in vitro tests were useful in predicting the rates of release into soil with some accuracy. However, estimates of biological efficacy based on laboratory data were not so predictable and formulations of aldicarb displaying relatively slow rates of release did not always have extended biological activity against the boll weevil.

Stokes et al. (1973) also reported a difference in the efficacy of a controlled-release formulation of dimethoate against two pest species in the field. Such differences may be explained in terms of the relative - 143 - susceptibility of each pest to the compound and emphasises the need for preliminary bioassays.

Consequently, if controlled-release granules do become commercially available, the selection of a formulation for field use would have to consider expected soil moisture and temperature conditions as well as anticipated pest problems. The practicality of producing such formulations with a wide range of release constants is uncertain

(Osgerby, 1972). It appears preferable, therefore, that a range of formulations are selected for field evaluation with a variety of release rate characteristics, to determine which provides the most effective overall control in a number of situations (Marrs and Seaman, 1978).

For the 1984 field trial, six experimental formulations whose release rates approximated most closely to the zero-order process of an ideal controlled-release granule, were selected for field evaluation against

H.schachtii on sugar beet. The chosen formulations (Table 3.12) had release rates varying from 1.0% to 3.0% a.i. mirT1 under laboratory conditions; Vydate 10G, the commercial formulation, has a release rate of greater than 4.0% a.i. miff1 . Formulations with a rate greater than

3.0% were considered similar to the commercial granules, while those less than 1.0% were envisaged to be too slow to be able to maintain the

T MIC and could result in a level of pest control lower than would have been obtained with a fast-release formulation. The test granules were prepared at 5% a.i. instead of 10%, with the aim of improving their distribution in the soil.

All of the formulation procedures employed in this work could realistically be conducted on an industrial scale: all solvents were 144 recoverable and re-usable and the polymer coating did not cause any agglomeration of the granules providing they were thoroughly agitated during drying.

4.3 Field studies

In an attempt to demonstrate the usefulness of a controlled-release nematicide against H.schachtii on sugar beet in the field, experimental formulations of oxamyl prepared in the laboratory displaying a variety of release characteristics were tested. Several existing formulations of oxamyl and aldicarb were also used both to re-assess their potential and as standards. These appraisal tests were in the form of pot or microplot trials or in large scale field trials on commercially cultivated land.

The results from the trials show that for the early part of the growing season (weeks 0-8) all the formulations tested were able to greatly reduce nematode invasion of the young beet seedlings, regardless of soil type. Such nematode control led to greater root weights compared with untreated controls by mid-season (weeks 8-16).

Good early control of H.schachtii has been reported by many other- workers using commercial formulations of oxamyl and aldicarb (Jorgenson,

1969; Steudel and Thielmann, 1970; Thielmann and Steudel, 1973; Griffin and Gessel, 1973; Steudel et al., 1978; Whitehead et al., 1979; Hartwig and Sikora, 1984), and low application rates of these compounds were often as effective as much larger amounts (Jorgenson, 1969; Cooke,

1975b; Whitehead, 1977; Whitehead et al., 1979).

As in the present studies, Jorgenson and Musselmann (1966) found weights 145 -

of non-invaded sugar beet seedlings were twice those of invaded

seedlings 4 weeks after germination and, on a much longer term basis,

Steudel and Thielmann (1967) estimated that 90% of final yield loss

could be prevented by protection of the young beet seedlings

demonstrating the importance of this early control.

In the present work, the invasion data for the controlled-release

formulation DPX 4702 showed it to give inconsistent control during the

early part of the growing season, and it was somewhat less effective

than the commercial formulation, Vydate 10G (Figs. 3.10, 3.17 and 3.18).

This may have been due to DPX 4702 releasing the a.i. too slowly,

thereby taking longer to achieve the MIC in the soil. Thus, J2 close to

the root may have invaded before sufficient toxicant levels were

attained, and since inhibition of development requires greater levels of

toxicant (Table 4.1 and 4.2), the J2 within the roots were able to feed

and moult before their development was inhibited. Certainly, the larger

granule size of the DPX 4702 formulation made it difficult to maintain

an even distribution in the soil and there would be a relatively smaller

surface area for release of a.i. compared with Vydate 10G. DPX 4702 has

also been found to give poorer early control than Vydate 10G against

froghoppers on sugar cane (S. Batterby,-personal communication).

Marrs and Seaman (1978) recognised the difficulty in developing a

formulation capable of giving both an immediate effect and also

long-term control and the potential problems which arise where losses by

leaching and degradation occur at the same rate as release. One solution

to this problem is to use mixtures of granules with different release

rates and Stokes et al. (1970) found that a mixture of fast and slow

release granules gave a greater initial uptake of a.i. into cotton \ 146 - plants when compared with a controlled-release formulation and a more prolonged uptake than a fast-release formulation. In the 1983 microplot trial, combinations of commercial and controlled-release formulations were also used and the Vydate DPX 4702/Vydate 10G combination was found to give the best initial control of invasion and development (Fig.

3.20). These experiments thus demonstrate that early attainment of an effective concentration of a.i. is a highly desirable property for a granular nematicide formulation.

All the formulations of oxamyl used in the 1984 trials had shown a rapid initial release of a.i. in laboratory elution tests and so insufficient immediate release was not thought likely to be a problem; this was confirmed by the results obtained for invasion and development (Fig.

3.13).

However, the incremental addition of Vydate 10G during the 1982 pot trial to simulate a controlled-release system presented a potential problem due to the difficulty in incorporating granules evenly around established seedlings. Den Ouden (1977) has shown that control of

G.rostochiensis on potatoes is less effective when aldicarb is not mixed adequately with the soil and the implications of the method of application on nematode control will be discussed later. The Vydate granules applied during the growing season in the field at Prickwillow

in 1982 were simply forked into the soil between the rows of sugar beet

seedlings; subsequent results confirmed that control was maintained and

root growth unaffected, showing that this sufficed. Batterby et al.

(1980) employing the same technique also justified its use. However, for

the addition of Vydate granules to the pots, the seedlings were removed to enable adequate mixing and subsequently replanted. Unfortunately, 147 - this disruption caused proliferation of the root system resulting in a greater density of roots per unit volume of soil, an increase in the number of suitable invasion sites and probably a greater concentration of hatch-promoting substances. This had the overall effect of substantially increasing root invasion and inhibiting subsequent root growth (Fig. 3.18). Results for the Vydate 106 Incremental treatments in the 1982 pot trial should therefore be viewed with caution. The effects of replanting could also account for the greater nematode density in plants grown in pots compared with plants in the field with the same initial nematode population, an observation also made by Whitehead et^ al_. (1972).

None of the formulations tested produced any visible signs of phytotoxicity. However, poor germination in the 1984 microplot trial suggests phytotoxicity as a contributory factor. In this trial, the application of nematicide was in-row as opposed to broadcast and as little rain fell in the first 4 weeks after sowing, a high concentration of toxicant would have remained in close contact with the germinating seed. Maughan et al. (1984) recognised the dangers of phytotoxicity with aldicarb in dry soils and Cooke et al. (1974) reported a 25% decrease in seedling numbers following aldicarb treatment of dry soil. In addition,

Maughan (1977) found that aldicarb applied at 5.7 g a.i. per 100 m row caused slight phytotoxicity.

In the present work, it is interesting to note that the controlled-release treatment in the 1984 microplot trial resulted in greater root weights compared with the commercial formulations at week 7

(4 weeks post-germination; Fig. 3.24), perhaps indicating that high

i rates of a slow-release treatment are less likely to be harmful to plant 148 - establishment than similar rates of a fast-release treatment, especially when applied in the seed furrow. Dunning and Winder (1974) have also

reported that smaller amounts of aldicarb gave better seedling establishment.

Poor seedling emergence in the 1984 microplot trial may also have been due to the fact that the variety sown, Regina, had a relatively poor germination efficiency giving only 47% establishment in the NIAB variety trials during the same season (Kimber and McCullagh, 1984).

By mid-season, control provided by the commercial formulations in field trials had decreased considerably. Thus, although nematode invasion was still less than in the control roots, particularly in the peat fen soils, the development of juveniles within the roots was now unimpaired

(Figs. 3.14, 3.15, 3.19, 3.21, 3.22, 3.25, and 3.26). Temik 10G appeared to be more effective than Vydate 10G at this time. The experimental controlled-release formulations faired little better with nematode invasion increasing rapidly in many instances. However, the controlled-release formulations used in the 1984 trials were as, or more, effective than the commercial treatment with the best control being provided by the formulation releasing 1.50% a.i. min’* (Figs.

3.25, 3.26, 3.14 and 3.15). Formulations releasing a.i. at slower or faster rates than 1.5% appeared to be increasingly less effective.

The above trends could have been predicted to some extent from the laboratory elution studies, especially in the case of Vydate 10G where all the a.i. was found to be rapidly released from the granules.

However, in the field the effectiveness of a soil-applied pesticide is influenced principally by: 1. the rate of chemical degradation to 149 - non-toxic materials; 2. the extent to which the concentration falls as a result of leaching by soil moisture; 3. by the degree of adsorption onto various components of the soil milieu, and to a lesser degree by root absorption.

Differences between soil types were apparent in the 1984 trials where the 1.25% release formulation appeared to be less effective on the sandy-loam at Silwood than on the peat fen at Prickwillow. However, the effect of soil type on nematicide efficacy was most clearly demonstrated in the 1982 pot trial (Fig. 3.19). In the latter experiment, while all pesticide treatments in the peat fen soil were able to greatly reduce nematode development, in the sandy-clay soil development progressed uninhibited in all treatments being similar to that in control roots.

The two compounds used in the sugar beet trials, oxamyl and aldicarb, have a similar effect on nematodes although there are differences in their degradation processes. Whereas oxamyl is the toxicant and its degradation product, the oxime, is non-toxic to nematodes, workers have shown that aldicarb is rapidly converted to the sulphoxide in the soil and then more slowly to the sulphone derivative, both of which are also inhibitors of AChE (Metcalf et al ., 1966) and toxic to nematodes

(Batterby et al., 1980).

The breakdown of oxamyl and aldicarb in the soil follows approximately first-order kinetics with a half life of around 10-14 days for oxamyl and slightly longer for aldicarb, although this may be extended under conditions of low rainfall and temperature and in a soil of high organic content (Bull, 1968; Kearby et al., 1970; Bromilow, 1973; Leistra et^ al., 1976; Bromilow et al., 1980; Gerstl, 1984). At commercial - 150 -

application rates for these compounds (5 Kg a.i. ha"*) toxic residues

have been found to persist for up to 11 weeks under the most favourable

conditions, with over half being lost in just 2-4 weeks (Bull, 1968;

Bromilow, 1973; Whitehead et al., 1979).

Control of H.schachtii will only last as long as the nematicide levels ‘

in the soil remain above the MIC and because the effects of carbamate

action are reversible, any formulation which rapidly releases ail the

a.i. will offer little protection against later invasion. The results

obtained in the field studies appear consistent with this theory.

Further evidence for the short life of a commercial application of

oxamyl in the field is provided by Griffin'and Gessel (1973) and Green

et al. (1981).

It has been suggested that the infective juveniles of plant parasitic

nematodes which recover from the effects of nematicide treatment could

have reduced infectivity (Wallace and Grandison, 1971; Steele and

Hodges, 1975). However, as discussed in Section 4.1, it appears that

such treatment does not affect subsequent infectivity of H.schachtii and merely delays invasion provided the oxamyl concentration in the soil is

sufficient to induce paralysis. This could also be the case in the

field, although low concentrations of oxamyl may encourage J2 to utilize

food reserves more rapidly and thus shorten their infective life.

Although it has been shown that there is a decrease in infectivity with

declining neutral lipid reserves in G.rostochiensis (Storey, 1984), it

has also been reported that infective nematode juveniles of some species are still able to invade when reserves are almost exhausted (Van Gundy et al., 1967). Thus, whether the need is for an extended period of nematode paralysis or just for low concentrations of toxicant to prevent 151

later invasion, both may be maintained by a suitable controlled-release

treatment.

Delayed invasion by nematodes is known to be of some benefit in that

nematodes cause less damage to seedlings that are well established

(Wallace, 1970; Brodie and Dukes, 1972; Griffin, 1981), but this may

have the opposite effect on population control since a larger root

system has a greater number of potential feeding sites as discussed

previously. For example, Steele (1975) found ten times as many adult

females of H.schachtii on sugar beet inoculated 40 days after

germination than on seedlings innoculated 10 days after germination.

When the a.i. falls below the MIC, infective juveniles may arise from

three possible sources: 1. Late-hatching juveniles; 2. Juveniles present

in the soil during the initial control period; or 3. A second generation

formed by those juveniles which were able to invade and develop during

the early part of the growing season. Other factors contributing to

late-invasion are the presence of hatch promoting substances from

actively growing roots and the stimulatory effect of low concentrations

of toxicant on hatching. Ultimately, the situation may arise where there

are more nematodes in treated roots than in controls; this occurred in

the 1982 field trial (Fig. 3.11) and has also been reported by other workers after oxamyl treatment (Overman, 1971; Bunt, 1973; Rhoades,

1975).

In the control roots at week 17 of the 1982 field trial (Fig. 3.11),

there were proportionately fewer developed stages than might have been

expected at this time (late July) which suggests that some adults or young cysts may have been lost during removal of the roots from the 152 - soil. Certainly, the soil was very dry during this period which made lifting the beet more difficult. This period may also have marked the beginning of a second generation of nematodes in the roots. Thus, Duggan

(1959) noted two distinct "flushes" of white cysts on sugar beet roots during the growing season; the first in mid-June and the second in early

September. After the appearance of immature cysts a maturation period of at least four weeks is required before the eggs are ready to hatch

(Jones, 1956; Duggan, 1959).

In nematicide-treated plots, the second generation of H.schachtii would be expected to occur somewhat later as a result of early inhibition. For instance, Hague (1979) found that up to an additional four weeks elapsed before a similar hatch of G.rostochiensis occurred in oxamyl-treated plots compared with controls. In the 1984 microplot trial, the appearance of a second generation of H.schachtii in treated plots occurred between weeks 15 and 21 (Figs. 3.26 and 3.27). During this period invasion in treated roots rose by up to 800%; in the same period control invasion increased by only 500%. As before, this difference was a consequence of the larger root system following nematicide treatment and emphasises the importance of control at this time.

The multiplication rate for H.schachtii is very dependent on environmental conditions (Steudel and Thielmann, 1967) and it is interesting to note that the period of greatest resurgence (late August/ early September) marks the end of the summer drought. The prevailing warm, wet conditions at this time would be ideal for rapid nematode reproduction and the completion of a second generation before harvest

(Wallace, 1956). It is suggested, therefore, that control influenced during late summer is just as important as that during the earlier 153 - period if we are to prevent nematode multiplication as well as losses in sugar yield. Indeed, tontaxis et al. (1976) found that an additional side dressing of aldicarb to the sugar beet crop after 21 weeks gave the best yield improvement and Steudel and Thielmann (1967, 1968) reduced or prevented nematode multiplication by spreading aldicarb on the sugar beet crop after sowing.

The above-mentioned differences in nematicidal effectiveness between the soil types, especially evident in the 1982 pot trial (Figs. 3.17 - 3.19) are probably due to the greater degree of adsorption of pesticide on organic soils (Bromilow, 1973). A sandy soil appears to be the more effective environment for nematicidal activity during the early part of the growing season, probably because oxamyl and aldicarb are only weakly adsorbed on this soil type and so remain potentially mobile (Bromilow and Lord, 1979). The a.i. will therefore.be more readily re-distributed by leaching and an equilibrium rapidly established between mobile and stationary phases (Leistra et al., 1980). Organic soils, on the other hand, tend to adsorb the a.i. more strongly permitting little mobility, but this does help maintain the pesticide in the rooting zone for a longer period of time compared.with sandy soils and losses by leaching are not likely to be as great in organic soils (Bromilow et al., 1980).

These differences probably account for the greater efficacy of nematicide treatments in peat soils by mid-season.

However, Woodham et al. (1973) noted that very little lateral movement of aldicarb took place in soils, while Harvey and Han (1978) found degradation of oxamyl in the field to be so rapid that few residues were found deep in the soil even under irrigation. A uniform distribution of nematicide granules is therefore essential to maintain an effective 154 concentration in all parts of the root zone. The increasingly popular method of in-furrow application, although more economical on pesticide usage and labour, only produces a high concentration of toxicant in the immediate vicinity of the seed. As the roots grow sideways and downwards, they eventually extend outside the protected zone. Such in-row treatments may also provide conditions favourable to hatching at sites distant from the points of application (Steele, 1983).

Broadcast application followed by rotivation is much the more efficient method for cyst-nematode control (Hague and Pain, 1972; Whitehead et al., 1975; Smith and Bromilow, 1977; Bromilow and Lord, 1979; Whitehead et al., 1981).

As mentioned previously, granule size may also be of importance in maintaining an even distribution of nematicide in the soil. A greater number of smaller granules not only have the advantage of achieving an even distribution more easily, but also present a larger surface area for release of a.i. The experimental controlled-release granules formulated in the laboratory, although being of a similar size to the commercial formulation (30/60 LVM), had 5% a.i. content instead of 10%.

It was hoped that this would give improved distribution of oxamyl in the field when applied at the same rate and should thereby increase its bioefficiency.

Despite the promotion of root growth up to mid-season by many of the experimental controlled-release formulations tested (Figs. 3.11, 3.15,

3.19, 3.21, and 3.27), none of these treatments significantly increased final root weight or total yield per plot at harvest when compared with untreated or commercially-treated plots. However, the Temik 10G - 155 - treatments in the 1984 microplot trial did give significantly greater root weights than controls (Fig. 3.28). It appears that because of the long vegetative life of the crop considerable compensatory growth of

"backward" plants occurs during the latter part of the growing season.

This occurs particularly in peat soils and where there is a greater growing area due to missing or small plants which compensate to some extent for plant loss (Jorritsma, 1972).

In the 1982/1984 field trials, yields were very good for both treated and untreated plots averaging 60-75 tonnes ha”* (25-30 tonnes acre"*).

However, in the 1983/1984 microplot trials root yields were much poorer, probably as a result of lack of deep cultivation of the soil and the summer drought conditions which affect crops grown in sandy soils much more readily. The 1982 pot trial was terminated after 12 weeks due to the restrictions in pot size. The harvested area in the field trials and in the 1984 microplot trial consisted of the middle three rows of each five row plot, as this has been reported to give the smallest coefficient of variance when compared with the same sample area in other plot widths (Jaggard, 1975).

Negligible differences in the yield of sugar beet crops f-rom treated and untreated plots have been reported by many workers (Dunning and Winder,

1974; Kontaxis and Thomason, 1978; Batterby et al., 1980). Yield losses are known to be dependent on the nematode population present at sowing

(Thielmann and Steudel, 1973) and Dunning and Winder(1974) and Whitehead et al. (1979) have reported that sugar yields were only increased by nematicide treatments in heavily infested soils; the same also applies to G.rostochiensis on potatoes (Moss et al., 1975). Where nematicide treatments were observed to cause an increased yield , the rate of 156 - application was found to have little effect; small amounts of compound often being just as effective as much larger amounts, especially with oxamyl (Whitehead et al., 1974; Cooke, 1975b, 1976; Whitehead et al.,

1979).

In the field studies, the greater initial population of H.schachtii at the start of the 1984 field trial (13 eggs g"*) resulted in greater yield responses than in 1982 trial (3 eggs g“*; Figs. 3.12 and 3.16), while the high population in the 1984 microplot trial on sandy soil (12 e99s g“M also resulted in increased yields with some treatments (Fig.

3.28). The results also suggest that H.schachtii is much more damaging in sandy soils. ADAS also recognise that a greater population of

H.schachtii is required in peat soils than in sandy soils per unit weight to incur an identical loss of yield. This is due in part to the difference in bulk density of the two soil types and so it is more practical to express populations per unit volume of soil, or as Moss et_ al. (1976) suggest to divide the number of eggs g*** peat soil by a factor of two.

Many similar studies of this nature put most emphasis on increased yield but, as discussed in the introduction, a successful formulation must also suppress nematode reproduction.

In the 1982, 1983 and 1984 field and microplot trials, all the experimental controlled-release formulations reduced nematode multiplication when compared with the control and commercially-treated plots; the slowest release rate in the 1984 field trial (1.00% a.i. mirr*

) had the least effect. Generally, the commercial formulation also marginally decreased nematode multiplication from that in control plots. 157

Control of H.schachtii populations by high rates of aldicarb has been reported by Heijbroek (1973), Dunning and Winder (1974; 1979) and by

Batterby et al. (1980), but there are few reports of oxamyl significantly reducing the reproductive rate of this species. In trials on sugar beet which have been conducted using oxamyl, final populations are often positively correllated with beet weights and low rates of oxamyl were often just as effective as much larger ones (Whitehead et_ al_., 1974, 1979).

From the field studies, it may be concluded that although the addition of oxamyl in controlled-release or simulated form successfully reduced

H.schachtii population increase, there was little evidence to suggest that the nematode was significantly affecting yield in control plots, except perhaps at an initial population of 12 egg g"l in a sandy-loam soil. In that instance the controlled-release formulation practically halved the final nematode population compared with untreated plots

(Table 3.19).

In the microplot trials, multiplication in control plots was poor (3-4 fold) making comparisons between treatments difficult, and because the pot trial was terminated at week 12 the results represented one generation only (Table 3.17). It is interesting to note that at week 12 all the reproductive rates were low, confirming that most of the population increase occured in the second generation..

The results from the trials also suggest that multiplication rates are greater in peat soils. This may be because of: 1. The greater root weights and more extensive root systems of plants grown in organic soils 158 -

providing a greater opportunity for invasion and non-competitive development; 2. Organic soils are more able to maintain an optimum soil moisture content; 3. Lower initial populations have the potential for greater percentage increases.

During the growing season, no other pests or diseases were seen to be affecting the trials apart from in the 1983 microplot trial where there was a heavy infestation of Aphis fabae at around 16 weeks. Although these aphids appeared to colonize control plots first, they quickly spread to the other treatments suggesting that little residual systemic pesticide activity remained.

The greater concentrations of soil oxamyl residues recovered from controlled-release treatments compared with the fast-release treatments

(Table 3.14 and 3.16), confirmed that the former showed a more prolonged release pattern in the field. In this instance, therefore, release rate characteristics observed in the laboratory were demonstrable in the field to some extent.

The more persistent release of a.i. was also correlated with more effective nematode control later in the season. This is in contrast to

Batterby (1978) who found that experimental formulations of aldicarb which showed controlled-release characteristics in the laboratory failed to give prolonged control in the field.

It has been shown that release rates of a.i. from various formulations increase with increasing soil moisture and temperature (Coppedge e t a l ., 1975a). Therefore, conditions of low rainfall would probably reduce the rate of oxamyl release from granules into the soil. 159 -

In the two years over which soil residues were monitored in the present study, drought conditions did not prevail nor was rainfall unduly heavy for any length of time and a continuous loss of a.i. from the granules would be expected to have occured.

With sugar beet, the increased persistence of oxamyl is unlikely to be a problem in the harvested crop. Dejonckheere et al. (1982) found residues of aldicarb and its toxic products to be very low in sugar beet at harvest after commercial treatment. Oxamyl is less persistent than aldicarb and because release of a.i. is not intended to extend into late season, the high rainfall in autumn and the relatively short half-life of oxamyl suggest that only negligible crop residues will be found.

The possibility of resistance developing in nematode populations due to the increased persistence of controlled-release nematicides is also unlikely. For example, there is no evidence of resistance to oxamyl in

G.rostochiensis populations with a history of pesticide treatment

(Whitehead et al., 1984). In fact, it has been suggested that with the very low selection pressure accompanying nematicide usage, mainly because only a small proportion of the nematode population is exposed to pesticide, significant nematicide resistance problems will never arise

(Wright, 1981).

As part of the field studies, microplots were established at Silwood

Park. Normal scale field trials have several disadvantages: 1. There is usually considerable variability in nematode population densities within trial plots; 2. There is the possibility of appreciable variability in soil structure and fertility in the plots; 3. They are relatively time consuming and expensive; 4. There is often difficulty in maintaining 160 - control over the management of the plots. Pot experiments, on the other hand, represent the other extreme and cannot be compared with normal field conditions of soil moisture, temperature and aeration (Jones,

1956) .

Thus, where a relatively large number of treatments were to be assessed, it was decided that the use of microplots was probably the most convenient, reproducible and cost-effective method to adopt. This allowed a much greater and more uniform initial soil nematode population to be used under less variable conditions than were possible in the field. For example, the initial population variance in the 1984 microplot trial was just a quarter of that in the concurrent field trial at Prickwillow and distribution of fertilizer, establishment of seedling density, suppression of weeds, plant and soil sampling and harvesting were all more controlled in microplots. Certainly in the microplot trials conducted in 1983 and 1984 yield responses between treatments were much more distinguishable (Figs. 3.23 and 3.28) than in full field trials. In 1984, this was despite part of the trial area producing very poor growth probably due to poor drainage providing less than optimal conditions for crop growth (see Webster et al., 1977) with the result that within treatment variance was found to be significant after 21 weeks (Appendix 46).

It is thought, therefore, that microplots are the most practical method for the secondary evaluation of nematicide formulations. The use of microplots for nematicide trials has also been endorsed by Jones (1956,

1957) and Sasser et al. (1982). - 161

5. CONCLUSIONS

In the laboratory experiments, although some systemic activity of oxamyl against H.schachtii within the roots was demonstrable, as Hague and Pain

(1973) have suggested, the greatest activity was against the free-living juveniles; oxamyl being particularly effective in reducing nematode infectivity where the MIC value was estimated to be 0.2 pg a.i. cm"?.

Elution studies showed that the commercial formulation of oxamyl, Vydate

10G, rapidly released all of its a.i. Thus, heavy rainfall soon after application coupled with the relatively short half life of oxamyl in the soil could markedly reduce the efficacy of such treatments in the field and it appears doubtful whether the commercial product at economic rates could achieve anything more than early control of the beet cyst nematode.

When tested in the field, Vydate 10G and all the experimental formulations of oxamyl provided protection of the crop during its establishment. However, unlike Vydate 10G, those experimental formulations which demonstrated a slow-release rate of a.i. under laboratory conditions also reduced the rate of population increase of

H.schachtii. The latter formulations were found-to give increased persistence of toxicant in the soil compared with the commercial treatment which related well with the elution results and clearly demonstrated that the nematicide should be continuously available to maintain control.

Laboratory studies on the effects of long-term exposure to oxamyl on the subsequent ability of J2 H.schachtii to migrate and to infect sugar beet - 162 - seedlings supported the above conclusion; nematodes exposed to low _3 concentrations of oxamyl (0.2 - 2.0 pg cm ) for up to 56 days remaining at least as active as untreated controls. A phenomenon which could be related to the preservation of neutral lipid (triglyceride) energy reserves in pesticide-immobilised nematodes.

While the value of controlled-release formulations remains to be determined it is likely that they will be most effective on lighter soils. It is known that pesticide adsorption onto soil particles regulates its availability; where the degree of adsorption is relatively low, as would occur in a sandy soil, the toxicant is leached much more rapidly. In such a situation, a controlled-release formulation would allow a desired concentration of a.i. to be maintained in the root zone for a longer period.

As agricultural intensification proceeds, it seems certain that chemical control of nematodes will be of increasing importance. However, in recent years, environmental and economic considerations have led to a search for more effective, inexpensive and safer compounds which are biodegradable or at least relatively non-persistent in the environment.

This has led to the increasingly restricted use of many of the fumigant nematicides, particularly the bromine containing compounds (Maddy, 1983;

Wybon and Homeyer, 1984) and is likely to lead to an increase in the use of non-fumigant compounds which couple effectiveness at low dosages with a relatively short residual persistence. The requirement for prolonged activity from such compounds may be accomodated for by repeated applications or by the adoption of a controlled-release system.

Today, up to 10 years of development and many millions of pounds are - 163 -

required to bring a new product to market, not least because of

increasingly more detailed registration requirements. New products must

also be formulated and manufactured in such a manner that they are cost

effective. As a result, research work on chemical control now includes

ways of making better use of products that already carry a

recommendation, either by improving application techniques or by

re-formulating the products. In this study, the re-formulation of the

oximecarbamate, oxamyl into controlled-release granules has demonstrated

some of the potential benefits of such a system.

However, despite its considerable activity against nematodes, a major

disadvantage of oxamyl and other similar nematicides is that their

reversibility of action may lead to a resurgence in the nematode

population later in the season. A more effective controlled-release

product may, therefore, be one incorporating an organophosphate, such as

fenamiphos, where recovery of nematode vigour is not so pronounced

although the compound retains the advantage of a relatively short period

of residual activity (Bunt, 1975; Hafez and Lear, 1981).

In addition, new products that are closely related to natural compounds

are especially appealing, as is the case with the recently discovered

avermectins, a group of macrocyclic lactones isolated from the

actinomycete, Streptomyces avermitilis (Burg et al., 1979). In the

laboratory they have been shown to inhibit nematode life cycle stages at very low concentrations (Birtle et al., 1983; Wright et al., 1983, 1984) while in the field, they have given good control of M.incognita at

0.16-0.24 Kg a.i. ha"* on soils of low organic content. These rates are

10-30 times as potent as commercial contact nematicides (Putter et al.,

1981; Preiser et al., 1981) and it would seem that avermectins are - 164 - excellent candidates for inclusion in a controlled-release formulation.

Finally, the concept of controlled-release provides a basis for a safer and more efficient use of nematicides and pesticides in general. While it is unlikely that the full theoretical potential of such formulations will be achieved in the near future, with existing technology it should be possible to produce formulations which are a substantial improvement on existing products. 165 -

REFERENCES

Abbott, W.S. (1925). A method for computing the effectiveness of an insecticide. Journal of Economic Entomology 18, 265-267.

Allan, G.G., Chopra, C.S., Maggi, M.W., Neogi, A.N. and Wilkins, R.M. (1973). Growth enhancement of a juvenile conifer forest by a controlled-release herbicide. International Pest Control 15, 8-13.

Allan, G.G., Chopra, C.S., Neogi, A.N. and Wilkins, R.M. (1971). Design and synthesis of controlled-release pesticide-polymer combinations. Nature, London 234, 349-350.

Allan, G.G., Chopra, C.S. and Russell, R.M. (1972) Selective suppression of weeds and deciduous brush in the presence of conifers. -International Pest Control 14, 15-20.

Allan, G.G., Friedhoff, J.F. and Powell, J.C. (1975). Growth enhancement of a juvenile conifer forest four years after application of a controlled-release herbicide. International Pest Control 17, 4-9.

Allan, G.G., Gara, R.I. and Wilkins, R.M. (1974). Field testing of insecticide-polymer combinations for the long term control of the mahogany shootborer. International Pest Control 16, 4-12.

Allen, S.E. (1977). Controlled-release fertilizers: theory and practice. Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp. 289-298.

Andrewes, G.L., Harris, F.A., Sikorowski, P.P. and McLaughlin, R.E. (1975). Evaluation of He!iothis nuclear polyhedrosis virus in cottonseed oil bait for control of Heliothis virescens and H.zea on cotton. Journal Economic Entomology 68, 87-90.

Anon". (1964). New products: Slow-release Vapona strip has many uses. International Pest Control 6, 24.

Anon. (1980). Beet Cyst Nematode. M.A.F.F. Leaflet No. 233, 5pp.

Anon. (1983). Effect of systemic nematicides on pineapples. Helminthological Abstracts B 52, 45.

Apt, W.J. (1981). The application of fenamiphos and oxamyl by drip irrigation for the control of the reniform nematode on pineapple in Hawaii. Journal of Nematology 13, 430 (Abstract).

Atilano, R.A. and Van Gundy, S.D. (1979). Systemic activity of oxamyl to Meloidogyne javanica at the root surface of tomato plants. In: The Soil-Root Interface, pp. 339-349 (Eds. J. Harley and A. Scott-Russel1), Academic Press, London.

Bailey, D.L., LaBreque, G.C. and Whitfield, T.L. (1971). Housefly control in a poultry house with insecticides in slow-release plastic formulations. Journal of Economic Entomology 64, 138-140. 166 -

Bakan, J.A. (1976). Controlled-release pesticides via microencapsulation Proceedings 1976 International Controlled Release Pesticide Symposium, Akron, pp. 1.1-1.33.

Baker, R.W. and Lonsdale, H.K. (1975). Principles of controlled release. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton, pp. 9-39.

Batterby, S. (1978). Studies on the control of Heterodera schachtii (Schmidt) using various formulations of aldicarb. Ph.D. Thesis, University of London. 168pp.

Batterby, S. (1979). Toxic effects of aldicarb and its metabolites on second stage larvae of Heterodera schachtii. Nematologica 25, 377-384.

Batterby, S., LePatourel, 6.N.J. and Wright, D.J. (1977). Accumulation and metabolism of aldicarb by the free-living nematodes Aphelenchus avenae and Panagrellus redivivus. Annals of Applied Biology 80, 69-76.

Batterby, S., LePatourel, G.N.J. and Wright, D.J. (1980). Effects of aldicarb persistence on field populations of Heterodera schachtii. Annals of Applied Biology 95, 105-113.

Beasley, M.L. (1970). Water-degradable polymers for controlled release of herbicides and other agents. Science 169, 769-770.

Beroza, M., Hood, C.S., Trefrey, D., Leonard, D.E., Knipling, E.F., Klassen, W. and Stevens, L.J. (1974). Large field trial with microencapsulated sex pheromone to prevent mating of the Gypsy moth. Journal of Economic Entomology 67, 659-664.

Birtle, A.J., Corps, A.E. and Wright, D.J. (1983). Biological effects of avermectins on nematodes. Nematologica 25, 137 (abstract).

Boparai, J.S. and Hague, N.G.M. (1974). The effect of thioxamyl on the development and reproduction of the potato cyst nematode, Heterodera rostochiensis (Woll.). Mededelingen van de Rijksfactulteit Landouwwetenschappen, Gent 39, 719-725.

Brodie, B.B. (1983). Control of Globodera rostochiensis in relation to the method of applying nematicides. Journal of Nematology 15, 491-495.

Brodie, B.B. and Dukes, P.D. (1972). The relationship between tobacco yield and time of infection with Meloidogyne javanica. Journal of Nematology 4, 80-83.

Bromilow, R.H. (1973). Breakdown and fate of oximecarbamate nematicides in crops and soils. Annals of Applied Biology 75, 473-479.

Bromilow, R.H. (1976). Determination of residues of oxamyl in crops and soils by gas-liquid chromatography. Analyst 101, 982-985. - 167 -

Bromilow, R.H., Baker, R.J., Freeman, M.A.H. and Gorog, K. (1980). The degradation of aldicarb and oxamyl in soil. Pesticide Science 11, 371-378.

Bromilow, R.H., and Lord, K.A. (1979). Distribution of nematicides in soil and its influence on control of cyst nematodes (Globodera and Heterodera spp.). Annals of Applied Biology 92, 93-107T

Brookes, T.W. and Swenson, D.K. (1970). Design considerations in the formulation of controlled release product forms based on hollow fibres. Proceedings 1976 International Controlled Release Pesticide Symposium, Akron, pp. 4.5-4.22.

Brown, R.A. (1983) Soil inhabiting pests of sugar beet and prospects for forecasting their damage. Aspects of Applied Biology 2, 1983. Pests, Diseases, Weeds and Weed Beet in Sugar Beet, 45-52~

Bull, D.L. (1968). Metabolism of U.C. 21149 (2-methyl 2-(methylthio) propionaldehyde 0-(methylcarbamy) oxime) in cotton plants and soil in the field. Journal of Economic Entomology 61, 1598-1602.

Bunt, J.A. (1973). Influence of temperature on the population density of Pratylenchus penetrans and the growth response of Pyrus malus (L.) seedlings, after a drench with S-methyl l-(dimethylcarbamoyl )-N- ((methylcarbamoyl)oxy) thioformimidate. Mededelingen van de Rijksfactwiteit Landbauwwetenschappen, Gent 38, 1259-1273.

Bunt, J.A. (1975). Effect and mode of action of some systemic nematicides. Mededelingen van de Landbouwhageschad Wageningen 75-10, 1-127.

Burg, R.Q., Miller, B.M., Baker, E.E., Birnbaum, J., Currie, S.A., Hartman, R., Kong, Y-L., Monaghan, R.L., Olson, G., Putter, I., Tunac, J.B., Wallick, H., Stapley, E.O., Oiwa, R. and Omura, S. (1979). Avermectins, a new family of potent anthelmintic agents: Producing organisms and fermentation. Antimicrobial Agents and Chemotherapy 15, 361-367.

Campion, D.G., Lester, R. and Nesbitt, B.F. (1978). Controlled release of pheromones. Pesticide Science 9, 434-440.

Cardarelli, N.F. (1974). Cercariacidal potency of slow release molluscicides. Proceedings 1974 International Controlled Release Pesticide Symposium, Akron, pp. 39.1-39.16.

Cardarelli, N.F. (1975). Controlled release pesticides: The state of the art. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton, pp. 1-8.

Cardarelli, N.F. (1976). Compounding methods for controlled release elastomers. Proceedings 1976 International Controlled Release Pesticide Symp., Akron, pp. 1.44-1.62. 168 -

Cardarelli, N.F., Evans, W. and Smith, D. (1981). Controlled release organotin pesticides biochemistry: toxicology: environmental factors. In: Controlled-release of Pesticides and Pharmaceuticals pp. 239-244 (Ed. D.H. Lewis). Plenum Press, New York.

Cardarelli, N.F. and Feldmesser, J. (1975). Controlled release nematicide development: Preliminary investigations. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton, pp. 386-392.

Cardarelli, N.F. and Walker, K.E. (1978). Development of registration criteria for controlled release pesticide formulations. Report, Environmental Protection Agency /540/9-77/016; Order no. PB-291755. 159pp.

Castro,C.E. (1964). The rapid oxidation of iron II porphyrins by alkyl halides. A possible mode of intoxication of organisms by alkyl halides. Journal of the American Chemical Society 86, 2310-2311.

Castro, C.E. and Belser, N.O. (1978). Intoxication of Aphelenchus avenae by ethylene dibromide. Nematologica 24, 37-44.

Caswell, E.P. and Thomason, I.J. (1984). The geographic distribution of Heterodera schachtii in the Imperial Valley of California from 1961- 1983. Proceedings 1st International Congress of Nematology, Guelph, Canada, p. 17 (absract).

Clarke, A.J. and Shepherd, A.M. (1966). Inorganic ions and the hatching of Heterodera spp. Annals of Applied Biology 58, 497-508.

Cooke, D.A. (1975a). Nematicide usage on sugar beet. Proceedings 8th British Insecticicide Fungicide Conference 1, 127-132.

Cooke, D.A. (1975b). Yellows and aphids - control by insecticides: Report Rothamsted Experimental Station for 1974, Pt 1, 55.

Cooke, D.A. (1976) Nematicide trials - beet cyst-eelworm. Report Rothamsted Experimental Station for 1975, Pt 1, 61-62.

Cooke, D.A. (1978). Beet cyst nematode: Its life history and control. British- Sugar Beet Review 46, 40-43.

Cooke, D.A. (1982). Beet cyst nematode. British Sugar Beet Review 51, 17-19.

Cooke, D.A. (1983). Distribution and pathogenicity of beet cyst nematode in England: Aspects of Applied Biology 2, 1983, Pests, Diseases, Weeds and Weed Beet in Sugar Beet, 53-57.

Cooke, D.A. (1984). The relationship between numbers of Heterodera schachtii and sugar beet yields on a mineral soil, 1978-1981. Annals of Applfed Biology 104, 121-129.

Cooke, D.A. and Thomason, I.J. (1979). The relationship between 169 -

population density of Heterodera schachtii, soil temperature and sugar beet yields. Journal of Nematology 11, 124-128.

Cooke, D.A., Dunning, R.A. and Winder, G.H. (1974). The effect of nematicides applied to the seed rows in spring on growth and yield of sugar beet in Docking-disorder-affected fields. Annals of Applied Biology 76, 289-298.

Cooke, D.A., Thomason, I.J., McKinney, H.E., Bendixen, W.E. and Hagemann, R.W. (1979). Chemical control of Heterodera schachtii on sugar beet in California. Journal of Nematology 11, 205-206.

Cooke, L.R., Clifford, D.R., Deas, A.H.B. and Holgate, M.E. (1982). Control of potato late blight using controlled-release granules containing ofurace. Pesticide Science 13, 686-692.

Cooke, L.R., Clifford, D.R. and Holgate, M.E. (1981). Control of potato late blight (Phytophthora infestans) with systemic fungicides. Pesticide Science 121, 6/8-684.

Coppedge, J.R., Stokes, R.A., Kinzer, R.E. and Ridgeway, R.L. (1974). Biological evaluation of slow release formulations of aldicarb. Journal of Economic Entomology 67, 292-294.

Coppedge, J.R., Stokes, R.A., Kinzer, R.E. and Ridgeway, R.L. (1975a). Effect of soil moisture and soil temperature on the release of aldicarb from granular formulations. Journal of Economic Entomology 68, 209-210.

Coppedge, J.R., Stokes, R.A., Ridgeway, R.L. and Bull, D.L. (1975b). Chemical and biological evaluation of slow release formulations of four plant systemic insecticides. Journal of Economic Entomology 68, 508-510.

Corbett, J.R. (1974). The Biochemical Mode of Action of Pesticides. Academic Press, London and New York. 330pp.

Cramer, R.D. (1976a). Controlled release pesticides - making the most of the benefits. Part I. International Pest Control 18, 1-3.

Cramer, R.D. (1976b). Controlled release pesticides - making the most of the benefits. Part II. International Pest Control 18, 4-8.

Curtis, 6.J. (1965). An improved method of hatching larvae of cyst-forming nematodes. Nematologica 11, 213-217.

Davies, K.A. and Fisher, J.M. (1976). Factors influencing the number of larvae of Heterodera avenae invading barley seedlings in vitro. Nematologica 22, 153-162.

Dejonckheere, W., Melkebeke, G., Steurbaut, W. and Kips, R.H. (1982). Uptake and residues of aldicarb in sugar beet leaves. Pesticide Science 13, 341-350. - 170 -

eggs in hatching and pot experiments with Heterodera rostochiensis. Nematologica 9, 225-230.

Den Ouden, H. (1977). The effect of different ways of watering pots after the application of aldicarb on control of the potato cyst nematode. Netherlands Journal of Plant Pathology 83, 1-4.

Dickson, D.W., Huisingh, D. and Sasser, J.N. (1971). Dehydrogenases, acid and alkaline phosphatases and esterases for chemotaxonomy of selected Meloidogyne, Ditylenchus, Heterodera and Aphelenchus spp. Journal of Nematology 3, 1-16.

Di Sanso, D.P. (1973). Nematode response to carbofuran. Journal of Nematology 5, 22-27.

Doncaster, C.C. and Shepherd, A. (1967). The behaviour of second stage Heterodera rostochiensis larvae leading to their emergence from the egg. Nematologica 13, 476-478.

Doney, D.L. (1980). Sugarbeet-fodder beet as a source of alcohol fuel. Proceedings Bio-energy 80, World Congress and Expo. Atlanta, Georgia, pp. 95-97.

Doney, D.L. and Theurer, J.C. (1980). Alcohol fuel from sugarbeets. Utah Science 41, 40-43.

Dorsey, C.K. (1966). Face fly experiments on quater horses, 1962-1964. Journal of Economic Entomology, 59, 86-88.

Dropkin, V., Martin, G.C. and Johnson, R.W. (1958). Effect of osmotic concentration on hatching of some plant parasitic nematodes. Nematologica 13, 115-126.

Duggal, C.I. (1978). Sex attraction in the free-living nematode Panagrellus redivivus. Nematologica 24, 213-221.

Duggan, J.J. (1959). On the number of generations of beet eelworm, Heterodera schachtii Schmidt, produced in a year. Nematologica 4, m - 2 44.

Dunn, R.L. and Strong, F.E. (1973). Control of catch-basin mosquitoes using Zoecan ZR-515 formulated in a slow-release polymer: A preliminary report. Mosquito News 33, 110-111.

Dunning, R.A. and Dyke, T.P.J. (1977). The Beet Cyst Nematode Order, 1977. British Sugar Beet Review 45, 13-14.

Dunning, R.A. and Heijbroek, K. (1981). Improved plant establishment through better control of pest and disease damage. Proceedings 44th I.I.R.B. Winter Congress, 37-58.

Dunning,R.A. and Winder, G.H. (1968). Docking disorder - Nematicide trials. Report Rothamsted Experimental Station for 1967, Pt 1, 275-278. - 171 -

Elson, J.E. (1982). Nematicide registration for minor crops. Journal of Nematology 14, 439 (abstract).

Engelhart, J. Beiter, C. and Frieman, A. (1974). Recent developments in slow release organotin antifoulants. Proceedings 1974 International Controlled Release Pesticide Symposium, Akron, pp. 18.1-18.17.

Evans, A.A.F. (1973). Mode of action of nematicides. Annals of Applied Biology 75, 469-473.

Evans, A.A.F. and Perry, R.N. (1976). Survival strategies in nematodes. In: The Organisation of Nematodes pp. 383-424 (Ed. N.A.Croll) Academic Press, London and New York.

Evans, K. (1984). Cyst nematode problems on oilseed rape. Aspects of Applied Biology 6, 275-279.

Evans, S.G. and Wright, D.J. (1982). Effects of the nematicide oxamyl on life cycle stages of Globodera rostochiensis. Annals of Applied Biology 100, 511-519.

Feld, W.A., Post, L.K. and Harris, F.W. (1975). Controlled release from polymers containing pesticides as pendent substituents. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton, pp. 113-122.

Feldmesser, J. (1974). Progress report on controlled release of a nematicide. Proceedings 1974 International Controlled Release Pesticide Symposium, Akron, pp. 1-32.11.

Feldmesser, J., Jaffe, H., Giang, P.A. and Wolford, G. (1981). Nematicidal amine in poly(vinylacetate) polymer. Proceedings 1981 International Controlled Release Pesticide Symposium., pp. 147-148.

Feldmesser, J., Jaffe, H. and Wolford, G. (1979). Nematicidal amine in cellulose acetate polymer. Proceedings 1979 International Controlled Release Pesticide Symposium, pp. IV-39-IV-43.

Feldmesser, J. and Shasha, B.S. (1977). Evaluation of controlled release DBCP-starch xanthide granules in laboratory and greenhouse tests. Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp. 205-213.

Feldmesser, J., Shasha, B.S. and Doane, W.M. (1976). Nematicides in starch for controlled release. Proceedings 1976 International Controlled Release Pesticide Symposium, Akron, pp. 6.18-6.29.

Fichtner, E., Grabert, D. and Fischer, W. (1983). Studies on the population dynamics of Heterodera schachtii. Helminthological Abstracts B 52, 42.

Fox, I., Rivera, G.A. and Bayona, I.G. (1969). Controlling cat fleas with dichloros impregnated collars. Journal of Economic Entomology 62, 1246-1248. 172 -

Furmidge, C.G.L. (1972). General principles governing the behaviour of granular formulations. Pesticide Science 3, 745-751.

Furmidge, C.G.L., Hill, A.C. and Osgerby, J.M. (1966). Physico-chemical aspects of the availability of pesticides in the soil: 1. Leaching of pesticides from granular formulations. Journal of Science, Food and Agriculture 17, 518-525.

Furmidge, C.G.L., Hill, A.C. and Osgerby, J.M. (1968). Phsico-chemical aspects of the availability of pesticides in the soil: 2. Controlled release of pesticides from granular formulations. Journal of Science Food and Agriculture 19, 91-95.

Garabedian, S. and Hague, N.G.M. (1981). The effect of oxamyl on the control of Heterodera sacchari on sugar cane. Journal of Nematology 13, 439 (abstract).

Garabedian, S. and Van Gundy, S.D. (1983). Use of avermectins for the control of Meloidogyne incognita on tomatoes. Journal of Nematology 15, 503-510.

Gauthier, N.L. (1977). Field testing experiments with slow release insecticide granular formulations to assist insect control. Proceedings 1977 International Controlled Release Pesticide Symposium Oregon, pp. 226 (abstract).

Gerstl, Z. (1984). Adsorption, decomposition and movement of oxamyl in soil. Pesticide Science 15, 9-17.

Glass, E.H., Roelofs, W.L., Arn, H. and Comeau, A. (1970). Sex pheromone trapping red-banded leaf-roller moths and development of a long-lasting polythene wick. Journal of Economic Entomology 63, 370-373.

Goffart, H. (1952). Ansteigen und abklingen der nematodenversenchung und ihre bewertung im rubenanbau. Zuckerrunbenbau 14, 315.

Golden, A.M. and Shafer, T. (1960). Survival of emerged larvae of the sugar-beet nematode (Heterodera schachtii) in water and in soil. Nematologica 5, 32-36.

Gowen, S.Pn. (1977). Nematicidal effects of oxamyl applied to leaves of banana seedlings. Journal of Nematology 9, 158-161.

Greco, N., Brandonisio, A. and De Marinis, G. (1982). Tolerance limit of the sugar beet to Heterodera schachtii. Journal of Nematology 14, 199-202.

Greco, N., Elia, F. and Brandonisio, A. (1984). Effect of DD and non-volatile chemicals on Heterodera schachtii, H.carotae and Meloidogyne javanica. Proceedings 1st International Congress of Nematology, Guelph, Canada, p. 37 (abstract).

Greco, N. and Thomason, I.J. (1980). Effect of fenamiphos on Heterodera schachtii and Meloidogyne javanica. Journal of Nematology 12, 91-96. 173 -

Green, A.A., Kane, J. and Gradidge, J.M.G. (1966). Experiments on the control of Ephestia elutella (Hb.) (Lepidoptera, Phycitidae) using dichlorvos vapour. Journal Stored Products Research 2, 147-157.

Green, C.D. (1966). Orientation of male Heterodera rostochiensis Woll. and H.schachtii Schm. to their females. Annals of Applied Biology 58, —

Green, C.D., Williamson, K., Dennis, E.B. and McBurney, T. (1981). The effect of oxamyl on the growth of peas attacked by the pea cyst nematode. Annals of Applied Biology 97, 303-309.

Griffin, G.D. (1975). Control of Heterodera schachtii with foliar application of nematicides. Journal of Nematology 7,~347-351.

Griffin, G.D. (1981). Pathological differences in Heterodera schachtii populations. Journal of Nematology 13, 191-195.

Griffin, G.D. and Gessel, T.G. (1973). Systemic nematicide control of Heterodera schachtii on sugar beet. Plant Disease Reporter 57, 942-945.

Guerrero, J., Rohovsky, M.W., Van der Westhuizen, B., Mellon, D. and Doddi, N. (1980). The use of mebendazole and levamisole in sustained release formulations. In: The Host Invader Interplay pp. 717-722 (Ed. H. Van den Bossche). Elsevier/ North Holland Biomedical Press, Amsterdam.

Hafez, S.L. and Lear, B. (1981). Action and longevity of some systemic nematicides in soil and roots of tomato for controlling Meloidogyne incognita. Journal of Nematology 13, 441-442 (abstract).

Hague, N.G.M. (1975). Nematicides - past and present. Proc. 8th British Insecticides and Fungicides Conference, Brighton 2, 837-851.

Hague, N.G.M. (1979). A technique to assess the efficacy of non-volatile nematicides against the potato cyst nematode Globodera rostochiensis. Annals of Applied Biology 93, 203-211.

Hague, N.G.M. and Pain, B.F. (1973). The effect of organophosphorus compounds and oxime carbamates on the potato cyst nematode Heterodera rostochiensis Woll. Pesticide Science 4, 459-465.

Hall, H.S. (1975). Controlled application of fungicide onto onion seed. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton, pp. 393-400.

Hall, H.S. (1977). Section of controlled release coatings - starches. Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp. 355-363.

Hardee, D.D., McKibben, G.H. and Huddlestone, P.M. (1975). Grandlure for boll weevils: Controlled release with a laminated plastic diffuser. Journal of Economic Entomology 68, 477-479. - 174 -

Harris, F.W. (1976). Preparation of plastic, controlled release coatings -starches. Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp. 355-363.

Harvey, J. and Han, J.C-Y. (1978). Deposition of oxamyl in soil and water. Journal Agriculture and Food Chemistry, 26, 536-541.

Harvey, T.L. and Brethour, J.R. (1970). Hornfly control with dichlorvos-impregnated strips. Journal of Economic Entomology 63, 1688.

Harvey, T.L. and Ely, D.G. (1968). Wax-bar applications of ciodrin for horn fly control. Journal of Economic Entomology 63, 1729-1730.

Heijbroek, W. (1973). Chemical control of beet cyst eelworm (Heterodera schachtii Schmidt). Proceedings 36th I.I.R.B. Winter Congress, Report No. 3 . 5 . 12pp.

Hb'gger, C., Estey, R.H. and Croll, N.A. (1978). Xiphinema americanum: cholinesterase and biogenic amines in the nervous system. Experimental Parasitology 45, 139-149.

Hooper, D.J. (1970) Preserving and staining nematodes in plant tissues. In: Laboratory Methods for work with Plant and Soil Nematodes, pp. 55-58 (Ed. J.F. Southey) M.A.F.F. Technical Bulletin No. 2 . , H.M.S.O., London.

Hough, A. and Thomason, I.J. (1975). Effects of aldicarb on the behaviour of Heterodera schachtii and Meloidogyne javanica. Journal of Nematology 7, 221-228.

Hylin, V., Hylin, J.W. and Apt, W. (1982). Nematicide residues in Pineapple culture following point source application. 5th International Congress of Pesticide Chemistry, Kyoto, Japan VIIg-4, 213-216.

Ivy, E.E. (1972). Pencap M: an improved methyl parathion formulation. Journal of Economic Entomology 65, 473-474.

Jaggard, K.W. (1975). The size and shape of plots in sugar beet experiments. Annals of Applied Biology 80, 351-357.

Janes, G.A. (1975). Controlled release copper herbicides. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton, pp. 325-333.

Janes, G.A. and Mansdorf, S.Z. (1977). Bioassay results of new aquatic, herbicide CR formulations. Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp. 10-18.

Johnson, W.L., McKibbon, G.H., Rodriguez, J.V. and Davich, T.B. (1976). Boll weevil: increased longevity using different formulations and dispensers. Journal of Economic Entomology 69, 263-265.

Jones, F.G.W. (1945). Soil population of beet eelworm (Heterodera schachtii Schm.) in relation to cropping. Annals of Applied Biology - 175 -

32, 351-380.

Jones, F.G.W. (1950). Observations on the beet eelworm and other cyst forming species of Heterodera. Annals of Applied Biology 37, 407-440.

Jones, F.G.W. (1956). Soil populations of beet eelworm (Heterodera schachtii Schm.) in relation to cropping: II. Microplot and field plot results. Annals of Applied Biology 44, 25-56.

Jones, F.G.W. (1957). Soil populations of beet eelworm (Heterodera schachtii Schm.) in relation to cropping: III. Further experiments with microplots and with pots. Nematologica 2, 257-272.

Jones, F.G.W. (1959). Beet eelworm and other root eelworms. In: Plant Nematology. pplOO-114 (Ed. J.F.Southey) M.A.F.F. Technical Bulletin No.7. H.M.S.O., London.

Jones, F.G.W. and Dunning, R.A. (1972). Sugar Beet Pests M.A.F.F. Bulletin 162, 3rd Edition. H.M.S.O., London, 113pp.

Jones, F.G.W. and Jones, M.G. (1984). Pests of Field Crops. 3rd Edition, Edward Arnold, 392pp.

Jorgenson, E.C. (1969). Control of the sugar beet nematode, Heterodera schachtii on sugar beets with organophosphate and carbamate nematicides. Plant Disease Reporter 53, 625-628.

Jorgenson, E.C. and Musselman, J.R. (1960). Influence of seedling age on susceptibility of sugar beet to Heterodera schachtii. Phytopathology 56, 883-884.

Jorritsma, J. (1972). Field experiments on plant populations and yield. Proceedings 35th I.I.R.B. Winter Congress, Rep No. 4.2. 18pp.

Kampfe, L. (1955). Die aktivitat von kartoffel und rubbennematoden bei verschiedenen temperaturen und ihre bedevtung fur die mittelprufung. Mitt. Biol. Zent. Anst. Berlin 83 139-142.

Kampfe, L. (1975). Untersuchungen zur wirking von aldicarb auf nematoden. In Vortregstagung (1) zu Aktuellen Problemem der Phytonematologie Am 29.5. 1975 Gesellschaft der DDR, Sektion Phytopathologie und Universitat Rostock, Rostock, DDR. "

Katz, S. and Fassbender, C. (1966). Biodegradability of ureaformaldehyde and related compounds. Journal of Agriculture and Food Chemistry 14, 336-339.

Kaul, R.K. and Sethi, C.L. (1984). Effect of soil application of certain systemic nematicides on emergence, penetration and development of Heterodera zeae in maize. Proceedings 1st International Congress Nematolo'gy, Guelph, Canada, p. 50 (abstract).

Kearby, W.H., Ercegovich, C.D. and Bliss, M. (1970). Residue studies on aldicarb in soil and scotch pine. Journal of Economic Entomology 63, 1317-1318. 176 -

Kibble, R.M. (1968). Effectiveness of dichlorvos impregnated collars in controlling fleas on dogs. Australian Veterinary Journal 44, 456-458.

Kimber, D. and McCullagh, S. (1984). Trials of commercial varieties of sugar beet. British Sugar Beet Review 52, 81-84.

Knapp, F.W. (1962). Fly control on cattle and horses. 75th Annual Report Agricultural Experimental Station, University of Kentucky, Lexington, ------

Knapp, F.W. (1977). An overview of controlled release pesticide systems in medical and veterinary entomology. Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp. 237-252.

Kontaxis, D.G. and Thomason, I.J. (1978). Chemical control of Heterodera schachtii on sugar beet production in Imperial Valley, California. Plant Disease Reporter 62, 79-82.

Kontaxis, D.G., Thomason, I.J., Hagemann, R. and McKinney, E. (1976). Chemical control of Heterodera schachtii on sugar beets in Imperial Valley, California. Journal of Nematology 8, 292 (abstract).

Kontaxis, D.G., Thomason, I.J., Yu, P. and Smith, B. (1975) Chemical control of the sugarbeet cyst nematode in Imperial Valley. California Agriculture 29, 6-7.

Kydonieus, A.F. (1980). Controlled Release Technologies: Methods, Theory and Applications Vol I and II C.R.C. Press, Boca Raton, 261 and 273 pp.

Kydonieus, A.F., Baldwin, S. and Hyman, S. (1976). Hereon granules and powders for agricultural applications. Proceedings 1976 International Controlled Release Pesticide Symposium, Akron pp. 4.23-4.35.

’.Kydonieus, A.F. and Beroza, M. (1982) Insect Suppression by Controlled Release Pheromone Systems. Vol I. C.R.C. Press, Boca Raton, Florida, 274pp.

Kydonieus, A.F., Beroza, M. and Zweig, G. (1982). Insect Suppression by Controlled Release Pheromone Systems. Vol II. C.R.C. Press, Boca Raton, Florida 312pp.

Lee, D.L. and Atkinson, H.J. (1976) Physiology of Nematodes. 2nd Edition, London and Basingstoke: Macmillan., 215 pp.

Leistra, M., Bromilow, R.H. and Boesten, J.J.T.I. (1980). Measured and simulated behaviour of oxamyl in fallow soils. Pesticide Science 11, 379-388.

Leistra, M., Smelt, J.H. and Lexmond, T.M. (1976). Conversion and leaching of aldicarb in soil columns. Pesticide Science 7, 471-482.

Le Patourel, G.N'.J. and Wright, D.J. (1976). Some factors affecting the susceptibility of two nematode species to phorate. Pesticide Biochemistry and Physiology 6, 296-305. - 177 -

Lewis, D.H. (1981). Controlled Release of Pesticides and Pharmaceuticals Plenum Press, New York 340 pp.

Lunt, O.R., Kofranek, A.M. and Oertli, J.J. (1961). Controlled availability fertilizers. Californian Agriculture 15, 2-3.

McFarlane, J.A. (1970). Treatment of large grain stores in Kenya with dichlorvos slow-release strips for control of Cadra cantella. Journal of Economic Entomology 63, 288-292.

McFarlane, N.R. and Pedley, J.B. (1978). Some fundamental considerations of controlled release. Pesticide Science 9, 411-424.

McGarvey, B.D., Potter, J.W. and Chiba, M. (1984). Nematostatic activity of oxamyl and N,N-dimethyl-l-cyanoformamide (DMCF) on Meloidogyne incognita juveniles. Journal of Nematology 16, 328-332.

McKibbon, G.H., Hardee, D.D., Davich, T.B., Gueldner, R.C. and Hedin, P.A. (1971). Slow release formulations of grandlure, the synthetic pheromone of the boll weevil. Journal of Economic Entomology 64, 317-319.

McLaren, D.J. (1972). Ulstructural and cytochemical studies on the sensory organelles and nervous system of Pi petalonema viteae (Nematoda: Filariodea). Parasitology 65, 507-524.

McLeod, R.W. and Khair, G.T. (1975). Effects of oximecarbamate, organophosphate and benzimidazole nematicides on life cycle stages of root-knot nematodes. Annals of Applied Biology 79, 329-341.

Maddy, K.T. (1983). Pesticide usage in California and the United States. Agricultural, Ecosystems and Environment 9, 159-172.

Marks, R.J. and McKenna, L.A. (1981). A simple procedure for the efficient estimation of potato cyst nematode larvae in lactophenol- treated roots. Annals of Applied Biology 97, 323-324.

Marrs, G.J. and Seaman, D. (1978). Practical considerations in the -control of bioavailability. Pesticide Science 9, 402-410.

Maughan, G.L. (1977). Temik-IOG and the sugar beet crop. International Pest Control 19, 4-6.

Maughan, G.L., Cooke, D.A. and Gnanasakthy, A. (1984). The effects of soil-applied granular pesticides on the establishment and yield of sugar beet in commercial fields. Annals Applied Biology (in press).

Metcalf, R.L., Fukuto, T.R., Collins, C., Borck, K.J., Burk, J., Reynolds, H.T. and Osman, M.F. (1966). Metabolism of 2-methyl-2- (methylthio)-propionaldehyde 0-(methylcarbamoyl) oxime in plant and insect. Journal Agriculture and Food Chemistry 14, 579-584.

Michaud, M.A. (1982). Slow release fertilizer composition. Chemical Abstracts 96, No. 21, 620 (abstract).

Miles, J.W., Pearce, G.W. and Woehst, J.E. (1962). Stable formulations 178 - for sustained release of DDVP. Journal Agriculture and Food Chemistry 10, 240-243.

Miller, J.A., Kunz, S.E. and Oehler, D.D. (1981). Sustained-release systems for livestock pest control. In: Controlled Release of Pesticides and Pharmaceuticals, pp. 311-318 (Ed. D.H. Lewis) Plenum Press, New York.

Miller, L.I. (1975). Sugarbeet (Beta vulgaris) as a host for the soybean cyst nematode Heterodera glycines. Virginia Journal of Science 26, 44 (abstract).

Miller, P.M. (1970). Failure of several non-volatile and contact nematicides to kill eggs in cysts of Heterodera tabacum. Plant Disease Reporter 54, 781-785.

Miller, T.A., Nelson, L.L., Young, W.W., Roberts, L.W., Roberts, D.R. and Wilkinson, R.L. (1973a). Polymer formulations of mosquito larvicides: I. Effectiveness of polyethylene and polyvinyl chloride formulations of chlorpyrifos applied to artificial field pools. Mosquito News 33, 148-155.

Miller, T.A., Nelson, L.L. and Young, W.W. (1973b). Polymer formulations of mosquito larvicides IV. Larvicidal effectiveness of polyethylene and polyvinyl chloride formulations of chlorpyrifos during an 18 month field test. Mosquito News 33 172-178.

Moje, W. (1960). The chemistry of nematicidal activity of organic halides. In: Advances in Pest Control Research 3, pp. 181-217 (Ed. R. Metcalf), Interscience, New York.

Montemarano, J.A., Cohen, S.A. and Fisher, E.C. (1975). Fouling: the problem and control with polymeric formulations. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton, pp. 268-275.

Moriarty, F. (1961). The effects of red beet and of Hesperis matronal is L. on a population of Heterodera schachtii Schm, Nematologica 6, 214-221.

Moriarty, F. (1963). The decline of a beet eelworm (Heterodera schachtii Schm.) population in microplots in the absence of host plants. Nematologica 9_, 24-30.

Moss, S.R., Crump, D. and Whitehead, A.G. (1975). Control of potato-cyst nematodes, Heterodera rostochiensis and G.pal 1ida, in sandy, peaty and silt loam soils by oximecarbamate and organophosphate nematicides. Annals of Applied Biology 81, 359-365.

Moss, S.R., Crump, D. and Whitehead, A.G. (1976). Control of potato cyst nematodes, Globodera rostochiensis and G.pa11ida, in different soils by small amounts of oxamyl and aldicarb. Annals of Applied Biology 84, 355-359.

Muller, J. (1979). On the number of generations of Heterodera schachtii produced in a year under field conditions on sugar beet. Nachrichtenbl 179 -

Deut. Pflanzenschutzd (Braunschv/eig) 31, 92-95.

Mumford, J. (1979). Pest control, what is worthwhile? British Sugar Beet Review 47, 14-15.

Mumford, J. (1981). Sugar beet weeds and pests in perspective. British Sugar Beet Review 49, 50-51.

Munneke, D.E. and Van Gundy, S.D. (1979). Movement of fumigants in soil, dosage responses and differential effects. Annual Review of Phytopathology 17, 405-429.

Nelmes, A.J. (1970). Behavioural responses of Heterodera rostochiensis larvae to aldicarb and its sulphoxide and sulphone. Journal of Nematology 2, 223-227.

Nelmes, A.J., Trudgill, D.L. and Corbett, D.C.M. (1973). Chemotherapy in the study of plant parasitic nematodes. In: Chemotherapeutic Agents in the study of Parasites pp. 95-112 (Ed. A.E.R. Taylor and R. Muller), Blackwell Scientific, Oxford.

Nelson, L.L., Barnes, W.W., Harris, F.W. and Lawson, M.A. (1970). Evaluation of release rates for Dursban from polyvinyl chloride formulations. Journal of Economic Entomology 63, 1870-1873.

Nichols, L.D. (1975). Poroplastic and sustrelle: controlled release vehicles having broad compatability with dissolved and precipitated pesticides. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton, pp.95-104.

Olthof, T.H.A. (1978). Influence of population densities of Heterodera schachtii on sugar beets grown in microplots. Journal of Nematology 10, 255-258.

Olthof, T.H.A., Potter, J.W. and Peterson, E.A. (1974). Relationship between population densities of Heterodera schachtii and losses in vegetable crops in Ontario. Phytopathology 64, 549-554.

Oostenbrink, M. (1958). An inoculation trial with Pratylenchus penetrans in potatoes. Nematologica 3, 30-33.

Osbourne, P. (1973). The effect of aldicarb on the hatching of Heterodera rostochiensis larvae. Nematologica 19, 7-14.

Osgerby, J.M. (1972). An assessment of the consequences of the controlled release of pesticides from granular formulations. Pesticide Science 3, 753-769.

Overman, A.J. (1971). Nematicides for control of Pratylenchus spp. in leatherleaf from plantings. Nematologica 1, 14 (abstract).

Park, R.H.N., Burgoyne, D. and Allison, D.A. (1982). Oxamyl (Vydate): different application techniques for nematode control. Journal of Nematology 14, 464-465 (abstract).

Parker, R.E. (1973). Introductory Statistics for Biology. Studies in - 180 -

Biology No. 43, Edward Arnold.

Parker, T.A. (1970) Effectiveness of 9.9 percent Dursban in polyethylene applied as a pre-season larvicide. USAEHA Special Study No. 31-005-71, Defense Documentation Center No. AD725573.

Pass, B.C. and Dorough, H.W. (1973). Insecticidal and residual properties of e.c. and encapsulated formulations of methyl parathion sprayed on alfalfa. Journal of Economic Entomology 66, 1117-1119.

Peacock, F.C. (1961). A note on the attractiveness of roots to plant parasitic nematodes, Nematologica, 6_, 85-86.

Pertel, R., Paran, N. and Mattern, C.F.T. (1976). Caenorhabditis elegans localization of cholinesterase associated with anterior nematode structures. Experimental Parasitology 39, 401-414.

Peters, B.G. (1928). Bionomics of the vinegar eelworm. Journal of Helminthology 6, 1-38.

Peterson, C.A., De Wildt, P.P.Q. and Edgington, L.V. (1978). A rationale for the ambimobile translocation of the nematicide oxamyl in plants. Pesticide Biochemistry and Physiology, 8 , 1-9.

Phillips, F.T. (1976). Controlling the persistence of insecticides with special reference to microencapsulation. Proceedings Symposium on Persistence of Insecticides and Herbicides, B.C.P.C., Reading, pp. 217-227.

Potter, J.W. and Fox, J.A. (1965). Hybridization of Heterodera schachtii and H,glycines. Phytopathology 55, 800-801.

Potter, J.W. and Marks, C.F. (1976a). Persistence of activity of oxamyl against Heterodera schachtii on cabbage. Journal of Nematology 8, 35-38.

Potter, J.W. and Marks, C.F. (1976b). Efficacy of oxamyl against Heterodera schachtii on cabbage. Journal of Nematology 8, 38-42.

Preiser, F.A., Babu, J.R., Dybas, R.A., Haidri, A.A. and Putter, I. (1981). Avermectins, a new class of nematicides. Journal of Nematology - 13, 457 (abstract).

Price, M., Caveness, C.E. and Riggs, R.D. (1978). Hybridization of races of Heterodera glycines. Journal of Nematology 10, 136-141.

Putter, I., MacConnell, J.G., Preiser, F.A., Haidri, A.A., Ristich, S.S. and Dybas, R.A. (1981). Avermectins: novel insecticides, acaricides and nematicides from a soil microorganism. Experientia 37, 963-964.

Quick, T.J., Caradarelli, N.F., Ellin, R.J. and Sherman, L.R. (1981). Controlled release temephos: laboratory and field evaluations. In: Controlled Release of Pesticides and Pharmaceuticals, pp. 275-286. (Ed. D.H. Lewis), Plenum Press, New York.

Quisumbing, A.R. and Kydonieus, A.F. (1977). Advantages of using 131

controlled release formulations for insect control in commercial food­ handling establishments. Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp. 216-225.

Radewald, J.D., Shibuya, F., Nelson, J. and Bivens, J. (1970). Nematode control with Dupont 1410, an experimental nematicide-insecticide. Plant Disease Reporter 54, 187-190.

Raski, D.J. and Johnson, R.T. (1959). Temperature and activity of the sugarbeet nematode as related to sugarbeet production. Nematologica 4, 136-141.

Rhoades, H.L. (1975). Pathogenicity and control of the sting nematode Belonolaimus longicaudatus. Plant Disease Reporter 59, 1021-1024.

Ridgeway, R.L., Jones, S.L., Coppedge, J.R. and Lindquist, D.A. (1969). Systemic activity of 2-methyl-2-(methylthiojpropioaldehyde 0- (methyl- carbomyl) oxime (UC-21149) in the cotton plant with special reference to the boll weevil. Journal of Economic Entomology 61, 1705-1711.

Rieley, C.E., Draper, S.G. and Brown, E.A. (1977) Sugar beet weed control - present and future requirements. International Pest Control 19, 16-18.

Riley, R.T. (1983). Starch-xanthate-encapsulated pesticides: a preliminary toxicological evaluation. Journal Agriculture and Food Chemistry 31, 202-206.

Ross, J.R. (1962). Physiological strains of Heterodera glycines . Plant Disease Reporter 46, 766-769.

Ross, J.P. and Brim, C.A. (1957). Resistance of soya beans to the soybean cyst nematode as determined by a double-row method. PI ant Disease Reporter 41, 923-924.

Sasser, J.N., Kirkpatrick, T.L. and Dybas, R.A. (1982). Efficacy of avermectins for root-knot control in tobacco. Plant Disease 65, 691-693.

Savitsky, H. (1975). Hybridization between Beta vulgaris and B.procumbens and transmission of nematode (Heterodera schachtii) resistance to sugarbeet. Canadian Journal Genetics and Cytolology 17, 197-209.

Schacht, E.H., Desmarets, G.E. and Goethals, E.J. (1981). Synthesis and properties of urea formaldehyde resins generating 2,6-dichlorobenzo- nitrile. In: Controlled Release of Pesticides and Pharmaceuticals.pp. 159-170 (Ed. D.H. Lewis), Plenum Press, New York.

Schaefer, C.H. and Wilder, W.H. (1972). Insect developmental inhibitors: a practical evaluation as mosquito control agents. Journal of Economic Entomology 65, 1066-1071.

Scher, H.B. (1977). Microencapsulated pesticides. ACS Symposium Series 53, 126-144. ACS Washington DC. 182 -

Seaman, D. and Warrington, R.P. (1972). Slow release pirimicarb granular formulations: measurements of release rate under field conditions. Pesticide Science 3, 799-804.

Seinhorst, J.W. (1965). The relation between nematode density and damage to plants. Nematologica 11, 137-154.

Sharma, G.C. (1979). Controlled-release fertilizers and horticultural applications. Scientia Horticulture 11, 107-129.

Shasha, B.S. (1980). Starch and other polyols as encapsulating matrices for pesticides. In: Controlled Release Technologies: Methods, Theory and Applications pp. 207-223 (Ed. A.F. Kydonieus), CRC Press, Boca Raton, Florida.

Shepherd, A.M. (1959). Increasing the rate of larval emergence from cysts in hatching tests with beet eelworm (Heterodera schachtii Schmidt). Nematologica 4, 161-164.

Shepherd, A.M. (1962). New blue R, a stain that differentiates between living and dead nematodes. Nematologica 8, 201.

Shepherd, A.M. (1970). Extraction and estimation of Heterodera. In: Laboratory Methods for Work with Plant and Soil Nematodes pp. 23-33. (Ed. J.F.Southey), M.A.F.F. Technical Bulletin 2. H.M.S.O., London.

Shepherd, A.M. and Clarke, A.J. (1971). Moulting and hatching stimuli. In: Plant Parasitic Nematodes, Vol. 2. pp. 267-287 (Ed. B.M. Zuckermann, W.F. Mai and R.A. Rohde). Academic Press, New York and London.

Shiff, C.J. (1974). Persective in the use of molluscicide. Proceedings 1974 International Controlled Release Pesticide Symposium, Akron, pp. 23.1-23.9.

Shiff, C.J. (1975). Field tests of controlled release molluscicides in Rhodesia. Proceedings 1975 International Controlled Release Pesticide Symposium, Dayton , pp. 177-188.

Simkin, J. and Galun, R. (1983). Microencapsulated natural pyrethrum - an improved insect repellant. In: Natural Products for Innovative Pest Management, pp. 151-163 (Eds D.L. Whitehead and W.S. Bowers), Pergamon Press.

Singhal, J.P., Khan, S. and Bansal, O.P. (1977). Spectrophotometric determination of oxamyl as copper dithiocarbamate. Journal Agriculture and Food Chemistry 25, 377-380.

Slack, D.A., Riggs, R.D. and Hamblen, M.L. (1972). The effect of temperature and moisture on the survival of Heterodera glycines in the absence of a host. Journal of Nematology 4, 263-266.

Smith, J. and Bromilow, R.H. (1977). Incorporation of jgranular nematicides in peat soils for control of potato cyst-nematodes (Heterodera rostochiensis, Wol1.). Experimental Husbandry 33, 98-111. 183 -

Smittle, B.J. and Burden, G.S. (1965). Dichlorvos as a vapour toxicant for control of roaches, bedbugs and fleas. Pest Control 33, 26-32.

Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods 6th Edition Ames: Iowa State Univ. Press. 593pp.

Somerville, G.R. (1977). Physical methods of producing microcapsules, Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp. 335-354.

Southey, J.F. (1965). Plant Nematology 2nd Edition, M.A.F.F. Technical Bulletin No. 7., H.M.S.O., London, 282 pp.

Southey, J.F., Alphey, T.J.W., Cooke, D.A., Whiteway. J.A. and Mathias, P.L. (1982). Surveys of beet cyst nematode in England 1977-80. Plant Pathology 31, 163-169.

Speight, B. (1980). In: Field Worker Exposure during Pesticide Application p. 29 (Eds W.F. Tordoir and E.A.H. Van Heemstra-Lequin), Elsevier, New York.

Spurr, H.W. (1966). Nematode cholinesterase and their inhibition by nematicidal carbamates. Phytopathology 56, 902-903 (abstract).

Spurr, H.W. and Chancey, E.L. (1967). Phosphate and carbamate inhibition of three nematode esterases. Phytopathology 57, 832 (abstract).

Steele, A.E. (1965). The host range of the sugarbeet nematode, ■Heterodera schachtii Schmidt. Journal American Society Sugar Beet Technology 13, 574-1T03.

Steele, A.E. (1975). Population dynamics of Heterodera schachtii on tomato and sugar beet. Journal of Nematology 7, 105-111.

Steele, A.E. (1983). Effects of selected nematicides on hatching of Heterodera schachtii. Journal of Nematology 15, 467-473.

Steele, A.E. and Hodges, L.R. (1975). In vitro and in vivo effects of aldicarb on survival and development of Heterodera schachtii. Journal of Nematology 7, 305-312.

Stephenson, J.W. (1972). Gelatin as a carrier for S -cyanoethyl N- [(methycarbomyl)oxy] thioacetimidate, and experimental molluscicide. Pesticide Science 3, 81-87.

Steudel, W. (1972). Versuche zum einfluss von aldicarb auf den schlupfuorgang bei zysten von Heterodera schachtii nach langerer einwirkung. Nematologica 18, 270-274.

Steudel, W. and Thielmann, R. (1967). Zur frage der wirking eines carbamoyloximgranulates auf die vermehring des rubennematoden 184 -

(Heterodera schachtii Schmidt) und den ortag von Zucherruben. Nachrichtenbl Deut. Pflanzenschutzd (Braunschweig) 19, 130-135.

Steudel, W. and Thielmann, R. (1968). Versuche zur frage der empfindlichkeit von zuckerruben gegen den rubenenematoden (Heterodera schachtii Schmidt). Meded. Rijks. Lanbouwweten, Gent 33, 707-718.

Steudel, W. and Thielmann, R. (1970). Weitere untersuchungen zur frage der empfindlichkeit von zuckerruben gegen den rubennematoden (Heterodera schachtii Schmidt). Zucker 23, 106-109.

Steudel, VJ., Thielmann, R. and Haufe, W. (1978). Der einfluss von aldicarb auf die vermehrung des rubenzystenalchens (Heterodera schachtii Schmidt) und den ertag von zukerruben in der Koln-Aachener Bucht. Nematologica 24, 361-374.

Stokes, R.A., Coppedge, J.R., Bull, D.L. and Ridgeway, R.L. (1973). Use of selected plastics in controlled release granular formulations of aldicarb and dimethoate. Journal Agriculture and Food Chemistry 21, 103-108.

Stokes, R.A., Coppedge, J.R. and Ridgeway, R.L. (1970). Chemical and biological evaluation of the release of aldicarb from granular formulations. Journal Agriculture and Food Chemistry 18, 195-198.

Storey, R.M.J. (1984). Relationship between neutral lipid reserves and infectivity for hatched and dormant juveniles of Globodera spp. Annals of Applied Biology 104, 511-520.

Taylor, C.E. and Alphey, T.J.W. (1973). Aspects of the systemic nematicidal potential of Du Pont 1410 in the control of Longidorus and Xiphinema virus vector nematodes. Annals of Applied Biology 75, 464-467.

Theeuwes, F. (1977). Delivery of active agents by osmosis. Proceedings 1977 International Controlled Release Pesticide Symposium, Oregon, pp." 36'4-381. ~ *

Theeuwes, F. (1980). Delivery of active agents by osmosis. In: Controlled Release Technologies: Methods, Theory and Applications pp. 195-206 (Ed. A.F. Kydonieus), CRC Press, Boca Raton, Florida.

Thielmann, R. and Steudel, W. (1973)..Neurijahrige erfahrungen mit monokultur von zukerruben auf mit Heterodera schachtii (Schmidt) verseuchtem boden. Nachrichtenbl Deut. Pflanzenschutzd (Braunschweig) 25, 145-1497

Thomason, I.J., Caswell. E.P. and McKinney, H.E. (1984). Efficacy of annual surveys for Heterodera schachtii and enforced rotations in maintaining sugarbeet production potential. Proceedings 1st International Congress of Nematology, Guelph, Canada, p. 102 (abstract).

Thomason, I.J. and Fife, D. (1962). The effect of temperature on development and survival of Heterodera schachtii Schmidt. 185 -

Nematologica 7, 139-145.

Thompson, H.E., Allan, G.G. and Neogi, A.N. (1981). The control of pine tip moths by using sustained release systemic insecticides. International Pest Control 23, 10-11.

Thompson, L.S. and Willis, C.B. (1976). Efficacy of multiple applications of oxamyl and fenamiphos for control of Pratylenchus penetrans in birdsfoot trefoil. Journal of Nematology 8, 342-346.

Thompson, W.E. (1974). Field tests of slow release herbicides. Proceedings 1974 International Controlled Release Pesticide Symposium, Akron pp. 15.1-15.3.

Turnipseed, S.G. and Reed, J.K. (1963). The rate of leaching of dieldrin from attapulgite clay granules. Journal of Economic Entomology, 56, 410-412.

Van Berkum, J.A. and Hoestra, H. (1979). Practical aspects of the chemical control of nematodes in soil. In: Soil Disinfestation pp53-134 (Ed. D.Mulder), Elsevier, Amsterdam, Oxford and New York.

Van Gundy, S.D., Bird, A.F. and Wallace, H.R. (1967). Ageing and starvation in larvae of Meloidogyne javanica and Tylenchulus semipenetrans. Phytopathology 57, 559-571.

Van Gundy, S.D., Garabedian, S. and Nigh, E.L. (1982). Alternatives to DBCP for citrus nematode control. Proceedings International Society for Citriculture.

Viglierchio, D.R. (1960). Resistance in Beta species to the sugarbeet nematode, Heterodera schachtii. Experimental Parasitology, 10, 389-395.

Viglierchio, D.R. (1961). Attraction of parasitic nematodes by plant root emanations. Phytopathology 51, 136-142.

Wade, R.S. and Castro, C.E. (1973). Oxidation of iron(II) porphyrins by alkyl halides. Journal American Chemical Society 95, 226-230.

Wallace, H.R. (1955). Factors influencing the emergence of larvae from cysts of the beet eelworm, Heterodera schachtii Schmidt. Journal of Helminthology 29, 3-16.

Wallace, H.R. (1962). Observations on the behaviour of Ditylenchus dipsaci in soil. Nematologica 7, 91-101.

Wallace, H.R. (1970). Some factors influencing nematode reproducation and the growth of tomatoes infected with Meloidogyne incognita. Nematologica 16, 387-397.

Wallace, H.R. and Grandison, G.S. (1971). The population ecology of the lesion nematode Pratylenchus thornei and Meloidogyne javanica. Biennial Report of the Waite Agricultural Research Institute, South Australia, 1970-1971, p. 83. 186 -

Webster, R., Hodge, C.A.H., Draycott, A.P. and Durrant, M.J. (1977). The effect of soil type and related factors on sugar beet yield. Journal of Agricultural Science 88, 455-469.

Whitehead, A.G. (1973). Control of cyst nematodes (Heterodera spp.) by organophosphates, oximecarbamates and soil fumigants. AnnalT~of Applied Biology 75, 439-479.

Whitehead, A.G. (1977). Progress in the chemical control of plant nematodes. International Pest Control 19, 14.

Whitehead, A.G., Fraser, J.E. and Storey, G. (1972). Chemical control of potato cyst nematode, Heterodera rostochiensis, in sandy clay soil. Annals of Applied Biology 72, 81-88.

Whitehead, A.G., Tite, D.J., Bromilow, R.H., Fraser, J.E. and Nichols, A.J. (1981). Control by nematicides - cyst nematodes. Report Rothamsted Experimental Station for 1980, Part 1, 150-151.

Whitehead, A.G., Tite, D.J., Finch, P.H., Fraser, J.E., and French, E.M. (1979). Chemical control of the beet cyst nematode, Heterodera schachtii, in some peaty loam soils. Annals of Applied Biology 92, 73-79.

Whitehead, A.G., Tite, D.J., Finch, P.H., Fraser, J.E., French, E.M. and Wright, S.M. (1974). Trials with nematicides. Report Rothamsted Experimental Stationn for 1973, Part 1, 160-163.

Whitehead, A.G., Tite, D.J., Fraser, J.E., French, E.M. and Smith, J. (1975). Incorporating granular neamticides in soil to control potato cyst-nematode, Heterodera rostichiensis. Annals of Applied Biology 80, 85-92.

Whithead, A.G., Tite, D.J., Fraser, J.E. and Nichols, A.J. (1984). Differential control of potato cyst-nematodes, Globodera rostochiensis and G.pallida, by oxamyl and the yields of resistant and susceptible potatoes in treated and untreated soils. Annals of Applied Biology 105, 231-244.

Whiteway, J.A., Alphey, T.J.W., Mahtias, P.L. and Southey, J.F. (1982). Computer mapping of records of beet cyst nematode (Heterodera schachtii) 1928-77. Plant Pathology 31, 157-162.

Whitlow, J.T. and Evans, S.E. (1968). Selected plastic formulations for use as mosquito larvicides. Journal of Economic Entomology 61, 889-829.

Wilkins, R.M. (1976). Design and evaluation of polymer combinations for the controlled release of herbicides. Proceedings 1976 Controlled Release Pesticide Symposium, Akron, pp. 7.1-7.15.

Wilkins, R.M. (1978). The use of biodegradable polymers as formulating agents for controlled release of pesticides. Pesticide Science 9, 443-444.

Wilkinson, R.N., Barnes, W.W., Gillogly, A.R. and Minnemeyer, C.D. 1 8 7

(1971). Field evaluation of slow-release mosquito larvicides. Journal of Economic Entomology 64, 1-3.

Winslow, R.D. (1954). Provisional lists of host plants of some root eelworms (Heterodera spp.). Annals of Applied Biology 41, 591-605.

Woodham, D.W., Edwards, R.R., Reeves, R.G. and Schutzmann, R.L. (1973). Total toxic aldicarb residues in the soil, cottonseed and cotton lint following a soil treatment with the insecticide on the Texas High Plains. Journal Agricultural and Food Chemistry 21, 303-307.

Wright, C.6. (1971). Efficacy of dichlorvos mini-strips for German cock­ roach control in enclosed kitchen cabinets. Journal of Economic Entomology 64, 278-280.

Wright, D.J. (1981). Nematicides: Mode of action and new approaches to chemical control. In: Plant Parasitic Nematodes Vol.III pp. 421-449 (Eds B.M. Zuckerman and R.A. Rohde), Academic Press, New York.

Wright, D.J. and Awan, F.A. (1976). Acetylcholinesterase activity in the region of the nematode nerve ring: improved histochemical specificity using ultrasonic pretreatment. Nematologica 22, 326-331.

Wright, D.J., Birtle, A.J., Corps, A.E. and Dybas, R.A. (1983). Efficacy of avermectins against a plant parasitic nematode. Annals of Applied Biology 103, 465-470.

Wright, D.J., Birtle, A.J. and Roberts, I.T.J. (1984). Triphasic locomotor response of a plant parasitic nematode to avermectin: inhibition by the GABA antagonists bicuculline and picrotoxin. Parasitology 88, 375-382.

Wright, D.J., Blyth, A.R.K. and Pearson, P.E. (1980). Behaviour of the systemic nematicide oxamyl in relation to control of invasion and development of Meloidogyne incognita. Annals of Applied Biology 96, 323-334.

Wright, D.J. and Rowland, A.J. (1982). Susceptibility of different developmental stages of the root-knot nematode, Meloidogyne incognita, to thenematicide oxamyl. Annals of Aplied Biology 100, 521-525.

Wright, D.J. and Womack, N. (1981). Inhibition of development of Meloidogyne incognita by root and foliar applications of oxamyl. Annals of Applied Biology 97, 297-302.

Yu, C.C., Kearns, C.W. and Metcalf, R.L. (1972). Acetyl-cholinesterase inhibition by substituted phenyl N-alkyl carbamates. Journal Agricultural and Food Chemistry 29, 537-540. 187a -

Cunningham, R.T. and Steiner, L.F. (1972). Field trial of cuelure-naled on saturated fibreboard blocks for control of the melon fly by the male- annihilation technique. Journal of Economic Entomology 65, 505-507.

Den Ouden, H. (1956). The influence of hosts and non-susceptible hatching plants on populations of Heterodera schachtii. Nematologica 1, 138-144.

Den Ouden, H. (1963). Comparisons between the use of free and encysted eggs in hatching and pot experiments with Heterodera rostochiensis. Nematologica 9, 225-230.

Dunning, R.A. and Winder, 6.H. (1974). Effects of aldicarb and some other nematicides on growth of sugar beet in Heterodera schachtii-infested soil. Plant Pathology 23, 1-8.

Ellenby, C. (1968). The survival of desiccated larvae of Heterodera rostochiensis and H.schachtii. Nematologica 14, 544-548.

Hartill, W.F.T. (1980). Spray and seed tuber/treatments for late blight control on potatoes. Plant Disease Reporter 64, 764-766.

Hartwig, J. and Sikora, R.A. (1984). Influence of the nematicides Lance, Temik and Curaterr or. Heterodera schachtii early root infection, nematode population dynamics and yield of sugar beet. Proceedings 1st International Congress of Nematology, Guelph, Canada, p.39 (abstract).

McKenry, M.V. and Buzo, T. (1984). Multitreatments of six nematicides coincidental with root flush of a vineyard. Proceedings 1st International Congress of Nematology, Guelph, Canada, p.63 (abstract).

Wybou, A.P. and Homeyer, B. (1984). Present day usage of nematicidal chemicals. Proceedings 1st International Congress of Nematology, Guelph, Canada, p.63 (abstract). 1 8 8

Appendix 1 Effect of oxamyl on hatching of H.schachtii J2.

No.of hatched juveniles (Mean ± S.E.)

Treatment Treatment Water Root diffusate (days) solution (UP cm"3) (7 days) (3 days) 7 14 28 Total

Oxamyl in 0 64 ± 10 10 ± 6 1327±156 577154 50H5 2028 (40) (2) (61) water (2) « d (17) 1.0 17 ± 5 21 ± 8 860± 66 45H40 28i 8 1378 0 ) (i) (31) (16) 0 ) (50) 2.5 7 ± 3 7 ± 2 13271212 480161 44123 1862 « d «i) (41) (15) 0 ) (58) 5.0 8 ± 1 40 ± 18 14381 99 285175 351 9 1805 « d (i) (42) (8) 0 ) (53) 10.0 2 ±<1 10 ± 5 524H18 282129 10919 927 « d « d (23) (12) (5) (40)

Oxamyl in 0 1563±132 158+ 35 115+ 50 88+12 6±<1 1930 (47) (5) (3) (3) (58) root «D 433±193 335±155 240± 81 181±57 50±19 1239 diffusate1*0 (18) (14) (10) (7) (2) (50) 2.5 126± 39 298+ 43 251± 21 158±55 33±11 866 (5) (12) (10) (6) 0 ) (34)

■ 5.0 18+ 16 207± 26 620±100 573±166 44±U 1402 (1) (7) (20) (16) 0 ) (45) 10.0 12± 4 73± 37 107± 35 195±81 93±38 480 (1) (4) (6) (ID (5) (26)

Means of four replicates, 10 cysts per replicate. Figures in parentheses represent percentage of total cyst contents. 1 8 9

Appendix 2 Effect of polymer coating on release rate of pesticide from

experimental formulations.

% oxamyl released from granules Vydate ^ Elution 10G CA CTA UFR PEG 1 time (min.) (1) (2) (3) (4) (5)

5 26.0 10.1 9.4 3.2 17.3 10 67.8 20.0 19. 22. 6.7 54.7 15 83.1 25.0 24.0 9.6 77.8 :20 88.7 28.8 27.3 11.4 87.8 25 92.0 32.1 30.1 12.5 91.2 30 94.2 35.2 32.7 13.2 92.5 35 95.6 38.0 35.4 13.6 93.2 40 96.7 40.6 38.0 14.0 93.6 45 97.6 42.9 40.2 14.2 93.8 50 98.2 45.0 42.2 14.4 93.9 55 98.7 47.1 44.2 14.6 94.0 60 99.1 49.3 46.2 14.8 94.0 65 99.3 51.3 48.2 15.1 94.0 70 99.6 52.9 49.8 15.3 94.0 75 99.8 54.7 51.5 15.5 94.0 80 99.9 56.4 53.1 15.7 94.0 85 100.0 58.1 54.6 16.0 94.0 90 100.0 59.5 56.0 16.1 94.0 95 100.0 60.8 57.3 16.2 94.0 100 100.0 62.2 58.4 16.3 94.0

+ 16% w/w

Commercial formulation.

Figures in parentheses refer to formulation number. 1 9 0

Appendix 3 Effect of plasticizers on release rate of pesticide from

experimental formulations prepared with cellulose acetate.

% oxamyl released from granules Elution time tppT DMpf DBP~f DBP(W) dbs"T DBS(1%) DMS"t" (min.) (6) (7) (8) (9) (10) (ID (12)

5 12.6 12.5 9.4 10.9 8.1 6.9 7.5 10 20.7 24.2 22.8 21.3 17.3 15.7 16.3 15 25.0 31.0 29.6 26.7 23.2 21.0 22.7 20 27.4 35.5 34.5 31.1 27.6 24.9 28.3 25 29.2 39.0 38.9 35.2 31.9 28.0 32.8 30 30.5 42.1 42.8 38.8 35.8 31.0 36.5 35 31.7 45.0 46.2 42.2 39.2 33.7 40.0 40 32.7 47.6 49.2 45.2 42.4 36.2 43.2 45 33.6 50.2 51.6 47.6 45.4-. 38.6 46.1 50 34.4 52.5 53.9 49.8 48.3 40.5 48.5 55 35.2 54.6 56.2 52.0 50:6' 41.9 50.9 60 36.0 56.6 57.9 54.3 52.8 43.3 53.5 65 36.5 58.5 59.4 56.3 54.8 44.7 56.2 70 37.1 60.3 61.0 58.4 56.4 46.1 57.9 75 37.6 62.0 62.6 60.3 58.0 47.6 59.4 80 38.2 63.7 64.1 62.2 59.6 49.2 60.9 85 38.6 65.1 65.7 63.9 61.0 50.6 62.4 90 39.0 66.4 67.2 65.4 62.5 51.9 63.6 95 39.5 67.8 68.5 67.0 63.8 53.4 64.8 100 39.9 69.1 69.7 68.5 65.0 54.8 66.0

0.5 % w/w. Figures in parentheses refer to formulation No. 1 9 1

Appendix 4. Effect of polyethylene glycol and Aerosol OT-B on release rate

of pesticide from experimental formulations prepared with

cellulose acetate.

% oxamyl released from granules CA+T Elution DMP(0.5%)+ DBP(.5%)+ DBP(.5%)+ DBP(.5°/)+ OT-B OT-B time PEG(1%) PEG(1%) PEG(0.5%) PEG(0.5%) PEG(.25%) PEG(.5%) {0.5%) i (0.25% (min.) (13) (14) (15) (16) (17) (18) (19) (2 0)

5 1 1 .2 18.3 15.2 14.5 8.7 7.3 . 5.8 9.7 10 30.5 44-4 31.0 31.8 25.3 . 16.8 14.8 19.1 15 44.0 58.7 40.4 42.7 35.8 23.0 22.2 24.3 20 . 53.4. 66.8 47-5 49.9 43.2 27.6 27.1 28.2 25 59.8 72.3 53.1 55.6 49.3 31.5 30.6 31.0 30 64.6 76.7 57.8 60.2 54.2 35.1 33.6 33.7 35 68.4 *80.0 61.6 63.8 58.4 38.2 36.5 36.3 40 71.6 82.8 65.1 66.8 62.2 41.0 39.3 38.7 ’ 45 74.2 84.9 68.1 69.3 65.2 43.9 41.8 41.2 50 76.1 8 6 .6 70.6 71.5 67.6 46.4 43.6 43.8 55 77.9 87.9 72.8 73.6 69.7 48.7 45.6 46.1 60 79.3 89.0 74.6 75.4 71.6 51.2 47.3 48.3 65 80.6 90.0 76.1 76.8 . 73.0 53.6 49.0 50.4 70 81.7 90.8 77.7 78.1 74.1 55.8 50.5 52.5 75 82.8 91.5 79.1 79.4 75.0 57.8 51.8 54.2 80 ... 83.7 92.2 80.2 80.5 75.9 59.5 53.1 55.8 85 84.6 92.7 81.1 81.5 76.8 . 61.2 54.3 57.0 90 85.5 93.3 82.0 82.5 77.7 62.6 55.4 58.1 95 8 6 .0 98.7 82.6 83.4 78.4 64.0 56.4 59.2 100 86.5 94.2 83.2 84.0 79.0 65.3 57.2 60.3

^ Second coating of CA solution giving 5% w/w of polymer.

Figures in parentheses refer to formulation no. 1 9 2

Appendix 5 Effect of plasticizers and polyethylene glycol on release rate

of pesticide from experimental formulations prepared with

cellulose triacetate.

% oxamyl released from granules

DHp(" Elution + time TPpt DMpt DBpt PEG(1%) PEG(n) (min.) (2 1 ) (2 2 ) (23) (24) (25)

5 8 .0 12.0 5.2 16.8 12.8 10 19.6 21.1 • 15.3 42.1 31.8 15 24.6 25.6 23.5 54.1 42.5 20 27.6 28.8 29.2 62.3 49.4 25 29.8 31.5 33.7 68.5 55.0 30- 31.7 33.9 37.4 73.5 59.4 35 33.3 36.5 40.7 77.4 63.1 40 34.8 39.3 43.8 80.7 66.6 45 36.0 41.6 46.8 83.3 69.5 50 37.1 43.8 49.4 85.2 71.9 55 38.0 45.6 51.6 8 6 .8 73.9 60 38.8 47.3 53.8 8 8 .2 75.5 65 39.7 49.0 55.9 89.4 77.0 70 40.6 50.5 57.8 90.3 78.2 75 41.5 52.0 59.5 91.2 79.2 80 42.1 53.7 61.2 91.8 80.2 85 42.8 55.0 62.7 92.5 81.1 90 43.2 56.3 64.2 93.2 81.8 95 43.7 57.6 65.6 93.6 82.6 100 43.9 59.0 66.9 94.0 83.2

^ 0.5 % w/w. Figures in parentheses refer to formulation No. 1 9 3

Appendix 6 Effect of urea-formaldehyde resin on release rate of pesticide

from experimental formulations prepared with cellulose acetate

and triacetate.

% oxamyl released from granules Elution DMpf+ DMpt+ CA DMp1+ CA DMP^+ DMpl+ CTA time CA CA + PEG(H) + PEG(2%) CTA CTA + PEG(1%) (min.) (26) (27) (28) (29) (30) (31) (32)

5 2 .0 2 .6 1.5 3.6 3.0 2.5 3.4 10 5.0 6 .2 5.0 7.9 5.1 5.1 8.1 15 7.6 8.5 7.5 10.1 6 .8 6.7 11.7 20 9.4 9.7 8 .8 11.6 8 .2 7.6 13.6 25 10.6 10.4 9.7 12.4 9.2 8 .1 15.1 30 11.3 10.9 10.3 13.1 1 0 .0 8 .6 16.1 35 11.8 11.1 10.9 13.8 10.4 8 .8 16.8 40 12.2 11.3 11.2 14.2 10.7 9.0 17.5 45 12.6 11.6 11.6 14.6 10.9 9.2 18.0 50 12.9 1 1 .8 11.9 15.0 11.1 9.5 18.6 55 13.1 11.8 12.0 15.3 11.3 9.7 19.0 60 13.3 1 1 .8 12.2 15.5 1 1 .6 9.7 19.5 65 13.4 1 1 .8 12.3 15.7 1 1 .8 9.7 19.9 70 13.5 11.8 12.3 16.1 1 1 .8 9.7 20.4 75 13.5 11.8 12.3 16.3 1 1 .8 9.7 20.8 80 13.5 11.8 12.3 16.5 1 1 .8 9.7 21.1 85 13.5 11.8 12.3 16.6 1 1 .8 9.7 21.4 90 13.5 11.8 12.3 16.7 11.8 9.7 21.7 95 13.5 11.8 12.3 16.7 1 1 .8 9.7 21.9 100 13.5 1 1 .8 12.3 16.7 1 1 .8 9.7 22.1

t 0.5% w/w.

Figures in parentheses refer to formulation no. 1 9 4

Appendix 7 Efficiency indices for experimental formulations.

Index Groups (Relative release rate: % oxamyl min"*)

Formulation 1 2 3 4 5 6 No. (3.00) (2.25) (1.75) (1.50) (1.25) (1 .0 0)

i$ 58 48 42 39 36 31 2 38 43 45 46 49 52 3 35 40 , 42 44 46 ' 49 4 14 . 14 15 15 16 16 5 60 50 41 36 31 26 6 32 32 31 31 30 29 7 45 48 48 49 51 53 8 46 49 51 52 53 55 9 42 48 48 50 53 57 10 39 45 50 53 55 57 11 34 . 39 42 43 46 49 12 40 46 51 55 57 58 13 64 64 6.0 57 55 52 14 66 60 55 53 50 47 15 61 60 59 58 58 55 16 62 60 57 57 56 53 17 57 60 60 60 58 55 18 38 44 48 52 55 57 19 37 42 45 47 49 50 20 -36 41 44 46 49 51 21 33 36 35 35 35 34 22 37 42 42 43 45 48 23 41 47 50 53 55 58 24 65 63 59 57 55 51 25 59 60 58 57 56 53

t Vydate 10G * Efficiency indices of all the CA and CTA granules coated with UFR (Formulation Nols 26 to 32) were less than 25. 1 9 5

Appendix 8 Release rate characteristics of experimental oxamyl formulations used in 1983 microplot trial.

% oxamyl release from granules LIUl IUII Experimental formulations ^ time (min.) Fast Medium Slow

5 29.6 24.8 13.3 10 55.6 47.9 27.9 15 71.9 59.4 36.5 20 83.3 67.3 43.1 25 90.6 73.3 48.5 30 95.0 78.1 53.3 35 96.9 81.9 58.1 40 97.5 85.6 62.5 45 97.5 8 8 .8 66.2 50 97.5 91.2 70.0 55 97.5 93.8 73.8 60 97.5 96.2 77.5

* Slow to fast release rate. Appendix 9 1982 Field trial: Effects of various formulations of oxamyl (Vydate) on root growth of sugar beet

and invasion and development of H.schachtii.

Mean ± S.E. Vydate 10G Week t Untreated Vydate 10G Vydate DPX 4702 Incremental

Plant weight 8 2.64 ± 0.21a 2.26 + 0.24a 2.12 + 0.18a 2.54 ± 0.34a (g) r

Root weight 17 352 ± 40a 533 + 96a 541 + 76a 570 ± 89a + ± i (g) 28 * 1425 ± 43a 1345 54ab 1240 26b 1293 ± 17b 196

Nematodes/root 8 26 ± 12 a (l)a 2 + lb (0)a 5 + 4b (7)a 1 ± lb(0)a i system , 17 258 ± 105a (24)a 347 + 86a (2 )b 304 + 62a(10)b 414 ± 136a(2) (% saccate) *

Nematodes/g. root 17 0.74 ± 0.25a 0.65 + 0.18a 0.55 + 0.04a 0.83 ± 0.25a

+ 1 After application of pesticide. Harvest at week 28.

^ Mean weight per beet at harvest.

^ Percentage of nematodes which had developed from a vermiform J2 to a saccate stage.

All figures in horizontal rows with the same letter are not significantly different at P>0.05. Appendix 10 1984 Field trial: effects of various formulations of oxamyl (Vydate) on root growth of sugar beet

and invasion and development of H.schachtii.

Mean ± S.E.

Experimental formulations31

Week t Untreated Vydate 10G 3.00 2.25 1.75 1.50 1.25 1.00

Plant weight 8 4.36±0.60ab 5.34±0.58ab 5.06±0.60ab 5.18±0.52ab 5.90±0.85a 4.56±0.38ab 4.64±0.47ab 4.26±0.42b (g)

Root weight 8 0.40±0.043 0.51±0.03a 0.44±0.07a 0.47±0.03a 0.51±0.06a 0.44±0.07a 0.49±0.09a 0.40±0.05a abc ac abc be be (g) 12 23.5±5.9a 40.1±5.7 37.7±5.3 40.3±6.3 49.1±5.2 58.0±7.5b 52.6±3.5b 42.7±7.6

16 316±77a 419±98ab 460±119ab 335±48a 610±99b 625±78b 532±82ab 501±65ab

28 f 1093±33a 1132±45a 1099±53a U83±26a 1138±35a 1170±32a 1125±32a 1114±29a 197

b, «ac Nematodes per 8 83±24a(18)a 33±8b(8)aC 34±5b(0)b 34±5b(0)b 30±4 (9) 30±4b(l)b 26±5b(3)bC 35±6b(l)b root system 12 94±16a(47)a 46±13b(48)a 37±15b(54)a 41±12b(45)a 40±12b(30)a 38±12b(37)a 39±lb(29)a 37±8b(59)a (% saccate) 16 510±62a(54)ab 250±41b(58)b 250±25b (57)b 213±33bC(48) abC177±26bC(42)abC146±21C(34)C 151±27C(36)aC 183±45bC(50)

t

-1 a b .b b b b b Nematodes g 8 205±52 65±14 83±14 71±12 61±10 71±10 58±14 92±20

root 12 5.26±1.36a 1.08±0.22b 0.90±0.23b 0.98±0.25b 0.78±0.17b 0.66±0.19b 0.78±0.25b 0.94±0.16b be 16 2.00±0.47a 0.64±0.07bC 0.86±0.37C 0.66±0.15 0.30±0.05b 0.24±0.05b 0.30±0.06b 0.34±0.07b

After application of pesticide. Harvest at week 28.

+ Mean weight per beet at harvest.

^ Release rate: % oxamyl min~*. All figures in horizontal rows with the same letter are not significantly different at P>0.05. \

Appendix 11 1982 Pot trial: Effects of various formulations of oxamyl (Vydate) and aldicarb (Temik) on the root growth of sugar beet

and invasion and development of H.schachtii.

Mean ± S.E.

Soil type: Peat fen , Vydate 10G Week ' Untreated Vydate 10G Vydate DPX 4702 Incremental Temik 10G Aldicarb VYHD

Plant weight 4 1.22 ± 0.22a 1.48 ± 0.32a 0.78 ± 0.1ia 1.08 ± 0.14a 1.15 ± 0.313 1.55 ± 0.37a (g)

be Root weight 8 0.75 ±.o.ioa 1.48 ± 0.16 1.05 ± 0.33ab 0.80 ± 0.15a 1.22 ± 0.283b 2.10 ± 0.09C (g) 12 26.4 ± 6.ia 41.9 ± 2.1b 45.8 ± 5.4 26.8 ± 4.4 29.6 ± 4.0 37.0 ± 4.iab I i ±

cd Nematodes/g 8 79.7 ± 8.83 21.1 ± 2.6bC 35.8 ± 6.6 55.6 ± 18.5ad 21.1 ± 5.7bC 7.8 ± 1.3b root ab be 12 31.7 ± 9.53 12.1 ± 2.3bC 9.8 ± 1.8( 19.8 ± 4.5 12.5 ± 2.4 8.1 ± 2.1°

t After application of pesticide.

All figures in horizontal rows with the same letter are not significantly different at P>0.05. Appendix 12 1982 Pot trial: Effects of various formulations of oxamyl (Vydate) and aldicarb (Temik) on the root growth of sugar beet

and invasion and development of H.schachtii.

Mean ± S.E.

Soil type: Sandy-clay. . Vydate Week ' Untreated Vydate 10G Vydate DPX 4702 Incremental Temik 10G Aldicarb VYHD > ab Plant weight 4 0.30 ± 0.10a 0.58 ± 0.14ab 0.75 ± 0.16b 0.70 ± 0.15b 0.52 ± 0.10 0.68 ± 0.11b (g)

ab ab ab Root weight 8 0.10 ± o.oia 0.45 ± 0.18 0.32 ± 0.11 0.20 ± 0.04 0.35 ± 0.13ab 0.50 ± 0.17b (g) ab ab 12 9.3 ± 1.8a 17.1 ± 5.1 23.0 ± 6.4b 11.8 ± 1.1 13.9 ± 4.8ab 24.2 ± io.oab I 199

Nematodes/ 4 23 ± 6a(29)a ob(o)b 1 ±

o o bC Nematodes/g 8 125.0±32.2a 4.8 ± 2.9b 5.2 ± 3.1b 62.5 ± 21.7C 6.7 ± 3.9b 27.7 ± 8.8 root . be 12 36.9 ± 3.3a 30.2 ± 8.4aC 16.8 ± 4.3bC 11.7 ± 2.7b 18.6 ± 5.5bC 18.5 ± 4.7

^ After application of pesticide.

All figures in horizontal rows with the same letter are not significantly different at P>0.05. Appendix 13 1983 Microplot trial: Effects of various formulations of oxamyl (Vydate) on root growth of sugar beet and invasion

and development of H.schachtii.

Mean ± S.E.

Experimental formulations 1 Vydate DPX Week^* Untreated Vydate 10G Fast Medium Slow DPX 5578-2 5578-2/10G DPX 4702/10G

Plant weight ab ab (g) 6 1.24±0.13a 1.43±0.11 1.46±0.10ab 1.46±0.13 1.55±0.12ab 1.50±0.06ab 1.60±0.17b 1.40±0.16ab

ac be ab be Root weight 10 8.8±1.0 6.5±0.3a 11.8±1.5 9.7±1.1 u 10.7±1.8 13.2±2.6b 13.3±1.8b 12.0±0.8 ab (g) 16 184±32a 176±10a 132±19a 147±25a 156±30a 153±28a 156±18a 197±34a ab ab ab ab 28 ^ 405±48ab 440±62ab 370±25 370±34 410±42 335±24a 410±19 460±37b | 200 O ac. .ab a, .ab ac, .ab be, . b ac, .ab b, ,ab o Nematodes per 6 39±4a(42)a 33±3aC(18)b 28±5 (29) 41±7 (27) 35±7 (30) 26±5 (19) 28±2 (29) 13±1 (19) w root system ab, .a 10 83±14ab(45)a 52±9ab(42)a 44±14ab(36)a 85±8a(47)a 42±7b(24)a 54±20ab(39)a 76±25 (20) 70±13ab(25)a (% saccate)

New cysts/root 16 48±6a 18±3b 18±3b 15±2b 17±4b :"17±3b 23±3b 19±3b

bd Nematodes/g 6 465±69a 346±54aCd 274±59bd 397±50ad 329±60aCd 256±50bC 263±27 158±13b root ab ab 10 10.3±2.4a 7.9±1.4ab 3.6±0.8b 9.3±1.6aC 4.4±0.9b 5.1±2.5bC 6.2±2.1 5.8±1.1

^ After application of pesticide. Harvest at week 28.

^ Mean weight per beet at harvest. '

$ Slow to fast release rate.

All figures in horizontal rows with the same letter are not significantly different at P>0.05. Appendix 14 1984 Microplot trial: Effects of various formulations of oxamyl (Vydate) and aldicarb (Temik) on root growth of sugar beet 1 * I and invasion and development of H.schachtii.

Mean ± S.E.

Experimental $ Week1 Untreated Temik 10G Vydate 10G formulation

Plant weight 7 2.20 ± 0.48a(65)3 3.08 ± 0.69a(57)a 3.12 ± 0.33a(77)a 3.72 ± 0.5ia(64)a (g) (% germination).

Root weight 7 0.23 ± 0.053 0.31 ± 0.07ab 0.32 ± 0.04ab 0.43 ± 0.05b I

(g) 201 11 5.5 ± l.l3 15.2 ± 1.8b 11.2 ± 2.1b 10.1 + 2.iab

15 48 ± 10a 160 ± 27b 133 ± 28b 112 ± 20b I 21 142 ± 34a 332 ± 27b 342 + 78b 296 ± 42b

^28 * 456 ± 26a 662 ± 72b 625 + 63ab 556 ± 62ab

ab, .b Nematodes/ 7 42 ± 13a(3l)a 10 ± 5b(8)b 26 ± 12 (9) 9 ± lb(2)b root system 11 91 ± 20a(50)a 30 ± 7b(63)a 65 ± 27ab(39)' 40 ± 7b(31)a (% saccate) 15 550 ± 49a(67)a 170 ± 16bC(62)a 235 ± 45C(64)a 140 ± 13b(62)a

21 2835 ± 334a(30)a 1425 ± 169b(43)a 1516 ± 217b(38) 1175 ± 214b(47)a

Nematodes/g 7 171 ± 26a 32 ± 9b 90 ± 48ab 21 ± 3b root 11 17.1 ± 3.4a 2.1 ± 0 .6b 6.0 ± 1.8b 4.3 ± 1.0b

15 13.6 ± 3.8a 1.1 ± 0.2b 2.0 ± 0 .6b 1.3 ± 0.1b

21 23.2 ± 4.6a 4.4 ± 0.6b 4.9 ± 0.8b 4.0 ± 0.5b Appendix 14 Symbols

^ After application of pesticide. Harvest at week 28.

^ Mean weight per beet at harvest.

^ Release rate: 1.25% oxamyl min *.

All figures in horizontal rows with the same letter are not significantly different at P>0.05.

I 202

l Appendix 15 Meteorological data for the field trials. Prickwillow figures are from Met. Office Station No. 194297 (Mepal)

15 Km west of the trial sites. Silwood Park figures are from the Met, site at Imperial College, Silwood Park.

Site Year March \ Apri 1 May June July August September October

Prickwillow 1982 Rainfall (mm) 16.6 9.7 69.2 112.9 22.8 81.8 46.0 63.7

Temperature Max(°C) 17.5 18.8 25.9 28.4 28.9 27.9 27.4 17.9

Mi n(°C) -0.6 -3.2 -4.5 7.6 7.5 5.5 2.6 6.5

Prickwillow 1984 Rainfall (mm) - 1.5 74.2 68.6 11.1 42.8 83.1 23.3 203 Temperature Max(°C) - 23.0 19.8 25.7 28.7 29.4 26.0 20.1

Min(°C) - -2.5 -1.8 4.5 5.7 5.6 3.8 4.0

Silwood 1982 Rainfall (mm) - - - 92.0 55.7 25.1 _ - Park Temperature Max(°C) - - - 28.3 27.5 26.5 - -

Mi n(°C) - - - 4.0 8.7 5.7 - -

Silwood 1983 Rainfall (mm) - 64.4 63.5 59.2 17.2 35.0 48.8 39.5 Park Temperature Max(°C) - 17.7 18.3 25.5 32.0 29.5 23.7 22.5

Mi n(°C) - -2.0 2.5 5.0 6.7 4.3 3.5 -3.5

Silwood 1984 Rainfall (mm) 4.0^ 113.7 39.8 22.1 28.2 84.9 64.4 Park Temperature Max(°C) - 22.7 22.3 26.3 29.7 29.5 21.5 19.0

Min(°C) - -0.3 1.3 2.3 5.7 5.3 5.0 1.7

t Irrigated. Figures are for the duration of the trial only. 2 0 4

Appendix 16 Statistical analysis of results for week 8 of the 1982 field trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Plant weights

Treatments 0.874 3 0.291 0.94 >0.25 Error 4.964 16 0.310 Total 5.8?8 19

Nematodes per root

Treatments 2126 3 709 3.74 >0.025 Error 3035 16 190 Total 5161 19

Proportions saccate

Treatments 282.3 3 94.1 1.42 >0.25 Error 1062.8 16 66.4 Total 1345.1 19

(Arcsin transformation of data) 2 0 5

Appendix 17 Statistical analysis of results for week 17 of the 1982 field trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Probability Variation Squares Freedom Square Ratio

Root weights

Treatments 148701 3 49567 1.62 <0.25 >0.10 Error 490601 16 30663 Total 639302 19

Nematodes per root

Treatments 65641 3 21880 0.43 >0.25 Error 818528 16 :51158 Total 884169 19

Proportions saccate

Treatments 1684.1 3 561.4 6.73 <0.005 Error, 1335.0 16 83.4 Total" 3019.1 19

: (Arcsin transformation of data)

Nematodes per g root

Treatments 0.209 3 0.070 0.35 >0.25 Error 3.183 ^ 16 0.199 Total 3.392 19 2 0 6

Appendix 18 Statistical analysis of harvest root weights for the 1982 field trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 92772 3 30924 4.33 <0.025>0.01 Error 114382 16 7149 Total 207154 19

Appendix 19 Statistical analysis of final H.schachtii populations for the 1982 field trial 28 weeks after application of nematicide (5.6 kg a.i . ha *).

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 376 3 125 1.10 >0.25 Error 1828 16 114 Total 2204 19 2 0 7

Appendix 20 Statistical analysis of the soil oxamyl residue concentrations for weeks 2, 4, 8 and 17 of the 1982 field trial after treat-

ment with nematicide (5.6 kg a.i. ha1 1 1 .

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Week 2

Treatments 2.250 2 1.125 3.74 <0.10>0.05 Error 2.706 9 0.301 Total 4.956 11

Week 4

Treatments 0.856 2 0.428 1.27 >0.25 Error 2.030 6 0.338 Total 2.886 8

Week 8

Treatments 5.403 2 2.701 4.62 <0.05>0.025 Error - 5.258 9 0.584 Total 10.661 11

Week 17

Treatments 0.002022 2 0.001011 10.11 <0.025>0.01 Error 0.000600 6 0.00 0 1 0^ 0 Total 0.002622 8 2 0 8

Appendix 21 Statistical analysis of the change in soil oxamyl residue concentrations during the 1982 field trial after application of 5.6 kg a .i. ha"* of the three oxamyl formulations.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Vydate 10G

Time 20.643 3 6.881 50.30 <0.005 Error 1.368 10 0.137 Total 22.011 13

DPX 4702

Time 12.997 3 4.332 11.82 <0.005 Error 3.665 10 0.367 Total 16.662 13

Vydate 10G Incremental

Time 10.455 3 3.485 7.02 <0.01>0.005 Error 4.961 10 0.496 Total 15.416 13 2 0 9

Appendix 22 Statistical analysis of results for week 8 of the 1984 field trial •

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Plant weights

Treatments 10.90 7 1.56 0.96 >0.25 Error 52.12 32 1.63 Total 63.02 39

Root weights

Treatments 0.0687 7 0.0098 0.60 >0.25 Error 0.5277 32 0.0165 Total 0.5964 39

Nematodes per root

Treatments 11875 7 1696 3.35 <0.01>0.005 Error 16228 32 507 Total 28104 39

Proportions saccate

Treatments 2378.7 7 339.8 6.95 <0.005 Error 1564.9 32 48.9 Total 3943.6 39

(Arcsin transformation of data)

Nematodes per g. root

Treatments 82389 7 11770 4.70 <0.005 Error 80122 32 2504 Total 162511 39 2 1 0

Appendix 23 Statistical analysis of results for week 12 of the 1984 field trial •

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 3892 7 556 3.08 <0.025X1.01 Error 5770 32 180 Total 9662 39

Nematodes per root

Treatments 13190 7 1884 2.40 <0.05X1.025 Error 25170 32 787 Total 38360 39

Proportions saccate

Treatments 3160 7 451 1.01 >0.25 Error 14357 32 449 Total 17517 39

(Arcsin transformation of data)

Nematodes per g. root

Treatments 84.76 7 12.11 9.00 <0.005 Error 43.05 32 1.35 Total 127.81 39 2 1 1

Appendix 24 Statistical analysis of results for week 16 of the 1984 field trial •

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 464120 7 66303 1.81 <0.25>0.10 Error 1173356 32 36667 Total 1637476 39

Nematodes per root

Treatments 488020 7 69717 9.96 <0.005 Error 224030 32 7001

Total 712050 39 -

Proportions saccate

Treatments 1138.8 7 162.7 2.08 <0.10>0.05 Error 2500.2 32 78.1 Total 3639.0 39

(Arcsin transformation of data)

Nematodes per g. root

Treatments 11.868 7 1.695 6.66 <0.005 Error 8.140 32 0.254 Total 20.008 39 2 1 2

Appendix 25 Statistical analysis of harvest root weights for the 1984 field trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 35557 7 5080 0.76 >0.25 Error 213876 32 6684 Total 249433 39

Appendix 26 Statistical analysis of final H.schachtii populations for the 1984 field trial 28 weeks after application of nematicide (5.6 kg a.i. ha 1).

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability t iS t.

Treatments 9098 7 1300 0.89 >0.25 Error 46528 32 1454 Total 55626 39 2 1 3

Appendix 27 Statistical analysis of the soil oxamyl residue concentrations for weeks 1, 2, 4, 8 and 12 of the 1984 field trial after treat­ ment with nematicide (5.6 kg a.i. ha ).

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Week 1

Treatments 0.365 3 0.122 0.14 >0.25 Error 6.995 8 0.874 Total 7.360 11

Week 2

Treatments 5.27 3 1.76 0.67 >0.25 Error 21.04 8 2.63 Total 26.30 11

Week 4

Treatments 1.312 3 0.437 1.62 >0.25 Error 2.166 8 0.271 Total 3.479 11

Week 8

Treatments 0.0573 3 0.0191 1.10 >0.25 Error 0.1386 8 0.0173 - Total 0.1959 11

Week 12

Treatments 0.022533 3 0.007511 8.50 <0.01>0.005 Error 0.007067 8 0.000883 Total 0.029600 11 2 1 4

Appendix 28 Statistical analysis of the change in soil oxamyl residue concentrations during the 1984 field trial after application of 5.6 kg a .i. ha of four oxamyl formulations.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Vydate 10G

Time 31.378 4 7.845 17.05 <0.005 Error 4.601 10 0.460 Total 35.979 14

2.25% oxamyl min”*

Time 14.345 4 3.586 3.82 <0.05>0.025 Error 9.390 10 0.939 Total 23.735 14

1.50% oxamyl min *

Time 30.716 4 7.679 8.65 <0.005 Error 39.597 10 0.888 Total 39.597 14

• . -1 1 .00% oxamyl min

Time 17.346 4 4.336 5.80 <0.005 Error 7.472 10 0.747 Total 24.818 14 2 1 5

Appendix 29 Statistical analysis of results for week 4 of the 1982 pot trial: Peat fen soil •

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Plant weights

Treatments 1.588 5 0.318 1.48 >0.25 Blocks 1.728 3 0.576 2 .6 8 <0.10>0.05 Error 3.222 15 0.215 Total 6.538 23

Nematodes per root

Treatments 3129.8 5 626.0 10.81 <0.005 Blocks 134.3 3 44.8 0.77 >0.25 Error 869.2 15 57.9 Total 4133.3 23

Proportions saccate

Treatments 65.27 5 13.05 9.00 <0.005 Blocks 4.36 3 1.45 1.0 0 >0.25 Error 21.81 15 1.45 Total 91.44 23

(Arcsin transformation of data). 2 1 6

Appendix 30 Statistical analysis of results for week 4 of the 1982 pot trial: Sandy-clay soil.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Plant weights

Treatments 0.5337 5 0.1068 1.39 >0.25 Blocks 0.0413 3 0.0138 0.18 >0.25 Error 1.1513 15 0.0767 Total 1.7263 23

Nematodes per root

Treatments 1594.7 5 318.9 12.91 <0.005 B1ocks 115.8 3 38.6 1.56 <0.25>0.10 Error 370.5 15 24.7 Total 2081.0 23

Proportions saccate

Treatments 4100 5 820 4.27 <0.025>0.01 Blocks 426 3 142 0.74 >0.25 Error 2877 15 192 Total 7402 23 2 1 7

Appendix 31 Statistical analysis of results for week 8 of the 1982 pot trial: Peat fen soil •

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 5.058 5 1.012 6.45 <0.005 Blocks 0.677 3 0.226 1.44 >0.025 Error 2.358 15 0.157 Total 8.093 23

Nematodes per root

Treatments 5471 5 1094 1.70 <0.25>0.10 Blocks 1742 3 581 0.90 >0.25 Error 9671 15 645 Total 10883 23

Proportions saccate

Treatments 4747.1 5 949.4 18.22 <0.005 Blocks 276.2 3 92.1 1.77 <0.25>0.10 Error 781.7 15 52.1 Total 5805.0 23

(Arcsin transformation of data).

Nematodes per g. root

Treatments 14163 5 2833 7.80 <0.005 Blocks 605 3 202 0.57 >0.25 Error 5451 15 363 Total 20219 23 2 1 8

Appendix 32 Statistical analysis of results for week 8 of the 1982 pot trial: Sandy-clay soil.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 0.4521 5 0.0904 1.33 >0.25 Blocks 0.0846 3 0.0282 0.41 >0.25 Error 1.0229 15 0.0682 Total 1.5596 23

Nematodes per root

Treatments 467.7 5 93.5 3.04 <0.05>0.025 Blocks 119.8 3 39.9 1.30 >0.25 Error 461.5 15 30.8 Total 1049.0 23

Proportions saccate

Treatments 4697 5 939 1.08 >0.25 Blocks 3611 3 1204 1.39 >0.25 Error 13036 15 869 Total 21343 23

(Arcsin transformation of data)

Nematodes per g. root

Treatments 45858 5 9172 8.30 <0.005 B1ocks 2864 3 955 0.86 >0.25 Error 16568 15 1105 Total 65290 23 2 1 9

Appendix 33 Statistical analysis of results for week 12 of the 1982 pot trial: Peat fen soil.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 1453 5 291 1.85 <0.25>0.10 Blocks 502 3 167 1.06 >0.25 Error 2352 15 157 Total 4307 23

Nematodes per root

Treatments 544300 5 108860 1.21 >0.25 Blocks 550533 3 183511 2.05 <0.25>0.10 Error 1344167 15 89611 Total 2439000 23

Proportions saccate

Treatments 3437.0 5 687.4 15.38 <0.005 Blocks 317.4 3 105.8 2.37 <0.25>0.10 Error 670.4 15 44.7 Total 4424.8 23

(Arcsin transformation of data)

Nematodes per g. root

Treatments 1737 5 347 1.63 <0.25>0.10 Blocks 131 3 44 0.21 >0.25 Error 3199 15 213 Total 5067 23 2 2 0

Appendix 34 Statistical analysis of results for week 12 of the 1982 pot trial: Sandy-clay soil.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 900 5 180 0.66 >0.25 Blocks 1387 3 462 1.70 <0.25>0.10 Error 4072 15 271 Total 6360 23

Nematodes per root

Treatments 162771 5 32554 3.06 <0.05>0.025 Blocks 59213 3 19738 1.85 <0.25>0.10 Error 159712 15 10648 Total 381696 23

Proportions saccate

Treatments 261 5 52 0.24 >0.25 B1ocks 325 3 108 0.50 >0.25 Error • 3226 15 215 Total 3811 23

(Arcsin transformation of data)

Nematodes per g. root

Treatments 1846 5 369 1.96 <0.25>0.10 Blocks 89 3 30 0.16 >0.25 Error 2818 15 188 Total 4753 23 2 2 1

Appendix 35 Statistical analysis of final H.schachtii populations for the 1982 pot trial 12 weeks after application of nematicide: Peat fen soil.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 28.23 5 5.65 0 .6 6 >0.25 Blocks 24.32 3 8.11 0.95 >0.25 Error 128.44 15 8.56 Total 180.99 23

Appendix 36 Statistical analysis of final H.schachtii populations for the 1982 pot; trial 12 weeks after application of nematicide: Sandy-clay soil.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 208.0 5 41.6 3.38 <0.05>0.025 Blocks 32.9 3 11.0 0.89 >0.25 Error 184.1 15 12.3 Total 424.9 23 2 2 2

Appendix 37 Statistical analysis of results for week 6 of the 1983 microplot trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Plant weights

Treatments 0.4003 7 0.0572 0.72 >0.25 Error 2.5540 32 0.0798 Total 2.9544 39

Nematodes per root

Treatments 2707 7 387 3.65 <0.01>0.005 Error 3394 32 106 Total 6101 39

Proportions saccate

Treatments 1221 7 174 1.26 >0.25 Error 4422 32 138 Total 5643 39

(Arcsin transformation of data)

Nematodes per g. root

Treatments 314064 7 44866 3.48 <0.01>0.05 Error 411971 32 12874 Total 726035 39 2 2 3

Appendix 38 Statistical analysis of results for week 10 of the 1983 microplot trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 191.2 7 27.3 2.38 <0.05>0.025 Error 367.3 32 11.5 Total 558.5 39

Nematodes per root system

Treatments 10528 7 1504 1.36 >0.25 Error 35350 32 Total 45878 39

Proportions saccate

Treatments 2039 7 291 1.05 >0.25 Error 8837 32 276 Total 10876 39

(Arcsin transformation of data)

Nematodes per g. root

Treatments 198.5 7 28.4 1.93 <0.10>0.05 Error 470.3 32 14.7

Total 668.8 39 224

Appendix 39 Statistical analysis of results for week 16 of the 1983 microplot trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 15696 7 2242 0.69 >0.25 Error 104478 32 3265 Total 120174 39

New cysts per root

Treatments 4050.8 7 578.7 9.64 <0.005 Error 1920.8 32 60.0 Total 5971.6 39

Appendix 40 Statistical analysis of harvest root weights for the! 1983 microplot trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 57250 7 8179 1.08 >0.25 Error 241500 32 7547 Total 298750 39 2 2 5

Appendix 41 Stati stical analysis of final H.schachtii populations for the 1983 microplot trial 28 weeks after application of nematicide (5.6 kq a.i . ha *).

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 277 7 40 0.20 >0.25 Error 6388 32 200 Total 6665 39

Appendix 42 Statistical analysis of the percentage germination of sugar beet seed for the 1984 microplot trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 308 3 103 0.61 >0.25 Blocks 504 3 168 0.99 >0.25 Error 1523 9 169 Total 2335 15

(Arcsin transformation of data) 22 6

Appendix 43 Statistical analysis of results for week 7 of the 1984 microplot trial.

Analysis of variance

Source of . Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Plant weights

Treatments 4.732 3 1.577 2.16 <0.25>0.10 B1ocks 6.277 3 2.092 2.87 <0.10>0.05 Error 6.566 9 0.730 Total 17.574 15

Root weights

Treatments 0.0788 3 0.0263 2.33 <0.25>0.10 Blocks 0.0376 3 0.0125 1.11 >0.25 Error 0.1019 9 0.0133 Total 0.2182 15

Nematodes per root

Treatments 2903 3 968 2.16 <0.25>0.10 Blocks 4 3 i: <0.01 >0.25 Error 4036 9 448 Total 6944 15

Proportions saccate

Treatments 1897 3 632 6.08 <0.025X3.01 Blocks 673 3 224 2.15 <0.25X3.10 Error 940 9 104 Total 3511 15

(Arcsin transformation of data) 2 2 7

Appendix 43 (continued)

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Nematodes per g. root

Treatments 56930 3 18977 4.90 <0.05>0.025 Blocks 1311 3 437 0.11 >0.25 Error 34871 9 3875 Total 93112 15 2 2 8

Appendix 44 Statistical analysis of results for week 11 of the 1984 microplot trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 193.0 3 64.3 4.66 <0.05>0.025 Blocks 31.8 3 10.6 0.77 >0.25 Error 124.4 9 13.8 Total 349.2 15

Nematodes per root

Treatments 9017 3 3006 3.56 <0.10>0.05 Blocks 7217 3 2406 2.85 <0.10>0.05 Error 7602 9 845 Total 23836 15

Proportions saccate

Treatments 1331 3 444 1.52 >0.25 Blocks 1913 3 638 2.18 <0.25>0.10 Error 2632 9 292 Total 5876' 15

(Arcs in transformation of data)

Nematodes per g. root

Treatments 533.88 3 177.96 19.16 <0.005 Blocks 107.15 3 35.72 3.84 <0.10>0.05 Error 83.63 9 9.29 Total 724.67 15 229

Appendix 45 Statistical analysis of results for week 15 of the 1984 microplot trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Root weights

Treatments 27564 3 9188 5.67 <0.025>0.01 Blocks 9473 3 3158 1.95 <0.25>0.10 Error 14576 9 1620 Total 51612 15

Nematodes per root

Treatments 425875 3 141958 49.07 <0.005 B1ocks 32713 3 10904 3.77 <0.10>0.05 Error 26038 9 2893 Total 484625 15

Proportions saccate

Treatments 23 3 8 0.06 >0.25 Blocks 577 3 192 1.39 >0.25 Error 1246 9 138 Total 1846 15

(Arcsin transformation of data)

Nematodes per g. root

Treatments 441.1 3 147.0 9.48 <0.005 Blocks 35.0 3 11.7 0.75 >0.25 Error 139.9 9 15.5 Total 616.1 15 2 3 0

Appendix 46 Stati stical analysis of results for week 21 of the 1984 microplot trial.

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probabi 1 ity

Root weights

Treatments 103098 3 34366 8.77 <0.005 Blocks 82202 3 27401 6.99 <0.01>0.005 Error 35279 9 3920 Total 220579 15

Nematodes per root

Treatments 6670080 3 2223360 20.58 <0.005 Blocks 1821580 3 607193 5.62 <0.025>0.01 Error 972289 9 108032 Total 9463948 15

Proportions saccate

Treatments 232 3 77 0.39 >0.25 B1ocks 491 3 164 0.82 >0.25 Error 1795 9 199 Total 2518 15

(Arcsin transformation of data)

Nematodes per g. root

Treatments 1053.5 3 351.2 16.33 <0.005 Blocks 79.4 3 26.5 1.23 >0.25 Error 193.4 9 21.5 Total 1326.3 15 2 3 1

Appendix 47 Statistical analysis of harvest root weights for the 1984 microplot trial,

Analysis of variance

Source of Sum of Degrees of Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 98438 3 32813 3.69 <0.10>0.05 Blocks 82813 3 27604 3.11 <0.10>0.05 Error 80000 9 8889 Total 261250 15

Appendix 48 Statistical analysis of final H.schachtii populations for the 1984 microplot trial 28 weeks after application of nematicide (5.6 kg a.i . ha *).

Analysis of variance

Source of Sum of Degrees < Df Mean Variance Variation Squares Freedom Square Ratio Probability

Treatments 1398.6 3 466.2 10.11 <0.005 Blocks 129.2 3 43.1 0.93 >0.25 Error 414.6 9 46.1 Total 1942.5 15