"Some Effects of Methyl Bromide on Aphids and Whiteny and their Host Plants."
by
Anthony P. Mainwaring, B.Sc. (Lona.)
A Thesis subadtted for the degree of Doctor of Philosophy in the Faculty of Science in the University of London.
Imperial College Field Station, Silwood Park, Sunninghill, Ascot, Berkshire. August, 1961. ABSTRACT.
The toxicity of methyl bromide to six species of aphids and the greenhouse whitefly at different stages in the life-histories has been stud- ied. The effect of a number of extrinsic and intrinsic factors on suscep- tibility has been determined.
The effects of the `_as on reproduction and development and rate of water loss were also studied with particular reference to aphids, and some interesting observations on the behaviour of the insects after fumigation were made.
The temperature and humidity during the post-fumigation period had an important influence on the length of time which elapsed before the insects succumbed to the effects of a treatment.
Presence of visible tissue damage proved to be an unsatisfactory criterion for assessing the limit of tolerance of plants to methyl bromide.
In young plants particularly, severe physiological disturbances occurred at doses much lower than those causing only slight visible damage.
Generally, however, the susceptibility of the insects was far greater than, that of the plants and fumigation with methyl bromide was shown to be a practicable method of control.
The rate of water loss from the shoot of a plant and the rate of water uptake by the roots decreased after fumigation. Growth was apprec- iably retarded at many doses and the plants displayed signs of mineral deficiency a few weeks after treatment. Flower production, however, was stimulated. The effect of methyl bromide on the roots of a plant was shown to be particularly important and protection of the root-system during fumigation considerably alleviated some of the adverse effects mentioned.
Some comments on the possible mode of action of methyl bromide are included in the thesis in relation to both insects and plants and a short account of the practical application of the work to aphid control in marigold clamps is appended.. ACTIOWLEDGNEIUS.
The author wishes to express his thanks to Professor 0.W.Richards, F.R.S., for permission to work in his department and to Dr.A.B.P.Page for supervising the work.
Thanks are also due to Dr.Page and Dr.O.F.Lubatti for their sustained interest and encouragement and to Dr.Lubatti for his advice and assistance with the technical aspects of fumigation. The work was carried out during the tenure of a postgraduate studentship from the Agricultural Research Council.
TABLE OF CONTENTS. Page 1. Introduction 1
,Insect Fumigation 2. Review of the Literature 3. Life-Histories of Aphids and Whitefly 6 4. Materials and Methods 13 5. Factors affecting the Dosage-Mortality-Response 27 6. Some Effects of Methyl bromide on the Physiology and Behaviour of the Insects 14.8 7. Discussion 62
Plant Fumigation
8. Review of the Literature 77 9. Materials and Methods 81 10. Plant Tolerance to Methyl Bromide and Factors affecting Tolerance 87 11. Some Effects of Methyl Bromide on the Physiology of Plants: (a) Water Balance 106 ( b ) Stomatal Opening 128 (c) Growth 132 (d) Flowering 149 12. Discussion 155
13. Practical Applications 170 14. Bibliography 175 1. INTRODUCTION.
The control of insect pests on various plant materials by fumigation has been well established and many different chemicals have been used. Among these hydrogen cyanide and nicotine particularly deserve mention.
However, since 1932, when Le Goupils first reported its insectic- idal properties, methyl bromide has rapidly gained in importance and has largely replaced these others. Its extensive use in the treatment of infested stored grain and seed needs no emphasising. In accordance with the quarantine regulations of many countries methyl bromide is also used to fumigate bulbs and tubers and other prop- agative parts including cuttings of woody perennials.
Fresh fruit and vegetables are fumigated with methyl bromide in certain countries, e.g. France, Germany, South Africa and. the U.S.A., and the control of pests on ornamental plants and flower crops by this means is also extensively employed.
On a smaller scale, methyl bromide fumigation is used in laboratories and Research Institutes for herbaria and where plants free of insect pests, are required for use in, for example, work on plant viruses.
It is, however, the fumigation of non-dormant plants, and especially herb- aceous ones, which particularly concerns us here.
The work described in this thesis originates from an enquiry from Rothamsted Experimental Station, Harpenden, concerning the fumigation of lettuce plants with methyl bromide to control aphids. Comparatively 2.
little was known at that time of the effects of this fumigant on herbaceous plants and their aphid parasites and the investigation was undertaken to supply some of the information required.
For experimental purposes the plant and insect aspects were treated separately. A final analysis of control measures, however, must take into consideration the interaction of both sets of results and this is done in a separate section at the end of the thesis. In all twelve species of herbaceous plants and six species of aphids were used and the Scope of the work was extended to include some of the effects of the gas on the greenhouse whitefly. A short note is also included on the practical application of some of the results to the control of aphids in marigold clamps in the Lincolnshire and Cambridgeshire areas. Aphids and whitefly are notorious pests of a wide range of plants and are of very great economic importance, not least because of their ability to transmit several important strains of virus. The large populations which rapidly build up, especially under glass or indoors, constitute a serious drain on the resources of an infested plant and yield is correspondingly reduced. In addition, the copious honey dew with which the insects may cover a plant renders it and its fruit unsightly, particularly when the excrement is supporting a fungal growth, as frequently happens. Fumigation, where practicable, has the great advantage of ensuring the even dosage of every individual with the insecticide. The rapid rate of reproduction and great fecundity of most of these insects make it essential 3- that as near complete control as possible be obtained at each treatment. Methyl bromide was the only fumigant used in the experiments and, in addition to determining the lethal concentrations for insect and plant, an investigation was made of some of the physiological effects which occurred at lower doses. Doses are expressed throughout the thesis as the product of the determined concentration in mg. per litre and the duration of exposure in hours, i.e. as the concentration time product (abbr. C.T.P.). This unit is inappreciably different from the unit oz.hrs. per 1,000 cu. ft. commonly used in commercial practice and in America. Thus a C.T.P. of 50 mg.hrs. per litre can be read also as 50 oz.hrs. per 1,000 cu.ft. Because of the nature of a fumigation treatment this work is particularly relevant to plants grown in glasshouses etc. or where plant material can readily be brought to a suitable chamber. However, fumigation of plants in the field under sheets is also feasible on a small scale. 4.
IUSECT FUEIGLTION.
2. Review of the literature.
The literature on the chemical control of aphids and, to a lesser extent, whitefly is considerable. References to the use of almost every known insecticide can be found.
Jmo*ng the fumigants which have been used against aphids are hydrogen cyanide (Brinley and Baker, 1927)0-00fdichlorethyl ether (Wileoxon and Hartzell, 1938) and fluoro-acetamide (Gentle, 1957) in addition to compounds such as BHC which have a slight secondary fumigant action. Nicotine, however, has been the most widely used (Alsterlund and Compton, 1937; Richardson and Busbey, 1937; Richardson (nicotine and napthalene mixture) 1940; Richardson and Casanges, 1942; Richardson, Bulger and Busbey, 1943) and is still probably so today. The control of the greenhouse whitefly in all its stages by fumigation was investigated by Lloyd (1922) who found that poor control was obtained with napthalene and nicotine, whereas tetrachlorethane and hydrogen cyanide vapour gave good results. Tetrachlorethane, however, is too expensive for general use and cyanide has been widely employed ever since.
Despite the many applications of methyl bromide as a fumigant for a variety of pests of plant materials (Ilackie, 19414 Hawkins, 1942; Steinweden, 1945) a systematic study of its effects on either aphids or whitefly has not, to the author's knowledge been published. 00 Richardson et al. (1943) reported briefly on the relative susceptibilities of a number of insects to methyl bromide, including the chrysanthemum aphid, Macrosiphoniella sanborni (Gill.), and Trialeurodes vaporariorum. Mackie (1941), Hawkins (194.2) and. Steinweden (1945) also included brief allusions to aphid and whitefly susceptibility to the gas. Otherwise little information exists. 6.
3. The Life-Histories of Aphids and Whit efly. In view of the complex and unusual nature of the life-histories of these two groups of insects, a brief resume of the essential features may be of value in relation to the work to be described..
CLASSIFICATION. Order: BEMIP2ERA Sub-order: HOMOPrERA STERHORRITICHA.
Super-family: APHIDOIDEA Family: APFTDIDAB. Aphids.
Super-family: ALEYRODOIDEL
Family: ATE ODIDAE (= ALWRODIDLE). Whitefly. Aphids and. whitefly belong to the same order of insects which is characterised by having piercing mouthparts. They are external parasites
of plants, feeding on the sap, and are capable of both sexual and asexual
reproduction. They commonly secrete wax. Excess food is got rid of as honeydew, a sticky exudation largely carbohydrate, forcibly ejected from
the anus.
(a) Life-History of Aphids. Aphids are highly polymorphic and are common both out of doors and under glass.
A typical species has both a primary, winter host-plant (usually a woody perennial) and a secondary, summer (herbaceous) host or hosts. Few aphids have only one host. Indoors, however, the life-history may be considerably modified. Some aphids also attack roots and bulbs etc. while a few produce galls. One species feeds on woody stems but the great majority are found on the foliage of growing plants. One or two species are essentially glasshouse pests only.
Metamorphosis is incomplete and the adult is preceded by a succession of similar instars (nymphs). The life-history of a typical outdoor species with an alternation of hosts, e.g. A.fabae, is as follows.
The eggs are laid in the autumn on the winter host and are the form in which the majority of species overwinter. (Iyzus persicae and
Brevicoryne brassicae, among others, have been known to overwinter as viviparous females). In the spring, hatching occurs and apterous, parthenogenetic, viviparous females emerge. These are the fundatrices and are characterised by poorly developed sense-organs, legs and antennae, compensated by great fecundity.
The progeny of the fundatrices are the fundatrigeniae which are also apterous, parthenogenetic, viviparous females. A few to several generations of fundatrigeniae are produced during which increasing numbers of winged migrantes appear. Again parthenogenetic and viviparous these forms migrate to the secondary host where they give rise to the alienicolae.
The latter are the aphids commonly met with during the summer months. They frequently differ in details of appearance from the other forms and give rise to both apterous and elate forms by parthenogenetic viviparity. Many generations of alienicolae are produced and at the end of 8. the summer alatae occur which migrate back to the primary host. These are the sexuparae.
These, also parthenogenetic and viviparous, produce the sexuales which is the only time males are produced. The females are almost always apterous and are oviparous and are frequently characterised by thickened tibiae on the hind legs. Males are either alate or apterous. Pairing occurs and eggs are laid.
The causes and physiological mechanisms involved in the production of the various aphid forms are still imperfectly understood. The quality of the food, water-content of the host-plant, starvation and population density all seem to be involved. Photo-period and temperature also play an important part.
The biology of aphids has recently been reviewed by Kennedy and
Stroyan (1959) while Johnson (1958) has suggested that the same hormone system involved in insect metamorphosis may be involved in the determination of the alate or apterous condition in aphids.
Under glass the favourable conditions of temperature and humidity considerably modify the life-cycle of aphids. Continuous production of parthenogenetic females by viviparity may be almost indefinite. The forms produced are of the alienicolae type and may be apterous or alate.
It is these forms which have been used in the present work and the species involved were the following. 9.
1. Acyrthosiphon pisum. Mainly a pest of Papilionaceae out of doors, especially peas, clover and lucerne but also beans. 2. Aphis fabae. An important pest out of doors on beans and sugar-beet. Also occurs at times in glasshouses. The winter host is the spindle tree, Euonymus europaeus.
3. Aulacorthum circumflexum. A very common pest in glasshouses occur-ing on a wide variety of plants, particularly members of the Orchidaceae and Liliaceae. Reproduction is said to be entirely parthenogenetic. Sexuales
apparently do not occur. 4- lyzus Persicae. A very important species economically both out of door and under glass. Has a very wide variety of secondary hosts. The winter host is peach but the genus is also known to overwinter out of doors as viviparous females on brassicas. It is a vector of several important plant viruses such as "breaking" in tulips, sugar-beet yellows and some potato
viruses. 5. Rhopalosiphoninus latysiphon. Is occasionally a serious pest on stored bulbs and root vegetables and may occur in small numbers on other plants. The closely related species R.staphyleae is frequently found in very large numbers on clamped marigolds. 6. Sappaphis tulipae. fn important pest of stored bulbs, corms and tubers.
(b) Life-History of Whitefly. The best-known and most important species economically is the greenhouse whitefly, Trialeurodes vaporariorum (Westw.) and the following description applies to it in particular. There is no alternation of hosts 10. and the insects feed exclusively on the foliage of growing plants, especially tomatoes, cucumbers and beans. The eggs are cigar-shaped and about 0.25-0.30 mm. in length. They are laid in groups on the under-surface of young leaves and are frequently arranged in an arc or semi-circle when the leaf is not hairy. At first they are pale yellow-green in colour and have a covering of wax Particles.
The leaf surface in the immediate area of the eggs is also covered with wax. After a few days the eggs begin to darken in colour and become brown and finally purple-black. Several days before hatching, the waxy covering is shed leaving them smooth and shiny. Each egg is provided with a small stalk or pedicel which penetrates the leaf epidermis and acts as an organ for the uptake of water. At 20°C., hatching occurs about 10-12 days after laying. split appears at the free end of the egg and the first-instar "larva" emerges. This is pale green in colour, oval, flattened and about 0.3 mm. in length.
The larva has rudimentary legs and antennae and differs from later larvae in being exploratory. For up to three days after emerging it may wander about the leaf surface but finally settles permanently, close to a vein, inserting its stylets into the phloem. The duration of the instar is usually 7-8 days. Whitefly larvae have four pairs of ventrally placed spiracles and a simple tracheal system. There is also a structure known as the vasiform orifice on the dorsal posterior surface through which the honeydew is elimin- ated from the anus. A number of wax-covered spines occur laterally and dorsally and tend to become more numerous in successive stases. 11.
The second-iristar larva differs from the first in being almost completely transparent and much. larger (0.35-0.40 mm. in length).
It, also, is a much flattened, oval scale and occupies more or less the same position as the first-instar. It has a waxy covering which is secreted by a series of about 12 pairs of dorsal glands and, as in later larvae, the legs and antennae are vestigial and functionless. The general structure of the thirdinstar larva resembles that of of the second-instar larva but the marginal waxy fringe is denser and narrower. The larva is, however, larger, being about 0.50 mm. in length. A pair of ventro-lateral grooves are present (and in the next stage also) in the thoracic region and facilitate gaseous exchange via the spiracles. The second-instar lasts about 3-4 anys and the third 5-7 days at 20°c.
The fourth and final larva is frequently referred to as a 'pupa', which it resembles in several respects. At first the pupa is semi-transparent but as it increases in size it becomes more opaque. It varies in length from 0.70-0.85 mm. and. is considerably thicker than the previous larvae. Fully-formed it is a pearl- coloured, oval, box-like structure, elaborately adorned with waxy spines and processes of various sizes. There is an additional breathing fold round the spiracles lateral to the vasiform orifice. The duration of the pupal stage is about 10-12 days towards the end of which the adult, nearly ready to emerge, becomes visible. The adult emerges by a T-shaped rupture in the dorsal wall of the pupa and within an hour or so the wings are fully erect. Soon afterwards 12. the insect begins to acquire a loose covering of wax threads over the entire body.
The female varies in length from about 1.0-1.5 mm. and. the male is somewhat smaller.
By the time the adults emerge, which is about 40 days after err- laying, the leaves upon which they are found are becoming mature. Newly- emerged adults therefore generally migrate to the younger leaves where copulation and egg-laying take place.
Parthenogenesis is quite common in whitefly and it has variously been reported that unfertilised eggs give rise solely to males, solely to females, or to either sex. In the present cultures there was a high proportion of males and copulating pairs of insects were frequently observed.
The length of life of adult females is generally reported as being
30-40 days on averare and that of males somewhat less. Between 130-150 eggs may be laid by one female.
(Both Lloyd (1922) and Trehan (1940) reported the appearance of larvae lacking dorsal wax tubes and having marginal wax processes which were longer than usual. This type of larva has been identified as belonging to a different species, T.sonchi (Kotinsky), and apparently occurs quite commonly in association with T.vaporariorum.
In consequence, the larvae produced from the small number of adults used to start the present culture were carefully examined at frequent interv- als. Only typical T.vaporariorum larvae were found). 13.
4. Materials and Methods. (a) Insect Culture. The culture details for the seven species of insect are given in Table I. With the exception of Rhopalosiphoninus latysiphon, which was obtained from the Dunholme rield Station Laboratories in Lincolnshire, the
cultures were started from insects occurring naturally on available plant materials. Identification of the aphids was confirmed from various manuals (notably Theobald's "The Plant lice or Lphididae of Great Britain" 1926, 1927, 1929) and a key to economically important species by Stroyan (1952).
The aphid cultures were each started from one apterous female alienicola kept under conditions (Kenten, 195) ensuring the continued
production (by parthenogenesis) of this form only. By so doing a geneticall:? homogeneous population could be expected during the few months the cultures were maintained. This was considered to be of advanta6e in insects used in bioassay experiments. The same degree of uniformity, however, could not be hoped for in
the whitefly culture where reproduction was at least partly a sexual process. Consequently, to minimise deterioration in the stock due to inbreeding among the descendents of one pair of adults, four original pairs were selected,
each from a different plant. The procedures for setting up a culture and its routine management
were such that a continuous supply of insects of known age and stage was available. The principles involved were the same for both aphids and
Table I. Details of the insect cultures maintained.
Species Host-Plant Source of Insects Culture Conditions used in used to start the Culture Culture
Acyrthosiphon Broad bean Broad bean plants 20°C. and 65% R.K . pisum (Harris) Fl_asshouse in growth room. 18 hr. daylength.
Aphis fabae Broad bean. Broad bean plants I t (Stop.) out of doors Aul;lcort hum Tulip Tulip ulants in It circumflexum glasshouse (Buckt.)
1yzus persica° Turnip Sugar-beet pinnts I t (Sulz.) out of doors Rhooalosiphoninus Potato Dunholme Field At 20°C. in dimly- latysiphon Station (Lincs.) lit constant temp- (Davidson) erature room
Sappaphis tulipae Sprouting Stored tulip II (Ponsc.) tulip bulbs bulbs
Trialeurodes Broad bean Tomatu plants At 20°C. and 65%1/.11. vaporariorum in glasshouse in growth room. (Vestw.) 18 hr. daylength. 15.
whitefly but the details of the techniques, of necessity, differed. Those
for aphids will be outlined first.
A 'reservoirs population of several hundred aphids of all ages
was maintained, being kept at a manageable size by discarding a proportion
at regular intervals. From this population upwards of 100 mature females
were removed and transfe=ed to the young leaves of a new food-giant where
they were allowed to remain for 24 hours. The adults were then removed to
another plant leaving behind the nymphs born within the same period of
1 day‘ Repetition of this routine produced a series of plants each
supporting a culture of insects whose age was known.
The procedures involved in the whitefly cultures differed slightly
because the adults were winged. Infesting a plant necessitated enclosing
it first in a small muslin cage under which the adults were released. Egg
laying was then allowed to continue for 24 hours before the adults were
removed.
Handling of the insects was kept to a minimum. Aphids were
easily dislodged from a plant with a fine paint-brush and collected in a
small glass beaker held underneath. Transference to a new plant was effect-
ed by tapping the beaker and tipping the insects out onto the young leaves.
Whitefly adults were collected in an aspirator made from a small
glass specimen tube.
At all times careful attention was given to the conditions of
culture so that as nearly "standard" insects as possible were produced.
This was particularly important in the bio-assay experiments. 16.
To eliminate variations in susceptibility due to differences in nutrition one only of the known species of host-plant of a particular insect was used. The plants were young and with one exception were growing active- ly. Details of their cultivation are given in Table :VIII. (.82). In order, however, to produce tender shoots lacking in chlorophyll, which was the condition of the host-plant which the insects preferred aegowski and Gough, 1953), the potato plants used in the culture of
R.latysiphon were grown in near-darkness. The culture of Sappaphis tulipae was the only one maintained on non-growing plant tissues; sprouted tulip bulbs with green shoots l-12? in length were used. With the exception of the two aphid species just mentioned the cultures were housed in a plant growth-room specially constructed for the purpose. The temperature was kept constant at 20°C. and the humidity at
65% R.H. An 18 hour daylength was provided by a battery of ten 8' daylight fluorescent tube lights giving an intensity of illumination sufficient to maintain good growth of the plants. Mixed infestation was avoided by breeding not more than two insect species at a time and by keeping a close watch on the condition of the
'reservoir' cultures. There necessary, groups of plants were isolated on raised stands, whose legs rested in containers of diluted castor oil. Plants were isolated individually by placing them on up-turned
Petri-dish halves in a large tray of the diluted oil. The two cultures not kept in the growth room were kept in a poorly 17. lit constant-temperature room at 20°C. where there was no humidity control.
(b) Fumigation Procedure. i. Chamber techniques. The fumigations were performed in cylindrical steel chambers with a capacity of 20 litres. The inside surfaces were coated with a non-sorp ive paint and a large, freely movable piece of sheet aluminium was attached to the vertical wall. The chambers were closed by screwing down a steel 110. against a rubber gasket. Ampoules containing. a measured amount (see 0.25) of methyl bromide were broken in a plunger device in the lid and the gas was circulated by movinc the chamber onto its side and rocking it to cause movement of the aluminium vane. The doses produced inside a chamber by breaking ampoules containing different amounts of methyl bromide were thoroughly investigated before beginning experiments. 50 ml. samples of the fumigant-air mixture were withdrawn in evacuated glass sampling flasks and analysed by the combustion method of Lubatti and Harrison (1944). The mixture was drawn over heated platinum wire at about 700°C. and the combustion products collected in a mixture of Ny110 sodium hydroxide and hydrogen peroxide solution. Liter evaporation to dryness and acidification with acetic acid the bromide was estimated by titration against silver nitrate solution. The analyses confirmed the concentrations calculated from the volume of the chamber and the amount of methyl bromide in the ampoule and were omitted in subsequent fumigations. (Sorptive material inside the 18. chambers during fumicfation was negligible). The duration of the fumigations was always 1 hour and the chamber: were aired by simply removing the lid and withdrawing the insects. Control of the fumigation temperature was achieved by performing the fumigations in a constant-temperature room. Generally, the humidity Inside a chamber was that of the atmcspbere and fluctuated about 60% R.H. Men required, however, it was controlled by circulating the air in the chamber through solutions of sulphuric acid of appropriate density (Solomon, 1951). Immediately after leaving the chamber the air passed through a glass jar in which a piece of B.D.H. cobalt thiocyanate paper was suspended The jar could be removed from the circuit and the paper taken out and immers- ed in liquid paraffin and mounted folded over between two microscope slides. The humidity of the air in the chamber was estimated by comparing the colour of this paper with that of similarly mounted papers which had been hanging for many hours over sulphuric acid solutions giving several humidity values in the particular range (Solomon, 1957). This gave a means of checking the value of the humidity attained. ii. Exposure of insects. Aphids. The exposure of aphids to the fumigant while still on their host- plant proved impracticable mainly because of the behavioural responses to the gas, which differed with the dose, reported later (p.S8). Instead the insects were exposed in small glass specimen tubes 19. which were sealed with coarse muslin held in place by a rubber band. Another piece of muslin, in the shape of a narrow strip, was enclosed in the tube to provide an additional, usually more acceptable, surface for the insects to climb upon. The tube was fastened to a hook on the inside wall of the chamber by a loop of copper wire and the fumigation begtIti. The number of insects per tube was 30 and 2-3 replicates were exposed per treatment. Whiteflv. Adult whitefly were also fumigated in small glass specimen tubes closed with muslin, the tube initially forming part of the aspirator in which the insects were collected. The number of insects per treatment was variable. The sedentary habit of immature whitefly necessitated their exposure vrhilst still on host-plant material but at the same time presented no behavioural obstacles . A broad bean leaf was cut from the plant, together with most of the internode below, and placed in a specimen tube, the bottom of which was just covered by 3 ml. of water. The unit stood on the floor of the chamber (which could not, therefore, be tipped onto its side) during fumigation. Since sorption of methyl bromide by the leaf and water surfaces would occur the concentration of gas inside the chamber during these experiments was determined by the chemical analysis of samples. After airing, the leaf was transferred to a larger vessel of nutrient solution (formula p. 81 ) in the growth room. 20.
The number of eggs and larvae exposed at a time was difficult to regulate. They were counted under the microscope before treatment and any individuals of the wrong age were removed. when the population on a leaf was large the two leaflets were regarded as separate replicates. Most treatments had two replicates each of upwards of !.0 insects. iii. Pre-Fumigation treatment of insects.
For the majority of experiments the insects were bred in the growth room at the same temperature as that at which they were later fumigated.
On occasion, however, experimental temperatures differed from those at which the insects were cultured and an extended period of conditioning to the new temperature was then necessary.
Placts bearing the developing insects were removed from the growth room to a constant-temperature room a few days prior to treatment. The temperature of the room could be altered to correspond to the experimental temperature and an 18 hour photoperiod was provided. Although the intensity of illumination was rather low it was sufficient to maintain the plants in a more or less normal condition for at least the two days required.
It was believed that the change in light intensity did not greatly affect the insects as long as the photoperiod did not alter.
When it was desired to accustom insects in tubes to a given humid:- itys they were kept for two hours in a fumigation chamber in which the hum- idity had been standardised by circulating the air through sulphuric acid solutions. Circulation of the air was con:t.inodd for about fifteen minutes after enclosing the insects to facilitate the equilibration of the 21. humidity in the tubes with that of the chamber. iv. Post-Fumic7ation Treatment of Insects.
Lphids.
The obvious procedure after airing was to confine treated insects to a leaf of their host-plant. Several types of cage were constructed for this purpose but all had a number of disadvantages. Finally, the under- lying principle was rejected and a totally different procedure adopted. The reasons for this decision were the following:
(1) Fumigated insects showed a greater reluctance to settle on the
leaf than did control insects (p.(00), preferring the sides of
the cage. This behaviour, furthermore, changed with dose and
temperature.
Even when conditions were identical the proportion of insects on
the cage walls was extremely variable.
Small cages were necessary to increase the relative area of leaf
surface over cage-wall and in the confined space contamination
from water condensation and honeydew was rapid and the insects
soon spoiled.
Results obtained using leaf cages were very unsatisfactory and, since the degree of variability seemed dependent upon the dose and the post- fumigation conditions, had little value for comparative purposes.
The best results were obtained by the following post-treatment procedure.
The tubes of insects were removed from the fumigation chamber and 22. suspended over sulphuric acid solutions in bottles giviiv a relative humidity of 94 For 45-60 minutes after the addition of the tubes the air inside the bottles was bubbled throuc,h more acid of the appropriate density in another part of the circuit.
By so doing any alterations in humidity which occurred when the bottle was opened would be corrected and also the air in the tubes would be brought to the same humidity.
The purpose of the acid in the cage-containing bottles was to ensure the desired humidity was maintained after they were removed from the circuit and sealed.
This procedure ruled out any possible variations between successive sets of results attributable to sporadic feeding end made possible the
study of the effect of different post-fumigation humidities on survival.
A high humidity was essential to keep the rate of loss of water
as low as possible and 9 R.H. was chosen since by experiment it was the value found best to suit the insects.
The effect of starvation after fumigation on susceptibility will be discussed later (p.33 ). Whilst in the constant temperature rooms the insects were in dark- ness almost the whole time and unless otherwise stated the temperature was that at which fumigation took place.
Whiteflv.
Adult whitefly were treated in a similar manner to aphids after fumigation except that they were kept at 8071e R.H. At higher humidities 23. many of the insects stuck to the sides of the tube by their wings and died there.
Immature stages presented no special problem. They were kept in the „growth room at 20°C. regardless of fumigation temperature. v. Assessment of results.
Aphids.
At the end of the recovery period the insects were tipped onto a filter-paper in the bottom of a petri-cli sh. Those insects which were able to leave the spot where they fell and walk about the dish were recorded as 'survivors'; those unable to move at all and those lying ventral surface uppermost, feebly waving one or more legs, were recorded as 'knocked down'. Experience taught that those insects which, although upright, could only occasionally raise a leg and made hardly any progress forwards should also be recorded as knocked-down. The optimum time for assessing results in A. pisum was found to be
24 hours after the end of fumigation and in the other species 48 hours.
The validity of these criteria for separating survivors from the other treated insects was the subject of considerable experimentation and is dealt with in detail in the sections on behaviour on pp. Ss-6o.
The characteristics expected of a survivor were that it should be able to settle and feed on a leaf when given the opportunity and to reproduce.
It is sufficient here to record that all those aphids able to walk away at the end of the specified recovery period were capable of so doing 211..
but not the others.
Results obtained in this wry were repeatable many times.
An unfumigated control group of insects was kept under the same
conditions as the fumigated insects in each experiment. :However, deaths
in the controls, especially aphids, were so rare that these results have not been included in the tables in the next section. On the few occasions
when control mortality did occur the figures presented have been corrected
for this using Abbott's formula. They concern almost entirely the
results for adult vinitefly where insects were sometimes killed almost cert-
ainly as a result of being sucked into the aspirator.
Whitefly.
The tube of winged adults was tapped smartly so that the insects
fell into a petri-dish, the lid of which was quickly replaced. The
insects which flew to the sides and lid of the dish were those which given
the opportunity, settled on a broad bean leaf; they were called survivors.
Those remaining immobile on the floor of the dish were recorded as knocked-
down. Assessment was made 24 hours after the end. of fumigation.
"Larvae" regarded as survivors of a treatment were those which
continued their development to the emerged adult. All other larvae died•
Dead first, second and third-instar larvae could be recognised under
the microscope as discoloured, shrivelled scales. Dead fourth-instar larvae
were characterised by discolouration and the collapse of the dorsal body
wall.
Assessment of the effect of the fumigant on eggs was based upon 25.
whether or not hatchin-: took place and the larvae developed to at least the
third instal. Occasionally, eggs hatched but the larvae died before
completing emergence.
The adverse effect on freshly laid eggs could also be recognised
by their failure to darken in colour. Similarly, older eggs, if affected,
never shed their waxy covering and remained dull in appearqnce.
(c) Other Techniques.
i. Manufacture of ampoules.
Small glass phials of the sort generally used by the medical
profession for vaccines were used for making the ampoules in an apparatus
designed by Lubatti.
A small cylinder of methyl bromide was used to fill a fine-bore
burette from -which measured amounts of fumigant were run into weighed
ampoules cooled in solid carbon dioxide and alcohol. A small gas-flame
was used to seal the ampoule and the whole, including the glass fragment,
was weighed again.
The amount of fumigant in the ampoule was the difference between the two weights.
By chemical analysis it was shown that the liquid in the ampoules
could be recovered entirely as methyl bromide. There was no condensed we:ter present.
ii. Determination of the water content of ayhids.
A weighed quantity of aphids was transferred to a small porcelain crucible with lid and, dried to constant weight in an oven at 105°C. This 26. usually took 6-8 hours. The chine in weight was assumed to be entirely due to loss of water and the percentage chane in weiht was used as a measure of the oriinal water content of the insects. 27.
5. Factors affectinfr, the rosare-Mortalitv Response.
The results of the fumi,_;ation experiments with insects are presented in two parts. The present section deals with some of the factors affecting the susceptibility of aphids and whitefly to methyl bromide and the following section with some of the effects of the '.:as on the physiology and behaviour of these insects. .f PHIDS
Unless otherwise stated adult apterous alienicolae which had not begun to reproduce were used in the experiments.
For purposes of comparison and subsequent reference a composite diagram of the regression lines for the six species; fumigated at 15°G., is given in figure I (from anta in Tables Il, III) VI, VIII, IX and X).
The LD50 at this temperature ranged from about 15 mg./1. for 1 hour down to 5 mg./1. for 1 hour. iliuus persicae was the most resistant and
Acyrthosiphon pisum the least resistant.
The slope of the regression lines in most of the experiments was fairly steep, suggesting uniformity of resistance among individuals of the populations tested. )c 2 tests gave a value in every case less than p at was very small. the 0.5 level. The variance of the estimated ID50 (a) kaa and Form. Preliminary tests with each species showed conclusively that immat• ure forms of both alate and apterous alienicolae were more susceptible than the adults to methyl bromide poisoning. A precise determination of the level of susceptibility of each instar, however, was not attempted. Lny 28.
Table II. The susceptibility of :tpterae of M.persicfte at 150c.
C.T.P. % Knockdown Log.C.T.P. Pi-obit
1 2 Mean
13.14 26.67 23.33 25.00 1.1186 4.33 15.11 40.00 4.6.67 43.33 1.1793 4.83 16.61 70.00 73.33 71.67 1.2204 5.57 18.90 93.33 93.33 93.33 1.2765 6.50 19.83 96.67 96.67 96.67 1.2973 6.84
Regression Equation : y 14.169x - 11.674 LD50 = 15.02 (C.T.P.) LD90 = 18.50 (0.T.P.)
Table III. The, susceptibility of Apterao of A.fabae at 15°C.
C.T.P. 7 Knockdown Log.C.T.P. Probit 1 2 Mean
6.277 20.00 23.33 21.67 0.7978 4.22 7.600 53.33 4.6.67 50.00 0.8808 5.00 8.452 73.33 70.00 71.67 0.9270 5.57 9.810 96.67 96.67 96.67 0.9917 6.84 10.01 100.00 96.67 98.33 1.0005 7.13
Regression Equation : y = 13.128x - 6.410 LD50 . 7.40 (C.T.P.) ; LI0190 = 9.26 (C.T.P.) FIG. I. THE SUSCEPTIBILITY OF ADULT AP TER A E OF SIX SPECIES OF
0 APHIDS AT 15 C
vi
6
5
4
PROS! T O A. PISUM 3 • A. FABAE ;i; x R. LATYSIPHON .• 0 S. TULIPAE 2 v • A.CIRCUMFLEXUM v; o M.PERSICAE
1
0 0.5 0.7 0.9 1.1 1.3 LOG. CTP 29. concentration which vas just sufficient to kill the most resistant adult also complete kill of all nymphal stav es. In general, the youngest instars had the least resistance.
The possibility of a change in susceptibility within an instar was not easy to study in insects which moulted almost daily. Experience with adults, however, suggested that such changes may occur at this stage, which is of much lorloDr duration than any other.
The observation was tested with A.pisum.
Adults were removed at intervals from a large culture of insects born on the same day, and fumigated. The first batch was taken soon after the last moult and the second batch two days later, when reproduction had just begun. A third batch was taken when the adults were 52 days old and considerable reproduction had taken place.
The results are shown in Table IV and figure II.
There was no appreciable difference between the susceptibility of newly-emerged adults and that of adults two days older which had just begun to reproduce. Susceptibility, however, was much higher towards the end of adult life when considerable reproduction had taken place.
Concentrating next on insects of the same age but different form, the susceptibilities of recently emerged apterous and alate alienicolae were compared at the adult stage. Table V contains the results of fumigationn. with A.Pisum and
Table VI with S.tulipae. The regression lines based on the tables are shown in figures III and IV. 30.
Table IV. The effect of ae (and reproductil-e state) on susceptibility in
adult Jab erap of Pisum c t 20°C.
Insects C.T.P. o, KmoCKbov.MO Log. Probit C.T.P. 1 2 ;lean
Young adults 3.033 10.00 10.00 10.00 0.4818 3.72
No reproduction 3.4.27 16.67 13.33 15.00 0.5349 3.96
4.171 80.00 86.67 83.34 0.6202 5.97 5.051 100.00 96.67 7.12 -- 98.34 0.7034 Mature adults 3.170 20.00 16.67 18.34 0.5011 4.10 just beginning 3.379 30.00 30.00 30.00 0.5288 4.48
to reproduce 4.007 90.00 80.00 85.00 0.6029 6.04. 5.371 96.67 96.67 96.67 0.7301 6.84
Old adults after 2.700 46.67 46.67 46.67 0.4314 4.92 considerable 2.887 53.33 46.67 50.00 0.4605 5.00 reproduction 3.481 83.33 90.00 86.67 0.54.17 6.11 3.980 100.00 96.67 98.34 0.5999 7.12
Insects Regression Equation 1450 LD90 (C.T.P.) (C.T.P.)
Young adults y = 17.261x - 4.877 3.73 4.43 Mature adults y = 14.352x - 3.005 3.61 4.44 Old adults y = 12.4.80x - 0.599 2.81 3.56 FIG.5. THE EFFECT OF AGE (AND REPRODUCTIVE STATE) ON SUSCEPTIBILITY IN ADULT APTERAE OF A. PISUM AT 20C°
8 1ii
6
PROBIT
4 I YOUNG ADULTS
I' MATURE ADULTS W OLD ADULTS 2
O 0 0.2 0.4 0.6 0.8 LOG. CTP
FIG.111. THE SUSCEPTIBILITY OF APTERAE AND ALATAE OF o A. PISUM AT 20 C
8
6
PROBIT
4 / o APTERAE x ALATAE FROM PLANT • ALATAE FROM CAGE WALL 2
O 0 0.2 0.4 0.6 0.8 LOG.CTP 31. Table V. The susceptibility of Lpterae and Alatae of A.pisum at 20°C.
Insects C.T.P. ;;Hnockdown Log.C.T.P. Pi-obit 1 2 Mean _
Apterae 2.760 10.00 6.67 8.34 0.71)1 09 3.62
3.549 40.00 40.00 40.00 0.5501 4.75 3.924 66.67 73.33 70.00 0.5937 5.52 4.621 90.00 96.67 93.33 0.6647 6.50
Alatae from 2.850 13.33 20.00 16.67 0.4548 4.03 plant 3.200 46.67 53.33 50.00 0.5051 5.00 3.428 90.00 86.67 88.34 0.5350 6.19 3.890 90.00 96.67 93.33 0.5899 6.50
Alatae from 2.402 10.00 6.67 8.34 0.3806 3.62 cage wall 2.984 53.33 60.00 56.67 0.4746 5.17 3.232 90.00 83.33 86.67 0.5095 6.11
Insects Regression Equation LD50 (CTO 13190 (CTP)
Apterae y = 13.084x - 2.294 3.61 4.52 Alatae (plant) y = 20.567x - 5.259 3.15 3.64 Alatae (cage) y = 18.803x - 3.621 2.87 3.36 32. Table VI. The susceptibility of Apterae and Alatae of S.tulipae at 15°C.
Insects C.T.P. % Knockdown Log.C.T.P. Probit 1 2 3 Mean
Apterae 6.301 0.00 0.00 10.00 3.33 0.7994 3.16 6.590 13.33 3.33 6.67 7.78 0.8189 3.58
10.02 70.00 63.33 63.33 65.55 1.0009 5.40 10.80 96.67 96.67 100.00 97.78 1.0334 7.01
Alatae 5.761 10.00 16.67 - 13.33 0.7605 3.89 7.671 66.67 63.33 - 65.00 0.8849 5.39 8.523 86.67 83.33 - 85.00 0.9306 6.04
9.287 96.67 96.67 - 96.67 0.9678 6.84
Insect Regression Equation LD50 (C.T.P.) LD90 (C.T.P.)
Apterae y = 12.793x - 7.035 8.73 11.00 Alatae y = 13.253x - 6.245 7.06 8.81
In both species the apterae were less susceptible than the winged forms. Furthermore, in A.,pisum, alatae which had spent some considerable time off the plant on the cage wall were even less resistant. Experiments with sexual forms and eggs were not performed with any of the species. FIG. IV. THE SUSCEPTIBILITY OF APTERAE AND 0 ALATAE OF S. TULIPAE AT 15 C
B
6
PROBIT 4
x APTERAE 2 0 ALATAE
O
0.2 0.4 0.6 0.8 1.0 LOG. CTP 33. (b) Food.
The experimental procedure followed excluded the provision of
food for the aphids during and after fumigation. (The reasons for this
have already been given on p.2.2.).
It was therefore important to determine what effect the lack of
food had on the value of the results obtained. Experiments were performed
in which some replicates of treated insects were provided with food after
fumigation and some were not. The important point to keep in mind is that although fumigated
insects may be provided with perfectly acceptable food it is by no means
certain that they will settle and feed. (This will be appreciated more
fully after reading the account of the behavioural resnonses of aphids to
methyl bromide onpp.s3-60). Even under identical conditions the number
which settles is extremely variable. Adults of A.pisum and A.circumflexum were fumigated at a number
of C.T.P.s at 15°C. and afterwards some were confined to leaves of their
host plant.
The broad bean leaves for A.pisum were young ones cut from the
plant: together with the internode below, and corked into phials of nutrient
solution. The tulip leaves for A.circumflexum were whole ones pulled from
young plants and similarly maintained in nutrient solution. Both sets were kept in a dimly-lit constant-temperature room at
15°C. under a polythene cage in which a relative humidity of c.80%; was
maintained. 3i.
The insects were confined to the leaves by muslin sleeves.
No Oifference in mortality was detected between the starved and un-starved insects. This was largely because so very few of the insects provided with food were feeding so that in effect both groups were starved.
Furthermore, it was only at the low doses that insects were known to feed.
Unfumigated aphids used as controls in these experiments readily settled and fed.
(c) Water Content.
The water content of the adults of three species was determined by drying samples of 200 or more insects to constant weight in an oven at 10500.
The LD50 at 15°C. and 90% past-fumigation R.H. was also determined for the same batches of insects.
The results are given in Table VII.
From the limited data obtained a correlation between water-content and susceptibility to methyl bromide did not appear to exist.
(d) Temperature.
The effect of temperature on susceptibility in the three species
A.Pisum, R.latysiphon and A.circumfleXum is shown by the results of fumigations given in Tables VIII, IX and X (Figures V, VI and VII).
As the temperature was increased susceptibility also increased, the increase being roughly proportional to the rise in temperature. 35.
Table VII. The water content of adult Lpterae of three species of Aphid and the LD50 at 15°C.
Species LD50 Water Content (o Total Wt.)
1 2 3 4 Mean
A.pisum 5.012 78.3838 79.2841 78.8930 _ 79.0110
A.circumflexum 12.45 74.3615 73.2017 73.7147 .. 73.7593 S.tulipae 8.610 68.4068 61.5123 63.7147 62.4041 64.0095
Approx. vt. 200 adults Aopisum = 1.2 gm.
" 300 L.circumflexum = 0.3 Gm.
tt " 250 S.tulipae = 0.2 Gm. 36. T4b1e VIII. The effect of temperature on the susceptibility of adult APterae of A. piston.
C.T.P. Fumigation % Knockdown Log.C.T.P. Probit Temp. 1 2 3 Mean
3.891 15°C. 10.00 10.00 10.00 10.00 0.5900 3.72
5.500 fl 60.00 63.33 70.00 64.44 0.7404 5.37 5.890 it 70.00 70.00 66.67 68.89 0.7701 5.49
6.303 ti 93.33 93.33 90.00 92.22 0.7995 6.4.2 7.000 It 100.00 93.33 100.00 97.77 0.8451 7.01
2.985 20°0. 26.67 13.33 16.67 18.89 0.4749 4..12
3.550 p, 33.33 4.3.33 36.67 37.78 0.5502 4..69 4.170 it 73.33 76.67 76.67 75.56 0.6201 5.69 4 .521 t, 86.67 80.00 90.00 85.56 0.6552 6.06
5.309 /7 100.00 100.00 93.33 97.77 0.7250 7.01
2.649 2500. 4.0.00 33.33 56.67 4.3.33 0.4231 4..83
3.055 ft 63.33 60.00 60.00 61.11 0.4.850 5.28 3.312 ,, 83.33 80.00 80.00 81.11 0.5201 5.88 4..370 ti 100.00 96.67 100.00 98.89 0.64.05 7.29
1.590 30°C. 10.00 3.33 3.33 5.55 0.2014. 3.41 1.821 it 20.00 20.00 16.67 18.89 0.2603 4.12 1.958 tt 20.00 30.00 26.67 25.56 0.2917 4.34
2.600 /I 90.00 93.33 90.00 91.11 0.4150 6.34. 3.020 ,, 100.00 96.67 96.67 97.77 0.4.800 7.01
CONTINUED ON NEXT PAGE. 37. Table VIII Cont.
Temp. Regression Equation LD50 (C.T.P.) LD90 (C.T.P.)
15°C. y = 12.092x - 3.535 5.08 6.48 20°C. y = 11.552x - 1.485 3.64 4.70 25°C. y = 10.949x + 0.124 2.79 3.65 30°C. y = 13.776x + 0.488 2.13 2.63
Table IX. The effect of temperature on the susceptibility of adult Apterae of R.latysiphon.
C.T.P. Fumigation % Knockdown Log.C.T.P. Probit Temp. 1 2 3 lean 8oc. 11.42 20.00 30.00 30.00 26.67 1.0577 4.38 12.09 TI 50.00 33.33 60.00 47.78 1.0824 4.95 13.04 ti 80.00 76.67 76.67 77.78 1.1152 5.77 13.46 t, 96.67 96.67 90.0o 91.11 1.1290 6.35 13.74 ,, 96.67 loom 100.00 98.89 1.138o 7.29
7.37o 1500. 16.67 23.33 13.33 17.78 0.8675 4.08
8.140 II 30.0o 33.33 30.00 31.11 0.9106 4.51
8.470 33.33 33.33 40.00 35.56 0.9279 4.63 9.W ,. 80.00 70.00 66.67 72.23 0.9611 5.59
9.622 II 86.67 93.33 80.00 86.67 0.9833 6.11
I Temp. Regression Equation LD50 (C.T.P.) LD90 (C.T.P.) 8°C. y = 29.215x - 26.619 12.09 13.37 1500. y = 17.922x - 11.712 8.56 10.09 1 38. Table K. The effuet of temperature on the susceptibility of adult Apterae
of A.4rq1.151flexum.
C.T.P. Fumigation 5 Knockdown Lo ;.C.T.P. Probit Temp. 1 2 3 Mean 1 10.* 15°C. 0.00 10.00 3.33 4.bh 1.0216 3.29
11.47 11 20.00 20.00 20.00 20.00 1.0596 4.16 12.32 t? 53.33 56.67 50.00 53.33 1.0906 5.08 12.70 " 70.00 60.00 60.00 63.33 1.1038 5.34
13.47 t, 93.33 96.67 90.00 93.33 1.1287 6.50
8.011 20°C. 16.67 10.00 26.67 17.78 0.9037 4.08 8.355 " 16.67 10.00 13.33 13.33 0.9220 3.89 8.701 ,, 23.33 26.67 20.00 23.33 0.9395 4.27
II 9.441 43.33 40.00 36.67 40.00 0.9750 4.75 10.17 ?, 63.33 66.67 60.00 63.33 1.0073 5.34 11.09 ,/ 90.00 93.33 loom 94.44 1.0449 6.59
Temp. Regression Equation LD50 (C.T.P.) LD90 (C.T.P.)
15°C. y = 28.315x - 25.781 12.22 13.56 20°C. y = 17.100x - 11.737 9.53 11.31
THE EFFECT OF TEMPERATURE ON THE SUSCEPTIBILITY OF ADULT APHIDS (APTERAE)
FIG.V. A. PISUM FIG.TI• R. LATY51PHON •
8 30° 25 20° 15°C 8 15 8C
6 6
0 cr 0 a. 4 cr 4 a.
2 2
O O 0.1 0.3 0.5 0.7 0.9 06 0.8 1.0 1.2 1.4 LOG. CTP LOG.CTP
FIG. VII. A.CIRCUMFLEXUM
6
o— cra 0 4 ct a
0 0 6 0.8 1.0 1.2 1.4 LOG. CTP 39. (e) Humidity.
The effect of the humidity of the air immediately surrounding the
insects on susceptibility must be considered separately for each of the three
phases of fumigation viz. (i) the pre-fumigation period, (ii) the duration
of the fumigation itself and (iii) the post-fumigation period.
Pre-treatment of insects at different humidities necessitated
their removal from the plant and it was found that periods of up to 12 hours
at atmospheric humidity in the laboratory 'Produced little or no change in
the susceptibility of A.circumflex-um or M.persicae. Severe dessication at
25% R.H. at 17-18°C. for 15 hours, however, resulted in a very slightly
increases susceptibility and the appearance of poisoning symptoms sooner
than usual after fumigation.
In a series of experiments with A.circumflexum taken from wilted
tulip leaves no evidence was obtained of a change in susceptibility due to
a deficit of water in the food plant.
To test the effect of different relative Humidities inside the
chamber during fumigation the three species A. pisum, A. circumflexum and
A.fabae were used.
The experiments were performed at 15°C. and 20°C. at relative
humidities of 90%, 65% and 25%. The post-fumigation R.H. was in each case
90%.
When the duration of the fumigations was 1 hour it was not possible
to demonstrate any difference in susceptibility at the different humidities.
The experiments with A.circumflexum at 2000. were repeated and
the duration of the fumigations extended to 3 hours. 40. Again no difference in susceptibility at different humidities was detected.
The importance of humidity was greatest in the recovery period after fumigation.
A.circumflexum was fumigated at a number of C.T.F.s at 15oC. and kept afterwards at several different humidities.
As customary with this species the results of the fumigations were assessed after L8 hours and are given in Table XI (Figure VIII).
A low humidity after fumigation had the effect of increasing the knockdown at a given dose. This was particularly marked after high doses.
The survivors of this series of fumigations were kept under observation for a further two days. They were transferred to tulip leaves at 15°C. and although many took several hours to settle all of them did so eventually. The majority began to reproduce. Very few died.
Aphids thus seem better able to survive the effects of moderate fumigation if kept afterwards under conditions where water loss is reduced to a minimum.
The length of time which aphids remained capable of making spont- aneous movements of the legs and antennae after being knocked down by the fumigant was also affected by the post-fumigation humidity at certain doses.
Keeping aphids at a high humidity after doses slightly in excess of the
LD99 considerably delay ed their death. Similarly low humidities hastened death. At C.T.P.s in the region of 30-40 at 15°C., however, insects of all the species tested were usually completely paralysed even by the end of 41. Table XI. The effect of post-fumigation humidity on survival in A.circumflexum at 15°C.
Knockdown R.H. 95% 80% 7o% 6o% 45% 25%
C.T.P. 16.67 20.00 23.33 23.33 4.0.00 60.00 11.17 13.33 23.33 26.67 33.33 40.00 53.33 15.00 21.67 25.00 28.33 4.0.00 56.67 Mean
30.00 36.67 4.6.67 63.33 93.33 100.00 11.99 36.67 4.3.33 50.00 63.33 86.67 100.00 r-- 33,33 4.0.00 4.8.33 63.33 90.00 100.0o Mean
76.67 80.00 93.33 100.00 100.00 100.00 12.71 66.67 80.00 96.67 loom 100.00 loom 71.67 80.00 95.00 100.00 100.00 100.00 Mean
fumigation and died soon afterwards regardless of humidity. Although at first this observation may not appear of more than passing importance it was made the subject of a series of experiments in view of its relevance to practical fumigations. At the time, the experimental fumigations of marigold clamps (see p. p.) were being performed and some doubt was at first expressed by some observers about the efficacy of the treatments prescribed in view of the fact that several days after treatment very large numbers of insects could be found which were still moving. FIG. VIII.THEEFFECTOFPOST-FUMIGATIONHUMIDITYON
KNOCK DOWN 100 80 60 40 20 0 0
SURVIVAL INA.CIRCUMFLEXUMATI5 (iii) CTP:12.71 20 CTP: 11.17 CTP: 11.99
• 40
60 •
80
100 ° ° C / ( 0
) t2. The feeding and reproductive behaviour of these insects will be discussed in the next section (p.50A and only the experiments to determine the time of death under different conditions will be dealt with here.
Between 150-200 adults of S.tulipae and A.eircumflexum, together with a large number of immature stages, were confined in glass cylinders closed at both ends with coarse muslin. The cylinders contained additional strips of muslin for the insects to disperse upon and were suspended over sulphuric acid solutions in jars.
First, however, some of the insects were fumigated. at 1500. at doses slightly in excess of the LO99.
Insects were kept at three temperatures and three humidities at each temperature and the maximum survival time at each was determined.
This was done by examining the insects in the morning and evening of successive days and noting the first occasion when no further movement could be detected.
The results are given in Tables XII and XIII (Figures IX and X).
The survival time increased both with humidity and a fall in temperature and was always greater in control than fumigated insects. At
800. and 90% R.H. movements were still detectable a week and more after fumigation.
It should be stressed that in toxicity tests performed simultan- eously with these experiments, using adults from the same cultures, all the insects were recorded as 'knocked down' 48 hours after fumigation at these doses. 43.
Table XII. The survival times of fumigated and unfumigated adult Apterae
of S.tulipae at different temperatures and humidities.
Temp. 22°C. 15°c. 8°C. R.H. 90% 60% 25% 90% 60% 25% 90% 60% 25%
Fumigated 96 70 47 128 96 58 170 130 81 Control 156 110 78 265 174 110 410 270 202
C.T.P. = 15.52 at 15°C. ; LE90 = 11.00
Table XIII. The survival times of fumir;ated and unfumigated adult ApLerae
of A.eircumflexum at different temperatures and humidities.
Temp. 22°C. 15°C. 8°C. R.H. 90% 60% 25% 90% 60% 25% 90% 60% 25%
Fumigated 87 76 63 107 96 80 194 158 105 Control 160 117 87 259 173 101 380 191 217
C.T.P. = 1(1%17 at 15°C. LD90 = 13.56 FIG. IX.THESURVIVALTIMESOFFUMIGATEDANDUNFUMIGATEDADULT et SURVIVAL T IME - 411 280 400 200 240 320 360 120 160 80 40 0 APTERAE OFS.TULIPAEATDIFFERENTTEMPERATURESAND 22 HUMIDITIES. CTP.,15.52AT15
FUMIGATED 15
8 ° C
IMP 22 UNFUMIGATED
° C. Is
7 8 °C 111 ' 90 60% R.H 25 JoR.H. ° c 'h, R.H. SURVIVA L TIME• HRS, 200 280 400 240 360 320 160 120 40 THE SURVIVALTIMESOFFUMIGATEDANDUNFUMIGATEDADULT 80 0 AP TERAEOFA.CIRCUMFLEXUMATDIFFERENTTEMPERATURES 22 AND HUMIDITIES.CTP*19.17AT15 FUMIGATED
1 IS
8 ° C
22 UNFUMIGATED
IS °
C. 8 ° C 25% R.H. 90% R.H. 6 0%R.H Whitefly.
(a) The susceptibility to methyl bromide of all the stages in the
life-history from egg to adult was tested. The fumigations were performed
at 15°C. and the insects kept afterwards in the growth room at 20°C. (except the adults, which were kept in humidity bottles in a constant temperature
room, also at 2000.). The results are given in Table XIV and Figure XI.
The order of increasing resistance was as follows: adults - first
instar larvae - eggs - second and third instar larvae - fourth instar
larvae ("pupae").
The adults tested were taken 3-5 days after emergence, when still
young. An equal susceptibility was found in the two sexes in preliminary
tests and consequently mixed adults were used in all subsequent experiments.
First, second and third instar larvae were fumigated as soon as
possible after ecdysis. Fourth instar larvae were fumigated about the
middle of the instar just before the eyes of the developing adult became
heavily pigmented. Eggs were fumigated at two stages; when newly-laid and therefore
yellow in colour and when much older and darker and about to shed the waxy
covering. The younger eggs were slightly more susceptible. 45.
Table XIV. The susceptibility of the ft fferent st ;es of Whitefly to Methyl bromide at 15°C.
Stage C.T.P. Nb. No. Do. No. Mean. Log.C.T.P. Probit Tested Killed Tested Killed 5, Kill
Young eggs 14.12 50 26 - - 52.00 1.1498 5.05 (yellow) 15.45 47 36 - - 76.60 1.1889 5.73 19.10 33 31 - - 93.94 1.2810 6.55 21.32 41 40 - - 97.56 1.3288 7.00
Eggs (black 14.12 29 10 36 12 33.91 1.1498 4.59 and shiny) 19.10 51 43 35 31 86.44 1.2810 6.10
21.32 43 40 33 32 95.00 1.3288 6.65 23.17 37 35 29 29 96.97 1.3649 6.87
Instar I 7.584 27 6 37 9 23.27 0.8799 4.27 8.581 33 11 30 11 35.01 0.9336 4.62 12.60 38 37 27 25 94.98 1.1004 6.65 15.91 30 29 33 33 98.34 1.2017 7.13
Instal- II 17.77 33 9 34 10 28.34 1.2497 4.43 27.60 36 29 47 40 82.84 1.4409 5.95 33.02 47 45 - - 95.75 1.5188 6.73
Instar III 18.20 40 10 - - 25.00 1.2601 4.33 21.51 36 18 - - 50.00 1.3326 5.00 24.20 41 35 - - 85.37 1.3838 6.05
29.00 40 38 - - 95.00 1.4624 6.65
COPT INIJED ON NEXT PAGE . 46.
Table XIV Cont.
Stage C.T.P. No. No. No. No. Mean Log. Probit Tested =led Tested Killed % Xill C.T.P.
Instar IV 28.22 37 10 27 7 26.48 1.4505 4.37 32.36 41 23 24. 14. 57.22 1.5100 5.18 40.80 50 47 25 23 93.00 1.6107 6.48 47.86 39 35 33 33 94..87 1.6799 6.64
Adult 2.505 81 6 80 6 7.4.5 0.3988 3.56 3.320 74 4-1 76 4.0 54.02 0.5211 5.10 3.4.86 128 91 114. 76 68.88 0.5424 5.49 3.997 151 121 123 99 80.32 0.6018 5.85
Stage Regression Equation LD50 (C.T.P.) LE90 (C.T.P.)
Eggs (yellow) y = 10.832x - 7.304 13.68 17.95 Eggs (black) y = 11.283x - 8.384 15.35 19.94 Instar I y = 10.076x - 4.674 9.12 12.22 Instar II y = 8.157x - 5.774 20.94 30.05 Instar III y = 12.409x - 11.355 20.80 26.38 Instar IV y = 10.924x - 11.377 31.56 41.34 Adults y = 11.207x - 0.753 3.26 4.24 FIG. XI. THE SUSCEPTIBILITY OF THE DIFFERENT STAGES OF WHITEFLY TO METHYL
• BROMIDE AT 15 ° C
7
6
5 I 0 4 cC a. 3
2
0 0 3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 LOG. CTP
I ADULTS V INSTAR II II INSTAR T VI INSTAR III III EGGS- YELLOW VII INSTAR IV IV EGGS- BLACK 47.
(b) Temperature.
Adult whitefly were fumigated at 15°C. and 25°C. and kept after- wards at the same temperatures and 8O5 R.H.
The results are given in Table XV and Figure XII.
Susceptibility increased with temperature. The effect of other factors on the susceptibility of whitefly was not investigated.
Table XV. The effect of temperature on the susceptibility of adult Whitefly%
C.T.P. Fumigation No. No. %Ian Log.C.T.P. Probit Temp. Tested Killed
2.531 15°C. 157 16 10.19% 0.4033 3.73 3.010 t, 165 61 36.97 0.4.786 4.67 4.523 t, 168 146 86.91 0.6554 6.12 5.056 II 162 158 97.53 0.7038 6.96
1.542 25°C. 74 15 20.27 0.1881 4..17 1.992 It 82 57 69.51 0.2993 5.51
2.570 1, 66 63 95.4.6 0.4099 6.70
3.020 It 70 68 97.14 0.4800 6.90
Temp. Regression Equation LD50 (C.T.P.) Lt90 (C.T.P.)
15°C. y= 9.621x - 0.033 3.34 4.53 25°C. y = 10.323x + 2.324 1.82 2.42 FIG. XII. THE EFFECT OF FUMIGATION TEMPERATURE
ON THE SUSCEPTIBILITY OF ADULT WHITEFLY
8 25°C 15 °C
6
PROBIT
4
2
0 0 0.2 0.4 0.6 0.8 LOG. CTP 48. 6. Some effects of Methyl bromide on the physiology and behaviour of the
insects.
(a) Development and Ecdysis.
After exposure to a very low concentration of gas, aphid nymphs
of all species appeared to continue their development normally, if settled
on a leaf, moulting at frequent intervals. At higher doses, however,
including those in the lower part of the lethal rangei insects were commonly
found which failed to moult though they remained alive for several days.
They were apparently able to withdraw from the old exocuticle but were
unable tb emerge. Such insects did not feed and had to be kept at a high
humidity.
The length of time a partially-moulted nymph remained alive varied
with age among other factors. Older and larger nymphs survived in this
condition longer than the very young nymphs.
The effect of methyl bromide on the emergence of alate A.pisum
was also studied.
Final-instar nymphs were fumigated at 15°C. and kept at a R.H.
of 90;, at the same temperature afterwards.
Following doses which were just sub-lethal adults emerged but
1-2 days later than from unfumigated nymphs of the same age. (The term
"sub-lethal" in this thesis refers to doses which are insufficient to cause
the knock-down of even a single test insect but are nevertheless approaching
the order of magnitude of doses which do cause some knock-down i.e. lethal
doses). These retarded adults commonly had crumpled wings which were never
erected even though they seemed to be quite normal morphologically. Most 49. of the insects could walk properly and a few, at least, were observed with their stylets inserted in a leaf. Out of 47 adults with crumpled wings in an experiment, 21 also had at least two segments of each antenna missing and 2 had no antennae at all.
Seven of those with malformed antennae also had great difficulty in walking, especially on glass. The tarsi of these insects were found to be vestigial or, in some cases, non-existant.
Such insects frequently remained alive for several days but did not reproduce.
In whitefly, low (sub-lethal) doses appeared to have no effect on the hatching of eggs and development time was little affected.
The development of whitefly larvae after fumigation was not easy to follow; they either became discoloured and shrivelled and died or con- tinued apparently normal development. Only occasionally were partly- emerged larvae found and these were always in the third instar. Fourth instar larvae, however, commonly developed to the stage where a future adult with wing-buds and pigmented eyes could be recognised inside but the adult generally failed to emerge.
Dissection of such larvae exposed a small but well-formed adult insect capable of movement when stimulated. On a few occasions emergence began but was never completed and the withered, half-emerged insect lived only a short time.
Twice, fully-emerged insects were found which had the wings on the right side missing. 50.
The arrest of development in whitefly eggs of different ages at the doses specified has already been referred to (p.2.5). Mention has
also been made of the failure of some first-instar larvae to complete emerg- ence from the egg after fumigation at certain doses.
(b) Reproduction. Mature aphids continued to produce nymphs for at least a few
hours after fumigation, even at doses slightly in excess of the 10199.
However, by the time toxic symptoms were developing reproduction was nil.
The greatest number of nymphs was produced at high humidities and
after a few hours at R.H.s below 50% dessicated, cigar-shaped capsules were
laid. The doses which were just sufficient to inhibit completely the
production of nymphs after fumigation at 15°C. by insects kept at 90% post
fumigation R.H. were determined for a number of species. The results are
given in Table XVI. The doses required to prevent reproduction entirely were appreciab
greater than the LD90. The viability of nymphs laid by fumigated aphids was very high,
unless they were born in a dessicated state or during fumigation at a
lethal concentration. In an experiment, every one of the nymphs produced at 90% R.H.
by a group of 50 adults of A. circumflexum after fumigation at the 1080 at
15°C. and 20°C. settled and fed and grew to maturity when transferred tO a
tulip leaf. At no time was there any evidence that methyl bromide increased 51.
Table XVI. The minimum C.T.P. tote ley inhibiting reproduction in three
species of Aphid p.t 15°C.
Species L.D.90 (C.T.P.) Minimum dose (C.T.P.) inhibiting reproduction
A. pisum 6.48 12.5
A. circumflexum 13.56 22.0 R. latysiphon. 10.09 18.5
the amount of reproduction. Those insects which remained permanently settled on a leaf after fumigation continued to reproduce at a rate similar to unfumigated aphids. The rate of reproduction of poisoned insects however, decreased rapidly as toxic symptoms appeared.
The reproduction of fumigated whitefly was not studied.
(c) Water loss.
Because water loss played such an obviously important part in determining survival, a short investigation of the rate of water loss by aphids after fumigation was made.
Day-old apterous adult alienicolae of A.pisum were used.
The insects were fumigated at 15°C. and 90;;', R.H. and after airing numbers of them were transferred to small muslin cages and suspended in a series of bottles through which air of 60%R.H. was circulated. 52.
The cages were supported by a wire frame and when empty weighed about 7.0 gm.
(Metal-wire cages had the disadvantages that unless the mesh was
very fine, newly-born nymphs escaped, also the adult insects tended to crowd together in one corner.
In the muslin cages the nymphs could not escape and the adults dispersed well).
Prior to the experiment, the cages were kept in air of 6070 R.H.
for 24 hours at 15°C. They were then removed and weighed in a close-
fitting weighing-bottle at the same temperature.
200-250 fumigated insects (average wt. per aphid = 6 mg.) were weighed in a small weighing-bottle and transferred to a cage which was quickly returned to its humidity bottle. After 2L hours the cage and insects were removed and weighed again.
The loss in weight was regarded solely as loss of water from the insects and the percentage loss in weight of each group was taken as a
measure of the extent of water loss. The results are given in Table XVII.
Insects were fumigated at three C.T.P.s and an unfumigated control was set up.
The amount of water lost by the aphids receiving the sub-lethal dose (C.T.P. = 1.621) and those the threshold dose (C.T.P. = 3.317) was almost the same as the amount lost by the unfumigated controls. In the aphids which received the dose approaching the LD90, however, the amount of water lost was greater. 53. Table XVII. The % loss in weight of adult A.pisum at 1500. and 60% R.H.
C.T.P. Loss in Weight
2 Mean
1.621 27.01 26.69 26.85
3.317 28.77 26.22 27.50
6.024 28.89 30.04 29.47
Control 27.34 28.01 27.68
ID90 at 15°C. = 6.48
(d) Activity Level.
This is the first of the behavioural reactions to methyl bromide
to be examined and the observations relate entirely to aphids, in particular
A.pisum and A.circumflexum, Unless otherwise stated they were made on
adult apterous alienicolae in petri-dishes illuminated. from above. Each
dish had a piece of white filter-paper in the bottom and was kept at a
constant temperature of 20°C.
Batches of 30 insects were fumigated simultaneously at a number of
C.T.P.s and after airing were transferred to petri-dishes. At the same
time 30 unfumigated insects were placed in another dish. The behaviour of
the insects was observed continuously for 2-3 hours and thereafter at
regular intervals, observations being recorded as they were made.
The following is a summary of the sequence of events in a typical
experiment with A.pisum. 54. There were three dishes. The insects in one (A) had been
fumigated at 20°C. at a C.T.P. of 1.54 (sub-lethal) and those in another (B)
at a C.T.P. of 5.32 (LD98). The insects in the third dish (C) were
unfumigatea.
Four minutes after transferring the insects to the dishes those
(B) which received the lethal dose began to disperse even more rapidly than
the controls. Those (A) which had received the sub-lethal dose, however,
showed few signs of movement.
After thirteen minutes the high-dosed insects were extremely
active, rapidly crossing and re-crossing the lid and floor of the dish and
were widely dispersed.
The control insects were likewise widely dispersed but had a
noticeably slower rate of progress.
The insects in dish A, on the other hand, were still largely
quiescent and very few had dispersed. Those which had, displayed an
un-naturally clumsy gait.
Twenty minutes after the start of the experiment the high dosed
insects in dish B had begun to quieten and tended to wander more slowly
around the Tim of the dish. The controls maintained their steady explor-
ation of all surfaces. Sluggish, laboured movements characterised the few
insects which were moving about in dish A.
After thirty-five minutes activity was greatest in the controls,
all of which were actively exploring.
At the end of two hours and a half the type of activity of the
high-dosed insects was very different from what it had been earlier. More 55. than half were restlessly "marking time" at the dish edge whilst the remaind- er made sporadic journeys across the dish at an un-naturally fast pace but with properly co-ordinated leg movements. The same steady exploration of the dish continued in the controls. A few of the low-dosed insects were sluggishly moving about but the others were lying listlessly in the centre of the dish where they had fallen originally.
The controls remained active for twenty-four hours and, as a rule, much longer, but within this time complete paralysis and death overtook the high-dosed insects.
The first symptoms of poisoning in insects receiving the LL60-99 could be seen between 5-9 hours after fumigation. If, at about this time, all the insects in the three dishes were shaken to the sides of the dishes it was 3-4 minutes before general dispersal took place in the controls. In the fumigated insects, however, it was very much longer, being about 10 minutes in the high-dosed group and considerably more in the others.
From about 5 hours onwards, however, there was a steady increase in the amount of activity in the low-doseci insects although a few usually remained semi-paralysed at the edge of the dish.
When the number of doses per experiment was increased so that doses in the range LD20-30 were included, behaviour intermediate between the two extremes just described was witnessed.
Not only A.pisum but also A. circumflex= and. R.latysiphon displayed this same sequence of responses to successively increasing doses of fumigant.
Very low concentrations had little or no effect on the activity level but slightly higher ones, still in the sub-lethal range, had a depressing effect. 56.
This was followed at threshold concentrations by a return to a more normal activity level and was superceded by periods of hyperactivity at lethal doses.
The duration of these periods of hyper-activity rapidly decreased, however, as the dose rose above the LD99 until at C.T.F.s of 30-40 the insects were completely paralysed when they were removed from the chamber
(fumigation time being one hour).
It must also be added that temperature and humidity had an import- ant controlling' effect on the activity level of both fumigated and unfumigat- ed insects.
The activity level of aphids was greater at low than at high humidities both in unfumigated and fumigated insects. At R.H.s of 855 or more the insects tended to be rather sluggish and in toxicity tests it was frequently a few minutes before it was possible to decide with certainty which of a group of fumigated insects were truly 'knocked-down'. The sudden drop in humidity, however, was always a sufficient stimulus to cause the survivors to disperse about the petri-dish.
A rise in temperature likewise favoured a higher activity level in both types of insect.
(e) Locomotion.
Aphids poisoned by methyl bromide had a characteristic gait. This was first seen as a gradual loss of co-ordinated limb movement which gave way to complete inability to make forward progress. Total paralysis and then death followed. 57. The range of symptoms was best studied in adults exposed to a concentration about the IJD80 and kept afterwards at a R.H. of 80485%. (In an atmosphere which favoured rapid evaporation of water from the insect the syndrome appeared sooner, and was less prolonged, while at very high humidities the insects did not move about sufficiently for adequate observations to be made).
The following is a detailed description of the locomotion of
adult apterous alienicolae of A.pisum under the conditions detailed above.
It could equally well apply, however, to each of the five other species, but with one reservation. It was normally only about 16 hours before the symptoms began to appear in A.pisum and 30 hours in the others.
On the day following fumigation the progress of an aphid (across
a filter paper in a petri dish) was marked by a series of abrupt halts.
These were caused by the insect falling forwards onto its front legs,
almost as though it were trying to move at a faster pace than possible and
stumbling in so doing.
Several hours later, attempts at walking were impeded by an in-
capacity to move the legs in a co-ordinated fashion. Forward movement at this time was typified by a pronounced pitch.
As paralysis began to take hold the insect could be seen labouring to move a stiffened leg into the air and forwards and failing. This
sometimes continued for many hours (especially at high humidities, see P.O.)
until the insect fell over onto its back eventually to die. Even then,
however, feeble spontaneous limb movements continued, often for a consider-
able time, depending upon the conditions. 58. (f) Settling and feeding.
A knowledge of the feeding habits of fumigated aphids is important
in relation to virus transmission.
When an untreated aphid is transferred to a new host it 'probes'
the plant at intervals) inserting its stylets a short way only into the
tissues but soon withdraws them and moves on. Eventually, however, it
chooses to settle (usually on the undersurface of a leaf) and the stylets
then penetrate much deeper, usually to the phloem.
An aphid carrying a plant virus is capable of causing a new site
of infection with each probe.
Broad-bean plants bearing large numbers of settled adults and
nymphs of A.pisum were fumigated under bell-jars in daylight at 15°C. (for
details of these chambers see p.128). At C.T.P.s of 5 (LD50) and less, no marked reaction to the fumig-
ant by the insects was observed until 20 minutes after the start of the
fumigation when a few adults began to free themselves and wander about the
plant. A short time later many more did the same.
At C.T.P.s of 11-18 (2-3 x LD90), however, the response :was more
rapid and within 10-15 minutes most, if not all, the insects were active,
being seen not only on the plant surface but on the pot and walls of the
bell-jar as well.
Many fell to the pot and base of the jar but others reached there
by walking down the plant stem. Generally, the plant was more or less
91k It is very characteristic of A.pisum that when disturbed the insects
readily fall from the plant. 59. devoid of aphids by the middle of a fumigation lasting 1 hour.
Insects which left the plant rarely returned and died elsewhere.
These observations were also true for plants infested with each of the other species of aphid.
In general, the insects which adhered longest to the plants were nymphs of the middle instars. Adults invariably deserted.
To establish that the cause of the insects' leaving the plant was not in fact a change in the plant itself due to the fumigant, un-treated insects were transferred to plants which had been fumigated at a C.T.P. of 45 at 20°C. Even a short time after airing the insects displayed no objection to the treated plants as sources of food and settled as readily as un-treated insects on un-treated plants.
Fumigated insects were also transferred to unfumigated plants.
A.pisum, R.latysiphon and A.circumfleram were used and a series of C.T.P.s were given. The same responses to different dose levels as were obtained in the absence of plant material (p.64) were observed.
Insects exposed to just sub-lethal concentrations remained quiescent on the leaf for a period of 5-8 hours before most of them found a suitable place and settled to feed and reproduce. Little exploration of the plant took place and those insects which fell from the plant rarely re-gained their position.
On the other hand, insects activated by high doses, especially adults and near adults, dispersed rapidly over the plant surfaces, many falling to the ground (in A. pisum and R.latysiphon especially) when trying to pass from the upper surface of a leaf to the lower surface. 60.
Always a strong tendency of the fumigated insects to leave the plant was observed.
The feeding behaviour of aphids activated by fumigation in this way was studied. Probing was not uncommon during the insects' induced wanderings over the plant in the immediate post-fumigation period but they never settled to feed properly, even for a short time, in a great many tests.
On plants where crevices and cavities (e.g. at leaf bases) existed, it was not uncommon to find large numbers of the bodies of the younger instars.
According to the dose and the post-fumigation conditions, these insects would or would not still be capable of move-lent a day or so after fumigation
(1"1.14- ). However, any insect remaining on a plant and showing, the obvious sympLoms of poisoning outlined earlier was never found to be feeding. If an insect was found settled and feeding 10 hours or more after fumigation it could be assumed with certainty to have survived the treatment and would go on feeding to grow and to reproduce.
Nothing has been said so far concerning the behaviour of fumigated whitefly adults, largely because such studies have been brief.
However, insects observed during fumigation under glass were very sensitive to methyl bromide, soon becoming agitated and flying from the plant to the chamber walls.
Whether the insects fed after fumigation was not determined.
Doses only slightly in excess of the IlD99, however, resulted in a quite rapid immobilisation of the insects which then fell to the ground. 61.
(g) Phototropism.
During the conduct of some of the foregoing experiments a marked
positive response to light was confirmed in unfUmigated apterous and alate
adult alienicolae of A.fabae and adult whitefly when one half of the dish
was shaded. The response in apterous adult alienicolae of A, pisum was also
positive although less marked.
Provided that the insects were able to walk properly, fumigated
apterae and alatae of A. fabae showed no change in their response to light
after a wide range of doses and neither did whitefly adults.
With apterous A.pisum, however, some rather conflicting and not
always repeatable results were obtained
On foUr different occasions; using different batches of insects,
a quite definite negative response to light was witnessed after doses
ranging from the LD40 TD80.
It was found that different batches of insects, although of the
same age and. cultured under the same conditions, varied somewhat in the
degree to which they responded to light before fumigation. On three of the
four occasions mentioned it was those insects which showed an almost complete
indifference or at most a weak positive response to light which, after
fumigation, sought out the shaded part of the dish. On the fourth the
positive response of the control insects was quite marked.
It must be pointed out, however, that the number of times this
reversal of response did not take place greatly outnumbered those when it
did. 62. 7. Insect Fumigation Discussion.
The susceptibility of six species of aphid and the greenhouse whitefly to methyl bromide was influenced by a number of factors.
Both types of insect differed in susceptibility from instar to instar and, in aphids, from one form to another. Changes with age and reproductive state were also detected in adult aphids and in whitefly eggs at different sta;es of development. Many workers, using a large number of different insects and insecticides, have reported changes in the susceptibility of insects which have been attributed to a wide variety of factors. Most of these have ultimately been associated with attendant differences in the structure and metabolism of the insects and sometimes in behaviour as well. Busvine (1938) quotes work on the bed-bug, Cimex lectularius, in which the eggs were the least resistant stage to ethylene oxide and hydrogen cyanide vapour, followed by young nymphs, adults and fourth and fifth instar nymphs (most resistant). A similar order of resistance to hydrogen cyanide and sulphur dioxide was found in the louse, Pediculus humanus (Busvine, 1943). Gough (1939) showed that the order of increasing resistance to hydrogen cyanide vapour in Tribolium confusum was egg-larva-adult-pupa, and Sun (194.7) obtained similar results using methyl bromide. A correlation between the metabolic rate of insects and their susceptibility to fumigants was suggested by Cotton (1932) who was supported
in this view by Lindgren (1935). They showed that the stages with the lowest rate of metabolism (pupae) were usually the most resistant to fumig- 63. ants whereas those (larvae) with the highest rate were least resistant.
The theory was further supported by Gough (1939) for Stages other than the egg in holometabolouS insects and by Busvine (1957). According to the insect species and the insecticide used, the egg may be either the most or the least resistant stage.
More recently Bond (1956) has shown that susceptibility to methyl bromide in larvae of Tenebroides mauritanicus is directly related to respiratory rate. It must also be kept in mind, however, that marked changes in metabolic rate within a particular stage, particularly the egg and pupa, may occur and with these will presumably be associated further changes in susceptibility to fumigants. In particular, newly laid eggs and eggs near to completing their development may have a lower rate of metabolism than eggs at intermediate stages in development (Gough, 1939). If whitefly eggs also have different metabolic rates at different stages in development it would help to explain the different levels of susceptibility to methyl bromide found in these eggs. In this connection attention is once again drawn to the three visibly recognisable phases in the development of white- fly eggs (p.10). In aphids, which have an incomplete metamorphosis (hemimetabolous), the resistance of alienicolae to methyl bromide increased from one instar to the next, the young adult being the most resistant. In the absence of a stage (e.g. a pupa) radically different from any other in the life-history it might be expected that sharp differences in susceptibility in aphids would not occur. 64.
Unfortunately, no definite information on the resistance of the aphid egg was obtained but in brief tests (unreported here) with A. fabae, it appeared that the egg was more resiotant than any of the active stages.
Whitefly, on the other hand, differ from aphids in that the immat- ure stages are sessile scales with reduced morphological differentiation.
The order of increasing resistance of whitefly stages to methyl bromide was: adult, instarl, egg, instar II, instar III, instar IV.
Disregarding the resistance of the egg for a moment, it can be seen that larval resistance increased successively with each instar. While metabolic differences must form part of the explanation of this it may be that the increase in thickness of the cuticle and waxy covering and, in particular, the progressively greater inaccessibility of the spiracles might be involved, both tendin to reduce the uptake of fumigant. The resistance of whitefly adults to methyl bromide was much less than that of the egg or larvae. Considering the sedentary habit of the larvae in comparison to the activity of the adult it is perhaps not unreas- onable to expect a lower rate of metabolism in the larvae and that this should be associated with a correspondingly greater degree of resistance.
The absence of detectable difference between the susceptibilities of male and female whitefly adults is consistent with other work on fumigants,
Gough (1940) fumigated bed-bugs with sulphur dioxide and found the two
sexes equally resistant. Similarly Sun (1947) was unable to find a difference in susceptibility between the sexes in Tribolium confusum and
Spermophaus subfasciatus when exposed to carbon disulphide vapour.
However, although young adults of T.confUsum were about equally resistant 65. in the two sexes to hydrogen cyanide vapour, in elaor adults the males were more resistant than the females (Gough, 1939).
Park (1936) found that females of T.confusum have a higher respir- atory rate than males and it was assumed that this was contributory to their (females) lower resistance.
Differences in metabolism must also be assumed to explain, at least in part, the different susceptibilities of winged and wingless aphids to methyl bromide.
Johnson (1958) has shown that alate aphids which have spent several hours on the sides of cages or on glass, after a very brief flight showed a strong tendency to settle. Such a tendency has been shown by this author (1953) to be accompanied by parturition (if the insect is on a plant) but also by autolysis of the flight muscles.
Thus although increases susceptibility due to depletion of food and water reserves may be part of the explanation of the decreased resistance of alate A.pisum collected from the cage-walls in the present experiments, more fundamental metabolic changes may again be involved.
Similarly, the decline in resistance in apterous alienicolae of
A.pisum as reproduction proceeded was almost certainly due to associated physiological changes.
From the few measurements made there did not appear to be any marked correlation between the water-content of settled aphids and their susceptibility to methyl bromide. However, the water content of insects, even under identical conditions, is subject to considerable variability and many more determinations would be necessary beeore a definite statement 66. could be made.
Unfortunately, time did not permit an investigation of whether or not the susceptibility of aphids and whitefly is affected by the nature of the host-plant. Both types of insect are highly polyphagous and some at least feed upon a number of botanically widely different types of plant.
Richardson and Casanges (1942) showed that the resistance of
M.persicae to nicotine vapour was different according to whether the insects were reared on nasturtium, climbing dahlia, lettuce or turnip. Potter (1957), working with A.oisum, also found a difference in susceptibility to rotenone in insects reared on clover and broad bean.
It is possible, therefore, that the susceptibility of aphids and whitefly to methyl bromide might also differ slightly according to the host- plant.
The increased susceptibility of aphids and whitefly to a particular dose of fumigant with en increase in temperature is in agreement with the early results of Fisk and Shepherd (1938) who showed that the susceptibility of other insects to methyl bromide increased with temperature.
Numerous similar observations with this fumigant have since been (b) made and Richardson et al. (1943) have reported along the same lines for the Mexican mealybug and common red spider.
When considering the effect of temperature on susceptibility it is very important to bear in mind that the actual temperature measurements which are made relate to the air surrounding the insects and that no matter how accurate such measurements might be they give a very incomplete picture of the true temperature relationships of the insect. Furthermore, due 67. account of the humidity of the air must be taken into consideration at each temperature.
Gunn (19/4.2) has reviewed the subject of body temperature in insects. He points out that in a moving stream of dry air at a high temp- erature the body temperature of an insect may be a degree or so less than that of the surrounding air as a result of cooling caused by the evaporation of water. On the other hand, the production of metabolic heat at times of activity may raise the body temperature quite considerably above that of the surrounding air. This would particularly apply to aphids away from the plant at temperatures of 20 - 3000. in air.of moderate - low humidity, when activity is usually increased.
As the temperature increases the rate of metabolism will also increase and the greater chemical reactivity which can be supposed to occur would largely account for the apparently increased toxicity of methyl bromide with a rise in temperature.
A faster rate of gaseous exchange in the tracheae at a higher temperature either by a faster rate of diffusion or by increased tracheal ventilation by active means will also contribute tv a greater upl,ake of fumigant into the insect body.
The fact that susceptibility to methyl bromide did increase with temperature emphasises the chemical nature of its action as opposed to a purely narcotic mode of action in which susceptibility would be greatest at low temperatures.
Finally, when measuring the responses of insects to poisons by bio-assay techniques, it is important to consider the temperatures at which 68.
the test insects were reared. A difference, albeit slight, may be found
between the susceptibility of insects reared at low temperatures and those
reared at high temperatures. Both rate of development (and therefore
usually size) and the chemical composition of the tissue are affected by temperature and these may in turn affect resistance to insecticides.
Munson (1953, 1954) showed that the composition of lipoids in
Periplaneta americana was dependent upon the breeding temperature and he
related differences in susceptibility to DDT between groups of insects reared
at high and at low temperatures to differences in lipoid composition. He
also showed that differences in lipoid composition between males and females
could be correlated with differences in susceptibility to LET.
With regard to the effect of humidity on susceptibility, Fisk and
Shephard (1938) stated that methyl bromide is "definitely more effective
when it is in the presence of moisture". They showed that adults of
Tribolium castaneurn were more susceptible to the fumigant at relative
humidities of 80 - 90% -than at 10 - 205. (6) Richardson et al. (1943) confirmed this observation but at the
same time found no change in the susceptibility of the Mexican mealybug and
common red spider at different humidities when the exposure period was six
hours or more.
Richardson and Busbey (1937), on the other haad, found that
M.persicae and A.pisum were more sensitive to short exposures to nicotine
vapour at very low than at high humidities although the difference disappeared
with lung exposures.
No difference in susceptibility to methyl bromdie was found in 69.
adult aphids at different humidities, however, in the present work when the
exposure period ,gas 1 - 3 hours at 15° and 20°C. This was surprising since
aphids tend to be more active in dry than in wet air. Assuming an increased rate of respiration with a rise in the activity level and a correlation between respiratory rate and the uptake of fumigant, a higher kill would
be expected under dry conditions especially at the higher temperature.
It was after fumigation that the atmospheric humidity was of great-
est importance. The degree of saturation of the air surrounding an insect
can be supposed to govern, more or less, its rate of loss of water. In
air of a high humidity aphids survived fumigation better than did insects
kept at a low humidity. It seemed that insects whose level of resistance was equalled but not exceeded by a given dose were more likely to be 'knocked.
down' by the treatment if they experienced a rapid rate of water loss. If water loss was kept to a minimum, however, by keeping the insects in wet
air the ability to survive of a few insects was thereby increased. In
other words, slightly more insects succumbed to a particular dose of fumigant
in dry air than did so in wet air.
Consider next the disturbances in development which took place
when aphids and whitefly were fumigated.
The inability of immature insects to moult successfully after
fumigation cannot, for the present at least, be satisfactorily explained.
It may be that some part of the hormonal system controlling moulting was
affected. Alternatively, failure to moult may have been a consequence of
the general poisoning of the insect.
The failure of adult insects fumigated as final instar larvae to 70. expand the wings may have been due to the poisoning of the neuro-muscular system involved in forcing haemolymph into the wing veins. The fact that the same insects could walk about, however, does not support the idea of impaired. muscle action.
Shortage of water in the haemolymph is another possible explanation.
The nymphs were kept in air of 90 R.H. after fumigation and did not have the opportunity to feed. Consequently, the adults which emerged may have been suffering from a water deficit. However, the unfUmigated nymphs were kept under identical conditions and never failed to erect their wings as adults, even in the absence of food.
Although the un-expanded wings of fumigated insects appeared to be complete in terms of gross anatomy when opened and examined microscopically it is not impossible that there was an undetected defect in their structure which prevented their being erected.
Finally, disinclination rather than inability of the poisoned insects to erect the wings must not be ruled out as an explanation.
Kundu (1957) reported wing abnormalities in adults of Tenebrio which emerged from pupae fumigated with methyl bromide. He also found fused abdominal segments in larvae emerging, from fumigated eggs and a postponement of moulting and pupation roughly proportional to the dose at sub-lethal concentrations.
Abnormalities following fumigation were also found in aphids and whitefly. Adults of both groups of insects fumigated as final instar nymphs lacked or had malformed wings. In aphids tarsi and antennal segments were also missing. 71.
Goldschrnidt (1927, 1932) proposed a theory of development in which the genes act by accelerating or retarding chains of reactions responsible for the production of a particular character or set of characters. He postulated a 'genic balance' in which the final characters are the resultant of reactions initiated by genes of different tendencies. Interference with one or several of these reactions by altering either its rate of progress or by interfering with the production of che:lical substrates involved, results in the appearance of abnormalities in the fully-developed animal. Short exposures to high temperature in Drosophila and Ephestia are sufficient to cause such changes. (Review Sinnott and Dunn, 1935).
Gene action, however, is also intimately dependent upon enzymes.
Perhaps even very low concentrations of methyl bromide are capable of inter- fering with an enzyme or enzyme system or associated substrates upsetting slightly a normal series of reactions leading to the formation of a particular morphological character e. f;. antennae.
An insect in a physiologically sub-normal condition frequently has a sub-normal rate of reproduction and it is therefore not surprising to find this is true also of fumigated aphids. The interesting point is that the fumigant does not appear to affect the nymphs ready to be born at the time of fumigation and which are born soon afterwards. (The significance of this will be discussed later in the section on the practical aspects of this work). The apparent increase in the rate of water loss from adults of
A. pisum following fumigation at the LD90 at 15°C. was interesting.
Bond (1956) has shown that the respiratory rate of larvae and 72.
adults of Tenebroides mauritani(ms :Is little affected by methyl broml.de and
remains almost constant for long periods after fumigation, even through
phases of hyperactivity and paralysis.
The respiratory rate of fumigated aphids is unfortunately not known. If it also remains unchanged after fumigation then the extra water
lost cannot be due to an increase in the amount of water lost as vapour
simultaneously with the gaseous exchange via the spiracles and tracheae.
Perhaps, instead, the basic assumption of these experiments was
at fault and insufficient attention to weight loss due to metabolism was
given. Doses of the magnitude of the LD90 have been shown to induce
considerable hyper-activity for a time after fumigation which must make
some additional demands on the food reserves of the insects.
Nevertheless it is worthwhile considering at least the possibility of a direct effect of methyl bromide on the permeability of the insect
cuticle. Although such an effect may not be very important at the low doses required to kill aphids and whitefly it may be relevant to, say, stored
product beetles which require a much higher dose of fumigant for control.
(It will be remembered that the chemistry of insect fats associated with the cuticle does not differ greatly from one group to another).
Methyl bromide is highly soluble in fats (Mackie 1938; Fisk and
Shephard, 1938).
KI:thnelt (1928, 1939) recognised that the impermeability of insect
cuticle to water was clue to the lipoid nature of the epicuticle and this has
since been confirmed for many insects and the subject reviewed by Wiggles-- worth (1945, 1948). 73. The epicuticle is a thin layer, 1 - 4r thick, overlying the rest; of the cuticle. It consists of at least four layers. The innermost is a thin layer of tanned. lipoprotein (cuticulin) above which is a layer of prot- ein-bound polyphenols. This is overlaid by a monolayer, generally 0.25 -
0.5ir thick;, of crystallised waxes which is in turn covered by a protective
'cement' layer similar in composition to the cuticulin layer.
It is the waxy monolayer which is responsible for the impermeability of the cuticle and the nature of the waxes composing it has been investigated by Beament (1945, 1955). Both Wigglesworth and Beament stressed the importance of the compactness and orientation of the long wax molecules in conferring impermr, eability on the cuticle. They also showed that the vapours of fat solvents such as chloroform, benzene or ether, increase the permeability of insect cuticle to water at room temperatures. This they attributed to a permanent disorientation of the important wax monolayer in the epicuticle.
It would be very interesting to know whether methyl bromide at insecticidal concentrations can cause a similar change.
Kundu (1957) showed that mirdworms in contact with a film of water took up water and swelled to a greater extent after fumigation than could be attributed to the normal abrasion of the cuticle by soil particles. He also showed that Tenebrio larvae lost considerably more water in un-saturated air after fumigation than before. The C.T.P. range was 30 - 80.
The behaviour of insects fumigated. with methyl bromide has been reported briefly by a number of authors. Bond (1956) described a period of hyperactivity followed by a steady decline to paralysis in Tenebroides 74. mauritanicus after fumigation.
Behavioural changes, however, are not easy to interpret. The tendency of aphids and adult whitefly to leave their host-plants and cease feeding after fumigation must be largely interpreted as due to the induced hyperactivity and also as an irritant response.
The occasional reversal of the posit3ve response towards light in A.pisum after fumigation was peculiar. It • still not possible to explain fully, in biochemical terms, the mode of action of methyl bromide on living tissues. However, a brief review of what is known is useful. Lewis and Eccleston (1946) and Winteringham and Harrison (194.6) showed that the decomposition of sorbed methyl bromide took place largely in the protein fraction of wheat flour. Blackburn and Phillips (1944) also showed that irreversible methylation of carboxyl groups and peptide linkages occurred when aqueous suspensions of wool, silk, fibroin, collagen and gelatin were treated with liquid methyl bromide. Blackburn, Consden and Phillips (J.944) and Lewis (1948) demonstrated the methylation of the al- groups of cysteine and reduced glutathione as well as of methionine and histidine. Irreversible inhibition of urease, succinic dehydrogenase (but riot cytochrome oxidase), reduced papain and yeast respiration were also shown by Lewis. All Lhese enzyme systems are dependent upon the presence of SH- groups for their activity. Loved*. and Winteringham (1951) carried the work further and con- firmed the rapid blocking of free SH- groups at high concentrations of methyl bromide in vivo in the larvae of Calliphora erythrocephala. 75. On the basis of these results it was for some time believed that methyl bromide acted by inhibiting sulphydryl Froulos and this would indeed be a very important reaction in view of the widespread importance of SH - systems in metabolism. However, all the work involved very high concentrat- ions of fumigant (C.T.P.s of 165-500 and. more).
More recently Winteringham, Hellyer and McKay (1958) have suggested that the inhibition of SH- groups is not the primary mode of action of methyl bromide. Using 32,E isotopes they showed that brief exposure of adult female houseflies to saturated atmospheres of methyl bromide resulted in a reversible breakdown of adenosine triphosphe (ATP) which coincided with temporary immobilisation of the flies. Longer exposures were associated with the irreversible depletion of ATP, arginine phosphoric acid and phospho- glyceric acid and the immediate and permanent immobilisation of the insects.
These authors put forward the view that methyl bromide could only be active as an al - enzyme inhibitor at hi0a concentrations. They hold that, the main effect of the. gas is on. phosphorus metabolism.
The fact. that respiration contimos at a near-normal rate. in. several insects for many hours after. exposure even when symptoms of paralysis are evident (Bond, 1956; Winteringhaxn et al. 1958) is consisted with the idea that SH - enzymes have not been affected.
However, there is also a. major difficulty in interpreting the action of methyl bromide on phosphorus metabolism. Many insects, aphids included display periods of marked hyperactivity after fumigation, even at doses of the order of the,LD90, which seems directly opposed to any rapid decline inATP. 76.
Nevertheless temporary depletion followed by a return to a more normal level of LTP in aphids fumigated at sub'-lethal doses would provide an explanation of the slow resumption of activity observed in these insects after an initial depression of activity. 77.
PI iLisiT FUILTGAT ION.
8. Review of the Literature.
Mackie (1938) sumaarsed the applications of methyl bromide as a fumigant in the six years since its use for this purpose was first described by Le Goupils. Iie listed a number of herbaceous plants, such as Begonia,
Coleus and Cyclamen, which had been successfully treated at reduced pressures for the control of mealybugs and the cyclamen mite. The fumigation of imported orchids was also mentioned. Since then a number of scattered reports have appeared concerning attempts, successful or otherwise, to control
pests on herbaceous or growing woody plants.
Hamilton (1941) described the successful control at high temperat- ures of the common red spider, Tetranyphus telarius (L.), on six varieties of roses in a glasshouse in Lmerica. In the same year Ritcher studied the fumigation of strawberry plants infested with the strawberry crown-borer
Tyloderma fragariae (Riley) and Kid() investigated the control of the straw- berry rootworm Paria canella var.quadrinota.ca (say). (6) Richardson et al. (1943) reported on the glasshouse and vault
fumigation of a large number of plants, mostly flowering ornamentals, for
such pests as thrips, mites, red spider, scales and mealybugs, whitefly and
aphids. Bulger (1946) and Richardson (1949) in Lmerica and Griffin, Lubatti
and Harrison (1956) in Great Britain also demonstrated that at least some
varieties of orchids would withstand fumigation at fairly high doses when
in the non-dormant condition. 78.
Obviously, from a practical point cf view, the information most urgently required is the amount of fun .ant at a given fumigation time which
a particular species of plant will tolerate. Such particulars, based almost
entirely on the presence or absence of visible shoot damage are available in the papers referred to and in publications of the various Plant Quarantine
Bureaux. The relationship of this dose to that required to kill the Pest
in its various stages is discussed.
A few papers also contain information on the effect on tolerance
of temperature (Hamilton, 1941; Richardson et al., 1943(WGriffin et al.,
1956), humidity (Hamilton, 1941), and the plant variety (Hamilton, 194.1; (6) Blanton, 1942; Richardson et al., 1943; Griffin et al., 1956).
However, no reference to a systematic study of methyl bromide
fumigation of non-dormant plants in purely physiological terms was found in the literature; only a very few isolated observations and a few concerning the after-effects on material fumigated in the dormant stage.
Lange (1940) fumigated artichoke stem bases at reduced pressures
prior to planting out. At doses required for control of the artichoke plume
moth, Platyptilia carduidactyla (Riley), there was a reduction in the amount of growth compared with the controls. After 10-20 days wilting and death
followed fumigation at higher doses.
Hitcher (1941) showed that at a C.T.P. of 96 at temperatures below
80°F., subsequent growth of dormant strawbc,cry plants was not affected. At
87-92°F., however, and at higher doses the blossom buds were killed in some
varieties and two growing points developed. 79.
This author also reports "stimulation of growth" in some fumigated plants. In Lmerican literature this phrase is commonly used in reference to fumigated plants but is never fully investigated.
Kido (1941) also reported "definite stimulation of growth" after fumigation at control doses in strawberry plants treated when dormant. lie showed that the number of runners produced by fumigated plants exceeded that of untreated plants and that furthermore, in one variety, the weights of the plants when dug out at a fixed time were greater after fumigation to an extent proportional to the dose increase.
The susceptibility of non-dormant plants was greater than that of dormant ones.
Latta (19)10 showed that the susceptibility to damage of a large number of conifers increased from February to April. The greatest tolerance was in mid-winter, when there was least cellular activity, and was preceded and followed by periods of successively lowered resistance. Foliage injury ceased to show after several frosts but began to show again with the first signs of growth next spring. Varietal differences were explained by the fact that some break dormancy sooner than others.
In later work with freshly cut fir and spruce Latta (1945) reported that in every instance fumigated trees retained their needles and colour longer than controls; keeping quality increased with dose. The effect was more noticeable in fur than in spruce.
All too frequently statements such as "nothing is known of the after-effects of treatment (with methyl bromide)" can be found in even fairly 80. recent literature on plant fumip:ation, and it is hoped that the work described. in the followingt section will provide some much needed information. In
particular, caution will be urged in accepting the appearance of visible shoot
damage as a criterion for assessing the tolerance limits of a particular
species of plant.
It is also much to be deplored that so many specifications for treatment ignore the effect of temperature and. even, at times, the placing of a definite time limit to the duration of exposure at a recommended concentration. 81.
9. Materials and Methods.
(a) Plant celture.
Plants were used for two purposes; one was the provision of food for insects, of which were external plant parasites, and the other was the investigation of the phytotoxic effects of methyl bromide,
In the course of the work twelve species of plants were grown and with the exception of potato and tulip they were raised from seed. Table
XVIII summarises the species and varieties and the type of soil in which each was grown. Because plants were mostly required singly they were grown in individual red whalehide pots of 5" and q" diameter. These were light to handle and allowed large numbers of small plants to be kept compactly. For some plants, however, these pots were not large enough and bigger, clay pots were then used.
The sand w.as ordinary fine potting sand and the loam a local one which had been passed through a coarse sieve. The sand-loam-fibre mixture was made up in the proportions 1 part sand : 3 parts sieved loam : 3 parts damp potting-fibre and proved an excellent growing medium.
To maintain a sufficiency of ions in the soil the pots were watered at intervals with a nutrient solution made up as follows (from Hoagland and
Lrnon, 1938) :-
Solution A.
K2 PO 1 ml. H ENO 5 ml. per 1 litre of 3 21 Ca (E03)2 5 ml. distilled water.
M ngSO4 2 ml. 82.
Table XVIII. Details of the plants used in the experiments.
Species Variety Soil Appearance of plants used in tolerance tests
Broad Bean Seville Sand. 10-12" in heioht. 6-7 leaves (Viola faba) Longpod expanded.
Pea Sutton's San loal2,7 12-14" in height. 8 leaves (Pisum arvense) Improved fibre mixture expanded. Pilot
Runner Bean Sutton's 11 Stem 18-20" in length. Several '(Ehaseolus Challenge leaves formed. in addition to multiflorus) cotyledons.
Lupin Russell IF Well-established plants with (Lupinus sp.) (mixed) many leaves. Potato (Solanum Lrran Loan 7e:11-established plants, 10-12" in tuberosum) Pilot height with considerable foliage. Tomato (Lyco- Ailsa Sand/loam/ 12-15" in height. 7 leaves persicon Craig fibre mixture expanded. esculentum) Lettuce Ylay King Well-established plants with a (Lactuca sativa) (Cabbage) large number of leaves. Nasturtium Dwarf Young plant s just beginning (Trooaeolum Crimson to branch. ma-zFiLn'T-
Tobacco 12-15" in height. (Nlcotiana t abacum)
Tulip (Tul*a Just coming into flower. gesneriana) Cocksfoot grass Blades 6-8" in length. (Dactylis olomerata)
Rye-grass tf Blades 5-7" in length. (Lolium perenne)
83.
Solution B.
H3BO 2.86 gm.
MnC12.24H20 1.81 gm. dissolved in
7E 0 anS02-1: 2 0.22 gm. 1 litre of CuS0:511 0.08 am. distilled water 20 H.1o0 11 0 0.09 gm. 4. 2
To each litre of solution Awas added 1 ml. of solution B and 1 ml. of 0.55 iron tartrate solution. The pH was adjusted to a value of 6.0 by adding 0.1 NH SO 2 4. Occasionally, 'Tomorite' was mixed in the recanmended quantities with tomato soil prior to potting but it was rover necessary to add fertiliser to broad bean plants :rowing. in sand, presumably because of tie store of ions in the seed. Broad bean, pea, runner bean, nasturtium, cocksfoot and rye-grass seeds were sown in the pots of soil themselves, singly, with the exception of the grass seed, which was scattered on the soil surface and covered.
Potato tubers and tulip bulbs were likewise sown directly, one to each pot of soil. The other seeds, lupin, tomato, lettuce and tobacco, were sown in seed-boxes and the seedlings planted out at a suitable stage. The plants used in the phytotoxicity tests were grown in the glass- house during the summers of 1958 and 1959 021l many of the other exneriments were also conducted there. However, as has already been explained, considerable difficulty was
experienced in raisin satisfactory plants for the culture of insects during
.the winter months. This was overcome by the provision of the growth-room
in Which broad bean, turnip and potato plants were satisfactorily reared.
• Jill the plants used in the experiments were of good appearance,
being vigorous in growth and perfectly healthy.
(b) N-niation. Procedure.
Plants were usually fumigated in a steel chamber of a capacity of
500 litres. Occasionally, however, this was too small and a much larger
chamber of 5,000 litres capacity was then used. The temperature of the
chambers could be controlled and tests were normally at 22°C., each fumigation
being of 2-3 hours duration unless exceptionally high C.T.P.s were required
when fumigation was extended to 4 and rarely 5 hours.
Prior to fumigation plants were kept for several hours at the
temperature at which they would later be fumigated.
Each chamber was fitted with a fan which provided a moderate rate
of circulation of the fumigant-air mixture throughout a fumigation.
Dosing was accomplished with the aid of a permanent dosing device
connected to each chamber. The pressure inside the chamber was reduced by
a few cm. of mercury and from a storage cylinder the required quantity of
liquid methyl bromide was measured into a partially evacuated glass measur-
ing cylinder graduated in ml S. The measuring cylinder formed an integral
part of the apparatus and was also connected to a vapouriser whicn consisted
of a copper coil surrounded by an electrically heated water bath. When the 85„ temperature of the water bath was about 30-40°0. the reduction in pressure in the chamber was ased to draw the liquid fumigant through the copper coil, where vapourisation was complete, and thence into the chamber.
Samples of the gas mixhire inside the chaMber were taken at intervals for analysis using the combustion method of Ilubatid mentioned earlier (page
11). Normally two samples were taken soon after the beginning of a fumigation and two more about the middle and two towards the end. After some experience this was :bund- to be sufficient for deciding exactly the overall concentration, and hence the duration of exposure needed to give the required C.T,I7.
Sorption of the fumigant on the materials inside the chamber was studied and occurred largely in the early stases of a treatment Because of the sorptive nature of soil-filled pots etc. excess fumigant was required over the quantity calculated for an expty chambex. Preliminary tests indicated the excess required for a particular loadine-.
Due to the large volume of the chambers it was also possible to use the thei- co-conductivity method for determining concentrations. The particular instrument used was the Pumiscope junior of U.S. manufacture.
This instrument operates upon the principle that when the fumiant-air mixture is made to pass across a thereto-couple a small electric current is generated. This current can then be used to deflect the needle of an ammeter which is calibrated directly in %. per litre (oz. per 1,000 cu.ft.).
The instrument proved very satisfactory in many of the routine phytotoxicity tests but the chemical method was retained fc: determining concentrations in important experimerts. 86.
The chaMbers were aired after fumigation by simply opening them and allowini:: the fan to clear away the fumiF,cant.
After airinn the plants were returned to the glasshouse and watered
if necessary.
(c) Determination of the water-content of plants. Subsequently, several references will be found to the water content of various plants. This was determined by drying the cut-up material to
constant weight in Etn oven at 105°C. and calculating the percentage change in weighty which was assumed to be due solely to loss of water. Unless otherwise stated only the shoot tissues were used and generally separate
determinations. were made for the stern and the leaves. (The significance of this will be appreciated later). 87.
10. Plant Tolerance to ZethyI Bromide and factors affecting. tolerance.
Before a satisfactory scheme for the control of any pest on growing plants can be laid down it is necessary to know not only how much insecticide is required but what effect, if any, the recommended dose has on the olant.
It is also useful to know to What extent the dose can safely be increased.
The criteria upon which a treatment is judged safe or not vary.
Generally, however, the safe dose-range for plants is considered to be that in which no visible damage to the shoot develops.
This has been called the plant tolerance-range and the determination of its upper-limit for several aphid and whitefly host-plants formed the early part of the present work.
The limit of tolerance was measured at 22°C. by determing the C.T.P. in a 2-3 hour fumigation which just failed to cause damage to any of a group of 10-15 plants. The approximate value was determined by preliminary experiments and plants were then fumigated at several C.T.P. s ranging closely
about this value. The results, which apply to plants kept in a glasshouse during
spring and summer, are given in Table XIX. For the majority of species the tolerance limit was determined on
a number of occasions during the growing season but was found to vary only
Very slightly. Similar values also applied later to plants raised in the
growth-room.
The length of time which was allowed to elapse before plants of a
particular species were judged to have survived a fumigation varied consider- ably. 88.
Table XIX. The maximum C.T.P. tolerated by some herbaceous plants at 22°C.
without visible damar;e occurring; to the shoot.
Plant Tolerance Limit (C.T.P.)
Broad Bean 60
Pea 80
Runner Bean 82
Lupin 80
Tomato 80
Potato 85
Tobacco 65
Lettuce 30
Nasturtium 83
Tulip 96
Cocksfoot Grass 90
Rye Grass 90
With some species it was only a matter of 48 hours but with others up to
12 days. Table XX contains the time intervals after fumigation at which moderate damage to the various plants first became evident.
However, the appearance and distribution of damaged tissues was never exactly the same from one species to another and consequently a brief 89.
Table XX. The length of Uwe after ft/mip7ation at which moderate damage to
plants first became evident.
Plant Time
Broad Bean 18 hours
Pea 6 days
Runner Bean 10 days
Lupin 36 hours
Tomato 20 hours
potato 40 hours
Tobacco 24 hours
Lettuce 6 hours
1Tasturt ium 3-24. days
Tulip 30 hours
cocksfoot grass 5-6 days
Rye [rass 5-6 days
account for each species is included below.
Broad Bean.
18-20 hours after fumi7otion affected leaf tissue was recognisable
by its dullness and lack of turuor. Affected areas were confined at first
to the margins of a few of the older leaves but at C.T.P.s of 70-75 whole
leaves were damaged. These were usually on the lower part of the stem. 90.
30-36 hours after fumi'ation or sooner, dependinc upon the condit- ions (see later), the damaged tissues were dry and brittle and black in colour.
:Tart from the leaves other parts of the plant were damaged. The
stipules were usually readily affected and areas of brown-black tissue on the stem and petiole were common althouFh not readily associable with the dose.
Unlike most of the other species tested, broad beans snowed. a somewhat wide variability in response, so much so that in a batch of 10 plants some were very seriously damaged by a C.T.P. of 75-80 whilst a few were only slightly damaged.
Occasionally, at lower doses, in the absence of any other stem damage, an entire internode was killed and became black and constricted.
The leaves and stem distal to this region then hung vertically downwards.
However, this in no way seemed to impair the continued Erowth and normal appearance of the inverted Part of the plant.
(ii) Pea.
Li average of 6 days elapsed before the gradual discolouration of affected leaves bec&me obvious above a C.T.P. of 80, startini: always with the oldest leaf at the stem base and extendinc, upwards according to the dose. The leaves slowly dried out and assumed a pale straw colour.
The tissues of the lowest internodes were similarly affected. 91.
(iii) Runner Bean.
10 days after fumigation at a C.T.P. of 105 the gradual discol- ouration of all the exposed leaves began. Within a week those became light brown in colour and developed a papery texture.
At C.T.P.s of 85-105 proportionately less damage occurred but Wqe always the pair of large cotyledons vows* seriously affected.
Stem damage was never very marked but a severe inhibition of the characteristic elongation was observed. The plant continued to produce normal leaves, however, except at very high doses.
In plants which had had all the leaves killed at C.T.P.s of 115-
120, out ,rowth of the buds in the axils of the cotyledons was observed after about 2 weeks.
Still higher doses killed the plants entirely.
(iv) Lupin.
Damage was normally visible 36-48 hours after fumigation. Accord- ing to the dose parts or entire leaflets beeale dry and brown in colour.
The oldest leaves were affected first.
(v) Tomato. Affected tissue was recognisable 20-24 hours after fumigation by its dull, dark green colour and wilted appearance. Such areas rapidly became dry and rich brown in colour. Lt C.T.P.s of 80-86 only the tips of a few leaflets were affected but at 95-100 whole loaves were killed, beginning with the oldest. 92.
Dry, irridoscent patches of tissue on the stems and petioles became evident at a C.T.P. of 100 and when the stem apex was killed at 110 lateral buds began to grow out on the lower Parts of the stem.
Occasionally, a petiole collapsed when the leaf was only slightly damaged so that the latter hung vertically from the plant. Living tissue remained healthy for a long time under such conditions.
(vi) Potato.
Damage developed within 56-48 hours of fumigation. Commonly the tips of the youngest leaves were the first to turn brown and dry.
Above a C.T.P. of 92 patches of brown tissue were also present on the stem.
(vii) Tobacco. Damage first became apparent after a C.T.P. of 65 but was never more than slijot until a C.T.P. of 80 or more.
Twenty-four hours after fumigation patches of dark green tissue were evident on the upper surface of some of the older leaves and the areas concerned developed into dry, liht-brown patches of dead cells. These, however, were confined to the upper surface of the leaf. The entire lower surface remained alive and green.
At a C.T.P. of about 84 old, senescing leaves were killed and occasionally a younger petiole withered leaving an otherwise undamaged leaf suspended in a vertical position. Stem damage also occurred at this level and increase in height of young plants was affected producing stunted plants. 93.
(viii) Lettuce.
These were by far the most susceptible plants tested and damage became evident within a matter of hours after fumigation. 6 hours after fumigation at a C.T.P. of 50 the leaves of a plant were found collapsed in a flat rosette on the soil surface. They never recovered and became dry and brown.
At C.T.P.s of 30-40, however, the reaction was less striking.
Areas of dark green tissue were visible 5-6 hours after fumigation which rapidly dried. to a golden-brown colour. The plant as a whole remained erect.
L1thouL;n the exposed tissues were so readily affected by fumigation it was noticeable that even at a C.T.P. of 65 new leaves began to grow within 7-10 days of treatment. Dissection showed that these were growing from the stem apex and were not axillary.
(ix) Nasturtium.
The susceptibility of the 2-! oldest leaves of these plants was considerably greater than that of any others. Within a C.T.P. range of
48-78 a few leaves at the base of the main stem were almost invariably killed althow:h those on control plants usually remained. alive and green.
Above a C.T.P. of 83, however, damage to the other leaves occurred.
Affected leaves gradually lost their colour and either remained
pale yellow or became dry and very pale brown in colour. Damage first
became evident 3-4 days after fumigation. 94-
(x) Tulip.
These plants were among the most resstant.
It C.T.P.s of 90-96 no actual damage occurred but it was noticed that in nlants in flower the peduncle exhibited a marked tendency to bend
over throelh an angle of 900 or more about half" ay along its length.
Lbove a C.T.P. of 96 small streaks of dead tissue appeared towards the margins of some loaves and at the tips. These became more extensive at higher doses, especially at the tip. (In monocotyledonous plants the
leaf grows from the base and the tip is therefore the oldest part).
7-36 hours after fumigation at a C.T.P. of 110 or more considerable
areas of leaf tissue become very limp and dull and dark green in colour.
These areas dried and turned a light-brown colour.
The flowers themselves were very resistant being un-damaged by
a C.T.P. of 120 but they had a just-perceptible unpleasant odour.
(xi) Cocksfoot and Rye grasses.
ist C.T.P.s exceeding 90 the tips of leaves slowly turned brown
from about the fifth day after fumigation, and the extent of the damage
increased with the dose. However: even plants which had all the leaves
destroyed by a C.T.P. of 120 were not completely killed and new growth
began after about 15-20 days.
The effect of a number of factorslacting during fumigation7on the
amount of damage produced was investigated. In order to be able to express the results in other than descriptive terms groups of plants were fumigated at a series of C.T.P.s between the 95. tolerance limit and that causing death of the entire plant and a comprehen- sive picture obtained of the successive effects of steadily increasing doses. Using this information a key was constructed in which the rlifferent degrees of damage were allotted a score of 1-10. The key was then used in experiments to assess the extent of the damage caused by different 0.2.P.s under various conditions.
Between 7-le plants were used per treatment and the mean score was plotted against the C.T.P.
Broad bean, tomato and lettuce plants were used and the appropriate keys are given below.
However, one feature of the results obtained in this way was pecul- iar. Always straight line graphs were produced when the damage score was plotted against the C.T.P. In biological material a linear response of this nature is unusual and it is thought that the keys took insufficient account of the various degrees of "slidht damage" and "serious damage"Itending to group them at the same score. This would Produce an unintended trans- formation of the data in a logarithmic fashion.
BROAD BEAN.
Score
0 No visible damage.
1 Slight blackening of few, sometimes all, stipules. No other
visible damage except occasional slight flecking of part of the
stem or petiole. 96.
Score
2 'Bruising' of the stem and petiole slight, frequently confined to petioles. Stipular damage moderate. Laminar damage very slight or negligible. 3 Tissues round veins at junction of petiole and lamina brown on leaflets of 1 - several leaves but never more than 25 of total
leaflet area. Youngest cluster of leaves rarely affected. 4 Laminar damage 50-60%;total leaflet area in 1-3 leaves. Brown tissues round majority of main veins. May also be slight marginal
blackening. Plant erect. Stipules and petioles, and usually
at least part of stem, affected also. 5 Damage 2-4 leaves more than 607 total leaflet area. Stipules dead and frequently stem and petiole damage. Still about 25%
entire shoot unaffected. 6 Entire shoot seriously damaged. Majority of tissue brown black.
All leaves showing extensive damage, many dead.
TOTI'TO.
Score
0 flo visible damage.
2 Few isolated areas of dead laminar tissue, rarely) 1 cm. in
diameter except occasionally at leaf margin. May be areas of
dead cells on upper surface of oldest petioles. 3 Laminar damage no more extensive than for 2 above but 1 or more of oldest petioles constricted and leaves pendant.
97.
Score Lreas of dead tissue not extensive but several exceeding 1 an.
in diameter. Never more than 4 leaves (usually oldest) seriously
affected. 6 Lreas of dead tissue fairly extensive and some (usually oldest)
leaves completely killed. 4-7 leaves seriously affected. Terminal cluster of young leaves unaffected or only very slightly so.
8 Ls for 6 above but terminal cluster showing damage. Up to 25
of entire shoot unaffected still.
10 All leaves dead. It most only Part of main stem still alive.
LETTUCE.
Score. 0 No visible damage.
2 Damage very slight. Confined to 1-3 leaves of outer whorl and
seen as few isolated areas of dead tissue) usually only a few
mm. in diameter.
Lreas of dee tissue found on up to 7 leaves. Rarely exceeding 1 cm. in diameter and not extending over more than 25 of a
particular leaf area. 6 Damaged areas still rarely exceeding 1 cm. in diameter but forming
an extensive speckling over a large area of the majority of
leaves. Plant at all times erect.
8 Plant showing signs of collapse whilst still green. Few leaves
flaccid. Up to 75% of plant becoming dull green then brown as tissue dies. 98.
Score
10 General collapse of entire plant to form a flat rosette on soil
surface. Never recovers and usually all the leaf tissue dies.
Subsequently, after 1-2 weeks, new leaves may begin to grow.
The effect on the amount of damage of factors acting during fumigation.
(a) Temperature.
In experiments with broad bean, tomato and lettuce the amount of
damage caused at a number of C.T.P.s increased with a rise in temperature
(Tables XXI - XXIII and Figures XIII, XIV, and XV).
(b) Humidity.
The effect of high and low atmospheric humidities during fumigations
lasting 3 hours was investigated using broad bean and tomato plants.
near-saturation humidity was easily attained by enclosing an evaporating'
water-surface in the 500 litre chamber with the plants. L. low humidity
in the presence of transpiring plants, however, was more difficult. The
5,000 litre chamber was used and the plants were fumigated when the soil
was barely damp. The humidity during fummigation was measured with a
calibrated thermo-hydrograph and varied from 37-455 R.H. at 22°C.
Pto difference could be detected between the amount of damage
occurring at the two extremes of humidity at several different C.T.P. s.
(c) Illumination. The effect of fumigating plants in daylight as opposed to the
usual total darkness of a metal chamber was investigated by fumigating 99.
Table XXI, The effect of tempe:i•a:ture on the aii.ount of damaF,e caused to Broad bean plants bzjumdation.
15°C. 22°C. C.T.P. Mean Score C.T.P. 'lean Score 90 0 58.5 100 0.9 70.0 0.6 110 3.2 75.0 2.9 110 4..0 80.0 3.1
130 6.0 92.5 5.7
Om. 100.0 5.8 102.0 6.o
Table XXII. The effect of temperature on the amount of damaRe caused to Tomato plant s 13/ fumir;ation.
15°C. 22°c. C.T.P. Mean Score C.T.P. Mean Score
114.0 0 75.0 0 120.0 2.7 80.0 0.2 130.0 5.8 88.0 3.5 140.0 7.2 95.o 4.o
147.0 10.0 100.0 6.5 105.0 8.o 111.5 10.0 THE EFFECT OF TEMPERATURE ON THE AMOUNT OF DAMAGE CAUSED TO PLANTS fre FUMIGATION tr) Z ' < 6 a 22C
0 4
2 2 z ta
0
50 70 90 110 130 CTP FIG. XIII. (ABOVE) BROAD BEAN
IO N z
a 8 0
0 6
0 2 4
< 2 2
0
70 90 110 130 150 CTP FIG, XIV. TOMATO 100.
Table XXIII. The effect of temperature on the amount of damag e caused to
Lettuce plants by famirration.
15°C. 22°C.
C.T.P. :lean Score C.T.P. .lean- Score
50.0 0 27.5 0 55.0 1.6 31.0 0 69.o 3.8 36.5 2.6 74.5 8.L 44.5 5.9 81.5 10.0 55.0 7.5 56.5 10.0
ar••••-••••••••
tomato plants under glass. This was done out of doors in Juno 1959 when
the sunlight was strong but at the same time insufficient to cause any
appreciable rise in temperature inside the chamber.
very large bell-;jar was used which was clamped tightly by the
rim at its base to an aluminium base-plate. L rubber :asket covered with
thin polythene sheeting separated the two.
The chamber was Provided with a sampling point and dosing was by
means of ampoules which were placed in a small, deep metal dish on the floor
of the chadber and were broken by dropping a metal weicht onto them. The
weight was suspended from a length of cotton thread passing through a short
niece of glass tubing in the stopper and at the time of fumigation the thread was cut, releasing the weight, and the tube was then sealed with a
short piece of glass rod. Because of the small size of the chamber no fan FIG. XV. THE EFFECT OF TEMPERATURE ON THE AMOUNT OF DAMAGE CAUSED TO LETTUCE PLANTS BY FUMIGATION 10
NTS 8 PLA
/10
E 6 COR
4 AGE S AM
N D 2 A E M
0 0 20 40 60 80 CTP 0 FIG.xvi. THE EFFECT OF FUMIGATING TOMATO PLANTS IN SUNLIGHT AT 17 C. 10 TS N
A 8 PL
/6 6 CORE S 4 DAMAGE 2 AN E M
O
70 90 110 130 ISO CTP 101. was provided.
Athermameter was used to measure the slight temperature increase of 1-3°C. which occurred during exposure. The humidity soon became very high as a result of transpiration and evaporation of water from the soil.
Owing to the limited space inside the bell-jar plants were fumig- ated singly. Twelve fumigations in all were Performed, six at a C.T.P. of 110 and six at a C.T.P. of 120. The temperature was 17°C. and the dur- ation of exposure was li- hours.
A parallel series of experiments was performed in the steel chamber, the treatments differing only in the absence of light.
At both doses more damage occurred to the plants fumigated in pAaq daylight althourh the difference was not very great (Table XXIV, Figure
XVI).
( a) Water availabilkty in the soil. Some experiments were performed to ard out whether water-avail- ability in the soil affected the amount of damage.
Tomato plants were fumigated at 22°C. and C.T.P.s of 85 and 100.
The soil in some of the pots was still wet from a heavy watering the previous evening but in others it was beginning to dry out. The plants were not wilting however. After fumigation, some of the plants in the drier soil were watered and the others left another seven hours until wilting became apparent when they too were watered. 102.
the lower C.T.P. damage was only slight and vas similar in extent in the two groups of ?lants which had not wilted. There was possibly just a slight increase, however, in the amount of damage to the plants which had wilted, but it was not very marked. The most striking response of these plants was rather the great length of time taken to recover turgidity even though the soil was then very wet. 29 hours after fumigation 7 of the 12 plants in this category were still slightly wilted although they all recovered within the following 24 hours.
The amount of damage occurring at the higher C.T.P. was appreciably greater in the wilted plants than in the others. A few had recovered from the initial wilt in[ after 4.8 hours but the majority never did so entirely and the un-recoveref'_ tissues died.
In the two un-wilted ::roups of plants a similar amount of damage occurred.
(e) Age of the ELnt.
Tomato giants of different ages were fumigated. at 2200. and a series of C.T.P.s. Plants with 4, 7 and 11 leaves opened were used. Surprisingly little difference was found. between the doses at which damage first occurred in each group and in the relative extent of the damage. The youngest plants were most affected but this was more in regard to subsequent growth than damage to tissues (see later)..
Age differences were most imnortant within the plant itself.
During the conduct of the tolerance tests it was very obvious that the oldest leaves were in most cases the most susceptible to fumigant. This 103. was particularly true if the leaves were senescent. Younger leaves normally had a much treater resistance although in broad bean and tomato in particular, extremely young tissues frequently had less resistance than even old leaves.
(f) Health of the plant. Observations on the susceptibility of un-healthy plants were collected over a long period and this factor was not investi:ated by a specific series of experiments.
lMost plants which were of poor 7rowth or colour or attacked by fungi or heavily infested with auhids were somewhat more susceptible to damage at a particular C.T.P. than normal healthy ones.
(E) Water content of the plant. The relationship, if any, between the water content of a shoot and its tolerance to methyl bromide at '?2°C. was investigated.
The water-content of the shoots (see page86) of 6-10 plants of the same crop used in the phytotoxicity tests was determined separately.
For a few species independent determinations for the leaves and the stem were made. The results are given in. Table XXV.
The mean water content for each species was plotted against its tolerance limit at 22°C. (Figure :VII).
There appeared to be some correlation, apart from the figures
for tulip and lettuce, between water content and tolerance to methyl bromide.
Plants with a hic:h water content in general had the least resistance and
104.
Table XXIV 4 The effect of fumicatinr Tomato plants in sunlight at 17°C.
Sunlight Darkness
C.T P. Mean Score C.T.P. Mean Score
110.0 5.17 110.0 3.17 120.0 7.50 120.0 6.00
Table XXV. The ';5,;water content of the Dlants used in the tolerance tests. Plant `,:!; later Tolerance Cont ent Limit (C.T.P.22°C.)
Broad bean 90.49 Go Pea 83.02 80 Runner Bean (Leaves) 84-46 Runner Bean (Stem) 81.63 Runner Bean (Mole shoot) 83.92 82 Lupin 82.53 80
Tomato (Leaves) 86.63 Tomato (Stern) 93.25 Tomato (Whole shoot) 88.L3 80 Potato (Leaves) 78.04 85 Potato (Stem) 93.38 Tobacco (Leaves) 91.07 65 Tobacco (Stem) 93.08 Lettuce 35.28 30 Nasturtium 81.01 83 Tulip 86.24 96 Cocksfoot ,sass 77.72 90 Rye ryass 78.25 90 k, X v11 'HE RELATIONSHIP BETWEEN WATER CONTENT AND PLANT TOLERANCE
0 WHOLE SHOOT 0 LEAVES ONLY A STEM ONLY 0
90
• 0 • A 80 00 A 0 •
TOLERANCE LIMIT (CTP AT 22 C)
70
Li •
60 O
50 70 80 90 % WATER CONTENT 105. those with a low water content the r.eatest resistance.
The effect of methyl bromide on the appearance of the root astern of plants.
The appearance of the root system of broad bean plants after fumigation at doses which caused damage to the shoot was examined. The pots in which the Plants were :rowing were immersed in water and the roots carefully freed from the pot and sand and then washed.
Surprisingly little evidence could be found of damage even at
C.T.P.s of 90-100 at 22°C. Mat damage there was was confined to the root tip region whore from 0.5-1.5 cm. of tissue were blackened. In a rwoup of eight plants fumigated at a C.T.P. of 95, which caused a mean score for shoot damage of 4.8 out of a possible 6.0, only 13.7 of the total number of roots had the tip region killed.
Another experiment was performed in which broad bean seedlings which had germinated on moist black filter paper were fumigated. In this way the roots were freely exposed to the fumigant and could be examined. directly with a microscope at intervals after fumigation.
At a C.T.P. of 50 at 220C. no adverse effect on the roots could be detected. However, at C.T.P.s in the range 60-75 considerably more root tips were killed than in other experiments at the sane doses in which the plants were growing in sand.
Above a C.T.P. of 50 it was also noticed that the root hairs of a groat many of the roots rapidly perished. The root-hairs of control plants, meantime, remained alive. 106.
11. Some effects of Methyl bromide on the physiology of plants.
(a) Water balance.
In most of the species examined in this work the earliest visible sign that a fumigated plant had been damfted was that parts or entire leaves lost their turgidity. Normally the affected tissues never recovered and became discoloured and died.
Consequently, the investigation of the physiological effects of methyl bromide on plants was begun with a study of. their water-balance after treatment. Two main aspects were involved. One was water loss and the other water uptake and the effect of the fumigant on both of these was investigated. A line of research which led on from these investigations was then pursued. i. The rate of loss of water from the Shoots of fumizated broad bean plants.
Six broad bean plants with shoots 9-10" in height were selected.
Each had four fully expanded leaves and a terminal cluster of developing leaves. The plants were kept together for five hours at 22°C. after which three were fumigated at a C.T.P. of 88 for 2 hours, also at 22°C
For the experiment the shoots of all six plants were cut under water and sealed into glass bottles. The weight of the bottle and rubber stopper when empty and when almost full of water was known, also the weight of the shoot.
To secure the shoot in the bottle the rubber stopper was cut vertically into two and a groove made in each cut face so that when they were apposed a hole to contain the plant stem was formed. The cut end of 107. the stem dipped c. beneath the surface ()IL' the water in the bottle and. the unit was made water-tiOlt by applying melted candle-wax to the junctions of bottle and stopper and stopper and elant stem. The six units were weighed again and then transferred. to the glasshouse whence they were removed intervals for further weighi4:: which was done in the morning before the sun was very high and again in the evening when the sun was setting as well as twice auxins the intervenincr, period. The experiment lasted. three and a half days (in June 1959) during which the weather was brilliantly sunny. Day-time temperatures were consequently high in the glasshouse - about 30°C. at 1.0 p.m. - and. the relative humidity was down to 50;,-D at these times. During the nights the temperature never fell below 37°C. and the relative humidity was 85-90%. The un- furai. at ed. shoots D, E and F, remained healthy and of normal colour throughout the experiment but in the fumigated Shoots, A, B and C, areas of dead_ tissue could be seen 413 hours after the start of the experiment.
On the third. day the extent of the damage to these Plants was as follows:
A A few brown flecks on int e,rnodes 2 end. 3 and petiole 1. No leaflet azmaee, but all the stipules dead. and black.
B Leaf 24. with 75 of each leaflet dead. /^i.11 the stipules dead. C Plant heavily damaged. Between 30-60% of the leaflet area of leaves 1-4, dead and part of the unopened leaf 5. All the stipules dead and. the stem black and constricted in internode 3, the distal part of the shoot honeine inverted. from this point. 108.
.e,:t the end of the experiment the shoots were removed from the bottles and the wax fragments collected and. weighed. The stem bases were wiped dry and the wet weight of the shoot was determined. Each shoot was then cut up into pieces and dried to constant weight in an oven at 105°C.
Finally, the '.rreight of the bottle, stopper and remaining water was found. The results are given in Tables :CM and XXVII.
The experiment was based upon the assumption that the gradual loss in weight of the bottle/water/shoot unit could be attributed almost entirely to the lors, of water by evaporation from the exposed parts of the shoot. It was argued that if the change in weight was sufficiently great, changes due to respiration and growth could be ignored. The results in Table XXVI support this view. The weif-ht of water removed from the bottle by the shoot (vii) during the experiment was very nearly equal to the total loss in weight of the heft le/wat er/shoot unit (iii). The small difference was very nearly the same as the increase in wet weight of the shoot (v), except in Co which was heavily damaged, and corresponded to the retention of water by the shoot (viii) • Table TKVII contains the measurements of the amount of water lost up to the given time intervals during the exPeriment . They are expressed as grammes of water lost per gramme of the dry weight of the shoot at the end. of the experiment. The change in wet weight of the shoots (Table XXVI (v)) was so small that any dry weight differences arising during the experi- ment would be negligible. The data contained in Table XXVII have also been plotted -fraphicall,y 109.
Table ..xxvi. Details of the experiment to investi;7ate the rate of loss of water from fumigated broad bean shoots (C.T.P. = 88 at 2200.)
FumiEated Un-Fumi,sated L B C D E F
(a) Wt. Bottle + Stopper 156.10 160.39 178.44 132.64 178.27 180.87 (b) Vt. Bottle + Stopper + vlater 272.66 276.60 357.77 256.30 358.73 358.74
(1) Initial Vt. Water 116.56 116.21 179.33 123.66 180.46 177.87 (c) Wt.Bottle + Stopper + Water + Shoot + Wax 280.94 285.95 366.53 263.67 366.68 366.68 (ii) Vet 7t. Shoot Before Expt. 8.12 8.95 8.49 7.23 7.75 7.74
(a) Vt. Bottle + Stomper + Water + Shoot + Wax After c.80 Hrs. 258.40 254.20 314.75 223.18 322.50 336.10 (iii) Total Loss in Wt. of Bottle/Water/Shoot Unit during Expt. 22.54. 31.75 51.78 40.49 44.18 30.58 (e) Wet Vt. Shoot After Exot, 8.49 9.35 6.75 8.37 9.35 8.45 Wt. Bottle + Stopper + Water + Wax after Expt 248.90 244.77 307.87 214.72 312.97 327.49 7t. Wax 0.1608 0.4011 0.2737 0.14) 4 0.2019 0.2044 Dry Wt. Shoot at End of Expt. 0.89 1.01 0.90 1.04 0.97 0.96 11-20 Content Shoot at End of Expt. 89.52 89.20 86.67 87.57 89.63 88.64. Charce in Wet Wt. of Shoot Expt. +0.37 +0.40 -1.74 +1.14 +1.60 +0.71 (vi) it. Water Lost by Evaporation from. Shoot 22.54 31.75 51.78 40.49 44.18 30.58 (vii) Water removed from Bottle by Shoot 22.94 32.23 50.17 41.72 45.96 31.45 (viii) Wt. Water retained by Shoot 0.40 0.4.8 ••• 1.23 1.78 0.87
110. Table XXVII. The rate of lose of water ;roll rural( ate 7. anc".1 un-fumir;ated Broad bean shoots (0.T = 38 at 22°C.). C : Fumisa.tea. D : 1 B C Time Since Loss of Time Since Loss of Time Since Loss of Sealing Shoot Water/n. Dry Sealin Shoot Whter/m. SealinE, Shoot Whter/gm. in Bottle Wt. in Bottle Dry Wt. in Bottle Dry Wt.
2 hrs. 0 mins. 0.47 g. 2 hrs. Omins 0.42 c. 2 hrs. Omins. 0.36 g.
4 " 9 " 1.54 4 " 9 " 1.41 4 " 4 " 1.70 19 it 9 ,, 2.96 19 " 8 " 2.62 19 " 6 " 4.81 23 " 1 " 4.97 23 " 0 " 4.36 22 " 56 " 9.26 25 " 50 " 6.78 25 " 50 " 5.90 25 " 48 " 13.65
28 " 40 " 7.79 28 " 40 " 6.73 28 " 35 " 16.31 42 " 35 " 9.20 42 " 35 " 8.10 42 " 31 " 21.31 49 it 25 v, 16.13 49 " 25 " 16.88 49 " 21 " 37.76 52 it 53 " 17.56 52 " 53 " 18.67 52 " 48 " 41.15 66 " 50 " 18.99 66 " 49 " 20.79 66 " 45 fT 45.04 77 " 14 " 25.31 77 " 5 " 31.44 77 " 10 " 57.54
D E F 3 hrs. 37mins. 1.86 g. 3 hrs.36mins. 2.08 s. 3 hrs.39mins. 1.97 i.;. 18 " 37 " 3.37 18 " 37 " 3.90 18 It 40 " 3.65 22 " 28 " 7.04 22 " 26 " 7.38 22 " 28 " 7.11 25 " 20 " 9.97 25 " 19 " 10.52 25 " 21 " 10.23 28 " 17 " 11.56 28 " 16 " 11.91 28 " 18 " 11.59 42 " 3 " 15.02 42 " 4 " 14.93 42 " 7 " 14.80 48 " 54 " 27.65 43 " 53 " 28.49 48 " 56 " 25.24 52 " 19 " 29.93 52 " 17 " 31.52 52 " 19 " 26.38 66 " 17 " 32.62 66 " 15 " 35.18 66 " 18 " 28.11 76 " 42 " 38.94 76 " 40 " 45.54 76 " 43 " 31.85 FIG. XVIII. THE RATE OF LOSS OF WATER FROM FUMIGATED
BROAD BEAN SHOOTS
60
A-C : FUMIGATED D-F :UiFUMIGATED 50
40 D
GMS. WATER LOST / GM. DRY WEIGHT
30
20
10
O 0 20 40 60 • 80 TIME (HAS) 3.30 P M. NOON NOON NOON NOON 11 JUNE 12 JUNE 13 JUNE 14 JUNE IS JUNE
(Figure XVIII). The amount of water lost from the sheets during the day
was considerable whereas at night it was very small. Hence the 'stepped/
appearance of the curves.
Until the end of the second full day cf the experiment the rates
of loss of water from the un-fumigated shoots were almost identical but there-
after differed widely, presumably because of physiological changes attendant
upon keeping a cut shoot in a bottle of water. The rates of loss of water from the fumigated shoots A and. B were
also similar throughout the experiment but much less than the rate of loss
from the un-fumigated controls. On the other hand, the rate of loss from
C exceeded the rate of loss from both A and B and from the controls during
the first full day of the experiment and subsequent days.
These results were supported by those of a preliminary experiment.
The rate of water loss by evaporation from fumigated bean shoots was less thar.,
that from un-fumigated shoots under the same conditions unless very much tiss-
ue damage occurred. The rate of evaporation than exceeded even that of
un-fumigated shoots.
The amount of water lost by shoot C (Table XXVI (vi)) was in excess
of the amount taken up from the bottle (Table YXVI (vii)) but the difference
was almost the same as the decrease in wet weight of the shoot during the
experiment; it must therefore represent the water lost from the dead cells.
ii. The rate of uptake of water by the roots of fumigated broad bean Plants.
Six broad bean plants were fumigated at 22°C. with their shoots
enclosed in 500 gauge (5/1000" thick) polythene sheeting. Three received a 112,
C.T.P. of 30.0 and three a C.T.P. of 55.0. Alter treatment the polythene was removed and the plants, together with three un-fumigated plants, were partly immersed in water and the roots carefully washed free of sand.
Each plant was then sealed into a glass bottle containing water
(see 'figure XIX:). The bottles were made water-tight by applying melted candle-wax to the joints around the rubber stopper and were shielded from sunlight by covering them with black paper. From each bottle a glass tube dipped into a 25 ml. measuring cylinder, graduated in 0.2 ml., also contain- ing water. L. thin layer, of toluene on the water surface in the measuring cylinder prevented evaporation.
The apparatus was set up by completely filling the bottle with water. This was done by sucking out any air present by way of a third glass tube wnich passed only part of the way through the rubber stopper.
Water was drawn into the bottle from the measuring cylinder and when the bottle was full the mouth piece was closed with a screw-clip.
The nine units were transferred to the glasshouse. As water was taken up by the roots it was automatically replaced by water from the measur-
ing cylinder. Thus as the water level in the measuring cylinder fell it was possible to measure directly the quantity of water being taken up by the plant.
Readings were taken at hourly intervals throughout the day. It was not possible to continue an experiment longer with any particular set of plants because the rate of uptake became very uneven on the second day,
presumably because of the lack of oxygen in the water in the bottle. .FIG. XIX. THE APPARATUS USED TO MEASURE THE RATE OF UPTAKE OF WATER BY BROAD BEAN PLANTS
%my
/ I / 113.
When more water was required in the measuring cylinder it was added from a pipette vvith a drawn-out end which was pushed below the toluene layer.
Lt the end of a series of observations the plants were removed from the bottles and the dry weight of the roots and shoots was determined separately. A correction to the dry weight of the roots was applied, however) after determination of the weight of the small amount of sand which was still adhering to them. The dishes plus dry roots were placed in an electric furnace to drive off the carbon and the weight of the remaining sand was determined.
The results of a typical experiment are given in Table XXVIII.
The rate of water uptake is expressed in terms of ml./;. dry weight of both the roots and the shoot. The data are also expressed graphically in
Figures XX and XXI.
Since only the roots were exposed to fumigant (see footnote p.12o) the shoots were presumed to function normally. Fumigation caused a decrease in the rate of uptake of water by the roots which was greatest at the high dose. (In similar experiments in which plants were used 42 hours after fumigation at the same doses the rate of uptake was still very low in fumigated plants compared with un-fumigated plants). iii. The effect of Methyl bromide on the water content of plants.
Twelve broad bean plants were fumigated for 2 hours at 22°C. at
C.T.P. of 86. Afterwards the plants were transferred to the glasshouse and removed at intervals in pairs and the water-content of the shoots 114. Table XXVIII. The rate of uptake of water by Broad bean roots. A - C : UnFumigated. D F : Fumigated, C.T.P. = 30.0.
G I : C.T.P. = 55.0.
(a) ml. water taken up / g. dry wt. of the shoot. Time Interval A B C D E F G H I (Hours)
1 3.62 3.26 3.02 2.92 2.93 1.50 1.43 0.92 1.74 2 6.59 6.46 5.98 5.10 5.53 2.82 2.27 2.02 2.69 3 11.30 11.28 9.65 8.74 8.80 5.47 4.53 3.39 4.86 4 13.77 14.69 13.02 10.93 11.87 7.06 5.96 4.22 6.08 5 18.04 19.23 16.39 14.21 14.73 9.53 7.55 5.50 7.90 6 20.43 22.21 18.70 16.83 17.05 11.82 8.76 6.87 9.55 7 22.90 24.06 20.06 18.95 19.44 12.88 9.89 7.79 10.68 8 24..64 25.48 21.54. 21.35 21.69 14.47 10.64. 10.08 11.38 9 25.87 26.26 22.08 22.37 22.44 15.44 11.55 11.92 12.16
(b) ml. water taken up / gm. dry wt. of the roots. Time Interval A B C D E F G H I (Hours) 1 6.76 6.66 6.20 5.07 4.81 2.69 2.71 1.83 3.28 2 12.31 13.18 12.27 8.87 9.07 5.06 4.28 4.03 5.08 3 21.10 23.03 19.80 15.20 14.4/1 9.81 8.55 6.78 9.18 4 25.70 29.99 26.73 19.00 19.47 12.65 11.26 8.43 11.4.8 5 33.68 39.26 33.65 24.70 24.17 17.08 14.26 11.00 14.92 6 38.14 45.78 38.39 29.26 27.98 21.19 16.54. 13.74 18.04 7 42.74 48.82 4.1.19 32.93 31.90 23.09 18.68 15.58 20.17 8 4.5.98 52.01 44.22 37.11 35.59 25.94 20.10 20.16 21.48 9 48.28 53.60 45.32 38.89 36.82 27.68 21.81 23.82 22.95
Continued. 115.
Table XXVIII Cont.
(c) The dry weights of the shoots and roots of plants 1.
Plant Dry 7t. Shoot Dry lt. Roots Sand Corrections
A 1.3802 g. 0.7394. g. 0.0500 g. B 1.4.092 0.6903 0.2208 C 1.6896 0.8231 0.0427 D 1.3723 0.7895 0.5891 E 1.4.660 0.8935 0.9856 r 1.1333 0.6323 0.2729 G 1.3249 0.7014 0.8368 H 1.0911 0.52457 0.3333 I 1.1513 0.6099 0.0317
Table XXIX. The % water content of the shoots of Broad bean plants at intervals after fumigation at 22°C. (C.T.P. = 86.0).
Time Since % Water Mean end of Content of Fumigation Shoot
3 hrs. 90.97 It 91.40 91.19
5 hrs. 90.70 91.55 91.13 8 hrs. 90.33 90.69 90.51
22 hrs. 91.10 it 90.53 90.82 27 hrs. 90.77 90.35 90.56 4647. hrs. 91.63 rr 91.30 91.47 Unfumigated 90.43 90.93 90.68 FIG. XX.THERATEOFUPTAKEWATERBYBROADBEAN / MLS. WATERT AK EN UP GM. DRYWT.
OFSH OOT ROOTS MEASUREDASMLS.WATERTAKENUP/GM. 30 25 20 15 I0
0
DRY WEIGHTOFTHESHOOT D-F :FUMIGATED.CTP•30.0 G-I :FUMIGATED.CTP.55.0 A-C :UNFUMIGATED 4
6
10 HOURS FIG. XXI. THE RATE OF UPTAKE OF WATER BY BROAD BEAN
ROOTS MEASURED AS MLS. WATER TAKEN UP/GM.
DRY WEIGHT OF THE ROOTS
A-C : UN FUMIGATED D- F : FUMIGATED. CTI2g30.0 G- I : FUMIGATED. CTP=55.0
O 2 4 6 8 10 HOURS 116. determined. At the same time as the first determination the water-content of two un-fumigated shoots was found. The results are given in Table XXIX.
The plants were not heavily damaged and only a small aznount of wilting occurred. The percentage water-content of the shoots hardly varied
from that of the controls during a period of 48 hours following fumigation.
The experiment was repeated at a C.T.P. of 116. Within 20 hours of the end of fumigation intact plants were seriously wilted and were dead
another 30 hours later. The wat er-cont ent of the shoots of other plants at successive intervals after fumigation is given in Table m.
A fall in the water-content of the shoots coinciding with visible
signs of wilting was demonstrated. Later, (p. i23), an experiment will be described in which ten lettuce plants were fumigated, also for 2 hours at 22°C., at a C.T.P. of 100. Pour
of these plants had a. piece of polythene sheeting fastened securely round the stem so that the pot, soil and root-system were protected from the
fumigant. .another four plants had the shoot protected while the remainder had both roots and shoots exposed. Only hours after the end of fumig-
ation different degrees of wilting could be recognised in the various groups of plants and the degree of wilting was reflected in the water-content of the plants at that time (Table XXXI). iv. The effect on survival of high doses of fumigant of cutting the shoot
after treatment.
In the experiments, already described, in which the rate of loss of water from fumigated shoots was investigated it was found that the shoots failed to develop the expected amount of damage after being sealed into the 117.
Table :Ca. The % Water content of the shoots of Broad bean plants at
intervals after ftaigation at 22°C. (C.T.P. = 116.0).
Time Since % '?latex' hean End of. Content of Fumigation Shoot
5 hrs 90.74 U 91.00 90.87 20:17 hxs. 89.30 89.07 89.19 25 hrs. 87.77
11 86.54 87.16
Unfumigated 90.72 90.13 90.43
Table =I. The $;vmter content of Lettuce shoots 5 hours after fumigation,
(C.T.P. = 100 at 22°C.)
Unfumigated Roots and Roots Shoots Shoots exposed Protected Protected
87.66 83.07 88.84. 84..88
88.94 83.47 88.60 86.49 85.79 87.22 87.45
83.86
Mean 88.30 84..05 88.22 86.27 118. bottle of water. In order to obtain shoots in which tissue damage occurred after cutting, increased concentrations of fumigant had to be used.
This observation has since been confLwed on many occasions and with several species of plant. If there is little damage to intact plants at a particular dose the cut shoots frequently show no damage at all when kept in water; when the intact plant is almost completely killed the cut shoot may retain a considerable amount of living tissue.
Eight young broad bean plants, each with six fully expanded leaves, were fumigated for 2 hours at 22°C. at a C.T.P. of. 93. Afterwards the shoots of four of the plants were cut at sand level and placed in a beaker of tap water which was left alongside the four pats of uncut plants in the glasshouse. Three days later the amount of damage to the eight shoots was as follows:-
A: Shoots Uncut. I: Entire stem black and constricted. Plant hanging over the side
of the pot. liost of the leaves black and dead except a small
amount of green tissue in the terminal cluster and small areas in leaves 3 and 4.
II: Proximal half of the stem blackened and the distal part of the
plant reflexed from the constricted third internode. Leaves 1, 2, 3, 5 and 6 black and dead. Patches of dead tissue in leaf 4 and the unopened terminal leaves.
III:Proximal third of the stem blackened. Leaves 1, 2, 3 and 6 black
and dead but leaves 4. and 5 and the terminal cluster only very slightly damaged. 119.
IV: Intornodes 3 and. 4 black and the plant reflexed about the middle
of internode Leaves 1, 4, 5 and 6 black and dead. Leaf 2
slightly damaged.
B: Shoots Cut and in Tater.
I: Parts of the stem slightly flecked with brown patches. Up to
7 of leaves 1 and 2 dead and extensive marginal damage in leaf
3. Part of the terminal cluster dead.
II: Scattered brown patches over about -1:5 of the stem. Leaves 2, 3
and 4. with marginal damage amounting to about one-third of the
total leaf surface. Part of the terminal cluster dead..
Internodo 3 blackened. Leaves I, 5 and 6 almost entirely killed.
IV: Proximal half of the stem blackened. Damage to leaves 3, 4-, 5
and 6 mostly marginal and about one--third total surface. Tips
of the leaflets of leaves 1 and 2 and the terminal cluster dead.
The presence of a greater amount of green tissue in the cut shoots was very obvious.
In another experiment eight tomato plants, between 12_15" in height and with 5-7 fully expanded leaves, were fumigated for 2 hrs. at 22°C. and a C.T.P. of 90. Again .'our of the shoots were cut after treatment and placed in tap water in the glasshouse. The amount of damage to each four days later was as follows: 120. A: Shoots Uncut.
I: Leaves 1, 2 and 4 dead. Tips of nearly all the leaflets in .Leave6
3 and 5 and parts of the terminal cluster dead. Petioles of leaves 3 and 4 with quite extensive dead tissue.
Leaves 2, 5 and 6 completely killed but leaves 1, 3 and 4 un- damaged.
III:Leaf 5 dead. About half of leaves 1 and 2 and the tips of most of the leaflets of the other leaves dead.
IV: Parts of the stem flecked with dead tissue and also the petioles
of leaves 4, 5 and 6. No leaf entirely killed but leaves 1, 2 and 3 heavily damaged.
B: Shoots Cut and in Water.
I: Only the tips of a few leaflets in leaves 3, 5 and 6 damaged.
II: Leaf 1 dead and a few leaflet tips in leaves 2, 4, 5 and 6.
III:Half the laminar tissue of leaf 3 dead and most of the terminal
cluster.
IV: The tips of a small number of ;eaflets of leaves 3, 4 and 5 dead.
Again the much heavier damage to the un-cut Shoots was obvious. v. The effect of protectin the root-system from fumigant.
Broad bean, tomato and lettuce plants were fumigated with a piece of 500 gauge polythene sheeting enclosing the pot, soil and root system.
* NDTE: An estimate of the amount of Methyl bromide which penetrated the polythene was obtained by constructing a metal-supported cylinder of the material which had a similar surface area to that of the pieces of polythene used to enclose the pots. The apparatus was suspended in an atmosphere of
Methyl bromide under the experimental conditions and the gas mixture inside was subsequently analysed. riven at higher concentrations and longer, exposures than those of the experiment the amount of Methyl bromide which penetrated the polythene was negligible. 121.
The polythene was tied securely round the base of the plant stem and melted candle—wax was used to seal any open passages which remained.
Protecting, the root system in this way considerably reduced the amount of damage.
Eight broad bean plants, four of which had their root systems protected, were fumigated at 2290. and a C.T.P. of 80 for two hours. After airing they were transferred to the glasshouse and the pieces of polythene removed. Each pot was well watered.
The following morning the shoots of the four plants which had hAd their roots protected during exposure to the fumigant were fully turgid and the leaves firm and fleshy. The shoots of the other four plants, however, were distinctly wilted and their leaves soft and flaccid and beginning to wrinkle.
On the third day (67 hours after the end of fuMigation) the amount of damage to the shoots was as follows:
A: Roots Exposed.
I: No stem damage. Leaves 1, 2 and 3 almost completely killed and
leaves 4, 5 and 6 and the terminal cluster with marginal damage
to the leaflets.
II: Scattered brown patches over most of the stem. Marginal damage
to the leaflets of leaves 3, 4 and 5. III:Internode 5 completely black and constricted. Up to q of the
total laminar tissue of leaves 1 and 2 killed. All leaves of the
terminal cluster killed. 3.22.
IV: Internodes 3, 11., 5, 6 and 7 with numerous patches of brown tissue.
Tissue at the junction of petiole and lamina in leaflets of leaves
3 and 5 black and also in leaves of terminal cluster.
B: Roots Protected. I: No stem damage. Small amount of dead tissue in the terminal cluster. II: No stem damage. Up to 714: of the total laminar tissue of the leaflets of leaves 3 and 4 killed. III:No stem damage. Small amount of marginal damage in leaves 4 and 5.
IV: No stem damage. A few patches of dead tissue in leaves 1, 2, 4 and 5. In a similar experiment with tomato plants some had their roots protected as before but this time plants were included which had their shoots protected and their roots exposed. Twelve plants, varying in height from 11-15" and having 6-7 fully expanded leaves, were used in the experiment. The C.T.P. was 101 at 2200. and the duration of the fumigation was 2 hours. After airing, the poly- thene was removed and the plants transferred to the glasshouse. The amount of damage to the plants 7.2 hours after the end of fumigation was as follows:
A: Roots and Shoots Exposed. I: Leaves 1, 2 and 3 dead and dry. Extensive damage to leaves 4 and 5. Very slight to leaf 6 and the terminal cluster. 123.
II: Leaves 1 and 2 dead. Prom one-third to two-thirds of leaves
3, 5 and 6 dead. Up to one-third of leaf 4. dead.
III:Leaf I dead. Extensive damage to leaves 3 and 24. but only a few leaflet tips killed in 5 and 6.
IV: Extensive damage in leaves 1, 2, 3 and 5. Only a few leaflet tips killed in 4, 6 and 7.
B: Roots Protected.
I: Leaf 1 dead. Fairly widespread damage to the tips of most of
the leaflets of leaves 2$ 3, 4., 5 and 7. II: A few of the tips of the leaflets of leaves 2, 3, 4 and 5 killed.
III:Leaves 3 and 4. with more than 2 of the laminar tissue dead. A few dead leaflet tips in 5 and 6 and the terminal cluster. IV: One dead leaflet in leaf 2. One-third of leaf 4 dead. A few dead leaflet tips in the terminal cluster.
C: Shoots Protected.
No damage to any of the plants, which are erect and turgid.
A decreased amount of damage in plants which had their roots
protected was again apparent. Protection of the shoots at this C.T.P.
completely prevented damage (cf. lettuce experiment below).
Whereas the descriptions of fumigated broad bean and tomato plants
just given are the results of only one of several similar exDeriments in which the same conclusions were reached, the following is an account of an un-repeated experiment using lettuces. At this particular time only a small number of lettuce plants were at hand. 124..
Ten plants were used three of them having the root system and. three the shoot enclosed in polythene in the usual way. They were fumigated together at a C.T.P. of 100 at 22°C. in a fumigation lasting 1 hour.
After airing, the polythene was removed and the plants transferred to the glasshouse. Four and a half hours later the appearance Of the different groups of plants was as follows.
A: Roots and Shoots Exposed.
The four plants seriously wilted. The leaves soft and flaccid.
B: Roots Protected.
The plants fully turgid. The leaves filn and erect.
C: Shoots Protected.
The plants obviously wilted but not to the same extent as those of
A. The leaves semi-erect and midway between those of B in regard to firmness when handled.
Since exposure to such a high C.T. P. was certain to be followed by total death of at least the plants in group A, none of them was kept longer. V. "7 Instead, the water-contents of the ten shoots were determined (Table MI). A (These measurements have been discussed in (iii) above).
Vi. The effect of a water-saturated atmosphere during the post-ftunigation
period.
Broad bean and tomato plants were kept after fumigation in a water-saturated atmosphere. The development of damage to the tissues at high doses was considerably delayed although not appreciably changed in 125. amount.
A wooden frame measuring 2' x 2' x 2' and covered with polythene sheeting was placed over a dish of hot water in a well-lit room at 17°C.
Within a short time condensation droplets covered the inside of the cage.
A thermo-hydrograph indicated that the air inside the cage rapidly approached saturation humidity which was maintained indefinitely at this temperature in the presence of the water surface.
Eight broad bean plants were fumigated at a C.T.P. of 93 at 22°C. for 2 hours. Afterwards four of the plants were placed in the wat er- saturated atmosphere of the polythene cage and the other four left un- covered on the same bench.
The following day the plants on the bench were seriously wilted although those in the cage were fully turgid. Purth„Irmore, the latter still had this appearance 0 hours after the end of fumigation. Not so, however, the plants on the bench in which a considerable amount of dead tissue was evident.
The plants from the polythene case were then removed to an un- saturated atmosphere and after 16 hours areas of dying tissue were to be found.
Seventy-two hours after fumigation the amount of damage to the shoots of both sets of plants was as follows:
Plants Kept in en Un-Saturated Atmosphere.
I: Proximal one-third of stein blackened. More than two-thirds of
the leaflets of leaves 3 and 4 dead and the entire terminal cluster. Small amount of damage to leaves 1 and 2. 126.
II: Stem more or less completely covered with patches of brown tissue.
Leaves 1 and 4 killed and up to one-third of the leaflets of
leaves 2 and 3.
III:No stem damage. Leaf 1 and the terminal cluster dead. Only
slight marginal damage to the other leaves,
IV: Internode 3 blackened. Up to two-thirds of the leaflets of
leaves 3 and dead. Slight marginal damage to leaf 2.
B: Plants Sept in a 7ater-Saturated Atmosphere for 48 Hours.
I: Few patches of brown tissue along the stem. Leaves 1 and 2
dead and also part of terminal cluster. Tips of leaflets of
leaf 4 dead. II: Patches of brown tissue on stem. Tips of leaflets of leaf 4
dead and most of terminal cluster.
III:Patches of brown tissue on stem. Marginal damage in leaves 2,
3 and 4 and terminal cluster. IV: Internode 5 blackened and terminal cluster pendent. Leaf 1
dead and marginal damage to leaves 2 and 4.
The amount of damage which finally occurred to the broad bean plants kept for part of the time after fumigation in a water-saturated atmosphere was less than that in plants which did not receive this treatment.
However, as the dose was increased the difference in the amount of damage to plants receiving the two post-fumigation treatments rapidly dwindled.
Again, similar experiments were performed with tomato plants.
Ten plants were fumigated at 22°C. at a C.T.P. of 108 for 2 hours. 127.
Five were kept in a saturated atmosphere and five in an un-saturated one
after airing. Within 20 hours of the end of fumigation the plants in the
un-saturated atmosphere were seriously wilted whereas those in the saturated
atmosphere were still turgid. (Three hours after putting the plants into the polythene cage droplets of water produced by guttation were evident at
several places at the edges of the leaflets).
Forty-six hours after the end of fumigation the plants on the
bench were almost dead whereas only two leaflets were affected among the
plants in the polythene cage. These leaflets were darker green than usual
and hung in a collapsed fashion from the leaf axis.
/It intervals of 20, 36, 48 and 72 hours after the end of fumigation
a plant was removed from the polythene cage and left on the bench.
Invariably the plant soon wilted and died.
Unlike broad beans, however, tomato plants, after being treated
at dosages causing only a small amount of damage and kept for up to 80
hours in a saturated atmosphere, did not show an enhanced recovery. 128.
(b) The effect of fumigation with methyl bromide on the stomata of broad
bean plants.
Following the observation of a reduction in the rate of transpir- ation from broad bean plants after fumigation the condition of the stomata under these circumstances was investigated. This was done by means of a modified version of Darwin's porometer.
The apparatus (Figure XXII) is based upon the assumption that when a portion of the undersurface (of a hypostomatal leaf) is enclosed and a gentle suction applied, air will be drawn out of the leaf and replaced by air enterinc the stomata on the remainder of the leaf undersurface. By measuring the rate of flow of air through the leaf en estimate of the degree to which the stomata are open under the given conditions should be obtained.
A slow rate of flow will indicate a greater resistance to the entry of air, and hence a state of near-closure of the stomata, and a rapid rate of flow will indicate that the stomata are widely open.
A porometer cup, 2 x 2 x 2 cm., was made from perspex. One rim was covered with thin sheet-rubber and was fastened (see Figure XXII b - c) against the leaf surface using a gasket cut from a gelatine plate, 2-3 mm. thick, prepared in a petri-dish.
At one side of the perspex cup was a short polythene tube (p) which was normally closed but which could be opened when required to allow the space enclosed in the cup to be flushed with a direct stream of air.
From the base of the cup another polythene tube was connected to a two-way glass tap. special Perspex adhesive was used to secure the FIG.XXII • DESIGN OF THE POROMETER USED TO MEASURE STOMATAL APERTURES (.)
METHOD OF FASTENING POROMETER CUP TO THE LEAF (s- c)
/ / / 129.
polythene tubes in place andito prevent movements fire cement, painted when
hard with shellac, was applied externally at the junction of the tubes and the outer surface of the cup.
!as° attached to the two-way tap was a lone Blass tube dipping at
its lower end into water in a beaker. The tube was filled by turning the tap so that the porometer cup was cut off and applying; suction by means of a mouthpiece (q).
When the water had been raised to a pre-determined level in the tube and the tap closed, the apparatus was ready for use. The side tube
(p) was closed and the porometer and water column put into communication.
The level of the water in the tube then becan to fall steadily as air passed through the leaf as a result of the suction arisinn from the weight of the water column. The time taken for the water in the tube to fall a distance of 20 cm. was then determined. In strong sunlight the time was about 1 minute.
Between Lroups of readings the porometer cavity was flushed with fresh air and the leaf left for a period of 10 minutes or so before further readings were taken.
In the first series of experiments the rate of flow of air through the leaves of fumigated plants was compared with that through similar un- fumigated plants under the same conditions. Observations were made simultaneously using two sets of the awaratus described.
Four hours after fumigation at 22°C. at a C.T.P. of 65 the rate of flow of air through fumigated leaves was considerably less than through the 130. controls. This was confirmed with several pairs of plants. The observ- ations were made in bright sunlight between 2.0 p.m. and 4.0 p.m. during
August 1959.
The apparatus was then modified for making observations on plants inside a chamber simply by increasing the lenth of the tube connecting the porometer cup to the two-way tap. The porometer was attached to a leaf in the usual way and the connecting tube led through an outlet in the side of the chamber to the water column outside. Readings were kept to a minimum because of the un-natural situation arising from the drawing of fumigant into the leaf tissues.
Observations on plants inside a closed chamber before the admission of fumigant confirmed that the stomata were closed. A reading was then taken 20, 40, 60 and 100 minutes after the start of fumigation for each of o three plants inside the chamber during a 2 hour fumigation at 22 C. at a
C.T.P. of 60. On each occasion the stomata were closed.
Finally, the behaviour of stomata during fumigation in sunlight was studied. Prior to breaking the ampoules the stomata of broad bean plants were open although for some reason never to quite the same extent as when the bell-jar was removed. Nevertheless it only took about 1 minute for the level of the water in the tube to fall completely.
If a reading was then taken five minutes after liberation of the fumigant the stomata were still open to about the same extent. Fifteen minutes after the start of the fumigation, however, the rate of fall of the water in the tube was noticeably slower and thirty minutes after the start of the fumigation the stomata were virtually closed and remained so for the 131. remainder of the fumigation (and indeed for very much longer).
Even in the absence of fumigant the stomata of broad bean plants kept inside the bell-jar chamber tended to close slightly after about an hour but the degree of closing was only small compared with that in plants exposed to fumigant.
It would appear, therefore, that the stomata of fumigated plants remain closed during fumigation in a normal un-lit chamber and close soon after the start of a fumigation performed in sunlight. Recovery is slow and at high doses the stomata may remain almost closed for several days after treatment. 132.
(c) The effect of Methyl bromide on growth.
During the course of the work reported so far there were several
indications that fumigation of plants at high doses resulted in an apprec-
iable check to growth. The stunting of young tobacco plants above a C.T.P.
of 85 was one such example and the failure of runner bean stems to elongate
above a C.T.P. of 100 was another.
In order to examine this effect systematically an experiment was
designed in which the growth of young seedlings could be measured after
exposure to different concentrations of fumigant. Peas were chosen for the
purpose because of their extensive growth in a vertical direction only which
therefore made measurement easier.
Twenty-four pea plants were selected which were identical in
regard to a:e, growth conditions, height and number of leaves un-furled.
They were divided into three groups of eight and labelled M, Td and 0.
GroupM was set aside as a control and the other two fumigated
at 22°C. on 10 August 1959, the duration of the fumigations being 2 hours.
The C.T.P.s were 25.0 (N) and 73.5 (0), the higher dosage approaching the maximum tolerated by mature plants and the lower being just sufficient to
kill most species of aphids.
At the time of fumigation the plants were 13.5 cm. in height and
had 3 leaves fully expanded. They were 13 days old.
After fumigation the plants were returned to the glasshouse along-
side the controls and at intervals of 2-3 days the height of each shoot was
measured. 1:1hile the plants were small this could be done directly but later 133. a more satisfactory method was to fit a piece of thin copper wire along the stem and then measure the length of wire against a metre ruler. The height was taken as the distance between the soil level and the tip of the stipular tissue enclosing the stem apex.
As growth progressed the number of fully un-furled leaves was also noted and later the number of flowers and pods (Table XXXIII).
The plants remained alive for the duration of the experiment
(59 days) during which time the growth of the controls was vigorous, produc- ing sturdy, healthy plants. As the plants increased in height it was necessary to provide them with a piece of thin glass tubing to which they could be fastened for support.
The mean height of each group of plants at intervals after fumig-
ation is given in Table XXXII and the results arc expressed graphically in Figure XXIII.
In Succeeding paragraphs this experiment will be referred to as experiment I.
Experiment I was repeated but with a few modifications. In order to be sure that the differences in growth rate of the three groups of
plants were the result of fumigation it was necessary to establish that without such treatment growth would not have differed very much. This was done by measuring the heights of the plants on alternate days for 8 days prior to fumigation, Fumigation took place on the ninth day (3 September
1959). Thirty plants in three groups of ten, labelled S, T and U, were used. Each was 15 days old and 13.0 cm, in height and had 3 leaves fully 134.
Table XLKII. The mean heights of eight plants at intervals after fumigation in experiment I.
Time since FumiL;ation M N 0
Aug.lOth. 0 13.50 13.45 13.47 7 20.16 18.51 14.94 11 25.01 23.44 18.14.
15 33.67 30.93 20.90 18 42.03 38.97 24.43 21 47.83 4.3.10 29.01 Sept.3rd. 24. 54.43 49.15 33.70 28 64..68 57.96 41.90 30 70.04 62.67 45.27
32 73.78 65.33 48.82 35 81.64. 72.51 55.15 38 86.24 79.21 60.57 4.2 92.75 86.88 67.5o
44 96.4.1 90.92 71.50 46 96.50 91.31 72.80
49 96.63 94.58 75.27 51 97.03 96.14 76.60
Oct.8th. 59 96.30 96.75 76.85
M: Unfumigated control. N: C.T.P. = 25.0 0: C.T.P. = 73.5 FIG. XXIII. THE GROWTH RATES OF PLANTS IN EXPERIMENT I
60 HEIGHT CMS. 50
20 30 40 50 60 DAYS 135.
Table VIII. The mean numbers of (a) leaves expanded (b) flowers opened (c) pods formed, for eight plants at intervals after fumigation in experiment I.
Leaves Expanded Flowers Opened Pods Formed Days since Fumigation II N 0 M N 0 M N 0
Aug. 0 3.000 3.000 3.000 10th. 7 5.125 5.250 4.875 11 6.125 6.250 6.000 15 7.750 7.750 7.250 18 9.000 9.000 8.250 t 2.5" 21 10.000 10.0ft 9.125 Sept. 24 11.000 10.875 10.000 3rd. 28 - - - 30 - - - 0.000 32 - - - 0.500 0.000 0.000 35 - - - 1.375 0.875 0.375 38 16.000 16.250 15.250 2.250 1.625 1.125 0.625 0.250 0.125 42 17.000 16.750 16.250 3.125 2.375 1.875 1.250 0.875 0.750 44. 17.625 17.750 16.750 3.500 3.375 2.625 1.625 1.250 1.000 4.6 18.000 17.875 17.250 4..125 4..125 3.125 2.625 1.750 1.375 4.9 4.500 4.375 3.625 3.125 2.125 2.000 51 4.625 5.000 3.750 3.875 3.125 2.625 53 4.625 5.250 3.750 4..000 3.375 3.000 Oct. 59 4..625 5.250 3.750 4.000 3.625 3.250 8th. 136. expanded. Group S was set aside as a control and T and U fumigated. Just before fumigation, however, one plant was removed from each group and the water content and dry weight of the shoot determined. As before, the fumigations were at 22°C. and of 2 hours duration. The C.T.P.s were
24.5 (T) and 70.5 (U).
The plants were allowed to finish their growth and the number of leaves, flowers and peas was again recorded. Dry weight determinations at the end of the experiment were not made, however, because by that time up to 11 leaves at the base of the stern in both controls and treated plants had died.
The results of experiment II are incorporated in Tables XXXIV -
XXXVI and Figure XXIV.
Simultaneously with these experiments results of other work (des- cribed previously) were being collected which indicated that the effect of methyl bromide on the roots of a plant modified the response of the shoot.
Consequently, a third experiment was set up in which the interaction of the effects on root and shoot was investigated in relation to growth in peas. rive groups of ten plants 6.5 cm. in height were selected and their heights measured every second day for eight days. On the ninth day, when the plants were 14.0 cm. in height, one plant was removed from each group and the water content and dry weight of the shoot determined. On the same day the fumigations were performed.
The five groups were labelled V - Z. V was set aside as a control and the other four fumigated at 22°C. on 3 September. The
fumigations were of 2 hours duration. 7 received a C.T.P. of 24.5 and 137. Table XXXIV. The mean heights of ten plants prior to fumigation and nine
plants after fumigation at intervals in ex eriment II.
Time Time since (Days) Fumigation S T U
Aug.25th. 0 13.90 13.06 12.94 S: Unfumigated Control. 3 17.15 15.92 16.15 T: C.T.P. = 24.5 6 20.04 18.97 18.66 U: C.T.P. = 70.5 8 22.13 21.65 21.24 Sept.3rd. Pumign.
13 32.88 30.10 26.11
15 6 37.35 33.28 27.44 17 8 41.91 37.18 29.88 20 11 48.76 43.86 34.17 23 55.00 50.17 38.24 27 18 65.16 58.94 43.70
29 20 70.39 62.50 4.8.06 32 23 76.72 69.72 53.63 36 27 86.69 77.87 59.76 Oct .2nd. 38 29
41 32 98.70 90.22 70.95 44 35 103.62 96.05 78.07 48 39 108.47 101.06 86.78
50 41
Continued on next page. 138.
Table XXXIV cont.
Time Time Since (Dogs) Fumigation S T U
52 4.3 110.47 103.86 93.17
55 112.97 105.73 98.56
59 50 115.15 105.91 102.28
64 55 105.62
66 57 116.65 106.18
Nov. 2nd. 69 60 1.1 41••• 011,
78 69 116.00 103.94 107.60 100
80
60 U
FIG. XXIV. THE GROWTH RATES OF La PLANTS IN EXPERIMENT II
40
20
FUMIGATION
0
0 20 40 60 SO DAYS 139. Table XXXV. The mean numbers of (a) leaves expanded (b) flowers opened (c) pods
azalql for nine plants at intervals after fumigation in experiment IT Leaves Expanded Flowers Opened Pods Formed Days since Days since beginning fumigation S T U S T U S T U experiment
27 18 11.556 11.222 10.556 29 20 32 23 0.000 36 27 0.333 0.000 Oct. 38 29 0.556 0.333 0.000 0.000 2nd. 41 32 2.333 1.W 0.000 0.667 0.333 44 35 3.111 2.667 0.111 1.667 0.889 48 39 4.333 3.667 1.01); 2.556 2.000 0.00( 50 41 4.778 4.222 2.111 2.778 2.556 0.33 5
52 43 1 55 46 5.000 4.667 3.667 3.222 3.778 1.33]
59 50 5.222 4.889 4.556 3.333 4.000 2.11:
64 55 5.222 4.889 5.556 - 4.111 2.66-1
66 57 ------
Nov. 69 60 16.889 16.556 18.))) 5.889 6.111 6.222 3.667 4.111 3.55(; 2nd. 78 69
2 fls./peduncle (2 pods/peduncle) on one plant. X40•
Table XXXVI. The dry- weight and 5 water content of a plant taken from each group at the time of fumigation (Sept.3) in experiment II. Mean Height 21.67 cm.
Plant Dry Wt. 'f'f) H2O Cont.
S J 0.4746 86.94 T J 0.5030 87.57 % U J 0.4861 87.55
Mean 0.4879 87.35(
X, Y and Z a C.T.P. of 71.0. At the higher dose, however, only the plants of group X were entirely exposed to the fumigant. Those of group Y had their roots protected by enclosing the pot and soil in a piece of 500 gauge (5/1000" thick) polythene sheeting ( see footnote p.1)43) and group Z had the roots exposed but the shoots protected. After fumigation the plants were returned to the glasshouse along- side the controls and the polythene coverings from Y and Z removed. The heights of all the plants were then measured in the usual way at intervals of a few days until 5 October, 32 days after the fumigations. By this time the height of the controls was 73.0 cm. and 13 leaves per plant were fully expanded. The first flower buds were just beginning to appear. Because it was desired to measure growth also by the change in dry weight of the plants during the experiment measurements of height were 10. terminated at an earlier stage than usual before death of the leaves at the base of the stern began. The shoots of all 45 plants were therefore cut and their dry weight determined 33 days after fumigation. The results are given in Tables )(XXVII - XL and Figure XXV.
Some time previously, however, striking differences in the appear- ance of the plants of groups V - Z, other than those of height, had become apparent which had not been noticeable in the other experiments. Group V, the controls, were healthy looking plants of good growth and appearance and so were the plants of group W (C.T.P. = 2..5). The general colour of groups X and Z, on the other hand, was much yellower. The group X plants were those which had had both roots and shoots exposed to fumigant at the high dose and the 2:plants those with only the roots exposed. The colour of plants in group Y, where only the shoots had been exposed, was very much nearer that of the controls but on the whole just a shade lighter.
The other striking difference between the plants was in the size and shape of the leaflets (Figure XXVI). A11 the leaves possessed the usual number of leaflets and were not deformed in any way except that the individual leaflets in the plants of groups X, Y and Z, but especially X, were very much smaller than those of the corresponding leaves in both the controls and group W.. The leaflets in group X also had a more serrated outline than any others.
Five plants were selected at random from each group and the length along the mid-rib and the maximum width of one of the leaflets of leaves 9,
11 and 13 were measured. (Each leaf has four leaflets and the one arbitr- arily chosen for measuring was the proximal left-hand side one as the leaf 142.
Table XXXVII. The mean heights of ten plants prior to fumiL,,ation and nine plants after fumigation at intervals in experiment III. V : Unfumigated control. Ti : C.T.P. = 24.5 X : C.T.P. = 71 (Roots and shoot exposed) Y : C.T.P. = 71 (Roots protected) Z : C.T.P. = 71 (Shoot protected).
Time Time since (Days) Fumigation V V X Y Z
Aug.25th 0 5.97 6.69 6.25 6.76 6.57
3 8.30 9.36 8.77 9.31 9.12 6 10.41 11.28 11.30 12.36 12.00
8 12.20 13.12 13.47 14.0o 13.77 Sept.3rd. 9 Fumigation
13 4 19.88 18.76 18.16 19.07 18.71 15 6 23.16 21.34 19.16 21.09 20.95
17 8 26.44. 24.15 21.05 22.71 21.47 20 11 31.54 28.42 23.41 25.84. 23.52 23 14 36.76 33.20 25.77 28,75 25.31 27 18 43.64 39.52 29.27 32.92 30.02 29 20 47.54 42.97 31.87 36.17 33.40 34 25 56.76 50.30 36.87 42.76 38.2o 36 27 60.92 54.45 39.97 45.84 42.10 Oot.5th. 41 32 72.73 65.23 47.50 54.17 52.05
V W X Oct.5th. No. Lvs. 14.778 14.556 13.111 14.000 13.556
No. Fls. 0.41l)1 0.222 - 0.111 FIG.XXV• THE GROWTH RATES OF PLANTS IN EXPERIMENT III
V- UNFUMIGATED CONTROL W. CTF3 24.5 X - CTP . 71.0 60 Y - CTP* 71.0 (ROOTS PROTECTED) Z - CTP. 71.0 (SHOOT PROTECTED)
0 I0 20 30 40 DAYS 143.
Table XXXVIII. The dimensions of a leaflet of leaves 9, 11 and 13 in plants in experiment III. (L = length of mid7rib, W = maximum width)
Leaf 9 11 13 Plant L W L/W L W L/W L W L/W
VA 3.7 2.9 1.276 3.9 2.7 1.44)1 3.8 2.4 1.583 VB 3.7 2.7 1.370 3.7 2.8 1.321 3.7 2.4 1.542
VD 3.3 2.6 1.269 3.6 2.4 1.500 3.4 2.2 1.545
VG 3.3 2.3 1.435 3.3 2.3 1.435 3.1 2.2 1.409
ITH 3.1 2.5 1.240 3.2 2..3 1.391 3.5 2.4 1.458
3.420 2.6000 1.318 3.5400 2.5000 1.418 3.5000 2.3200 1.507 Mean WA 2.2 1.7 1.294 2.5 1.9 1.316 2.5 1.9 1.316
WC 2.2 1.8 1.222 2.7 2.4. 1.125 3.2 2.4. 1.333 WD 2.4 1.8 1.333 2.9 2.1 1.381 2.8 1.9 1.474 WE 2.6 2.1 1.238 3.3 2.3 1.435 3.1 2.0 1.550 ti's 2.8 2.3 1.217 2.7 2.3 1.174. 3.1 2.3 1.348
2.4400 1.9400 1.261 2.8200 2.2000 1.286 2.9400 2.1000 1.404 Mean
XA 2.3 2.0 1.150 1.7 1.4 1.214 2.1 1.6 1.313 X0 2.0 1.5 1.333 1.2 0.9 1.333 2.1 1.5 1.400 XF 2.1 1.8 1.167 2.3 1.9 1.211 2.5 1.9 1.316 XG 1.9 1.5 1.267 1.8 1.5 1.200 2.4 1.8 1.333 XH 1.6 1.4 1.143 1.9 1.6 1.188 2.1 1.7 1.235
1.9800 1.6400 1.212 1.7800 1.4600 1.229 2.2400 1.7000_1.319 Mean
CONTINUED ON NEXT PAGE. Table XXXVIII Cont.
Leaf 9 11 13
Plant L Vi L/W L vr LA7 L W LATI
YA 2.2 1.4. 1.571 1.9 1.6 1.188 2.2 1.8 1.222 YB 2.0 1.5 1.333 1.7 1.5 1.133 2.1 1.9 1.105 YD 2.5 1.8 1.389 2.0 1.7 1.177 2.6 2.1 1.238
YG 2.7 2.4. 1.125 3.3 3.0 1.100 3.8 2.7 1.4.07 YH 2.3 1.7 1.353 2.0 1.6 1.250 2.5 1.8 1.389
2.34.00 1.7600 1.354. 2.1800 1.8800 1.170 2.6400 2.0600 1.272 Mean
ZB 1.9 1.5 1.267 1.5 1.1 1.364. 2.2 1.6 1.375 ZD 1.9 1.4. 1.357 1.6 1.3 1.231 2.1 1.5 1.4.00 ZF 2.0 1.6 1.250 1.9 1.7 1.118 2.3 1.9 1.211 03. 1.6 1.3 1.231 2.0 1.4 1.429 2.2 1.5 1.467 ZH 1.7 1.3 1.308 1.4 1.2 1.167 1.8 1.4 1.286
1.8200 1.4200 1.283 1.6800 1,3400 1.262 2.1200 1.5800 1.34.8 Meaa FIG. XXVI. THE ELEVENTH LEAF OF PLANTS IN EXPEbLIMENT III
4
(a) (b) (c)
(a) Unfumigated (V) (b) CTP = 24.5 (w) (c) CTP = 71.0 (X) (Roots and shoots exposed) 145.
Table XXXIX. The dry weight of the shoot of a plant from each group at
the beginninR of experiment III.
Plant Dry V/eight o Water Group of Shoot Content V 0.2439 87.97 0.2730 88.09 X 0.2227 88.70 Y 0.2263 87.35 0.2561 88.03
Mean 0.2/)14 88.03
Table XL. The dry weights of the shoots of plants at the end of
experiment III.
V W X Y Z
A 1.5348 1.0822 0.8926 0.9622 0.8415 B 1.3623 1.3994 0.6005 0.7940 0.9017 C 1.2754 1.2691 1.0975 1.0059 0.8025 D 2.4034 1.24.66 0.7174 1.3997 0.8958 E 1.0040 1.4984 0.9292 1.9551 0.7909 F 1.3309 2.2788 0.9034' 0,9375 0.7299 G 1.7840 1.4609 0.7425 1.6624 0.8733 H 1.5663 1.1504 0.5527 1.1363 0.5666 I 2.5242 1.3337 0.5308 1.0119 0.8204.
Mean 1.64.28 1.4133 0.7741 1.2072 0.8025 14.6. is viewed from above). Table =VIII contains the measurements made.
The results of the foregoing experiments indicated a number of effects of methyl bromide on the subsequent growth of young plants.
Firstly, the rate of growth was affected. This decreased for a period of several days following fumigation but then began to increase again approaching that of similar, un-fumigated plants growing under the same conditions (Pigs. XXIII, XXIV, XXV). The extent to which the growth rate was affected and the duration of the recovery period appeared to be proport- ional to the dose.
The reduction in growth rate at the higher C.T.P. in experiment III was modified by protecting parts of the plant. The plants (X) in which both roots and shoots were exposed suffered the greatest set-back but this was decreased (z) by exposing only the roots. By protecting the roots, however, and exposing only the shoots (`L) the adverse effect of the fumigant was alleviated still further.
That the changes observed were due to fumigation is supported by the growth curves obtained in experiments II and III which are almost identical prior to treatment. These curves also demonstrate the rapidity with which the effect occurs after treatment.
The plants in two of the experiments were allowed to complete their life-span and showed a number of differences in the mean maximum height attained. In neither experiment did the plants treated at a C.T.P. of 70-73 attain a final mean height greater than the controls although the difference between M and 0 was much greater than that between S and U. The plants treated at the lower C.T.P. attained more or less the same height 147. as the controls in experiment I (N) but in experiment II (T) they were
smaller than the controls and the plants receiving the high dose.
The number of leaves produced by the plants was very nearly the
same, regardless of height, and 13 leaves were always fully opened before
flower buds appeared.
The noticeably yellower green of the leaves of plants X and Z in
experiment III has already been mentioned.
The figures in Table XXXVIII show that the length and maximum width of the leaves produced 18, 24. and 30 days after fumigation in experiment
III were less in the fumigated plants than in the controls. Furthermore, this difference increased with the C.T.P. but was least at the higher dose in the plants (Y) which had had their roots protected at the time of
fumigation.
The rate at which flowers and Pods were produced in both un-treated and treated plants was virtually the same. However, the length of time after fumigation before reproduction began was different. In fumigated plants reproduction began later than in the controls, those plants receiving the high doses being the last to come into bloom. The delay was much greater in experiment II (U), however, than in experiment I (0).
In experiment I the mean number of flowers produced per plant exposed to the low dose was greater than in the controls but in the plants exposed to the high dose the nugber was less than in the controls. In experiment II the mean number of flowers produced was almost the same for every plant.
The only floral aberration noted in these experiments was in a 1143 . plant fumigated at a C.T.P. of 70.5 in experiment II. Two of the peduncles were forked distally, each bearing; a flower of normal appearance. All four flowers eventually gave rise to well-developed pods.
(Not all the flowers which were produced during these experiments remained alive. Although they opened normally, about 10% of the total died quite early, before pod formation. The reason for this was unknown but was thought to be related to the un-natural environment for pea plants of a glasshouse.
The remaining flowers all produced at least a rudimentary pod but only about half of these survived, the remainder dying before attaining a length of more than a few cm.
The mortality in flowers and pods was as frequent in control plants as in fumigated ones. When a pod did survive it usually grew to full size (between 7-9 em.) but rarely contained more than 5-6 seeds. These were collected, dried and sown. Only a very few germinated).
Finally, the measurement of the changes in dry weight of the shoots in experiment III underlined the magnitude of the effect of fumigation on growth. At the lower C.T.P. the average increase in dry weight was 16% less than in the controls. In plants fully exposed to the fumigant at the high C.T.P., however, and in those in which the shoots were protected the increase was W and 605 less than in the controls respectively. Protection of the roots reduced this difference to 3341,;. 149.
(a) The effect of Methyl bromide on Flowerinj.
Dwarf nasturtiums were chosen for studying the effect of methyl bromide on flower production. They produce a fairly large number of
flowers over a period of weeks and therefore seemed suitable for the purpose.
Two experiments were performed. In each three croups of twelve
plants were used, one to be set aside as a control and the other two to be
fumigated. The only major difference between the two experiments was in
the ages of the plants at the time of fumigation. Those in the first
experiment had just begun to produce branch shoots from the primary leaf-
axils and between 2-4 flower buds had appeared per plant. The plants were 71 weeks old.
In the second experiment the plants were much younger and there
were no branches or flower buds present at the time of fumigation. (it
was 15 drgs after the start of the experiment that branches first appeared in the controls.)
The fumigations were performed at 22°C. and lasted 2 hours. The C.T.P.s in the first experiment were 25.0 and 75.0 and in the second experi- ment 25.0 and 65.0.
After fumigation the plants were returned to the glasshouse along- side the controls and the number of flowers which had opened was recorded at weekly intervals. (Partly opened buds were not included). The results are given in Tables XLI and XLII and Figures XXVII and XXVIII.
Throughout both experiments the plants remained healthy, the only parts which died being some of the oldest leaves at the base of the main stem. This occurred in control and fumigated plants alike but took place 150. Table XLI, The number of flowers_aoduced by the groups of twelve nasturtium plants at intervals in flowering- experiment I.
Control C.T.P.=25.0 C.T.P.=75.0 Time Total Mean Total Mean Total Mean (Days)
23 July 0 (Fumigation) 7 4 0.333 0 0 0 0 13 28 2.333 24 2.000 5 0.417 18 38 3.167 43 3.583 9 0.750 25 54 4.500 62 5.167 45 3.750 32 73 6.083 82 6.833 85 7.083
31 August 39 84. 7.000 89 7.417 106 8.833 43 88 7.333 97 8.083 112 9.333 53 100 8.333 111 9.250 125 10.417 60 102 8.500 114. 9.500 127 10.583 28 September 67 102 8.500 114 9.500 128 10.667 FIG. XXVII THE RATE OF FLOWER PRODUCTION IN DWARF NASTURTIUM PLANTS FUMIGATED AT TIME OF FLOWER BUD FORMATION
CTP = 75.0
120
CTP % 25.0
100
UNFUMIGATED
80 NO. FLS. PER 12 PLANTS
60
40
20
O 6 20 40 60 DAYS FUMIGATION 151.
Table XLII. The number of flowers roduoed by the_groups of twelve nasturtium plants at intervals in flowering experiment II.
Control C.T.P.=25.0 C.T.P.=65.0 Time Total Mean Total Mean Total Mean (Days)
1 September 0 (Fumigation) 22 2 0.167 0 0 0 0 25 4. 0.333 6 0.500 0 0 29 10 0.833 8 0.667 2 0.167 5 October 34. 27 2.250 30 2.500 11 0.917 4.1 4.6 3.833 51 4.250 18 1.500 48 60 5.000 71 5.917 23 1.917 56 73 6.083 81 6.750 28 2.333 62 86 7.167 96 8.000 36 3.000 68 88 7.333 97 8.083 38 3.167 13 November 73 88 7.333 98 8.167 4.0 3.333 FIG. XXVIII. THE RATE OF FLOWER PRODUCTION IN DWARF NASTURTIUM
PLANTS FUMIGATED BEFORE FLOWER BUD FORMATION
100 CTP=25.0
SO
UNFUMIGATED
60 NO. FLS. PER 12 PLANTS
40
CTP: 65.0
20
O
9 20 40 60 DAYS
FUMIGATION 152. much sooner in the latter although rarely to a greater extent.
Flowers were produced continuously and were normal in all but the plants exposed to the C.T.P. of 65.0 in the second experiment. In this group 5 of the total of 40 flowers were about half the size of flowers opening at the same time on the controls. Morphologically, however, the two types of flowers were identical. Dwarf flowers appeared on 3 of the
12 plants and were the first to appear in two of them. In the third, 3 perfectly normal flowers were produced before the dwarf. The only other floral aberration recorded was in the second flower to appear in one of this same group of plants. The corolla in nasturtiums normally has five lobes but in this instance had only four, the left (looking at the flower from in front) of the three lower ones being absent and the corresponding sepal deeply bifia. The flower was of full size and the other structures were apparently normal.
The results of the experiments indicated a number of important effects on flowering which could be attributed to fumigation.
In experiment I where flower buds had already begun to appear on the plants by the beginning of the experiment, fumigation had a retarding effect on their further development. This was only slight at the low C.T.P. but at the high C.T.P. was considerable. Thirteen days after the first flower opened on a control plant•.only 9 flowers had opened among the twelve plants fumigated at the high dose although a total of 38 flowers had opened in the controls and 43 in those fumigated at the low dose. Once flowering had begun, however, the rate of flower production in both groups of fumigated plants soon exceeded that of the controls. The 153. total number of flowers produced was least in the controls and greatest in the plants which received the high close of fumigant. The number of flowers produced by the low-dosed group of plants was intermediate.
On the other hand, when much younger plants were treated, as in the second experiment, somewhat different results were obtained. There was again a delay in the time at which the first flower bud appeared and this delay was greatest at the high dose but once reproduction had begun it was only in the low-dosed group that the rate of flower production exceeded that of the controls. At the high dose many fewer flowers were produced and not all of these were of normal appearance (see above).
The amount of seed produced by the plants used in the experiments was small in each group and this was possibly associated with the lack of bees in the glasshouse. Those seeds which were produced, however, were collected, dried and sown. Although germination was poor it occurred in seeds from each of the three groups.
A series of experiments specifically concerned with the effect of methyl bromide on seed production in plants was not performed. A small amount of information on seed production in fumigated peas and nasturtiums, incidental to other observations, has already been mentioned, however, and to it may be added the following observation.
In the course of phytotoxicity tests some tobacco plants which had just begun to produce flowers were used. The plants were not of particularly good growth and the main object in using them was to ascertain whether their tolerance differed perceptibly from that of better plants, which it did only slightly. However, these plants were kept for some time after treatment 154.
and although the capsules they produced were outwardly quite normal they
did not contain any seeds. Unfortunately all the plants of this group were
fumigated and none were available as controls with which to make a comparison.
Whether the absence of seeds was due to fumigation was uncertain;
attempts to repeat the results failed. Perhaps instead the seedlessness was related to the original sub-normal condition of the plant or some other
factor and not the fumigation treatment. Similarly the poor seed production in fumigated peas and nasturtiums may have been at least partly a consequence of the unfavourable glasshouse environment. 155.
12. Discussion.
The maximum dose of methyl bromide tolerated by a number of species of aphid and whitefly host-plants without visible damage occurring has been investigated. It must be remembered, however, that the dose quoted is not the lethal dose in that the plant as a whole was not killed by it. It is based instead upon the requirements of commercial fumigations where visibly affected plants are unacceptable. The difference between the limit of tolerance on these grounds and the true lethal dose was shown to be, in fact, considerable.
The maximum C.T.P. tolerated at 22°C. by most of the plants was of the order of 80. For a few it was somewhat lower and for lettuce it was much lower.
Several factors acting during fumigation influenced the amount of damage caused to the plant.
As the temperature was increased from 15-50°C. a greater amount of tissue was damaged at a given dose. (6) Hamilton (1941), Richardson et al. (1943) and Griffin et al. (1956) also demonstrated an increase in damage to plants fumigated with methyl bromide as the temperature increased.
No appreciable difference in the amount of damage was detected, however, when the humidity of the air inside the chamber was increased from about 405 to 1005 R.H.
Fumigation under glass in bright sunshine caused only a slight increase in the amount of damage to plants over that which occurred to plants fumigated in total darkness at the same C.T.P. and conditions. 156. (b) Richardson et al. (1943), however, discussing fumigation experiments
with methyl bromide in a glasshouse state that presence or absence of sun-
light had no effect on plant tolerance providing that a temperature effect was not brought into play.
The age of a Plant influenced its susceptibility, in terms of
visible damage, to a small extent only. Young plants suffered just slightly
more damage at high doses than older plants although subsequent growth
processes were seriously affected in the young plants. However, plants
which had flowered and were fruiting were more heavily damaged at a particular
dose than those which were still growing.
The effect of age differences on susceptibility was frequently more
marked within a plant than between plants. The oldest leaves were normally
the first to show damage, particularly if senescent. Generally young,
established leaves were the most resistant although when very young,
especially in broad bean, they were the most susceptible.
There also appeared to be a correlation between the water content
of a plant and the limit of tolerance to fumigant. The more water a plant
contained, in general the lower was its resistance.
Diseased plants and those of poor growth had slightly less
resistance than healthy ones. This applied also to plants which were
un-healthy because they were supporting large populations of aphids.
Varying the pre-treatment conditions of a plant had no detectable
effect on susceptibility providing that the plants had been properly conditioned to the fumigation conditions.
The most important aspects of plant fumigation described in this 157. thesis are those concerned directly with specific physiological effects of
methyl bromide on plants. They form a basis upon which a plausible theory
of the mode of action of the gas can be based.
The stomata closed soon after admission of the fumigant in fumig-
ations performed in sunlight. This is interesting because only a slight
increase in the amount of damage to plants fumigated under these conditions
occurred over that occurring to plants similarly treated except for the
absence of light. It would be expected that in strong sunlight the stomata
would be open and that a much greater quantity of gas would therefore gain
access to the tissues during the fumigation period. A correspondingly
increased amount of damage would be expected. That this did not occur
must be related to the fact that the stomata were closed during the major
part of the fumigation. The reduced rates of transpiration in fumigated broad bean shoots
showing little or no damage as a result of treatment can likewise be attrib- uted to the fact that the stomata remained closed for long periods after
fumigation. However, whether this was due directly to an effect of the
fumigant on the guard cells or was a response to water shortage in the plant
due to impaired root uptake is not easy to assess. Stomata are known to
close when the plant is short of water.
The increased rate of water loss at high doses, however, was
almost certainly due to an adverse effect of an excessive amount of fumigant on the plant. Although this effect might conceivably have been partly a
direct one on the waxy cuticle no doubt the major factor was the water lost
from dying cells which would form areas in which the evaporation of water 158. would, be relatively unhindered. The breakdown of the water-proofing mechan- ism with the death of a cell is well known.
Turning aside for a moment to the effect of fumigation on roots two points need to be made. Firstly, examination of the root systems of fumigated plants showed that the root-tips were the parts most susceptible to damage. From 0.5 - 1.0 cm. of tissue at the root tip were killed in broad beans fumigated at high doses and the root hairb, where ion and water uptake occurs, were seen to be affected.
Secondly, the rate of uptake of water by fumigated roots was shown experimentally to decrease steadily as the dose increased until at very high doses uptake was negligible. The poor appearance of some of the pea plants in growth experiment III suggested an inadequate uptake of ions by the roots after fumigation.
An attempt can now be made to explain some of the changes which were observed in fumigated plants.
Provided that the dose is not severe we can expect that the decreased rate of uptake of water by the roots will be at least partly balanced by the lowered rate of evaporation from the shoots. This will hold true over most of the tolerance range of the plant.
A4 the highest closes, however, the situation will arise where the transpiration rate begins to increase again while the rate of uptake of water continues to fall. A water-deficit followed by wilting would then be expected - which is precisely what happens. The plant never recovers and the wilted tissues soon become discoloured and die.
The importance of water to a fumigated plant has also been demonstr- 159. ated in another way. It seems that where inter is readily available in the
shoot the resistance of the tissues is increased slightly.
This finding is supported by the results of experiments in which cutting a shoot after fumigation at a high dose and keeping it in water
decreased the amount of damage.
A similar effect was observed when the roots were protected from
exposure to the fumigant. Protection had the effect of preventing the roots from being damaged and thlis enabled them to function normally after treatment, presumably takinE up water at a rate sufficient to satisfy the needs of the shoot.
Protecting the shoot, on the other hand, served only to emphasise the effect of fumigation on uptake of water. Lettuce plants which had only their roots exposed nevertheless wilted.
The slow rate of uptake of water was no doubt also the reason why
plants which wilted in dry soil after moderate fumigation took such a very
long time to recover their turgidity after watering.
The results of the experiments in which plants were kept in a water-
saturated atmosphere after fumigation are perhaps not so significant as they might at first appear. Although the plants showed visible damage
only after removal to drier air the amount of the damage was in most cases
similar to that in plants which had not been kept at saturation humidity.
No increase in resistance was conferred by the treatment except possibly in
broad beans at low doses. The only effect of the high humidity seems to
have been the suspension of water loss from the damaged cells which, however,
had presumably ceased to function normally long before they were permitted 160.
to wilt.
The severe reduction in the amount of water being taken up by the
roots would still be in operation a few days after fumigation and consequently
the plants kept at saturation humidity would still experience the same con-
ditions of water shortage in unsaturated air as other treated plants once transpiration recommenced.
The interesting point is that temporary alleviation of this diffic-
ulty did not apparently increase resistance (unless the doses were too high
for a difference in the amount of damage to be detected). In this respect
these experiments are difficult to reconcile with those in which a decrease wcatec. in the amount of damage occurred to shoots cut after fumigation and kept inL
• Some authors e.g. Kido (1941), Ritcher (1941), have reported a
stimulation of plant growth as a result of fumigation with methyl bromide but this observation has not been confirmed. In fact, exactly the opposite has been demonstrated..
In the first instance growth was measured by following the rate of
increase in height of pea seedlings taken at the beginning of their 'grand
period of growth'. Fumigation, even at low doses, had a retarding effect, the magnitude of the initial suppression of the growth rate and the time taken before recovery occurred increasing with the dose.
Growth begins with the production of new cells by the meristematic tissue situated at the growing points of both roots and shoots. Vacuolation of the new cells then occurs after which differentiation is completed. The most important phase in regard to size increase, however, is vacuolation which is dependent upon the uptake of water. 161.
Water-shortage produced in growing plants by fumigation in the way
already established is one way in which the elongation of new cells and there-
fore growth might be impeded. Water-shortage would hardly be very great,
however: in plants which received only low doses of fumigant.
Methyl bromide might also affect growth by interfering with cell
metabolism or by preventing division of the meristematic cells or even by
interfering with the translocation of food.
Such theories, however, for the present at least, lack corroborat-
ing evidence. Consequently) it is propoSed to concentrate instead on the
one field of research in plant physiology which offers positive evidence
upon which a theory of the action of methyl bromide on plants can be based.
This is the study of plant growth hormones also known as auxins.
Many substances found in plants influence growth but not all of
them are essential. Of those which are essential auxins are probably the
most important. They are organic compounds which are produced in the grow-
ing apex and migrate basipetally to the embryonic cells where, in trace
quantities, they exert their effect. In shoots this is a stimulation of
vacuolation and therefore growth but in roots it is an inhibition. In the
sense that auxins are produced by tissues in one part of the plant and migrate
to other parts where they exert their effect at low concentrations they are
to be regarded as true hormones.
It is likely that more than one auxin or growth hormone exists but the best known and probably most important one is (3 -indole acetic acid, or IAA, derived from txyptophane.
Because of the great importance of the present knowledge of auxin 162. action to the interpretation of the results in this thesis it is now proposed to digress for a while to consider in outline some of the relative points.
Auxins are found in most parts of the plant but exert their effect on growth only at the meristems. They are known to stimulate or inhibit a wide variety of enzyme systems but no general conclusion concerning their mode of action can be drawn from such data. There is no evidence that they are themselves enzymes but it has been suggested that they might be co- enzymes.
In the presence of auxin, aerobic respiration is very much increased and so is active water absorption and salt uptake. Cell division is also stimulated and the elasticity of the primary cell-wall in vacuolating cells is decreased. As Bonner and Bandurski (1952) point out, the great diversity of processes in which auxins are involved, together with the apparent absence of any common feature which can be used to connect them, is strong evidence that auxins probably play an important role in some fundamental reaction in metabolism which is central to a large number of processes.
Stimulation of respiration by auxins in concentrations which stimulate ,growth is well established (Berger et al. 1946; Bonner, 1949(Q); Christiansen and. Thimann, 1950; Commoner and Thimann, 1941; Michel, 1951) but it is also true that increased respiration does not necessarily accompany the growth response (Bonner, 1936). Christiansen and Thimann (1950) showed that iocloacetate inhibits auxin-induced growth with only a slight reduction in respiration in the
Avena coleoptile. Iodoacetate is known to be a specific inhibitor of 163. enzymes containing a sulphydryl -Troup.
These authors also demonstrated a similar effect with arsenate but this was relieved by adding phosphate.
Arsenate is capable of replacing phosphate in reactions in which phosphate uptake is essential to substrate oxidation and this suggests a relationship between auxin metabolism and the utilisation of phosphate compounds produced in respiration.
2,4 dinitrophenol (DNP) is a compound known to inhibit the +1--; synthesis of adenosine Apr.-phosphate (ATP) thereby un-coupling respiratory oxidation from the production of energy-rich phosphate compounds (i.e.
ATP). Un-coupling with DNP produces an increased respiration rate (Loomis and Lipmann, 1948) and reactions which are dependent upon respiratory- produced .12P are inhibited.
DNP inhibits growth almost completely wUle causing an increase in respiration (Bonner, 191.9 (c and Avery, 1949).
Other processes inhibited by DNP are enzyme synthesis (Sussman and
Spieglemon, 1950), cell-division (Clowes and Krahl, 1936), active salt- uptake by roots (Robertson, 1951) and water-uptake by potato discs (Hackett and Thim,ann, 1950).
All these processes must require ATP. Furthermore they probably all require auxin.
Since auxin exerts no effect on respiration in the presence of
DNP, arsenate and other uncoupling, agents, auxin must, therefore, exert its effect by governing the utilisation of high energy phosphate compounds.
It is now possible to see how growth itself is affected by auxins. 164.
Commoner et al. (1943) found that potato discs in plasmolysing
sucrose solutions could actually take up water in the presence of auxin.
Commoner and Mazia (1942) also showed that at the same concentrations of
auxin upLake of KOl is stimulated.
Similarly, van Overbeck (19/l1,) found that during response to auxin
the osmotic concentration of the cell contents surprisingly decreased and therefore concluded that the effect of auxin on water uptake must be exerted throwh a non-osmotic mechanism. This he supposed to be active water
secretion, a phenomenon which has been shown to occur by Bennet-Clark et al.
(1936).
Active water-uptake is intimately dependent upon respiration, as
shown by Steward et al. (1940), Reinders (1938, 1942) and Van Overbeek
(1942) and it is concluded that it is in some way powered by ATP. It seems very likely, therefore, that auxin affects growth by
couplin:. ATP utilisation with active water uptake by the cell, resulting in
vacuolation and hence increase in size in embryonic cells.
Van Overbeek (1952) suggests that this process is accompanied by
an auxin-induced decrease in primary cell-wall elasticity rendering it more
plastic. A possible mechanism is the stimulation of pectin-methylesterase
leading to the breakdown of protopectins.
Returninc to the fumigation results perhaps some of the effects
of methyl bromide on plants can now be explained in terms of auxin action.
The predominant role of auxins in controlling growth has been
established as have the adverse effects on the same process of auxin inhibit-
ion. It therefore remains to show that methyl bromide could also be an 165. auxin inhibitor.
A possible mechanism is su&ested by the work with iodoacetate.
Iodoacetate is known to inhibit compounds containing sulphydryl (SH-) groups and the dependence of auxin action on the presence of SH- groups has been stressed by Thimann and W.D.Bonner (1948, 1949).
Muir, Hansch and Gallup (1949) demonstrated the importance of the ortho-positions in the benzene ring to auxin activity in both IAA and phenoxyacetic acid (another growth hormone). They suggested that the basic reaction of an auxin within a cell involves two sites. One of these is a carboxyl group and the other an prthR7croup. Hansch, Muir and Met zenburg (1951) further suggested that whereas the carboxyl group forms a peptide or other amino-type linkage, the ortho-position may react with a sulphydryl group such as that of protein-bound cysteine.
Methyl bromide has also been shown to inhibit SH- groups (Blackburn,
Consden and Phillips, 1944; Dixon and Needham, 1946; Lewis, 1948; Loved*. and Viinterinctham, 1951) but only at concentrations several times greater than the tolerance limit of any of the plants tested in this work viz. at
C.T.P.s of 165-500. The effect of lower doses on SH- groups seems uncertain.
Winteringham, Hellyer and McKay (1958), workirri with insects, were also of the opinion that inhibition of SH- groups by methyl bromide is only a secondary feature of its action occurring at high doses. The widespread importance of SH- group systems in metabolism would make this reaction an important one at lethal doses.
Using 32P Vianteringham et al. showed that even at low doses of methyl bromide there was an immediate and irreversible depletion of INP, 166.
arginine phosphoric acid and phospho-glyceric acid.
Thus an alternative and probably more likely way in which methyl bromide might affect auxin action is by the inhibition of its main substrate,
.ATP.
Nevertheless, the interesting possibility of a direct inhibition of auxins cannot be entirely ruled out. One such mechanism might be the
methylation of the carboxyl group whose importance to auxin activity was suggested byHansch et al. (1951).
In the experiment with peas in which growth was measured by changes in dry weight, the dry weights of fumigated plants at the finish were less than those of the controls, being least at the high dose. Furthermore, the plants exposed at the high dose showed poor leaf colour and dwarfed leaves, symptoms suggesting mineral deficiency.
The reduced assimilation may have been due to adverse effects of the fumigant on the photosynthetic tissue but this is not born out by the low weights of the plants whose shoots were protected.
An explanation which fits the facts much better is that methyl bromide affects the roots of a plant in such a way that ion uptake is severe- ly limited which in turn seriously limits growth. The strikingly better appearance and much greater assimilation in the plants whose roots were protected from fumigant in the pea experiment just referred to support this view.
The obvious way in which roots might be affected is by the death of the root hairs and this was indeed observed following moderate fumigation in broad. beans. Not only uptake of ions but water uptake as well would 167.
be affected and it has been shown that following fumigation water-uptake is
reduced.
However, at doses insufficient to kill the root hairs methyl bromide
might still have an effect on ion and water uptake. Both these functions
have been shown to be dependent upon the synthesis of ATP and auxins are also
involved. Thus by either inhibiting the synthesis of ATP or its utilisation
by inactivation of auxins methyl bror.ide would impair the functioning of even
intact root hairs.
Pursuing the theory of the inhibition of auxins by methyl bromide
still further, let us examine the remaining effects of the gas on plants.
Stimulation to increased flower production in nasturtiums by
fumigation was observed in plants fumigated at a time when flower buds were
just beginning to appear. High doses produced the greatest effect.
Similar results were obtained at low doses with much younger
plants in which there were no flower buds at the time of fumigation. High
doses, however, affected the general growth of the plants to a considerable
extent and a smaller number of flowers was produced than usual. Several of these flowers were dwarfed or malformed.
The physiology of flowering is still far from being fully understood but it is clear that a stimulus which initiates the formation of flower bud
orimordia acts on the young plant, and makes its effect, some considerable time before the buds actually appear. The production of such a stimulus is apparently intimately associated with photoperiodism and is located in the leaf tissues.
A flowering hormone, florigen, has been suggested but never isolated, 168.
It is certain, however, that auxins play a part in flower initiation
(reviews of Lang (1952) and Banner and Bandurski (1952)). Inhibition of floral initiation by applying auxin-paste to the leaves of various plants has been demonstrated (postal and Hosek, 1937 Harmer and Bonner, 1938;
Cholodny, 1939; Cooper, 1942; Galston, 1943; Thurlow and Bonner, 1947). High auxin levels the leaf of a plant at the critical time are unfavourable to flowering (Galston, 1943).
Conversely, stimulation of flower production has been demonstrated in the presence of auxin inhibitors such as tri-iodobenzoic acid and DCA
(Hamner and Donner, 1938; Bonner, 1949MBonner and Thurlow, 1949; Galston, 1947). Stimulation of flower production by methyl bromide could also then be explained by auxin-inhibition.
In apparent objection to the theory of auxin inhibition by methyl bromide is the observation of Latta (1945) that in cut fir and, to a lesser extent, in cut spruce branches fumigation with methyl bromide increased the length of time the plants retained their leaves Laibach(1933) and Gardner ane Cooper (1943) have shown that the presence of auxin delays the formation of the abscission layer. Therefore, by inhibitinL2 auxin action methyl bromide treatment would be expected to cause the leaves to fall sooner than usual and not later as observed.
Nevertheless, it seems extremely likely that by interferinc: with auxin action, possibly through the inactivation of essential sulohydryl groups and certainly by the inhibition of JPP, methyl bromide becomes respon- sible for a wide variety of effects on plants which cannot, for the moment 169. at least, be explained in any other way.
By no means all these effects of the fumigant have been fully explored in the present work and it is obviously now of considerable interest and importance to establish whether or not methyl bromide inhibits auxins directly, by methylation of some part of the molecule, or only indirectly by inhibition of JTP. 170.
13. Practical APPlications.
It has been shown that a C.T.F. of 25-30 at 15°C. is sufficient to control even the most resistant species of aphid (Yvzus persicae) and considerably more than is required to control several other species in the active stages. It is also sufficient to control adults, eggs and first- instar larvae of whitefly. It is not, however, advisable to exceed the minimum dose required to kill the insect pest because of the effect on the host plant. Although
all the pl-Aats, except lettuces, will remain free from visible damage
following treatment at a C.T.P. of at least 65-70 at 15°C. adverse effects on the roots, interfering with water and ion uptake, and on growth and
flowering will occur, especially in young plants.
Attention to a number of points of procedure, however, will help to increase the efficacy of a particular treatment. The susceptibility of both insects and plants to a given dose of fumigant increased with terricerature but the increase in susceptibility for a rise in temperature of 5°C. was
much greater in insects than in plants. Consequently, a fumigation temper-
ature of 20-25°C., or more, whenever practicable, would be an imprPvement
and the dose could be lowered slightly as well.
Because of the importance of the effect of methyl bromide on the roots of a plant it is desirable that plants should be fumigated with their root systems protected whenever this is possible. This procedure has been
shorn to lessen considerably the adverse effects of fumigation.
Allowing the plants to remain in warm, dry air after fumigation will hasten the death of at least the free-living insects and will also 171.
increase the kill slightly should the dose: for some reason, be insufficient
to provide complete control.
The length of time which poisoned insects will remain active after
fumigation will depend upon the temperature and humidity of the surrounding
air (p.lfo) and also, of course, on the dose. At the dose suggested above the most susceptible species of aphids, and adult whitefly, will die very
quickly and may be almost totally paralysed by the end of the fumigation.
It is unlikely that poisoned insects which take some time to die
will probe a leaf and settle to feed in a new place, however, and this is
important in that further dissemination of viruses by those species which
are vectors (and many are) is precluded.
On a number of occasions during experiments with aphids survivors
were found following treatments which should have given complete control.
Such insects were believed to have been born soon after the end of fumigation
and to have been unaffected by the fumigant. A dose sufficient to inhibit
reproduction should, therefore, be used against aphids. The increase
required, in terms of quantity of fumigant, is small (p.:51 ).
To control whitefly with a single treatment a higher C.T.P. than
that required for the control of aphids must be employed because of the
greater resistance of the third and fourth insters of whitefly. Alternative-
ly, repeated low doses sufficient to control adults and eggs and possibly
the first instar could be used.
The effect on plants of low doses repeated at, say, weekly intervals
is not known but is probably not serious particularly if the roots are
protected during treatment. 172.
The effect of the host plant on the susceptibility of the insects is also unknown. Both aphids and whitefly are highly polyphagous but although there might be a slight change in susceptibility according to the
host plant it is unlikely to be very important in practical fumigations where the dose used is generally well in excess of the minimum dose giving complete control in the laboratory.
The control value of a C.T.F. of 20-25 at 25°C. for aphids and whitefly (adults and eggs) has been widely tested on a laboratory scale and
very satisfactory results have been obtained. The plant roots were protect-
ed (by enclosing the pot in polythene) and the plants were kept in a warm, dry glasshouse after fumigation.
The humidity inside the chamber during treatment was not controlled
since it has no known effect on the susceptibility of either insects or
plants. Fumigation of aphids with methyl bromide has also been attempted
in the field in the areas of Lincolnshire and Cambridgeshire where sugar-
beet is grown. The work was supervised by Dr.A.B.P.Page, Dr.O.F.Lubatti
and Dr.A.Dunning and took place in the spring of 1960 and 1961.
During the winter months large populations of aphids (largely lazus persicae, RhoPalosiphoninus staphyleae and a few Aphis fabae) build up
in marigold. clamps (Broadbent et al. 1949) and form a major pest problem in
Eastern Enr;land the following spring when the clamps are opened since the winged aphids migrate to the neighbouring beds of sugar-beet stecklings
carrying with them the virus of sugar-beet yellows.
attempts were made to control these aphids by fumigating the clamps 173. with methyl bromide. The fumigations were carried out under polythene sheets thrown over the clamps and anchored at the edges with sand-filled canvas 'snakes'. The fum3,:ant was admitted as vapour through a rubber hose terminating in perforated metal pipes laid in the straw at the top of the clamps and the C.T.T. was of the order 50-100 in fumigations lasting 5-6 hours.
Laboratory tests showed that marigolds would withstand. very high doses of methyl bromide, up to a C.T.P. of 350 and higher, without showing any apparent ill-effect other than the death of the green tops at a C.T.P. of approximately 85. Furthermore, there was no evidence that fumigation hastened the decay of a heating clamp.
Control of aphids in the clamps was extremely good although occasional survivors were found. (The aphid population in the clamps was estimated by sampling both before and after fumigation by Dr. A.Dunning and his assistants at Dunholme Experimental Station near Lincoln). These survivors were thought to be either insects which had been produced soon after the end of fumigation by females which in some way had been Protected from the full dose of fumigant and were consequently still capable of limit- ed reproduction or insects which were already born at the time of fumigation but did not receive a lethal dose. The distribution of fumigant within a clamp during fumigation was studied in detail by analysing pas samples taken at regular intervals from a large number of Positions, with thermal-conductivity meters. Distribut- ion was uneven in the early part of a fumigation but was much better after about 1 hour. A minimum fumigation time of three hours is recommended 174.. to ensure the, best results.
The t9mperatures inside a clamp were also measured and on average
varied between 10-14°C. among the mangolds. Occasionally, higher temperat-
utes were found in clamps in which heatinE had begun.
The humidity inside the clamps was very high and this, combined
with the low temperatures, was believed to be the reason why apparent surviv-
ors could be found up to a week or more after treatment.
In all a total of between 15-20 clamps wore fumigated and a more
detailed account of the work will be published in due course when all the
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