The Ecology of the Aphid Hyperomyzus Lactucae (L) and The

WAITE INSTITUTE 19-6.82 LIBRARY

AND TFIE. EPIDEMIOI¡GY OF

I,ETTUCT¡ NI'CROTIC YETT,O\^/S VIRIIS

David K. Martin M.sc. (Lond.) ,B .Sc., Dip.Ed. (Adel.) D.I.C., Dip. T. (Se c.) .MIBo1.

A tl+eti's tubrrui-ttød (sot tl+ø degn-øe od Docf.on o{ Ph,LLotopltq in the Faculf4 o( AgnLeu,Ltatta,L Se-L¿nc¿ af. the UwLve¡,sÍfq o{ Ade,kLdø.

Departments of Entomology & Plant Pathology Vùaite Agricultural Research Institute, The University of Adelalde

NOVEMBER, L979

rl X¡c¡f"re'i l]!,u TASLE OF COIqTENTS

Page

SUMMARY i

DECI.ARATTON íii

ACKNOWLEDGEI'îENTS iv

GENERAL INTRODUCTION 1

t.r ftre aphid: Hyperomyzus lactucae (I,) 2 I.2 The thistle; Sonchus oleraceus (L) 4

SECTION A: BIOLOGY OF THE APHID 6 2.L Occurrence of aphids in relation to the growth stages of thistles in the field 2.L.I Results

2.2 Effect of density on emígration I4 2.2.L fntroduction

2.2.2 Methods 19

2.2.3 Results 20

(a) Effect of initial number of females on rate of reproduction (b) Effect of total nruriber of aphids on the percentage of aphids walking off a bud (c) The pattern in tÍme of nr¡nbers of nymphs walking off a bud 24 (d) Effect of crowding on alate formation 28

2.2.4 Discussion 29

2.3 The rate of increase 36 2.3.r Introduction

2.3.2 Methods

2.3.3 Results 37

(a) I xxand m in days (b) Intrinsic rate of Increase 4T 2.3.4 Discussion 42

2.4 Ernbryo number and potential fecundity 44 2.4.L Introduction

2.4.2 Methods 45

2.4.3 Results 46

2.4.4 Discussion 49

SECTION B: TRANSMTSSTON STUDIES 52 3.1 Feeding preferences of H. lactucae 3.1.I IntroducÈion

3.L.2 upÈake of 32P ¡y adult aphids 53

3.1. 3 Contamination of leaf by dead radioactive aphids 56 3.1.4 Transfer of radioactive tracer to thistle and lettuce leaves by radioactive aphids 58

3.I.5 Feeding preference 60

3.1.6 DiscussÍon 73

3.2 Detection of virus 76 3.2.I Introduction

3.2.2 Dilution of, the inoculum 77

3 .2.3 Inhibition of infection by thistle cell sap 79

3.2.4 Darkpretreatment of N . qlutinosa 84

3.2.5 Effect of light and dark on lesion number 87

3.2.6 Discussion 88

SECTION C: POPULATION STUDTES 94

4.r ÍLre occurrence of aphids, thistles and infected thistles in the field 4. r.1 Study area 94

4.L,2 Methods 97

4.r.3 Results 100

(a) llhe lrlhole Àrea

(b) Top Area I 107

4.L.4 Discussion 122

4-2 Age structure of aphtd populations L25 4.2.L Introd.uction

4.2.2 Methods L26

4.2.3 Discussion r34

SECTION D: THE INTERACTION OF THE APHÏD AND THE PLANT 136

5.1 llhe growth of the thlstle - stem elongation and number of buds produced 5.1.1 Introduction

5 .I.2 Methods

5.1.3 Results 140

(a) Stem Elongation

(b) Nr¡nber of flowers produced per plant I4L

5. I.4 Discussion r45 5-2 The length of time a bud remains favourable and the potential life of aphid 148 5.2.L fntroducÈion

5.2-2 Methods 150 5.2.3 Results

5.2.4 Di-scussion 156 5.3 H. lactucae as the only vector of LNW in lettuce 158 in S.A. 5.3.1 fntroduction 5.3.2 Further transmission studies 160 5.3.3 Aphid trap catches, the numbers of aphids on thistles and the incidence of LI{YV in thistles L62 5.3.4 Dístribution of infected lettuce wíthin crops r65

5- 3.5 Conclusions L67

DISCUSSION AND CONCLUSIONS 168

BIBLIOGRÀPHY L72

APPENDTCES 190

******

a l_

SUMMARY Lettuce necrotic yellows virus (LNfV), which is transmittêd by the aphid Hlperomyzus lactucae (t) causes major losses in lettuce crops in southern Australia. The main source of the virus is the co¡nmon sowthistle Sonchus oleraceus (t).

To better understand the epidemiology of LNYV a study was made of the population dynamics of the vector aphid species and of the reservoir host planÈs in the field over 3 years. Laboratory studies were also made of the biology of the aphid and of the growth of the thistle, and the results have been used to describe the build up and dispersal of the vector from sowthistles, and the spread of LNYV. New evid.ence is presented that H. Iactucae is the only vector of LNYV in lettuces in South a,rstr-fia. H. Iactucae occurs mainly on the flower buds of sowthistles, moving from these when the buds mature and begin to dry out. The percentage of nymphs in one generation that walked off young buds from colonies started by 2, 4, 6 ot 9 adults at each of 3 temperatures \^tas dependent on the interaction of the initial density and the temperature. However, the temperature and density treatments independently had no significant effect on the nu¡nber of nymphs that walked off. The effects of crowding and of temperature on the development of alate H. lactucae were found to be consistent htith the findings on other aphids and the rates of increase for both apterac and alatae increased with temperature and were higher for apterae than alatae at each of 3 temperatures. However, at each temperature there was no significant difference in the rate of reproduction of H. lactucae in colonies started by each of. 2t 4, 6 and 9 adults. rl-

In the field there were always sowthistles which were infected with

LNYV and were favourable to aphids. llhese plants were associated with recently cleared land and had a continually changing age distribution throughout the year. The flucÈuations in the number of plants which were favourable to aphids at any one time resulted in changes in the number which were infested with aphids, and in the densíty of the aphid infestations. Ttre numbers of aphids were highest in October L975¡

April L976¡ October 1976 and,lune 1977. Peaks in the nr¡mber of alate 4th ínstars generally coincided with peaks in aphid numbers except when temperatures were high and photoperiods long. H. IacÈucae was found not to achieve a stable age distribution in the field for a long enough period to enable population parameters to be calculated from the field samples.

!,lhen Sonchus oleraceus was gro$rn at each of 3 temperatures and 2 photoperiods the size of the plant was dependent on the photoperiod, with larger plants being obtained at long photoperiod. The number of buds produced by a plant was similarly dependent on the photoperiod but was also depend.ent on the temperature; and plants growing at 25oC and long daylength produced the most buds. Hor^tever, the relative time for which a bud remained favourable to H. lactucae at high temperatures was shorter than at low temperatures, especially at the short photoperiod, so that aphid colonies should theoretically be able to build up to much larger nr¡mbers at low tenperatures and short photoperíod.

This hypothesis explains why peak numbers of H. lactucae occur at relatively low temperatures (tso - tzoc) in the field. III

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ACKNOWLEDGEMENTS

I thank my suPervisors Dr. D.A. l4aetzet and Dr. J-!{. Randles for their hetpful discussion during the study; I particularly thank Dr. Maelzer for his constructive criticism and guidance during the writing up period.

I am grateful to ttre foltowing people at Hartley College of

Advanced Education who helped in the preparation of the ttresis' Dr. John Ottaway for advice and assistance with diagr¿¡¡ns, Eric Matson for the photographs, and to the technicians in the printerY'

I would like to extend my thanks and gratitude to Kate MacKenzie for the typing and for being so patient.

FinaIIy I am indebted to rny friends for their continual en-

couragement and understanding especially during the final period of writing. WAITE II.ISTITUTE LIBRARY

GE}]ERAL INTRODUCTION For any disease, the modelling of disease outbreaks is a complex undertaking. It requires quantificatíon of tJ.e interaction of virus host plants, the vectors that transmit the virus, climate and agricultural practices. In more detail, the parameters that influence this interaction can be listed as:

(i) relative abundance of different host species and their suitability as reservoirs of virus.

(ii) number of vector species - and for each vector species¡ its population dynamics, migratory and feeding behaviour, and transmission capability. (iii) temperature, wind, rainfall and photoperiod which influence

the numbers in time and space of reservoir virus plants,

host economic plants and vectors. (iv) modifications to the life-system of reservoir plants, economic host plants and vector species caused by agricultural practices such as spraying, burning off, and ploughing in.

The relative simplicity of the conceptual model of disease outbreaks of Lettuce Necrotic Yellows Virus (LNYV) in lettuce (Lactuca sativa L. ) first described by Stubbs Guy & Stubbs, (1963), has made this disease a useful model for studying host/virus,/vector interactions (Randles

& Crowley, L97O; Randles & Carver, 1-97J-¡ Boakye, 1973; Boakye & Randles,

19741 . As r,rrith most plant rhabdovirus diseases the main ef fort so far has centred on the identification of vectors, availability of virus,

its mode of acquisition and transmission by the vectors. There has been little aÈtention given to the virus host plants or to the vector population dynamics and feeding behaviour. Vector and vector-host 2 biotogy are briefly reviewed by Francki & Randles (1979).

This study investigates the population dynamics of the vector aphid species and of the reservoir host plants in the field so that the

epidemiology of LNYV can start to be quantified.

1.I THE APHID, HYPEROI{YZUS I,ACTUCAE L.

H. lactucae appears to be cosmopolitan (Hitle Ris Lambers, 1949¡

Cottier, 1953; Eastop, 1958); but is particularly co¡nmon in temperate

climates (Eastop, L966¡ Miyazake , L97L). Originally this aphid was palearctic or holarctic, (Eastop, L966) and since none of the attendant parasites of H. Iactucae occur in Australia (Stary & Schlinger, 196l¡ Stary, 1970), its migration or íntroduction into this country is probably relatively recent. In cold European and American climates H. Iactucae is holocyclic,

alternating between the summer host Sonchus and winter host Ribes (Hil1e Ris Lambers, 19491. In Australia, host alternation has not been recorded although the presence of male alatae has been noted in South Eastern Australia (Eastop, L966), and in South Àustralia (Carver personal communication). Carver has also recorded the presence of

fundatrices on Ribes in Canberra and Tasmania in late autumn. In South Australiar H. lactucae is normally anholocyclic on the sowthistle

Sonchus oleraceus L, reproducing parthenogenetically and viviparously

throughout the year, albeit in small numbers in winter and mid-srunmer.

Anholocyclic reproduction has also been noted on Reichardia tingiÈana L

and the endemic perennials Sonchus hydrophilus Boulos, Embergeria megaolocarpa, (Hook. F.) Boulos, (Randles and Carver, I97I). As the distribution of these latter plants is sparse, being confined mainly 3 to coastal regions (Black, 1957¡ Eichler, 1965) they would appear to be of littte importance as primary host plants to H. Iactucae. H. Iactucae is known to be a vector of LNYV (Str¡bbs & Grogan, 1963; Randles & Crowley, L97O). The virus is both circulative and propagative

(Stubbs Loughlin & Charnbers, L967) in H. Iactucae & Grogan, 1963; O. ' and the aphicl is not adversely affected by infection wÍth LNYV (Boakye , L973). The virus undergoes a minimum temperature dependent incr:baÈion period of 5 days at 28oC in the vector, so that nymphs which acquire the virus from the host plant after birth become infective approximateLy 24 hours after reaching the 4th instar (pre-adult stage), (Boakye & Randles , L974). A low rate of trans-ovarial transmission occurs in viruliferous viviparae apterae and adult apterae and alatae transmit more efficiently than nymphs (Boakye & Randles, 1974). In

Souttr Australia the economic importance of LNYV arises from the transmission of the virus to lettuce crops where symptoms and seasonal crop losses in excess of 508 can occur,

This thesis presents new evidence that H. lactucae is the only vector of LNYV in lettuces in South Australia and describes a study of ttre population dynamics of H. lactucae in preparation for the future introduction of a parasite for biological control of the aphÍd. Con- trol of the aphid may well be relatively simply achieved because it may be sufficient for the parasite to only reduce the aphid density on its reservoir host plant below the density at which alates are induced.

The ecology of H. Iactucae is described. as it affects its role as a vector of LNYV. It is hoped that after the parasite has been Íntro- duced, a similar study would enable the effectiveness of the parasite to be measured, not only in terms of reduction in aphicl nurnlcers, but 4

also in terms of reduction of viral infected thistles.

I.2 THE SO$ITITISTLE SONCHUS OLERACEUS L.

The source of LNYV and the main host of H. Iactucae is the co¡nmon sowthistle S. oleraceus L. (Stubbs & Grogan, 1963¡ Cottier, 1953).

S. oleraceus is one of the most conmon cosmopolitan weeds and has its origins in the Canary Islands (Boulos, 1960). In South Austrafia the

sowthistle is an extremely common weed found most abundantly near human habitation and along roadsides within the 15" isohyet (Lewin, 1948). ft grows on a variety of soils, with a pH range of 6.5-9.0

(Buckli, 1936) r ê.9. clay rich, Ioamy or sandy soils, and as it can tolerate salt, is often found in the first line of sandhills along the coast (Lewin, 1948). Germination of seedlings requires high iI- lumination and the plants do not survive beyond the seedling stage if,

they are shaded to any great extenÈ (Lewin, 1948). S . oleraceus is

therefore excluded from many dense, closed. comrnunities, but it will

quickly invade bare soil or soil with vegetation whích allows some

direct illumination (Lewin, 1948) .

Reproduction is by seed only, with a 1O0? germination rate in all

but the sunner months (cíll, 1938). The proportíon of fertile achenes produced in summer decreases, until by the end of the summer the germination rate is zero (Lewin , L948). 504 of fertilized seeds re- maín viable after I0 years (Dorph-Peterson, L924) thus there are always

viable seed in the vicinity of thistle plants, which can produce a population of plants on suitabl-e newly cleared soil once there is

enough moisture present for germination.

Francki & Randles (L979), consider LNYV to have had a long associ- 5

ation hrith s. oleraceus because the Èhistle shows no slmptoms of infection and no other plant or vector is involved in the survival of the virus. lltre virus is not seed transmítted (Boakye, L973) and is assumed to be spread by H. lactucae (Str¡bbs & Grogan, 1963). The objective of the work described in this thesis htas to investigate the ecolog'y of H. Iactucae and the epidemiology of LNYV. llhe investigations concentrated on:

i) Clarification of the role of H. lactucae as a vector and its choice of feeding sites on lettuce.

ii) Ttre growth pattern of the sowthistle and its availability as a food source for H. Iactucae in relation to the rate of increase of the aphid.

iii) Age structure, fecundity and dispersal behaviour of the vector on sowthistle.

iv) A continuous field survey over 30 months dealing with the changing numbers of aphids and sowthistles and the incidence

of thistles infected with virus in relation to the number of aphids.

The results obtained have been used to d.escribe the build up and dispersal of the vector from sowthisttes and the spread of LNYV. 6

SECTION A

BTOLOGY OF THE APHID

2.L OCCURRENCE OF APH IDS IN RELATION 1O THE GRO$ITH STAGES OF TÎIISTLES IN THE FIELD As a preliminary to the extensive field progranme, field observations were made of the growth of the thistle, Sonchus oleraceus and of the occurrence of aphids in relation to these growth stages so that an ade- quate sampling technique for both thistles and aphids could be determined. These observations r^¡ere made monthly for 12 months ín each of 2 market gardening areas one which l-ater formed the area of study (Section 4.1.1).

2.L.L Results

The sowtJristle, (S. oleraceus) is an annual whose growth can be conveniently divided into rosette, semi-erect and erect stages (Figure 2.L). The size and d.uration of each stage are determined

by environmental factors, mainly day length and temperature. The rosette stage precedes stem elongation and has leaves radiating from the apex of the stem. During stem elongation a bud head forms in

the leaves surrounding the growing tip. The plant may flower and

persist at this semi-erecÈ s tage. If further stem elongation occurs and the flower head is pushed well clear of the apical leaves the

plant is in the erect stage; in this s tage lateral flower heads develop sequentially. During the preliminary progranme of observation it was noted that H. lactucae generally occurred on the erect sowthistles rather

than on those which were in the rosetèe or semi-erect stage, and 7

FIGURE 2.Lz typical examples of rosette (A), semi-erect (B) and erect (c)

S. oleraceus P1ants- Þ

o 9

that these aphi,cls were most common on the developing flower buds and mature flowers, moving to the stems when crowded- To check that H. lactucae \dere most conìmon on the erect sowthistles aphids were collected from each of 30 plants at each of the 3 growth sÈages.

Ttre numbers of aphids which occurred on the different gro$tth stages of sowthistles on the 3 sampling occasions are shown in Table 2.1; they confirn that the erect growth stage is the one on which

E. lactucae most commonly occurs. To quantify the dispersion of aphids on the erect plants' the growth of a ùhisÈle was studied and the stages of growth of flower buds were classified. fhe sequence of gro$¡th of numerous bud heads on the same and on different plants was followed and five clearly defined stages of flower development \.¡ere identified: these stages are illustrated and described in Eigure 2.2. The inflorescence is cymose with 6-lO irunature buds first appearing terminally. Trr'ro or three of these immature buds rapidly develop through stage I to stage 2, anð, the stems within the bud-head elongate to form 2 or

3 heads each of which contains 2, 3 or 4 buds. The apical bud in each head develops first, its stem continuing to elongate as it grorÂrs through to stage 5. During this time the secondary and subsequent buds develop sequentially in the same way, the secondary to stage 4, and the tertiary to stage 3. During this growth, other heads form in the axils of thc leaves lower down, each in turn producing from 1-6 terminal flower heads as described. After the flower growth stages had been categorized, the infestation of aphids on each growth stage of the flower was recorded for each of the 30 field thistles on 3 sampling occasions. Ttre 10

TABLE 2.L

The nr¡ribers of aphids on different growth stages of thistle in three random samples of plants.

Sampling Date Thistle Growth Stages Rosette Semi-Erect Erect

23. 4.76 36 26 2,527 L5. 9.76 0 0 2rg8g LO.r2.76 o 0 2,354

TABLE 2.2 fnfestation of heads and flower buds of S. oleraceus by H. lactucae on three random samples of 30 plants.

Sarnpling Date No. fl-ower No. of each bud stage infested heads infested I 2 3 4 5

23. 4.76 26 9 10 7 9 0 15. 9 .76 45 16 15 4 5 0

IO .L2.76 30 13 10 10 3 0 II

FIGURE 2.2:

Growth stages of sowthistle flower: 1. Flower bud first evldent, tightly closed length equal to diameter; 2. Elongate with length greater than diameter; 3. Flowert sePals fol,decl back and coro]Ias appearr 4. Post-flower¡ ligules disappear, base starts to swell;

5. Seed head pappus and achene aPpear- V

Ì\)

(^)

(,| 13

results are shohrn in Table 2.2; they show t]:aL on each of the 3 samptlng occasions buds were favourable for ínfestatÍon and coloni- zation by H. lactucap from their first appearance in the bud head

(stage I) until the involucre bracts begin to dry out (up to and íncluding stage 4). rt was noted that at the corunencenent of

stage 5 the aphids migrated to favourable buds on the same head, different heads, or to different plants as does the rose aphÍd Macrosiphum rosae L on rose buds, (Maelzer, L977).

I L4

2.2 EFFECT OF DENSTTY ON EMIGRATION 2.2.L Introduction H. Iactucae does not breed on lettuce to which it transmits

LNW and to transmit the virus to lettuce plants the aphids must necessarity leave the host sowthistle plant. A knowledge of the

factors determiníng the movement of aphids from a sowthistle flower

bud may help in understanding the spread of LNYV and aid in its

eventual control. The d.ata discussed previously (Section 2.L.I)

show that the aphid is found on sowthistle flower buds at stages L - 4 but not on stage 5 buds; and the observations on the thistle growth (Section 2.1.1) indicated that on each plant, flowering

occurs over a long period of time, with new buds and flower heads

appearing sequentially. It follows that aphids can move from bud

to bud on one plant and/or emigrate from one plant and perhaps find another.

As with other aphid species, the nurnber of alate progeny pro-

duced is likely to be a function of the numbers of aphids on a bud

when the progeny are in the stage of growth (Ist instar) at which

wing formation may be induced (Hughes, L963; Johnson, L965i Shaw,

1970a). However, before first instars are induced to become alates,

the density of aphids on a bud may be reduced. by two other processes, narnely (i) aphids walking off a bud; (ii¡ the reproductive rate of

the female aphids being reduced because of their own densíty or

that of their progeny. An experiment was therefore designed to

measure the processes which influenced the growth of the aphid colonies, especially the influence of aphid density on the induct- ion of alatae. 15

FIGURE 2.3

Bud cage used to confine aphids to flower buds'

FIGURE 2.4

s. oleraceus plant showing position of sticky trap relati'-'e to the flower head. t 19

2.2.2 Methods

A series of aphid colonies were started on sowthistle buds with

2, 4, 6 or 9 adult females per bud. The aphids were obtained as 4th instars from uncrowded field colonies and were allocated at random to one of the 4 initial densities

Each aphid was then placed on its allotted bud within 12 hours

of the adult moult. There were 3 replicaÈes of each density, and

densities were further replicated at each of 3 temperatures. ftre

ttristles used were grown from field collected seed planted in U/C

mix (Baker, 1957) in sterilized 10cm plastic pots. After thinning to 1 plant per pot they were placed in consÈant temperature growth

cabinets at I5O lux, 12 hour photoperiod and at either 15o, 2Oo

or 25o where they remained throughout the experiment. Each bud that was seeded by aphids was at stage 2 at the start of tl¡e experi- ¡nent. Bud cages 4cm in diam (Figure 2.3) hanging from the roof of the growth cabinets were used to confine the aphids to a solitary bud for the first 24 hours whitst they settled. The cages were then removed, so that the aphids or their progeny

were free thereafter to walk to other buds of the flower head or to leave the flower head completely. Sticky traps were used to

estimate the number of aphids reaving the flower heads. Each trap

consisted of a card which v/as covered with two concentrÍc squares of double sided sticky tape (sellotape) and then sealed against the peduncre of the infested frower head with cotton woor (Figure

2.4). Daily counts were made of the number of adults and progeny:

(i) on the original bud; (ii) on ad.jacent buds of the same fl_ower head, and (iii) trapped on the sticky trap. The experiment was 20

terminated either I50 day degrees after seeding of the originally infested bud, or at the appearance of lst generation adults. At

its completion, counts were made of all aphids on the originat bud, on adjacent buds and on the traps. lltre 3rd and 4th instar nymphs from each of the three locations were subsequently classified as potential alatae or apterae to d.etermine the effects of density and/ot temperature on alate formation.

2.2.3 Results (a) Egle9!-e€-rlr!1eI-lsrþer-eI-lescIe:-9!-regg-e5-rspre9gg!i9s

To discover whether the rate of reproduction of the aphids was influenced by the original nr¡nber of females and their progeny, the number of progeny produced per tive female per day was calculated for each replicate and then accumulatecl till the end of the e:çeri-

ment. Tab1e 2.3 shows the mean number of progeny thus produced by

each adult aphid at the end of the experiment at each of r he ínitial

densities of 2, 4, 6 and 9 adults at each of the temperatures 15,

20 and 25oC. An analysis of variance applied to the data is shown in

Appendix 2.1. The analysis shows that the F ratios for the different densities and for the interaction of treatments are not sígnificant

at the P=0.05 level indicating that the rate of reproductÍon of females in this experiment was not influenced by the initial number of females or the number of their progeny. However, the F ratio for

Èemperatures was significant, as may be expected., with the rate of reproduction increasing as temperature increased.

(b ) -Eg19s!-eI-!e!el-lssþer-eg-epþrgg-el-!þ9-peree!!esg-eg-epþig: selbrsg-eII-e_þsÊ At the end of the experiment, the number of nymphs that had 2T

TABLE 2.3

Mean number of progeny per live adult H. lactucae at initial population densities of 2, 4, 6 and 9 adul_ts at I5o , 2Oo and 25oC.

Temp Replicates Mean for 2 4 6 9 Temperatures

I II. 5 25.5 l-9.2 25.r o 15 2 L2.5 2I.7 2L.4 20.o 3 11. 3 13.0 11.8 Mean for Density L2.O 19.5 L7.9 I8-4 17.0

I 33. O 27.O 16. 3 25.4 o 20 2 9.0 23.3 2L.O L7.L 3 L4.7 16.6 L6.9 25.9 Mean for Density 18.9 22.3 18.1 22.8 20.5

I 16. 3 I0. 3 23.2 31. 3 250 2 23.5 32.O 16.2 34.6 3 26 .3 29.I 26 .4 31.0 Mean for Density 22.O 23.8 2I.9 32.3 25.O Mean for Density over all 18.4 2L.9 19.3 24.7 temperatures 22

FTGURE 2.5

bud after Percentage number of H. lactucae nym¡)h8 that walked off a at one generation from colonies started by 2, 4, 6 and 9 adults no. each of 3 temperatures I5oC, 2OoC, and 25oC against the total

of nymphs produced in each treatment'

o o 15 c

o 2ooc

L 2soc

Regressíon of nymphs thaÈ walked off against total number of nYmPhs

I5 o c Y=55.9-0.90x o 20 c Y=70.9-0.02X o 25 c Y=19.9+0.02x 70 o

a o o óo o

¡Eso a

Þ a

9¿o6 a 3 ^ E i, 30 A ¡o E a ñ20 A

IO A

o 500 o roo 200 300 400

Total numbor of nymphs ol cnd of experiment 24

walked from the original bud to other adjacent ones or on to the trap was calculated as a percentage of the total number of nymphs produced. ftris was done for each Èemperature and initial density and the percentages \^lere plotted against the total nr:mber of nymphs at each density for each of the 3 Èemperatures (Figure

2.5). Ttre hypothesis "that the percentage of aphids that walked off was due to the total number of progeny produced" may be tested by regression. The nuII hypothesis is that the slope of the regression is zero. None of the slopes of the lines in Figure 2.5 were significanÈIy different from zero indicating that the total number of progeny produced by 2, 4' 6 and 9 adults in one generation had no effect on the percentage of aphids that walked off. However, a higher (constant) percentage of nymphs walked off at 2Ooc (70.98) compared with 15oc (55.9å) and 25oc (19.9%).

The pattern, in time, of aphids walking off a bud was determined for each density and temperatureby calculating the percentage of nymphs Èhat walked off the original bud each day.

The pattern was most conveniently expressed by plotting the log of the percentage of nymphs walking off the bud against the time in days for the duration of the experiment. The data could then be fitted by linear regïession equations. These are shown in Table 2.4 and. in Appendix 2.2, Figure 2.6.

In every colony the percentage of aphids that walked' off a bud increased with time. and the rate at which the aphids walked 25

TABLE 2.4

Tt¡e linear regressíon of (the percentage of progeny walking off a bud) on (days), for each of four differenÈ initial densities and three temperatures. Y = B no. that walked off; X = no. of days from start of experiment.

Initial Regression Lines S.E. of b Temp No. Adults

2 I.I4 + 0.075 X 0. 07 4 0.07 + 0.095 x 0.14 l-50 6 0.79 + 0.099 X 0.09 9 0.62 + 0.117 X 0.06

2 I.16 + 0.094 X 0.16

o 4 0.98 + 0.099 X 0.03 20 6 0.78 + 0.120 X 0.05 9 -o.L2 + 0.250 X 0.07

2 0.88 + 0.111 X 0.07

o 4 -o.20 + 0.315 X 0. t3 25 6 -0.35 + 0.397 X 0. 30 9 -o.22 + 0.369 X 0.34 26 off from colonies that had been started with one particular initial density, increased as temperature increasedr ê.9. at the initial density of. 2, the slopes were 0.075 at 15oC, 0.094 at 2qoc and 0.Ill at 25oc. V,rith the exception of the population started by 9 aphids at 25oc the rate of walking off atso in- cïeased as the size of the initiat population increased. fhj-s apparently contradicts the earlier conclusion (Table 2.3, Section 2.2.3 (a) ). In an effort to explain this contradiction, the nr¡mber of aphids that had walked off buds at the end of the experiment {ú were divided into 3 groups, ví2, firsÈ and second instars, apfr ous third and fourth instars, and alate Èhird and fourth instars' Percentages of those walking off in each of 3 replicates at each of the 3 temperatures and at initial densities of 4,6 and 9 adults \dere calculated and are shown in Table 2.5. Because of

the low numbers of aphids involved in colonies establíshed by 2 adults, the results from these colonies vtere omitted and an analysis of variance was applied to the rest of the data (Appendix 2.3).

The analysis shows that the F ratios for the different densities, temperatures and types of aphids are not significant at P = 0.05 and thus do not individually inf luence the nr¡nrber of aphids thaÈ walk off a bud. Simitarly, Lhe interactions bet\^7een (i) temperature and aphid type, (ii) density and aphid type, and (iii) temperature, aphid type and densiÈy are not significant.

Ho$/ever, the F ratio for the interaction of temperature and density was significant, disproving the null hypothesis that 27

TABLE 2.5

Mean percentages of apterous and alate fourth instar and first and second instar aphids walking off a bud at initial population densities of 4r 6, and 9 adults at 15o, 2Oo and 25oc.

Initial density of aphids

4 6 9 lleang TemP. RêpIi- ÀÞhial tvpe8 ÀDhId tvÞes ÀDhld t\/pes for (oc) cates tenP. 4rh 4rh lst & 4rh 4ttr lst & 4rh 4rh lst û Apterous À1âtê 2nð. Àlf,terous Àlete 2nd ÀÞtêrous AIate 2nð I r00 100 2.9 31.6 0 11.I 85. 2 43. I 47.L 15 2 100 100 IOO 57.L 27.6 24.8 0 0 L7.7

3 45 5 83.3 33. 3 85 .7 87.5 83. 3 54.5 93 .8 56. 3

Means aphid for 94.4 45.4 58. 38.4 41.1 46.6 45.6 40.4 typeg 81.83 I

Itleans for initial densities 73.9 45.9 44.2 54.6

I 33.3 66.7 7 4.4 77.5 0 26.L 40.5 42.9 59.2 20 2 23.4 11. I 35 .6 77.8 7L.4 78. 3 73.5 a2.6 94.9 3 62.5 90.2 7r.4 70.0 100 93.0 57.9 70.6 60 .0

Means for aphid 39 .9 56 .0 60. 5 75.2 57.r 65.8 57. 3 65.4 7L.4 types

Means for inltial densities s¡. I 66.O 64.7 60. 9

I 0 0 30 24.2 33. 3 38.3 86. 8 70.6 93.7 25 2 100 100 100 0 4.8 8.7 100 100 100

3 10.0 0 0 100 t00 100 100 100 r00

Means for aphid 70 33. 3 43.3 41. 4 46. O 49.0 95. 6 90.2 97.6 types

Means for initial deneities 48 .9 45. 5 94 .5 62.9

OVER,ALL MEANS FOR ÀPHID îYPES 63.9 6l .3 49.7 58.2 47 .2 51. O 66. 5 67.I 69.8

OVERÀLL MEANS FOR 58.3 52.2 67.8 INITIAL DENSIÎIES 59. 5 2A

"the interaction between the temperature and density of the initial colony does not affect the number of aphids that walk off a bud. " The result of this analysis confirms the findings described in Section 2.2.3(b) where it was shown that the number of aphids that walk off a bud is not determined by the density of their initial colony and it also explains why in

Section 2.2.3(c) the nrunber of aphids walking off a bud increased

with temperature and with density (Table 2.4). The interaction of these two factors were important although individually they were not. (d) Effect of on alate formation To test the hypothesis "that there will be an increase in percentage alate formation with an increase in initial population density" it was necessary to calculate the density of each population at the time when the stimulus for alate production

was present, i.e. when the alatiform nymphs were first instars'

Hughes (1963) and other aphid workers have estimated these effective densities by estimating the density of the aphid popu- Iation at a time 2 instar periods before the time at which the alates are observed. To similarly estimate the alate inducing densities, the

number of d.ays expected Èo occupy 2 instar periods at each of the 3 temperatures l¡rere taken from Boakye (1913, P.60). The

cumulative number of progeny \^¡ere then plotted against the time in days from the beginning of the experimenÈ for each temperature and initial density (Appendix 2.4, Ei-gute 2-7) - Curves were then fitted by eye to the points and the number of aphids 29

present two instar periods before the end of the experiment were estimated by interpolation. Ttrese estimates of aphid densíty were converted to logarithms and the percentage of 3rd and 4th instar alate progeny present at the end of the experiment were

plotted against them and regression lines fitted and drawn (Figure 2.8).

The graphs show that the percentage alate production increased

with increases in the log nunrber of aphids presenÈ at the tíme of stimulation for alate productio¡. They a]so show that at

15oC more alates were produced at any given density than at

either 2Oo or 25oc and that the l-east nurnber of alates were pro-

duced at 25oc at aII densities. The threshold density for alate

development on a single sowthistle bud was 19 aphids at 15oC,

22 aE 2Ooc and 2L at zsoc. The findings on the effect of

density and temperature on alate developmenÈ are consistent with those of other aphid workers e.g. Hughes (1963); Johnson,

(1966); Shaw (1970a).

2.2.4 Discussion

Population density has been shown to affect the rate of reproduction in Drepanosiphum platanoides (Schr). Aphis fabae (Scop),

Brevicoryne brassicae (L) and Hyperomyzus lactucae (L) (Dixon,

1963, L966¡ Vùay & Banks, 1967; Way, 1968; Boakye, 1973) when the number of progeny have been counted over several generations.

It is accepted that small individuals result when aphid nymphs develop under crowded conditions (Vfay & Banks, 1967: Murdie, L969a, b; Dixon, 1970) and that smal-I adults produce fewer and smaller nlrmphs than large ones (Murdie, I969b; Dixon, L97O; Dixon 6, 30

FIGURE 2.8

Regression of percenÈage of alate progeny on the density of the aphid population two instar periods earlier at each of l5oc, 2ooc

and 25oc.

Regression of percentage alate on density of aphid population of instar periods earlier'

r5oc Y = -62.37 + 48.87x '.(r=0-918) 2ooc y = -36.42 + 26.9Ox (5=O.925) 25oc y = -31 .69 + 25.32x (5=0.886) 50 a

E q, EÐ o 30 Ê

o o o À o A g g 20 o ^ o È: o a

to o

2'5 r.0 1'5 2.O

erperinenl log number of ophids 2 inslor periods before end of 32

ltlratten, 1971) . Although crowding of tlre sycamore aphid,

Drepanosiphum platanoides results in a reversa-b1e decrease in reproduction rate Ínitially, the effect is a lasting one when crowding is prolonged (Dixon, 1963). It is therefore not surprising that in this experiment with H. lactucae - in which the nu¡nber of progeny were counted in less than one generation, and in which the aphids were free to move from the infested bud - there v¡as no signifÍcant difference in the rate of reproduction between colonies started by 2, 4, 6 and 9 adults. Boakye (1973) calculated that 3.4 generations had elapsed in flte 22 days during which he measured the rate of reproduction of H. lactucae in the field. This is enough time for a permanent reduction in the size of adults and hence in the number of nymphs produced. Boakye (1973), also found that the total numbers of aphids that emigrated. from sowthistles increased with an increase in initial population density. Ho\¡/ever, the density of nymphs that walked off at each density were not significantly different. Maelzer (1977) counted the numbers of apterous adults and 3rd and 4th instars of the next generation of Macrosiphum rosae (L) which emigrated from colonies of different densities on rose buds after 70 Do, and also noted an increase in total emigration, with an increase in the density of the original population. Although the rate at which emigration occurred in the experiments described in this chapter \n¡as not directly depend.ent on the initial population density, the percentage of H. Iactucae that walked off the plants v/as dependent on the interaction of the initial density and the temperature. The rate of emigration also increased with time. H. lactucae, like M. rosae, only 33

utilizes flower buds at particular stages of development and mass migration of aphids occurs with the maÈuration of the flower or bud (section 2.L.I¡ Maelzer, 1977). In many of the replicates in the erçeriments described here, the thistle flowers matured and fruited before the completion of the et4>eriment, resulÈing in a mass exodus of the resident aphid colony. This wasmost noticeable at 25oC where, in 5 out of 12 replicates, fruiting occurred before the completion of the lst generation. For the purpose of calculations, the numbers walking off the day before fruiting occurred had to be used. this allowed. only 3 or 4 days in some cases during which thepossible effects of initial populatíon densities could be noted.

Apparently this \¡Ias not long enough although it was long enough to show that the percentage of H. lactucae that walked off the original bud increased with time even before the buds became unfavourable. That emigraÈion from the buds increased with time and with an ín- crease in the temperature and initial population d,ensity is not surprising, because senescence of the host sowthistle is a function of time and temperature (Section 5.2), and high aphid densities adversely affect thehostplants, eitherbya drain on nutrients' or by the injection of toxic saliva. (Kennedy & Stroyan, 1959i Noda'

1954) . As the host plant becomes less favourable and the supply of nutrients decreases, aphids become increasingly restless and are more likely to leave the planÈ.

In the fie1d, movement of nymphs and apterae from sowthistles, although no d.oubt effective in colonising adjacent sowthistles' is not of major importance to the spread of LNYV to lettuces' a spread which has been attributed to the flight of the alatae (Stubbs 34

and Grogan, 1963; Randles & crowley, 1970). The formation of alatae occurs as a result of changes in the endocrine activity of the corpus alIatr¡¡r¡. Ttris activity is brought about by the physical conditions within and around the developing aphid colony

(Bonnemaison, I95I; Kennedy & Stroyan, L959¡ Lees, L96L, L966¡

White, L968¡ Shaw I97Oc). Crowding of aphids on thehostplant is the most important stimulus, resulting in aLate production. In some species the sensitivity to crowding is confined almost wholly to the Ist instar (Bonnemaison, 195I; Noda, 1958; Kawada, L965¡

Toba et aI, L967) whilst in others, both prenatal crowding of mothers and post-natal crowding of larvae are important (Lees ' L96Li Noda, 196I; Johnson, 1965¡ Shaw, I970a; Dixon & GIen, 1971). In crowded colonies tactile stimulation appears to be the important factor in the determination of alatae (Johnson I L965; Lees , L967 ¡

Toba et al, 1967; Sutherl-and, 1969) . Tactile stimulation is implicated too, where alate formation has been attributed to nutritional changes which have been brought about by deterioration of the host ptant (Pintera, 1957¡ Johnson, 1966¡ Dixon & Glen, 1971). On wilt- itg, senescent, and unhealthy plants. aphids Èend. to be more restless and so move about and stimulate one another more than they do on healthy plants and this can Lead to alate production (Johnson' 1965; Murdie, 1969a). Diet and the nature of the food plant apparently exert some measure of direct control in some aphids.

(Johnson & Birks, 1960; Johnson, 1965; Mittler & Dadd' 1966).

The tendency of aphids to move about is reduced by the attend- ance of ants (Banks & Nixon, 1958; Banks, 1958; EI Ziady, 1960) and results in a decrease in alate formation (EI Ziady & Kennedy' 35

1956¡ Johnson, 1959; El Ziady, 1960). This decrease could be due to the lessened tactile stimuli resultant from reduced locomotor activity (Johnson, L966¡ Lees, 1966) but it is possible that the ants have a direct influence on the endocrine system through the aphid's éensory system (Johnson, 1959). Both temperature and photoperiod have been claimed by various authors to affect alate production with high ternperatures and long photoperiods having an inhibitory effect (white, L946; Johnson &

Birks, 1960; Lees' 1965, 1966¡ Lamb e lrlhite' 1966). fn the field' temperature and photoperiod together with humidity may indirectly affect the production of winged forms through an effect on the host plant (Shaw, 1970a), and although alatae are most important in the dispersal of an aphid species, (and hence in the spread of a virus), it should be remembered that not aII alates are obligate migrants (Shaw, 1970b; Dixon, L973), and the degree of overcrohtding during both prenatal and nymphal life of a potential alate may Ín- fluence how many will fly before reproduction takes place (Dixon

4, 1968) . Ttre effects of crowding and of temperature on the development of alate H. Iactucae are consistent with the findings of the aphid workers discussed above. 36

2.3 THE RATE OF TNCREASE 2.3.L Introduction

Laboratory measurements of the intrinsic rate of increase (r*)

have been published for many aphid species (Lamb, L96L¡ Barlow,

L962¡ Messenger, 1964; Sylvester & Richardson, 1966¡ Harrison,

L969¡ Dixon & Wratten, I97L¡ Erazer, L972a, br; Siddiqui et aI,

L973¡ Dean, 1974¡ De Loach, L974¡ Maelzer, l-977 r Wyatt & White, 1977). Although estinates of reproductive rates of aphids in the field from results obtained und,er constant physicat conditions in

the laboratory present problems (Dixon, L977), such estimates have

been useful in the assessment of the potential effectiveness of a parasite (Messenger, L964¡ Force, L97O); in the identification of biotypes (Erazer, L972a); and in comparing the reproductive potential

of different morphs of the same or of different species (Dixon c

Wratten, L97L; Dean, 1974). Primarily, however, rm has been used

to describe the effects of temperature on the population dynamics

of the aphids (Lanb, I96L¡ Barlow, 1962¡ Siddiqui et. al, L973¡ Dean, 1974¡ De Loach, 1974). Because estimates of the intrinsic

rate of increase can be usefur in herping to exprain the changes in

numbers that occur in field populations, rm was calculated for apterous and arate Hyperomyzus lactucae at each of three temperatures:

15o, 2oo and 25oc.

2.3.2 Methods

Aphids were obtained from smal-l colonies in the field as 4th

instar nymphs and were placed on sowthistle buds until they became adults. Vüithin 12 hours of the adult moult they were used in ex- 37

periments to obtain the 1* and m* data for the estimation of r*, the intrinsic rate of increase. All sets of data were obtained for aphids placed on sowthistles that had many young buds and that were groïrn in v/C mix in sterilized lOcm plastic pots within plant growLh cabineÈs at 150 tux and 12 hours photoperiod at each of 15, 20 and zsoc (att t loc). The plants were watered daily.

To were placed in pairs on Sonchus buds and obtain 1x data, adults left for 48 hours to reproduce. The adults were then removed and the number of progeny counted on that day and on every day thereafter until all the survivors had become adults. The proba-bilities of survival- were based on 63, 75 and 80 progeny initially at 15, 20 and 25oC respectively. To obtain m* datar adults were placed singly on younçJ Sonchus buds and the progeny each produced was counted and removed each day until the adult died. The m* data for apterae were based on the production of 37 adults at 15oc, 25 adults at 2OoC; and 73 adults at 25oC. For alatae there were 50 aduLts at 15oc; 48 at 2}oc,and 93 at 25oc. The values of r* were estimated by V'Iatsons (1964) method. 2.3.3 Results (a) -x1-_ -----xand m__ l!_qgyg The age specific survival (I*) and age specific fecundity

(m--) x of alate and apterous H. l-actucae at each of l-5oC, 2ooc and 25oc are shown in Figure 2.9. For the calculation of 1*

the nu¡nber of adult females alive each day has been plotted as

a percentage of the original number of nymphs.

The survivaf of the nymphs and the time taken in days to adult moult have been summarized in Table 2.6 together with 38

FIGURE 2.9

Age specific survival (1*) and age specific fecundity (m*) of apterous and alate H. Iactucae at each of 15oc , 2ooc and 25oc.

O-á - tsoc o.....o 2ooc A- -A - zsoc Apterae Alatae

Age spodfic survlvol (h)

l.o I'O

o.a o'8 E .¿ ¿ 6t E à ¡o -oo o2 À

o o o 5 to ts 20 25 6 10 f5 26 Time in days Time in dayr

Age spedfic fæundity (rnr)

I6 oE

o !

E c (¡: o ¡l

o 10 l5 20 o I t6 20 Time in days Tlmc ln day¡ 40

TABLE 2.6 Probabitity of survival to adult of immature g. lactucae; the time taken to adult moult; to 508 mortality of apterous and alate adults

(LTsO) and to the death of the last adult at each of 15oc, 2OoC and z5oc.

Temp Probability Days to LTso Days to oc) ( of nymPhs adult death of surviving moult last adult Apterae Alatae Apterae Alatae

T5 o.76 L2 6.7 6.5 20 T7 20 o.79 I 5.9 6.0 T4 23 25 0. 81 7 7.O 4.L 18 15

TABLE 2.7

The effect of 3 temperatures on the net reproductive rate (Ro) Genera- tion time (T) and inÈrinsic rate of increase (r_) of apterous and m alate H. lactucae.

o TYpe Temp. c No. of Components and rate of of aphids increase aphid tested

apterae 15 37 L4.2 16. 3 0.17 20 25 12.7 11.5 o.23 25 73 l-4.7 r0.8 o.26

alatae 15 50 7.8 16. 0 0. r3 20 48 8.3 r1.7 0.19 25 93 a.7 9.5 o.23 4L

the tine in days to 50t mortality of adults (denoted as LT59) and. to the death of the tast adult. the survival of irunature stages to the adult moult at the three temperatures !{as similar' and, as expected, the time up to the adult moult decreased with increased temperature. Except for alatae at 25oCrthe time to 508 mortality of adults was similar at the three temperatures.

The in Figure 2.9 show that there was generally mx data also a decrease in fecundity with an increase in the temperature.

At 15oc, for both apterae and alatae, the three m* peaks were generally higher and more clearly delineated than the peaks at the higher temperatures. For both morphs the prereproductive period was longer at 15oc than at 2ooc or 25oc. (b) lntrinsic rate of increase

The components of the rate of increase are sunmarised in Table 2.7. The net reproductive rate (Ro) of apterae was higher than

that of alatae but there was little difference in Ro for each morph at the three temperatures (Ta-ble 2.7Ì-. The generation time, however, was similar in both morphs at each temperature and decreased as the temperature increased. The r* values also increased with an increase in temperature, with the apterae having higher values at each temperature than the alatae.

The differences between values for r* at the different temperatures was mainly due to differences in the generation time of the aphíds. The longer generation times at low temperatures resulted in smaller r. values. The lower r* values of the

alatae resulted from their l-ower reproductive rate compared with apterae. 42

2.3.4 Discussion

The reproductive potential of apterous H. Iactucae at each constant temperature in the laboratory was higher than for alatae, and for both morphs it increased with increased temperature. fhis is also true of many other aphid species (Siddiqui et aI, 1973¡

Dean, L974¡ De Loach, 1974). Although the values of r* serve as a guide to what could be expected in field populations, there are many confounding factors.

For example, alternating temperatures (as could be expected in the

field), have been shown in the laboratory to increase rm above

that for a simil-ar mean constant temperature (tamb, L96L; Siddiqui et al, 1973). However, r. cannot be measured for a field population and the crude rates of increase that can be estimated for field poputations are likely to be l-ower than those achieved under ideal laboratory conditions, particularly as temperatures rise and

cause an increase in aphi¿l and predator acÈivity and deterioration

in the quality of the host plant. The effects of temperature on

the voracity of predators is well documented (e.g. Dunn, L952¡

Erazer & Gilbert, 1976; Maelzer, L978) and the effecÈs of temperature on the activiÈy of aphids - particularly in conjunction witÌ¡ nutritional changes brought about by deterioration in the quality of the host plant - have been discussed previously (Section 2.2.4\. Another possible reason whY r* values in the laboratory could bear little relation to field populations has been suggested by

Maelzer (L977'). He prop osed that for }4acrosiphum rosae (t) the

chance of a colony becoming intermediate or large was fess at high temperatures than at low temperatures because the relative 43

amount of time that rose buds were favourable to M.rosae was Less at high temperatures than at low temperatures. Since H, lacÈucae is similar to M. rqsae in that it selectively feeds on young

flower buds (Sectíon 2.I.L) the hypothesis proposed by MaeJ-zer

(L977) for M-. rosae on *osa sp. was tested for H. lactucae on Sonchus. Ttre results of this test have been discussed in Section 5.2.3.

.:.. i 44

2.4 EMBRYO NUMBER AND POTENTTAL FECUNDTTY 2.4.L Introduction It is useful in population studies to predict the number of progeny a newly moulted adult female aphid is likely to produce

based on some morphological characteristic of the aphid that is

easily measured.

Direct counts of the progeny produced are often tedious' and

assessment of potential fecundity can often be made by relating

nr:mbers of embryos and eggs to size (webber, 1955; Van den Heuvel,

1963), with the initial number of embryos contained in newly

moulted female adult aphids often characteristic of the morphs (Richards, L96I¡ Johnson, 1963). A direct relationship between size, usually weight, and fecundity has been demonstrated for a range of insect species, ê.g.

Psychoda sp. (Golightly, 1940); HofmannophíIa pseudospretella (Stainton), (lrloodroffe, I951a) ; Endrosis sarcitrella (L),

(Vtoodroffe, 1951b) ; Plutella maculipennis (Curtis), (Atwal, 1955) ; Cadra cautella (l{alker), (Takahashi , 1956). Fresh body weight is Èhe simplest criterion of size to measure and the fecundity of apterous virginoparae of Acyrthosiphon pisum

(Harris) and apterous and alate Aphis fabae (Scop¡ have been demonstrated to be positively correlated with their weight just after

moulting (Murdie , L969b¡ Dixon & ülratten, I97l-). Therefore experiments were conducted with adults of H. lactucae to determine

whether mean realized fecundity could be predicted from fresh weight, cube root of fresh weight, tibia length or antennal length of the aphid. 45

2.4.2 Methods

The aphids used in these experiments were collected as 4th in-

stars from both crowded and uncrowded colonies in the field in both mid February and late April so that the aphids were of different sizes. They were then placed on sowthistle flower heads in

1500 mm petri dishes lined with moist cotton wool and. kept ín the

laboratory under ambient temperature and light. The dishes were

examined at 4 hourly intervals and any newly moulted adults were

removed and weighed in¡nediately. I\À7o experiments urere conducted with these newly moulted adults gathered at the different times,

namely,

i) AO apterae and 52 alatae were weÍghed individually and were

then killed i¡nmediately by placing thern in a drop of 80g" eÈhanot on a microscope s1ide. For each aphid the lengths of the tibias

and of the antennae q¡ere measured and a count made of all of the

developing embryos.

ii) fff apÈerae and 60 alatae \^rere vreighed individualty and each was^then placed separately on a freshly cut individual sowthistle

Ieaúé disc floating upside d.own on 15 mls of modified Hoagland-

Snyd.er solution (Hughes & Woolcock, 1965) in 20 mm vials. Each

vial was placed in constant light at 2OoC and examined daily and the nurnber of progeny produced by each aphid were counted. After the counts on the Èhird day, each surviving adult was killed with

alcohol and dissected. The total number of embryos (i.e. those with eyespots and those yet to develop eyespots) hrere counted in

72 apterae and 41 alatae, and the number of embryos with eyespots only were counted in 61 apterae and 41 alatae. 46

2.4.3 Results

The raw data are given in Appendix 2.5. lltre mean nunber of embryos present in apterae and alatae at the adult moult and the mean number of progeny produced and embryos present 3 days after adult moult are shown in Table 2.8. Ta-ble 2.8 also shows the minimum, maximum and mean fresh weights of the newly moulted adults and the standard deviations of these measuremenÈs. Table 2.8 shows that, as with other polymorphic aphid species, the apterae were more fecund than the alatae (Mordvilko, 1928¡

Kennedy & Booth, L954¡ Dixon & tlratten, l97L). The table also shows that there \^¡as a large size range in both morphs and that the mean number of embryos at adult moult and the mean total number of embryos and, progeny after 3 days was higher when the mean weights of the adult aphids were high than when the mean adult weights were low. The smaller sizes and lower embryo nurnbers of the second experiment can be attributed to the crowding of aphids which occurred in the field at the time when the aphids were collected. Linear regression lines were calculated for each of: (i) regression of the number of embryos at the adult moult,

on each of: mean tibia length; mean antennal length; fresh

weight; the cube root of fresh weight; mean length of 3rd anten-

nal segment, mean cornicle length.

(ii) regression of the number of progeny plus the nunrber of enbryos with eyespots after 3 days on each of fresh weight; the cube root of the fresh weight.

(íii) regression of the number of progeny pl-us total number of 47

TABLE 2.8

Ttre minimum, maximum and mean weight, with standard deviations, of newly moulted virginoparae of H. Iactucaet the mean nurnber of embryos in newly moulted adults and the mean nr¡mber of embryos and progeny produced (per aphid) after 3 days.

wt. of adults in T\pes of No. of Mean no. of embryos Ug. adults adults at adult moult , min max mean S.D.

apterae 80 3I 352 r380 684 19. 3

I a1a tae 52 20 329 868 6r8 r5, 3

Mean total no. embryos + progeny after 3 days

apter ae 72 I7 155 900 404 14. t alatae 4t 10 259 680 45r 11.8 48

TABLE 2.9 Correlation coefficients between the nr¡mber of embryos and progeny of newly moulted virginoparae of H. lactucae and selected physical measurements. .Ihe asterisks mark the values for which the correlation coefficients themselves were not significant at P = 0.05.

Mean ProPerties of APhitts Nunbers of Iypes No. of Potential of Àphitls Length 3rd Progeny AduIts LA TiSia Antennal Àntennal Cornfcle r{eighr (¡{eíght) - length Iength Segrnent length

no. embryos apterae 80 0.575 o.572 0.545 -0.045* at adult 0. 391 o.525 o.489 noult alatae 52 o.625 apterae 25 0.4r8 0. 209* alatae 27 o.272* 0. 338*

no. Progeny apterae 61 0.732 0.691 + no. e¡nbryos ÍritJr eyespots after 3 day6 alatae 13 0.443* O.423*

. Progeny apterae 72 0.725 0.524 total no.

3 days aIatae 41 0.567 0.551 49

embryos after 3 days, on each of: fresh weight; the cu.be root of the fresh weight. The correlation co-efficients which indicate, for each regression, the percentage of the variance accounted for by the regression are given in Table 2.9. The raw data are given in Appendix 2.5.

Tab1e 2.9 shows that there were many correlations between the physical characteristic measured and the number of embryos or progeny + embryos counted. Although many correlations were significant there was too much variation within the sampfes to predict with any accuracy the potential fecundity of newly moulted females using fresh weight, tibia length, antennal length' Iength of 3rd antennal segrment or cornicle length. Nor is it possible to accuraÈely predict potential fecundity after 3 days by the weights of adults.

The highest correlations were found between the number of progeny + embryos and the fresh weight of the newly moulted adults, Fresh weight as a measure of fecundity was less reliable with alatae than with apterae. This is to be expected since alaLae produce fewer embryos per adult (16 against 20 per adult apterous female) and much of the weight of an alatae is in the wings. Hence a smaller percentage of the totat weight is due to a lower number of developing embryos.

2.4.4 Discussion

This experiment was done in 2 parts, the first, in which the number of embryos at the adult moult was counted,was done in mid

February 1976 when there were low numbers of favourable plants' i.e. the plants were either young or senescing (Section 4.1.3(a)' 50

(Figure 4.3). The second part, in which Progeny after 3 days + embryos were counted was done in late April of the s¿lme year hrhen there were large aphid numbers on mature thistles (Section 4.1.3(a) Figures 4.3 and 4.4). The resultant weight range of adults was remarkable. Aphids of several other aphid species also show a great range in weight. (Way & Banks, 1967¡ Murdie, 1969a, b; Dixon, 1970). Small individuals have been attributed to development under crowded conditions, or on mature host plants, and large aphids result when nymphs are reared ín isolation, or on young or senescent host plants ($Iay o Banks, 1967¡ Murd.ie, L969 â, b;

Dixon & Wratten, L9'7l-¡ Dewar, L976). The results for H. lactucae are consistent with those of other aphid workers. As with other polymorphic aphid species the apterae were more fecund than the alatae.

The potential fecundity at the aduÌt moul-t of H. lactucae taken from uncrowded colonies h¡as a reasonable estimate of the realized fecundity of aphids taken from similar colonies. this was 25 nymphs for each apterous adult and 17 for the alatae (Section

2.3.3). The potential fecundity of Megoura viciae (Buckton) at adult moult is also realized with I08 embryos giving rise to 97 nymphs (Lees, 1959). Aphis fabae (scop), however, has' at adult mou1t, only 36 embryos but produces 85 progeny (ganks, 1964). It

IS ovarioles vary in their productivity (webber, 1955), so in such cases embryo counÈs in newly moulted females are not good estimates of realized fecundity because new eggs and embryos are produced during the life of the adult (Banks , L964). The proportion of embryos produced after the teneral moult can also 5I

vary with the morph, as with A. fabae where alatae ovulate more often after the teneral moult anl-na"t"" (Dixon & Vüratten , I!9TL). Although for H. Iaetucae the nr:mber of potentíal progeny at the adult moult provided a good estimate of realized fecundity, the correlation between the physical characteristics of the aI¡hids and theír potential fecundity was not good enough for the accurate prediction of, the number of progeny that an individual aphid might have. 52

SECTION B

TRANSMISSION STUDIES

3.1 FEEDING PREFERENCES OF H. I,ACTUCAE 3.1.1 Introduction

OrLouglin & Chambers (1967) observed LNYV particles in the sal-

ivary grands of viruliferous Hyperomyzus lactucae (L) and Boakye &

Randles (L974') using Naito's (1965) stainihg techníque, demonstrated

that under laboratory conditions H. lactucae secretes saliva onto

both lettuce (Lactuca sativa L) and sowthistle (Sonchus oleraceus

L), with probing occurring in boÈh plants. rn the field, disease symptoms are easily recognised on rettuce

prants when they appear before "heading" conrmences. After headíng, characteristic external symptoms are not seen although internal

necrosis is thought to arise from rate infection (str¡bbs & Grogan,

1963). rt may be that these late infected. prants may have become resistant to inoculation by viruriferous H. ractucae. such resistance courd be due either to (i) aphids fairing to feed on older prants or (ii) the plant itself becoming resistant to infection

even though viruliferous aphids make an inoculative feed. To investigate the feeding behaviour of the vector on rettuce leaves of different ages, experiments using radioactive rabelting

methods were devised after Boakye (1973). Advantages of such Èech- niques are -

(i) sensitivity i.e. smarr amounts of radioactive isotope can be easily and reliably detected.

(ii) Ability to quantify sap ingested or secreted. 53

Van ünden (1973), says that "the isotation of the aphid body from its food source makes it a very suitabre insect for radioiso-

tope studies; body contamination is minimized". 1311 32p are "tt¿ particurarry usefur to raber aphid saliva as they are readiry taken up by plants and can be assessed with autoradiography or scintillation counting. since the rate of distribution ôf isotope within

a plant ís a function of the transpiration rate (vüright & Barton, 1955) , 32P may be simply and effectively used to 1abet plants (Matthews, 1960). The method results in a fairly even distributÍon

of the isotope throughout the ptant within 30-35 minutes. Aphids become radioactive when fed on prants containing either 32p or 86¡5 and the radioactivity of the aphid increases with feeding time (V'fatson & Nixon, 1953; Lamb et al , 1967). Lanb et aI (1967) also showed that the percentage of rad.ioactivity transferred to a reaf from an aphid during probing was a function of time.

The experiments described in this chapter were done to test the hypothesis that H. lacÈucae feeds preferentiarly on young rettuce tissue.

Three preliminary experiments Í/ere necessary:

(i) to measure the uptake by aphids, in time, of 32p from radioactive thistles;

(ii) to show that dead radioactive aphids did not contaminate fresh thistle and l_ettuce with radioactivity.

(iii) to show that reasonable amounts of rad.íoactive tracer hras transferred to thistre and rettuce by radioactive aphÍds.

3.L.2 uptake of 32p by adult aphids Methods To l-abel plants, glasshouse-gror¡rn sowthistle seedlings at 54

the 4 to 6 leaf stage were removed from their pots and their roots were washed free of soir and rinsed in distilled water. 25o milri- 32P curies of (as phosphate in HC.e. obtained from the Australian

Atomic Energy Commission) was applied directly to each seedling by allowing drops of the isotope solution to run down the exposed roots (Matttrews, 1960). After 20 minutes, the seedlings were placed in water and left for about 18 hours in iltumínated perspex cabinets before aphids were placed on them.

Forty apterous adult H. Iactucae collected from the field were pretreated by starving them in constant light at 25oc for 24 hours. Thirty of these aphids were allowed to feed o¡¡ a labelled sowthistle plant for either 1, 3, 4, 6 or 24 hours and then each aphid was killed - in ethyl acetate vapour - glued to a lcm square of graph paper, and oven dried at SOoC for about 4 hours. After drying, each aphid was placed in a vial with 5 ml of a standard volume -

P.P.O. - Popop scintillation fluid (Ajax Chemicals Ltd., Sydney) and the radioactivity of the vial and its contents was measured using a Packard Liquíd scintillation spectrometer model 3320. Each specimen was counted for 10 minutes with a gain setting of 18 and a window setting of 50-1000. Ten aphids v¡ere used as controls, five were kitred and praced on the l-abelted sowthistle plant and five were allowed to feed on a similar, unlabelled sowthistle plant.

Each control aphid \^¡as removed after 4 hours and treated in the same \^ray as the experimental aphids. Their radioactivity was also measured.

Resurts The background count, including the vial and its scintir- ration fruid was about 30 counts per minute, and was subtracted 55

from arl the readings. Tabre 3.1 shows the radioactivity of each

aphid; the data indicate that none of the dead aphids picked up

radioactivity from the radioactive thistle nor were any of the aphids feeding on the non-radioactive plant, radioactive. .\ \ One of the 30 )aphids given acquisition feeds was lost and of the

remaining 29,) O showed no radioactivity; it is assumed that these latter did not feed.

TABLE 3.1

Radioactivity of individual adult apterous H. Iactucae after acquisition feeds of 1,2,4,6 and. 24 hours. Five aphids sampred at each time except 24 hours.

Acquisition Counts per minute time in hours

I 89 26L 0* 113 28t 2 r009 1338 702 0* 4 0* 29676 0* 1831I 3402 6 765 958 3440 0* 35082 306303 33508 326797 308841 Controls Dead aphids on radioactive plant 4 0 o 0 0 0 Live aphids on non- radioactive plant 4 0 0 0 0 0

* Not radioactive - these aphids did not feed.

The data in Ta-ble 3.I show that dead aphids do not become radioactive when praced on a radioactive sowthtstle, nor are rive aphids feeding on non-radicective plants radioactive. 56

of the aphids which fed, most showed an increase in radioacÈivity

with time although much varÍation occurred between individuals. This

variabitity could be due to differential distribution of 32p in the in the leaves (wright & Barton, 1955), the site of feeding of the aphid' or whether the aphid probed nesophyll or vascurar tissue.

rt is arso possible that some aphids did not feed for the whore acquisition period because they either weren't hungry or the physical conditions or feeding site v/ere unsatisfactory. rt is also possible

that some aphids apparently did not feed at al.l, their stylets may

have been damaged, d.uring handring. Hoh¡ever, the 4 aphids that were

on the pJ-ant for 24 hours had imbibed similar anounts of. 32p. Boakye

& Randles (L974) showed that the depth of stylet penetration increased with the duration of probe and that after I hour of probing

H- lactucae had penetrated epidermar celrs but had not reached the vascular tissue. As the data in Tabre 3.r show a very great increase in radioactivity after the first hour of feeding, they suggest that in the second to fourth hours some of the aphids had reached a vein with their stylets.

Tt¡e data in Tabre 3.1 show that there is sufficient uptake of radioactive isotope by aphids in 4 hours to allow comparisons of counts between feeding and non-feeding aphids. Hovrever, it was necessary to also show that dead radioactive aphids do not transfer radioactivity to non- radioactive sowthistres or rettuce.

3. 1.3 Contamination of leaf bv de ad radioactive aphids Method To test the hypothesis that dead radioactive aphids do not contaminate/leaftissue ?Oaphi¿s ( were pretreated by starvation and 32p dehydration and given acguisition feeds of 4 hours, removed and 57

kilted as before. HaIf of them were placed on thistle leaves, the rest on lettuce. After 18 hours, the aphids were removed and lcm discs cut from the plant tissue on which the aphiils had been placed.

llhe dead aphids and the leaf discs were oven dried and independently

measured for radioactivity,

The radioactivity for dead H. lactucae and their respective plant

tissues are shown in Table 3.2. The data show that dead radioactive aphids did not contaminate the sowthistle or lettuce leaves.

TABLE 3.2 Radioactivity counts,/minute for dead, radioactive H. lactucae and the plant tissues on which they were placed.

THISTLE LETTUCE

Aphid Tissue Aphid Tissue

48s 0 48 0 138 0 88 0 545 0 105 0 130 0 160 0 480 0 444 0 230 0 45 0 47 0 T7 0 320 0

A possible source of variation in radioactivity counts in the experiments described in Sections 3.L.2 and 3.1.3 above was the time lapse between the count and the preparation of the aphids. This

time lapse was unavoidable because aII counts were taken for 20 58

minutes'and there r4tas thus many hours difference between the measurement of the radioactivity of the first sample and say the

30th one. An experiment was therefore done to check on the decay of the radioactive isotope in time within the aphids. 5 counts were made of each of 6 radioactive and non-radioactive aphids regularly over a period of 7 days. ft was found that the decay of the isotope in the aphids was no greater than the normal decay of the isotope

(Hollander et al, 1953), and was not great enough to have a marked dfect on the experimental results. To check that aphids would probe and secrete radioactive saliva onto leaf tissue the following experiment was done.

3.I.4 Transfer of radioactive tracer Èo thistle and lettuce leaves by radioactive aphids. Method Adult apterous H. lactucae were pretreated as described in

Section 2 above and placed on a sowthistle labelled with 32p.

Periodic checks were made to confirm settling. After 24 hours, 40 of the aphids that had settled were disturbed and transferred to fresh (unlabelled) Iettuce or sowthistle leaves in moist petri dishes, and l-eft for I hour to settle again, T\renty out of the 40 experimental aphids settled during the hour and the radioactivity of only these aphids was measured. Leaf discs lcm in diameter were cut from the leaves where aphids had settled, and each leaf disc and its respective aphid was individually treated and measured for radioactivity in the way previously described.

Results The number of counts per minute for radioactivity in aphids and the plant tissues on which they were ptaced are shown in

Table 3. 3. 59

TABLE 3.3 RadioacÈivity in counts per mÍnute for individual live radioactive H. Iactucae and their feeding sites I hour afÈer the aphids were placed on the thistle or lettuce plants.

THISTLE LETTUCE

Aphid Tissue Aphid Tissue

540 0 566 0 7OL2L 0 88 0 45569 0 9484 0 113750 o 5713 84 2L3747 0 41888 0 584386 0 16013 0 L96476 0 I2L756 6 2534 0 2875rO 67 4r887 0 5028 172 92773 0 1465I 51

Ttre data show that although the aphids feeding on thistle r^¡ere radioactive none of them secreted radioactive tracer onto the

leaves. Howevet, of. 10 radioactive aphids on lettuce leaves, 5

transferred radioactivity Èo the leaves. It is generally accepted

that aphids eject saliva during cell penetration (Miles, L959¡

Bradley, L952¡ Kloft, 1960; Lamb et aI, 1967) and Boakye & Randles

(1974) showed that, when H. lactucae were confined on the leaves of sowthistle and lettuce seedlings saliva was deposited on the

leaf surface of both plants after 30 minutes. However, t.lley were

not able to show whether cell peneÈration had occurred. Since 60

aphids may also secrete saliva without penetration (Van Hoof, 1961) it is possible that tittle or no penetration occurred in this experiment. lltris would account for the small amount of radioactive saliva detected on the lettuce tissue. As only 20 out of 40 aphids settled in this experiment, and 5 out of 40 tissues were probed,it is probable that the conditions of tl¡e experiment were unsuitable to mosÈ H. Iactucae for probing and settling. Because the aphids were transferred to non-radioactive leaves immediatety after their acquisition feed it Ís prob-

able that many of them were not hungry. The constant light in this experiment could also have inhibited some aphids from settling,

since lbbotson & Kennedy (1959) observed that Aphis fabae Scop. walked on a \i¡ax coated rim of a petri dish in conÈinuous light

overnight without stopping. Van Emden (L972) sunmarises some of

the problems involved in feeding e>

Since the preceding experiments showed that:

(i) an aphid could inbibe enough 32p in 4 hours to become radioactive; (ii) radioactivity was not transferred from aphid to plant or plant to aphid by contact alone; (iii) radioactive aphids secrete radioactive saliva onto lettuce tissue;

then an experiment to test whether aphids selectively probe 3

different ages of lettuce tissue could be done.

3.1.5 Feeding preference l,lethod To determine whether aphids probed. differentially on young' 61

FIGURE 3.1

Feeding preference disc showing sectors of lettuce leaves of three ages, young, semi-mature and mature as attached to a filter paper. Filter Paper ttuce leaf

Seml mturc

Yowg 63

semi-mature or mature lettuce tissue, I00 newly moulted adult H. Iactucae \itere starved and dehydrated for lg hours at 25oC and 5å R.H. before being given a 4 hours acquisition feed on a thistle labelled with 32p. After this feed they were similarly starved and dehydrated again and each aphid was placed in the centre of a 5cm diameÈer disc consisting of 3 contiguous leaf segments. Each disc was prepared by cutting a l2Oo segrment from a leaf of each of a younç¡, semi-mature and mature lettuce plant and sticking them onto a filter paper with double sided "sellotape" so that the 1200 angles met aÈ the centre (Figure 3.1). The aphid, when placed at the centre of the disc, thus had a choice of 3 different kinds of leaves to feed on.

Each disc was backed with an 8cm x 5cm piece of Kodirex medical

X-ray film wrapped in aluminium foil- and placed face up in a dry lOcm diam. petri dish. À needle was used as a punch to mark the boundaries on the X-ray film between each lettuce leaf seg,ment. The position of each aphid was recorded at 30 minute intervals over a 4 hour period. After 4 hours in the petri dishes, the aphids and the leaf segments were dried individually and the radioactivity of each aphid and each leaf segment was measured as before.

The films were developed to show the places where aphids had settled sufficiently long to produce an exposed area on the filn.

IWo control groups \^rere treated concurrently. In the first of these, 3 dead radioactive aphids were placed on each of Èhe 3 lettuce segments on 9 feeding discs in order to check that radioactive aphids would cause a spot on the X-ray film if left for 2 hours and to check that no radioactivity was transferred from the 64

dead aphid to the lettuce segments. In the second control çlroup' 10 live non-radioactive aphids were allowed to choose and probe in the same vray as the radioactive ones Èo check that no film spots d.eveloped nor radioactivity detected on the lettuce segments after probing. Atthough the aphicls were observed at 30 minute intervals, for the purposes of these e>çeriments settling has only been recorded where an aphid was observed in the one place on two consecutive 30 minute readings, i.e. the aphÍd must have been settled for at least 30 minutes so that it. probably probed. Results The data for the two control groups are sho\^rn in tables 3.4 and 3.5. Table 3.4 shows that in all but one case' film spots developed under the leaf segment on which the radioactive aphid

$ras pl-aced. On onty one occasion was there any contamination of the leaves by the radioactive aphid. It is possible that the aphid was damaged when it was handled and subsequently leaked saliva or body fluids containing the radioactive tracer. It is also possible t].at for this leaf, as perhaps with the apparently radioactive semi-mature lettuce tissue in the second control group (Tab1e 3.5) the su.btraction of the background count did not cover outliers in the distribution. Table 3.5 also shows that 8 of the 10 non-radioactive aphids were observed to settle. Íhe experimental results have been categorized into 3 categories according to the behaviour of the aphids and Èhe radioactivity of the leavesr namelY (i) aphids which were not observed to settle but which salivated onto the leaves (3 aPhids); 65

TABLE 3.4 fhe radioactivity of lettuce leaves of 3 ages and the presence or absence of spots on ttre X-ray film when dead radioactive aphids are placed on the leaves for 2 hours.

Dead Radioactive Aphids Radioactivity PIace- Film Aphids Young Semi Mature ment Spots of present Aphid

253 Y No 2895 Y Yes 2908 Y Yes LT62 S Yes r331 5 Yes 26640 S Yes r00 5 M Yes L874 M Yes 2A582 M Yes 66

TABLE 3.5

The radioacÈivity of lettuce leaves of 3 ages and the presence or absence of spots on the X-ïay film when ].ìve ncn-radioactive aphi

Líve Non Radioactive APhids

Radioactivity Film Obs. Aphids Young Semi Mature Spots St.lg. Present

4 Y No 0 M No 0 No 0 M No 0 I1 S No 0 Y No 0 M No 0 No 0 M No 0 S No 67

(ii) aphids which were observed to settle and e:çosed the film but for which no salivation was detected by the deposition of 32P on the leaves (30 aphids).

(iii) aphids which \^¡ere observed to settle and salivated on the Ieaves so that the leaves were radioactive (I8 aphids).

The results of these 3 categories are given in Table 3.6' 3.7 and 3.9 respectively. There were 19 radioactive aphids for which no settling was recorded and no radioactivity detected on the lettuce leaves, and lt aphids were lost. Table 3.6 shows that all the aphids salivated on the young leaves and two out of the three salivated on the semi-mature leaves. None salivated on the mature leaves. Although showing evidence of prob- írg, the three aphids did not settle long enough to be observed on 2 consecutive 30 minute observations or to expose the fíIm-

Table 3.7 shows that for t9'ãphids ouÈ of 30 there was a spot on the x-ray film under the leaf segrment on which the aphid was observed Èo settle and, for 4 aphids, spots developed under more than I kind of leaf. Since the presence of a spot on the film would be a better indicator of where the aphid spent most of its time, a spot has been the criterion for settling by an aphid' and observed settling has onty been scored as settling in the absence of spots on the film. Absence of spots could have occurred because (i) the aphid I^Ias not radioactive enough Èo expose the film or (ii) the aphid did not stay in one place Iong enough. The total frequencies of spots on the X-ray film and of observed settling with no spots on the x-ray film (Table 3.7) are summarízed in Table 3.8-

The total frequencies show that there was no discriml-nation be- 68

TABLE 3.6 Ttre radioactivity of each aphid and of lettuce leaves of 3 ages - for aphids which salivated but for which no settling r^ras observed

Radioactivity (C.P.M. )

Of Each Of LeÈtuce Leaves of Ages Aphid Young Semi-Mature Mature

2003 8 70 0

6822 2083 46 0

2L622 130 0 0

TABLE 3.7 lltre radioactivity of each aphid which did not make the lettuce leaves radioactive but which exposed the film and were observed settting on 3 ages of lettuce leaf, young (Y); semi-mature (S); Mature (M).

tivity Observed Film Radioactivity Observed FiIm of settling on spot on of settling on Spot on Aphid YSorM YSorM Aphid YSorM YSor

44 Y Y 2473 M 54 Y M 2670 Y Y 55 S I\,1 267 4 M r03 ¡4 2745 S S 260 M 3368 M Y 297 M 3803 Y Y i 513 MS 4526 Y Y I 568 YYS 5400 M Mi 7L5 YY 7052 s s : 732 SS 7L57 M Ì/tl

792 S- 7629 Y Y I I L346 M 8587 S S ,'l 1403 Y L429L Y Y 2I76 YY 20830 5

2270 Y 26640 Y Y I 69

TABLE 3.8

TÏ¡e total frcquencies of spots caused by radioactive aphlds on X-ray film and of observed settling on 3 ages of, lettuce leaf, young' semi- mature and mature.

Age of Leaves

Young Semi-Mature Mature

Frequency of spots on X-raY film 11 I I

Observed settling buÈ no spots on X-ray film 3 I 4

Total L4 9 L2 70

TABI,E 3.9

1¡rtre radioactivity of each aphid and of lettuce leaves of 3 ages young (y); semi-mature (S); and mature (M); and their observed and recorded settling behaviour.

Radioactivity (c.P.M. ) Observed Film settling spots Lettuce Leaves on on Aphid Young Semi-Mature Mature YSM YSM

55 13 0 74 SM 482 I 0 323 Þ Þ 568 39 0 58 Y Y: L923 78 0 IO Y 25]-9 4 3 25 Y M YM 259L 36 1090 0 Y Y 2670 6 0 0 Y Y 3478 7I 5 0 M M 3909 0 0 T4 M T,I 4302 29L 0 435 Y 68L2 0 365 0 S S 7052 0 6L9 0 S S 8539 0 LO7 0 Y M 9531 48 0 L2 Y Y M 9903 0 40 0 Y Y M L429I 0 8 0 Y Y r9819 0 0 50 M M 20830 0 10 0 S 7I

tween leaves of different ages (Table 3.8). lftre data for the aphids which were observed to settle and which salivated on the leaves Ieaving the tissue radioactive are shown in Table 3.9' Table 3.9 shows that spots on the film usually corresponded with the observed settling of the aphids and, for most aphids, radioactive saliva was secreted on that leaf. Out of the 18 aphids whích salivated 9 salivated on two or more leaves. For l0 aphids young leaves showed radioactivity and for 9 aPhids each of the semi- mature and mature leaves were radioactive' suggest,ing that the aphids salivateC indiscriminately on the 3 ages of lettuce leaves. The data for settling behaviour supports that shown in Tab]e 3.7 with no apparent preferences in the age of the leaf chosen. The data in Tab]e 3.9 show 9 film spots corresponding with the young leavesi 6 with the semi-mature onesi and I with the mature leaves. On 14 occasions radioactive saliva was secreted where no film spot developed.

The total frequencies of aphids which settled on each of 3 ages of lettuce leaf as sho$In in Tables 3-7 and 3.9 were the young, 24¡ semi-mature, 15; and mature, 20. To quantify feeding behaviour the radioactivity transferred to each lettuce segment by probing aphids was calculated as a percentage of the radioactivity of the aphid. llhe percentages were then grouped into three categories; 5% for each of the three ages of lettuce leaf and are shown in Table 3.I0. These data show that the aphids did not probe or feed differentially on the lettuce leaves of different ages. 72

TABLE 3.10 lltre frequencies of the relative percentage radioactivity of young, semi-mature and mature leaf segments after probing by radioactive aphids. Relative percentage radioactivity \^Ias expressed as the counts per rninute of the leaf tissue divíded by the counts per minute of the aphid.

Ages of Lettuce Leaves Relative Percentage RadioactiviÈy Young Semi-Mature 'Mature

<18 5 5 5 I-5% 2 1 0

>58 3 3 4 73

Discussion Behaviour in phytophagus insects usually appears to be highly adapted to recognizing the most suitable host plant for the species (Taylor, 1959). This is,,true of aphids that recognize potential host plants by their colour (Moericke, 1950, 196;; Kennedy, Booth

& Kersha\^r, 196l) or odour (Petterson, 1970). Many aphids, hourever, land and probe indiscriminately on host and non-host plants before they become exhausted (Dixon, L973).

The pattern of behaviour involved in host plant selection by both apterae and alatae of many aphid species, and especially A. fabae (Scop.) has been well studied (e.9. Kennedy & Booth, 1951; Johnson, 1958; Muller, 1958). Theories on mechanisms by which insects accept host plants after landing have been reviewed by Bech (1965) and Kennedy (1965). Kennedy (1958) proposed a "duaf discrimination" theory of host selection by aphids based on the aphídst response to "flavour" or "nutrient" stimuli. The flavour stimulus is due to botanicat biochemicals such as glucosides and alkaloids which act as arrestants to feeding (Vtensler, L962¡ Smith, L966¡ Klingauf, 1971). The nutrient stimulus consists of either

required or non-required nutrients (Mittler & Dadd, L964) - Selective feeding behaviour experiments are difficult to inter-

pret (Kennedy and Booth, 1954) and some elaborate experiments have

been designed to oveïcome some of Èhe difficulties and possible

criticisms (Kennedy and Booth, 1951; Russell ' L966). Hov'/ever, the data in Tables 3.6 to 3.lO show that, for H. lactucae probing on l-ettuce: (j.) there is no feedi,rg preference for three rJ.ifferent ages 74

of lettuce tissue; (ii) there is a good correspondence between the presence of radioactive saliva on the lettuce tissue and recorded settling behaviour; (iii) on very few occasions was one aphid recorded as settling

on more than one tissue in this experiment. so probing on lettuce tissue may be inferred to be a random process and once an aphid has probed and settled, a second probe was unlikely during the duration of the experiment. Boakye & Randles (L974) suggested that H. Iactucae have a threshold level of probing and demonstrated that the predisposition to settle and probe increased with time. Vlhen the aphids were starved for up Eo 24 hours in constant light, they had comparable stylet penetration into host and non-host tissue for the first 30 minutes' but after this periodthe threshold level of probing probably rose because withdrawal of the stylet occurred after 30 minutes.

v,Iensler (1962) showed that Brevicoryne brassicag (L) could distinguish quickty and clearly beÈween host and non-host leaves and would walk off non-host leaves (Vicia faba L.) withtn 3 minutes of probing. It is possible that, vrithout pretreatment, H. lactucae can also quickly distinguish between hosÈ and non-host leaves; buÈ its abílity to do so could be lost after pretreaÈment by starvation in constant tight. at 25oC because Boakye and Randtes (L974) found Èhat the frequency of setÈling increased with an increase in the duration of pretreatment. fn the field, migrating H. Iactucae witl be variously pretreated by starvation and constant light, their predisposition to settle 75

and probe increasing with the duration of their pretreatment' Boakye and Randles (1974) found that lettuces may be infecÈed with virus after a 5 minuÈe probe (albeit without subsequent development of symptoms), indicating that viruliferous saliva must be ejected in that tine. Hence the presence of radioactivity on tissues on which no settling was recorded would have resulted from aphids probing and moving off between sampling occasions. The method of recording settting i.e. aphids shown as having settled only if seen on the same tissue on tvfo cgnsecutive 30 minute observatiOns, allowed nearly an hour during which probing could have occurred without a record of settling being made. Lambetal(1967)alsoobservedthatsecretionofsalivamaybe intermittent or continuous and it is possible that in this experiment (Table 3.7), although settling was recorded no saliva was secreted, either for natural reasons or because the stylet of the aphid

had been damaged in the transfer from the radioactive plant' In the experiment (Section 3.1.5),of the 81 H. lactucae used after 24 hours of pre-treatmentrlS settled and probed indiscriminately on 3 kinds of lettuce leaves for at least 30 minutes; another 3 salivated but did not cause a spot on the film nor were they seen to settle. As the inoculation threshold of LNYV for viruliferous aphids on lettuce seedlings is between l,;and 5 minutes with a feed of in excess of 15 minutes necessary for the subsequent appearance of disease inoculation of symptoms in the lettuce (Boakye & Randles , L974) ' virus into lettuce can occur regardless of the age of the lettuce Ieaves. 76

3.2 DETECTION OF VIRUS 3.2.L Introduction

The main source of LNYV inoculum in the field is the sowthistle,

Sonchus oleraceus L. (Str¡bbs & Grogan, L963¡ Randles & Carver, L97L) and the estimate of nu¡nbers of infected sowÈhistle plants - which are crucial to the study of the epideniology of the virus - are dependent on the relia-bility of detection of LNYV in field infected thistles. rnfected sowthistles are symptomless and Crowley's (L967) technique of infecting Nicotiana glutinosa (L) was used for the indexing of field collections of sowthistles during preliminary field sampling'

LNyV inoculum (SE3 strain) was found to be most infective when mixed

with O.lM glycine buffer, PH 8.6, and the susceptibility of N' glutinosa plants was highest in the afternoon and early evening'es-

pecially when leaves which were between one-half and three-fourths

expanded \irere inoculated (Crowley, L967). Ttre SE3 strain l^las found to remain infective longer when chilled (crowley et al, 1965). At the start of the study two leaves were collected from each thistle - one fully expanded and the other not fully e>çanded. Both leaves \¡rere ground together wiÈh a few drops of cold glycine buffer {l and rubbed over the top/three-i. leaves of each of IO Nicotiana glutinosa plants which had been dusted with 50O mesh carborundum- However, this procedure often resulted in less than 50å of the N. glutinosa showing signs of infection, so the experiments described below were carried out in an attempt to improve the relia- bility of the test. 77

3.2.2 Dilution of the Inoculum Methods Roberts (L964) in his review on local lesion assay of ptant viruses,points out the value of dilution curves in the initial testing of virus infectivity and host susceptibilityr so a preliminary serial dilution experiment was carried out to examine the relationships between lesion nu¡nber and concentration of inoculum using SE3,which produces local lesions in N. Iutinosa

(Stubbs & crogan, 1963).

Ttre inoculum was prepared from N. glutinosa plants maintained in the glasshouse and infected with SE3 strain. AtI inoculations were done late in the afternoon. Infected leaves were thoroughly ground together with an equal weight of cold O.I.M. Glycine buffer, pH 8.6 and the leaf debris separated out by squeezing the sap through muslin. This inoculum was chilled and serially díluted immediately, from the original 1:1 extract down 1:2 (1 part exÈract:

I part buffer); I:I0; I:100; and 1:1000.

Four d,rops of a dilution of inoculum were put on each of the top three leaves of a N. glutinosa plant which had been dusted with 500 mesh carborundum. On each leaf the drops were rubbed over the Ieaf with 5 strokes of the fore-finger. Ten plants v¡ere used for each inoculum with another ten plants treated with buffer on1y, as a control. The plants were then lightly sprayed with water to minimize mechanical damage to the epidermisr and left in the glasshouse. To avoid increasing the dilutions of inocuLa by contamination, the control plants were treated first and, then the inocula were applied in order of decreasing dilution. The lesions l^rere counted after 7 days. 78

Results Using this whole leaf assay system ,the mean mr¡riber of lesions per leaf decreased with dilution (Tab1e 3.11). HoÌ^rever, the variation in lesion nunber was large as reflected ín the standard deviations. Crowley (L967), noted that the susceptibility of different leaves on any one N. glutinosa plant to a single in-

oculum of LNYV varied greatly, some leaves producing many lesions

and some producing none at aII. He also noticed that the variation

in lesion number was greater within the plants than between them.

A similar result htas apparent in this experiment.

TABLE 3.IT

Ihe mean nr:¡nber of loca1 lesíonsr/leaf produced by various dilutions of

SE3 strain of LNYV in O.lM glycíne buffer pH 8-6 when applied to

Nicotiana glutinosa plants .

Standard Dilution of Mean Inoculum Deviation

1:1 13. 3 11.04 Lz2 2.r L.87 1: I0 o.7 r.34 I: 100 0.1 0.43 I: 1000 0 Control 0

Variation in l-esion nuÍibers due to differences in the plants could have been reduced by the use of half leaf comparisons, i-e.

the pairing of different concentrations on half leaves of the same plant.

Since the highest number of lesions occurred when the extract of

leaf tissue l¡ras diluted with an equal weight of buffer, this dilution was the one generally used in subsequent experiments- 79

3.2.3 Inhíbition of Infection bv Thistle Cell SaP

Yarwood and Fulton (1967), sununarising other work, state that in general, inhibitors are the major cause of difficulty in mechanically transmitting plant viruses. An experiment was therefore carried out to find whether such an inhibitor of LNW infectivity occurs in the sap of s. oleraceus. / Method SelecÈed field thistles were py'otted and transferred to I the glasshouse where they were indexed on N. glutinosa L, N. glauca R. Graham, chenopodium amaranticolor, coste et Reyn,

Cucumis sP. L, and Datura stramoniumL. These tesÈ plants were ex-

amined for symptoms after IO days and thistles infected with

viruses other than LNYV were discarded. Only infected thistles and those with ¡nild isolates of LNYV (i.e. which show systemic symptoms but no local lesions) were used in this experiment.

Ttrirty leaf discs 5mm in diameter were punched from each of the infected (nild LIIYV thistle sap) and uninfect'ed thistles (thíst]e sap). Each sample of 30 discs was weighed separately and was mixed with an equal weight of N. glutinosa leaf discs to produce

one of two mixes, namelY, Uninfected thistle + infected thistle + N. glutinosa Uninfected thistle + N. slutinosa

The N. lutinosa had been infected with SE3 to produce local lesions for scoring in the experiment and at the time of incorporation into the mixes the plants were showing early systemic s)¡mptoms of SE3

,.infection. These plants were the source of SE3 sap. The groups of discs for each mix were Èhoroughty ground in an equal weight of cold distilled waÈer and the extracts were clarified 80 in a refrigeraÈed ultracentrifuge at 13009 for three minutes- Each clarified extract was divided into two equal aliquots, and the volume of each was measured and then treated further and/ot diluted to give a series of different treatments which comprised:

A. Thistle sqP¿-Ell9-!NIy-lb1g!19-geP-3!q-EE 3 93P seríallv diluted with 0.1M gtycine buffer pH 8.6, 1 part extract to I part buffer; ]-z4¡ 1:16; L¿64¡ I2256. B. yilg_!NIY-gl9-9Es prepared from thistle sap, mild LNYV thistle

sap and SE3 sap; centrifuged f,or 15 mins. at 801000 g to separate virus particles from the sap containing a possible in-

hibitor. the resultant virus pellet was L-esuspended to the original volume of tJ:e aliquot in distilled water, and a dilution series with glycine prepared as with A. c. I!lg!Ig_g3p¿_EE 3 g3p serially diluted with slycíne buffer in the same way as A.

D. qE glly prepared from thistle sap, SE3 sapt centrifuged, -- 3 ---- resuspended and. diluted in series in the same way as B.

Half leaf comparisons \¡¡ere made between treatments thus; A. opposite B; C to D; A to C; and B to D, by mechanically inoculating two drops of each inoculum to three half leaves of three

\. glutinosa plants. Results The results are shown in Figures 3.2; 3.3; 3.4¡ and 3.5-

FÍgure 3.2 shows the total nr¡niber of lesions produced at each dilution by SE3 sap alone (D) and mixed with mild strain LNYV (B) " As both inocula were diluted fewer lesions were produced. Ihere v/as an apparent synergistic effect between the SE 3 and mild strains of LNYV as shown by the higher numbers of lesions produced at the 8I

FIGURE 3.2

Loca1 lesíons produced by dilution with glycine buffer of: .< SE 3 strain of LIiIYV (Treatment D)

QroooorQ mild LNYV + SE3 (Treatment B)

FIGURE 3.3 Local lesions produced by clílution with glycine buffer of: .€ thíst1e sap + SE 3 strain of LNYV (Treatment C) $.ooooQ lltristle saP + milil LNYV + SE3 (Treatment A)

FIGURE 3.4 Local lesions proiluced by dílution with glycine buffer of: 04 SE3 straín of LNYV (Treatment D)

QooorooQ rhistle saP + SE 3 (Treatment C)

FIGUR.E 3. 5 Local lesions produced by dílution with glycine buffer of: .-.< ¡nild LNYV + SE3 strain of LNYV (Treatment B)

QorooroQ Thistle sap + mild LNYV + SE3 (Treatment A) tu

Total number of lesions ¿¿ NÀ6t@Oñ Ì\)ao)(Þoñò o aãooooo o ooooooo oN 8rÉÉË o Bå888

tr, c o a 83 fírsttwo concentrations by the inoculum containing both strains. To test if ûre difference in the number of lesions produced by the two inocula was significant, means were calculated for the number of lesions produced on each half leaf at the highest concentration in this paired treatment (Appendix 3.1). The mean nu¡hber of lesions/ half leaf for sE3 alone vras 6.9 (SD = 6.9) and for the mixture

9.4 (SE = 10.8) . This difference was not significant at P = O'05 when analysed with a student 'tr test (Appendix 3.1). To check that there were no oÈher viruses present which may have affected the number of lesions produced, both virus strains were indexed onto a range of test plants. No other viruses were detected'

Figure 3.3 showç the number of lesions produced at each dilution by sE3 with mild LNYV and thistle sap (A) and by sE 3 with thistle sap (c). Figure 3.3 shows further evidence of synergism where at the 2 highest concentrations there v¡ere more than twice the nrunber of lesions using mixed inoculum than with SE3 alone. Figure 3.3 also indicates that there were more Iêsions produced where the inoculum containing thistle sap I^tas diluted with buffer three times, than where no dilution occurred, suggesting that an inhibitor is indeed present in the sap and that its effect is

diluted out (Yarwood & Fulton, 1967) - Figures 3.4 and 3.5 represent the control comparison where inocula containing thistle sap were compared on half leaves with those not containing sap. Figure 3.4 shows the nurnber of local

Iesions at each dilution of SE 3 with thistle sap (C) and SE3 alone (D) whilst figure 3.5 similarly shows lesion nudbers produced by 84

the mixture of sE3 and LNYV with thistle sap (B) and without thistle sap (A). Bothfigures3.4and3.5showthatthethistleceltsaphad an inhibitory effect on the number of lesions produced and they also show that the inhibítor was effectively diluted out after the addition of three parts of glycine buffer. At this dilution (I:4) there were more lesions with the mixed virus strains than between the with SE 3 alone, again indicating apparent synergism two strains. 3.2.4 Dark Pretrea tment of N. qlutinosa

Tobacco necrosis virus (TNV) produces more local lesions in

beans when the test plants are kept in the dark Lot 24 hours before inoculations (Bawden & Roberts, Ig48; Matthews' 1953) ' However, fewer lesions are produced When test plants are placed

in the dark for shorter periods before (or after) inoculations. (Matthews 1953). An experiment was done to test the effect of up to 24 hours pretreatment in the dark on the susceptibility of N. glutinosa to LNYV. Although crowley (1967) found that freshly

prepared LNYV inoculum ln O.lM phosphate buffer did not vary great- Iy in infectivity for susceptibility tests, it was decided to use

the same inoculum preparation for all treatments since the effects of darkness pretreatment observed by lrfatthews (1953) were confounded with those of inoculaÈion at different times of the day, and

with Èhose of the number of hours of daylight or darkness inmedi- ately after inoculation. Method 6 matched groups each of 10 plants, were transferred to a culture room at 25oC under constanÈ light a week before the 85

experiment was commenced. Àt 48 hours ¡ 24¡ L6¡ I and 4 hours before inoculation one group of IO planÈs \das placed in a darkened cabinet. The inoculum was prepared (at time 0) from

SE3 strain in glycine buffer 1:1 and left chilled' For inoculation the 6 groups of plants \iìrere arranged in rows, dusted with carborundum, and inoculated in the usual way, working across the rows so that any drop in the infectivity of the virus with time did not influence the results due to the darkness pretreatment. The plants r^¡eïe then lightly sprayed with water and left in the glasshouse. Results Although this experiment vtas repeated several times, on all occasions the sE 3 strain failed to produce primary local Iesions. It is possible that the period of constant light in the culture room before the pretreatment made Èhe plants less likely to react with chlorotic local lesions as the leaves were noticeably darker in colour, and harder in texture than the glasshouse plants used in other experiments. This hypothesis is

supported by the very low incid.ence of disease sl¡mptoms seen in the control treatment (no darkness) when compared with glasshouse plants used without pretreatment in the previous experiments.

Because local lesions were absent, the effects of pretreatments on the severity and frequency of systemic symptoms were assessed'

They were classed as: i) Severe - Severe stunting, new leaves grossly reduced and de- formed, mottling of older leaves"

ii) ¡leaium - marked stunting, some reduction in size and deformity

of new leaves, some mottling of older leaves' 86

iii)Light - slighÈ stunting, downward cupping and yettowìng of the margln of new leavesr' no mottllng of older leaves'

iv) No apparent slzmptoms.

More N. glutinosa plants showed symPtoms when darkened before inoculations (Tabte 3.I2). More plants showed symptons after four hours pretreatment than with all other treatments. Ttre plants kept in the dark for forty eight hours were very fragile when they were inoculated and died subsequently. lltre low degree of infection in the untreated control plants

has been discussed above.

TABLE 3.I2

The frequencies of symptoms of LNYV infection in groups of 10 N' glutinosa plants darkened for various periods before inoculation'

Disease Hours of darkness before inoculation symptoms

Severe 0 7 I 0 0 A L Medium I 5 4 3 4 "o Light 2 I 4 1 I I No symptoms 7 0 I 6 5 E D

For practical purposes where field samples had to be indexed as soon as possible afÈer collection, it was not possible to pre- treat the test plants with tess than 8 hours of darkness' so a final experiment was devised using s4.p from tÌ¡istles known to be

carrying LNYV, and treating the N. glutinosa in ways which were feasible for a continuing field study progralnme' 3.2.5 Effect of Liqht and Dark on Lesion Nunber Method Late in the afternoon, three matched groups of I0 N. qlutinosa plants at the 5-7 leaf stage were placed in a culture room at 25oC. in constant light alongside three similar groups in a right proof blackened container. Duplicate groups were set up in a naturalty lit glasshouse which was maintained at a temperature range of between 20 and 25oC. There were thus 4 groups of 30 plants. After 24 hours each plant was inoculated with one of three dilutions of sap from infected field grown thisÈles, each made up as a chilted inoculum with gtycine buffer in one of the ratios; I part sap to t part buffer (1:1); I:10 and l:l0o- After Ínocu- lation and spraying with water the darkened prants were left un- covered and the 60 plants from the culture room were returned to that room and were therefore exposed to constant light- the 60 plants from the glasshouse lvere returned to the glasshouse where darkness had fallen and they therefore experienced 10 hours of darkness. Thus there were 2 groups of 30 planls pretreaÈed in the dark and another 2 groups of 30 plants pretreated in the light. Fotlowing inoculation, l group of each of the dark and light pretreated plants were placed in constant light and the other two groups were placed in the dark for 10 hours-

Tabl-e 3.13 shows the effect of pretreatment of light or dark followed by post-treatment of light or dark (I0 hours) on the symptoms produced by LNYV in N. glutinosa. 88

TABLE 3.13

LNYI/ slrmptoms in gïoups of 10 N. glutinosa plants pretreated for 24 hours in light or dark and kepÈ in light or dark (I0 hours) after inoculation.

Post- Culture Room Glasshouse Treatment ( right) (10 hrs dark) Pre Dark Light Dark Light Treatment Concn of I:I0 1:I00 1:1 1:10 1:100 I: I 1: 10 I :100 1:1 1:10 1:100 inoculum 1:I

ase s Severe 9 00 5 0 0 4 0 0 I 0 0 Medium I 70 5 5 0 3 2 0 6 0 0 Light 0310 0 5 t0 131 0 4 4 No s]¡mptoms 000 0 0 0 259 3 6 6

Table 3.13 shows that all plants kept in the culture room became infected whilst fewer of the glasshouse plants showed sl¡mptoms. Light or dark pretreatment before ínoculation appeared to have little difference in effect although the symptoms of the darkened

plants were moïe severer êrìd the s)¡mptoms of plants sr.rbjected to the dark-light cycle were also more severe than those symptoms of plants exposed to constant light. 3.2.6 Discussion It appears that Èhe sowthistle contains an inhibitor in its

sap which reduces Èhe effect of LNYV when sap is mechanically in-

oculated onto N. Iutinosa plants since there is a significant difference (at P = O.O5), between the means for the nu¡nber of lesions produced per half leaf by each of the SE3, thistle sap mixed inoculum (C) and the inoculum containing SEg alone (D) at 89

each of the first Èwo dilutions (1:1 dilution t26 = 2.382¡ L¡4 dilution |L2B = 2.930') (Appendix 3.1) The inhibitor can be effective- Iy diluted out by the addition of three parts of 0.1 glycine buffer pH 8.6. The presence of inhibitors in ceII sap and their removal- by dilution with buffer is not uncommon and has been fully discussed by Bawden (1954).

The infectivity of the SE3 strain of LNYV is apparently increased when it is mixed with a ¡nild field strain although the differences in means for lesion nurnbers per half leaf at the two highest concentrations of the paired inocula, SE 3 + mild strain (B) and, SE aLone (D), illustrated in Figure 3.2, were not significant at p = 0.05. (1:I dilution t26 = I.2I\¡ 1:4 dilution t2g + 0.847) (Appendix 3.1). There were, however, significant differences in the means for lesions produced per half leaf with inocula used in each of the two highest concentrations illustrated in Figure 3.3 (l-:l dilution t2g = 2.370¡ 1:4 dilution t26 = 2.103), and at the highest concentration illustrated in Figure 3.5 (t2g = 2.658)

(Appendix 3.I). The non significance between the means for the data illustrated in Figure 3.2 was probably due to the high variability and the small sample size. It is probable that a re- peat of the treatment in which SE 3 and mitd strain were compared with SE 3 alone on adjacent half leaves using a large nunber of rep-

Iicates would show that synergism occurs between the Èwo strains.

That synergism has occurred at all, poses some interesting questions and coul-d prove useful in the detection of small quantities of mi1d virus strains. Synergism was unexpected because two similar strains of virus usually exhibit antagonism. e.g. with Tobacco 90

Mosaic Virus (TMV) (Bennett, 195I); Passionfruit Woodiness Virus (Simmonds, 1959) and Tobacco Necrosis Virus (Kassanis C !'Ihite, L972). Synergistic effects are usually associated with simultaneous inoculations of dis-simifar viruses, ê.9" Tomato Aspermy

Vírus and cucurnber Mosaic virus (Holmes, 1956); Potato Virus X and Potato Virus Y (Damirdagh and Ross , L967); and Barley stripe

Mosaic Virus and TMV (Hamilton & Dodds, L97O) .

Ttre test plants, N. gfutinosa, when hardened by a week under constant light h¡ere more susceptible to infection after four hours of pretreatment in the dark than with no pretreatment. However, glasshouse gro\Á¡n plants always became infected with mild strain

LNyV when kept under constant light f.or 24 hours after inoculation, regardless of a pretreatment of 24 hours darkness. It is possible that the temperature in the culture room immediately after inocu- Iation was higher than that of the glasshouse and although' before inoculation, temperature effects when compared with 1i9ht' play a rel-atively small part in causing fluctuation in the susceptibitity of plants, (Matthews, 1953; Ross, 1953) it may be important after- wards in the development of symptoms (Ross, 1953). This could explain the higher evide¡¡ce of severe symptoms shol^/n in Table 3.13. As a result of these investigations, indexing of all thistles collectecl or used during the field survey and laboratory work was done in the late afternoon' ThIo ,\' glutinosa plants were always used for each inoculation alrd they were selected from glasshouse grown stocks at the 5 - 7 leaf stage. Before indexing, these test plants were left in the dark for at least 24 hours and whenever possible v/ere transferrefl to the culture room immediately after 9I

inoculation and spraying, otherwise, they were left under a light in the glasshouse. The inoculum was prepared by bJ-ending leaf with two to three parts of cold o.IM glycine buffer at pH 8.6. Using this method, transmission of LNYV to both tesÈ seedlings was normally achieved. only rarely was one of the plants infected. 92

FIGURE 4.I

Map of Adelaide showing the location of the coÍunercial market gardening area at Highbury (insert: part of South Australia showing location of Adelaide). ,:y

N T' F È I I

! EOUNDARY OF IHE ADELAIOE - I1ETROPOL¡1AI{ PLAXT{ING AREA

{ 20À8 ö SCALE ln XILoHEIRES o

.¡ J :

SCALE ln KlLoñETnES 94

SECTION C

POPULATION STUDIES

4.L THE OCCURRENCE OF APHIDS, THISTLES & INFECTED THISTLES TN THE FfELD

4.I.1 Study Area Field sampling was carried out once a month in a market garden

at Highbury in the Adelaide foothills (Figure 4.1) -

In Èhe study area' as in other areas of lettuce growing, the farmer rotated his lettuce planting on a six monthly basis' planting blocks of lettuces at 10-14 day intervals. Bet!,teen these blocks, the land which was unsuitable for lettuce growing became infested with thistles. This land was chosen for sampling thistles and aphids.

Hol^rever, since Stubbs and Grogan (1963) and Rogers and Baker (1970) demonstrated that the clearing of thistles in the irunediat,e vicinity of young lettuces reduces the incidence of LñYV signifi-

cantly, it has become agricultural practice to destroy thistles in land adjacent to young tettuce crops when the thistles reach

maturity and show evidence of aphid infestation. The farmer did this in the study area by mowing the plants off near the ground anfl by ploughing. Consequently, it was not possible to take continuous

samples within the total area and it was necessary to divide the intercrop area colonised by thistles into four separaÈe subareas and to sample from two or more subareas on each sampling date.

Four subareas have been utilized in this detailed study. TI^7o,

called "Top area l" and "southern roadside'rr \¡Iêfê sampled most reguJ-arly oveï a 30 month period; the others "Bottom area 1" and 95

FIGURE 4.2

Map of the study area at Highbury, south Australia, showing the contour lines and the 4 subareas utilized. z c e o F

4 E

Lc N fop orco I Lollucc crop

Soulh rocd¡ido Rood

Bottom oroo I Trcck

A¡cq ó F¡nce

SCAIE I ' 250o

50 0 50 t00 190

CONTOUR INTERVAT 2 METRES 97

,,Area 6" were utitized when the thistles had been destroyed in

Top area I or Southern roadside (Figure 4'2)'

4.1.2 Method Since the density of thi.stles varied along the length of each subarea but not across its width, on each sanpling occasion a tran- sect was taken along the full length of one or more sub-sampling areas to include a total of approxinately I10 thistles. The thistles sampled were divided into the growth stages outlined earlier. viz. rosette, semi-erect, an<1 erect (section 2.I)¡ then within each stage, size intervals were o,:vi;e''l and the number of plants in each were recorded. The sizes of rt:settes were: IeSs than 5cm, 5-10 cm, 10-15cm, and greater thall 'lscm; and for erect and semi-erect plants: less than l5cm, 15-30cm, and greater than 3Ocm. For each plant, the number of flower heads, number of flowers per head and the presence or abstlnce clf aphids were also recorded. A sub-sample of 30 plants was also taken which inclu'iled every thircl plant, and the flowers of each plant vtere Èhen classified into stages l-5 as outlined before (Figure 2'21 '

The occurrence of aphids on any of the flower stages was noted anrl the aphids collected. Leaf samples were also taken. rn the laboratory, Èhe collecLed aphids were sorted into their four separate instarsr âIìd alate nymphs and alate arld apt-erous adults were also separated. The leaves were tested for the presence of

LNYV (Section 3.2) and prcrlators were identified'

To examirre the interactiott t-¡e:t-ween aphids and plants, a planL was clescr:ibr:d as favc¡urable íf flower burls were present, i'"e' all

semi-err:ct or ereclt planLs (Ser:tion 2.1) . 98

FIGURE 4.3

to Log nr:mber of all thistte plants and of thistle Plants favourable

aphids per lOOrn2 at different tímes of the year at Híghbury' Qooorooe log total thistles o< log favourable thistles

FIGURE 4.4

Log number H. lactucae and of flower heads er lOOm2at different

times of the Year at HighburY.

Q orroooQ log number of aBhids

O- Iog nuriber of flower heads

FIGURE 4.5

Log nr:mber of infested flowerheads, of alate 4th instar H. Iactucae year and of thisÈIes with virus per I0O m2 at different times of the at Highbury. Qreoo-Q 1og number of infested flowerheads O-4 log number of alate 4th instar aphids A- -^ log nurnber of thistles \4tith virus o

A J J A s N A s o N F A J J A s o N J F

I(! o 6 .9 E -c. o .Ë (! t(') 6 l¡0, E z :'

!.' :.. 3 M A A s o N A s o N D F A J J A s o N J

î I .^ \ I to I \

J J A N o J M J A N J J A o N D J F o

19Tl 1975 1976

Months and y€ars expressed in accumulated day degrees abova 4C 100

The data from each subarea were standardized by converting raw scores to nr:rnbers per lOO m2. lltris was necessary because on each samplingoccasionthepercentageofeachsubareasampledchanged as the density of the plants and aphids fluctuated. To combine data from all subareas the standardized data were multiPlied by a number which represented the proporÈion of the sr:barea sampled in relation to the whole site. These numbers were then added to gíve the number of plants, flower heads or aphids for each loo n2 of the whole síte.

4.1.3 Results

(a) Ttre whole area

Tt¡e results for the whole area are summarized in Figures 4'3,

4.4 and 4.5. For the 30 month period from 20th June, L975 to 17th November, 1977, Figure 4.3 shows the log total nr¡mber of thistle plants and 1og number of favourable plants; Figure 4.4 ttre 1og number of flower heads and the log number of aphids; and Figure 4.5 the log number of infested flower heads, the log

number of alate 4th instar aphids, and the log number of plants infected with virus. AlL are expressed per IOO m2' Figure 4.3 shows that, throughout the sampling period, thistle

plant nu¡nlcers varied bet\4teen 6 and 591 plants with peak numbers

in November to December (early summer) each year' I'{inor peaks occurred in May-June (early winter), (Figure 4.3). Similarly, the numbers of favourable plants (erect and semi-erect) show main

peaks in Novemberr/December wiÈh smaller peaks in February' L977

and May,/June, :-:g77 . There was no apparent peak in the nrunber of WAITE INSTITUTE LIBRARY

favourable plants in May,/June, L976. Of all thistle plants, at least 508 were favoura.ble to aphids except in February, 1976 (40å favourable); November, Lg76 (2Oz) ¡ January, 1977 (40t);

April, lg77 (398); May, Lg77 (38s); November, ]-977 (458) ' Figure 4.4 shows that the numbers of flower heads were usually highest when the nr¡rnbers of favourable plants were highest

(Figure 4.3) viz. November, L975¡ December, L976¡ February, 1977i

June, 1977 and November, 1977.

Four major peaks in aphid nr¡nbers occurred (Figure 4'4) ¡

October , L975; April, L976¡ October, 1976¡ and May, L977 ' Ieast numbers were Present in January and october' 1977 ' Figure 4.5 shows that the highest number of infested flower

heads occurred in November, L975t April and september and

December, L976 and in May to June, L977.

and 1976 Tlre peak of alate 4th instars in september ' 1975 (Figure 4.5) preceded the peak log nuniber of aphids in october of those years (Figure 4.4) and the peak in May, 1977 (Figure 4.5) also preceded the peak log nurnber of aphids in June of that year (Figure 4.4).

The variation in numbers of alate 4th instars for the whole

area can be summarized as follows:

--ì _; i) Maxinum nrunbers occurred in september, L975, April, L976'

sepLember and December 1976 and lltay, 1977. High numbers also occurred in october to November, L975, and June to August' 1977 (Figure 4,5). At all these times the total number of aphids was high (Figure 4.4) and usually the number of infested flower heads was also high (Figure 4.5). ro2

FIGURE 4.6

Log number of H. lactucae per infested flower head and the log

nr¡¡nber of their alate 4th instars per 100 m2 at different times of the year at HighburY. o.....o log no. aphids,/infested flower head a- log no. alate 4th instars/IOO m2 I 0000

Io o t000 6 o .9 .'o E E, ..oj i o .Ë o o t00 Io) o o ¡(¡) o E z

N J J A o N D M A M A s o N D M A M J A s

1975 1976 19Tl

Months and years expressed in accumulated day degrees above 4C to4

ii) None were present in ,funer/Ju1y, L975i December, 1975¡

January/February, L976¡ May/July. L976¡ Januaryr/March, 1977

and SepÈember/November, 1977 when there were relatively

few infested flower heads present. fhe relation between the numbers of 4Èh instars, the nunber of infested flower heads and the numbers of aphids is best iI-

lustrated by plotting the nurnbers of aphids per ínfested flower head on the same graph as the numbers of 4th instar alates. This is done in Figure 4.6 which shows that there were three

very clear peaks of alate incidence, Se-ptember, 1975 anil 1976

and May L977. It also shows that there urere four period,s when there vrere no alates on infested flower heads. i) september to November, L977, when the nr¡ibers of aphids on infested flower heads were low.

ii) Juner/.ruly and December , L975; ilanuaryr/February and May/ July 1976; and January,/March, 1977¡ when the numbers of aphids per infested flower head were sufficiently large to

have produced new alate forms, if other stimuli (e.g. temperature photoperiod) had also been optimal for alate develop-

ment.

Figure 4.5 also shows that plants infected with virus were

found throughout the year. flre incidence of virus in plants was

híghest in December, 1976 and again in November, 1977 but was

also high in June, 1976 and June and August, L977. The highest

incidence of virus in plants generally occurred when there \^Iere

some alate 4th instar aphids in the population (Figure 4.5).

There is much variability in the data for the whole area, r05

FIGURE 4.7

Log nurnber of thistles and of flower heads per lOO m2 in subsampling area I'Top area 1" at dífferenÈ times of the year O.....O log no. thistles o.{ log no. flo¡rer heads

FIGURE 4.8

Log npmber of H. lactucae and of their alate 4th instars per lOO m2

in sr¡bsampling area t,TQp area Lt at different times of the year

QrrrroQ log no. aphids

O< Iog no. alate 4th instars

FIGURE 4.9

Iog number of all thistles with virus and of rosettes with virus per

lOO m2 in subsampling area "Top area l" at different times of the year

Q.oo.oQ Iog no. all thistles wittr virus

H 1og no. rosettes with vÍrus O..O

J F A M J A o N J A s o N J F A M J o

o o o o

o (! oa¡ O¡ o o .E(, E q.'

(ú o ôctt 'o = o ø ú, -ô E z

N D J F A M A s o N J A s o N D J F M A M J J A s o

o o o

D M A M J A o N J J A s o N o J M A J A o N

1975 1976 19TT

Months and years expressed in accumulated day degrees above 4oC r07 particularly the incidence of alates and virus and so to determine whether the relationship between the aphid, the plant and the virus could be more clearly seen, the data expressed in Fígures 4"3, 4.4 and 4.5 fot thewhole area were also plotted for the two subareas which had been most regularly sampled, i.e.

Top area 1 and Southern roadside.

(b) Tgp_eree_l Figures 4.7, 4.8 and 4.9 summarize the log number per IOO m2 of: total plants, flower heads, aphids, alate 4th instars. total planÈs with virus and rosettes with viv'rrs jn the sub-sampling area, Top area 1. There are gaps in this data for the reasons outlined in the method i.e. mowing or ploughing of plants and spraying for aphids. Plants and flowerheads

Figure 4.7 shows that the peak incidence of plants occurred

in November, L975, December, 1976 and November, 1977, times at which the number of flower heads were also highest (rigure 4.7)

Minor peaks in the nurnber of flower heads occurred in May, 1976

and L977. (Figure 4.7). Numbers were also high in January.

February, 1976 but were low in Januaryr/February, L917.

The decline to zero in February, L977 was due to the plants being ploughed in by the farmer, but the data for the previous

sampJ-ing date indicate that numbers were starting to decrease, probably because the thistles were dying off during the hot weather, and ít is likely therefore that they would have declined near to zero even if they had not been ploughed in. By contrast

the number of flower heads was high in January,/February' L976 108

FIGURE 4.10

Mean weekty temperatures in Adelaide from Julyr 1975 to November,

1977 showing the times of field saÍpling (toP scale) and the times at which the main decreases in aphid nu¡nbers occurred (arrows) ' 200 v, o o, + ++ oct) t, illll I lll llllllllllll I lllllll ïto 16() .; f o o CL E (D à o) It, 50 ì c o o E o F M A M J J A s o N D J ¡J A s o N D J F M A M J J A s o N Ð J 1975 1976 1977

Months and years 110

because the plant population \,{as a relatively young one as a result of the mowing in september the previous year. It is Iikely thaÈ if the plants were not cut in any subarea the

natural sequence would be low numbers in January'/February each year.

4pþi9: Figure 4.8 show that the nu¡nbers of aphids in Top area I fluctuated greatly, being highest in January/February L976, April

1976 and September to December, L976, and ifune, L977. There are indications of a rise in aphid numbers taking place in September

1977 shortly before the subarea was sprayed with an aphicide, metasystox, which effectively kilted off the aphids in Èhis

subarea. In order to explain the significant increases and decreases

in aphid numbers the mean weekly temperature records for the period were examined (Figure 4.10) and the periods when the

decreases in aphid nurnlcers occurred were marked with arro!{s 't. main in Figure 4.I0.

These data (Figure 4.10) can be used to help explain the signi-

ficant changes in aphid numbers.

fncreases in ePÞig-rsrþers i) High aphid nu¡nbers occur at times when the numbers of flower heads are increasing (Figure 4.7).

Decreases in numbers i) The significant decreases in aphid numbers from November to

December 1975 and December, 19'76 to January, 1977 (Figure 4'8)

probably occurred when the mean day temperatures were high 111

(Figure 4.10), and were due to a reduction in the nunber of favourable plants (Figure 4'7) as well as the killing of

Èhe aPhids bY high temperatures' ii) similar decreases occurred from April to June, 1976 and JunetoAugust¡Lg77(Figure4.8)whenthereweresignificant The mean numbers of favourable plants available (Figure 4.7). weeklytemperaturesatthesetimes\^'ereverylow(Figure4.10)

and would be expected to have caused a negative rate of increase of aPhids.

Alate aPhirls Figure 4.8 also shows the numbers of alate 4th instars for Topareal.Theiroccurrencescanbesummarizedasfollows: i)MaximumnumbersoccurredinthissubareainNovember,LgTS April, lt976, and September to December, 1976 with a few occurring in lvlay and July, 1977 generally times at which there were peaks in total aphid numbers (Figure 4'8) ' ii) Except in December, 1975 to February' L9'76' none occurred when aphid numbers were low (Figure 4'g) and when mean temper-

aÈures were either highest or lowest (Figure 4'10) i'e' May/ JuIy, it976. January,/March, 1977 and August/September' 1977' ontheotherhandnoneoccurredinDecember'1975whenaphid

numbers were high (Figure 4'8) and it is like1y that at this time high temperatures and tong photoperiods would have inhibited alate production despite the relatively high density of aphids' Inthelastperiodinwhichnoalatesoccurredi'e'september' Ig77, the farmer had sprayed with an aphicide' II2

Viral infected-Pl3t!9 Figure4.gshowstheincreasesanddecreasesinallviral infected plants and infecÈed rosettes in this subarea Top 1'

They can be summarized thust . -virus plants infected with v¡ere most abundant in June and

Deremtrer, 1976 and ilunêr Augqst and November' Iþ77 and peaks in infected rosettes occurred in fe¡ruàry, June and November

1976 and June and November, L977 '

In November, 1977 and r.¡rrr"ry, 1977 ùhere $tere no viral in- fecteci ¡rlants ín this area:. lltre apparent absence of infected plants in November, 1975 may have been due to a faulty indexing technique, while in February, 1977 there were no plants present in the area because the farmer had ploughed them in' Infectioninrosettes(Figure4.9)fgllowedpeaksofaphid incidence in this area (Figure 4'8) ' . The trend in nurnbers of plants lÀtith virus (Figure 4.9) are similar to those of the numbers of plants except in November'

1g75 and October, Ig77 (Figure 4.7), and a Log/Log plot of the two variables (Figurè 4.fl) indicates thataconstant proportion of favourable plants is infected with virus.thloügthout the year. InFigure4.gthechangesinnumbersofinfectedplantscan

be summarized thus:- r

i) Decreases In JuIy, 1976.and L977 decreases in viral infested plants (Figure 4.9) were probably due to the senescence of mature plantswhichþadgerminatedinsummerand'hadbecomeinfected

in Autumn. Each year these plants were partially replaced by 113

FTGURE 4.LT

Log number of favourable plants with virus against the log number of thistle ptants with virus.

Regression of log no. favourabLe plants with virus on log no. Plants with virus

Y = 0.227 + I.088X (¡=0.931) log numher of lovourobfc plonts wilh viru

o o o 0 o o

o o €o 3 o e o ã a ot o o o Ê o oa ! 6 o f - o o c o o a a o

q 115

rosettes infected in June (Figure 4'9)' HoÌ^tever' newly germinatedrosettesrarelyshowedinfectionintheperiod July to october in each of the years L976 and 1977 (Figure 4.9)intheseperiodsfewalatesv'eÏepresent(Figure4.8) and temperatures were low (Figure 4.to). Growth of thistles atlowtemperaturesisslow(Section5.l)andviraldevelop- ment (when present) is generally dependent on growth of the host plant (e.g. At1en, 1978) ' Hence it is likely that' evenifinfectedlrosetteswou]-dnotcontaindetectable virr¡s cluring the winter Period' In October, 1977 the decrease in nr¡nbers of viral infected plants (Figure 4.9) was probabty due to a marked fall in the nu¡ribers of alate aphids (cf . october, 1976) (eigure 4.8) after the farmer had sprayed with the aphicide in september. ii) rncreases Increases in numbers of viral infected plants can generally be explained. by the presence of alate 4th instar aphids in Èhe field,but the data of Figure 4.9 indicate that there were two period,s in which the number of infected plants increased, although there \^/ere no aLate 4th instars' cLl In March, L977 when the increase in viral infested plants

may be attributed to numbers of dispersing apterae. Boakye

(1973) showed that apterae dísperse from thistles by walking, effectively spreading LNYV in this way' This mode of dispersion is most marked when aphid populations are crowded.Aphidpopulations\ÁIerecrowdedinthissubareain 116

December, 1976 and after March, L977 (Figure 4.8). Crowd- ing of aphids also occurred in the adjacent subarea Southern roadside from February, 1977 until March, L977,

(trigure 4.I3) and aphids may þ¿1zs ¡er.rsd from this area.

b) rn November, 1977 when rosettes which had been dormant during the winter grew rapidly. Many of these may have been infected with virus by apterous aphids present in this subarea, Top area I in Septemberr 1977 (Figure 4.8) and alates from the adjacent sub-area Southern roadside from July to August, L9'77 (Figure 4.13).

The observations on this area suggest that the transmission of virus in a particular area is largely due to the alates produced in that area and adj.rc:ent areas rather than to alates flying in from further avtay. This supports

the observation by Stubbs, Guy and Stubbs (1963) and

Rogers & Baker (1970), that the incidence of viral infected lettuce is a function of the proximity to them of sowthistles, and is consistent with the general principles on gradients of viral infectíon into crops as discussed by Thresh (1976).

Data from the Southern Roadside subarea are shown in Figures

4.L2, 4.13 and 4"I4. This area was mowed twice by the farmer during the sampling programme:

i) ln May, 1976 when all of the thistles were destroyed (Figure 4.L2). Aphids and viral infected plants did not re-appear until September of that year (Figure 4.13 and 4.I4). IL7

FTGURE 4.T2

Log number of thistles and of flower heads per IOO m2 in su.bsampling

"So'uthern roadside" at different times of the year.

QoorrrQ log no. thíst1es

È"{ log no' flower heads

FTGURE 4.T3

Log number of H. Iactucae and their alate 4th instars per tOO m2 in subsampling area "southern roadside" at different times of the year.

oo....o log no. aphids o< log no. alate 4th instars

FIGURE 4.I4

Log number of all thistles with virus and of rosettes with virus per lOO m2 ir, subsanpling area "southern roadside,' at different times of the year.

o.....o log no. all thistles with virus

Iog no. rosettes with virus o ¡o cls o.' o

o o

M M J J s o J J A s o D J F A A s N D J A A

g o o v, .9 E E o .Ë o o) e ø o ll o E zt

j1 D F A M J J A o N J J A S o N D J F M A M J J A s o N

o o o. o o t o o 'rl \ o'o"o Í o tT

A J A o N JJ A s o J A J A s o N D J F

1975 1976 19TT

Months and yea;s expressed in accumulated day degrees above 4C 119

In September, 1976 there were new plants which were colonised by aphids (Figure 4.I2 and 4.13) and all Ì^tere infected with virus (Figure 4.L4).

ii) In October , Lg77 when a few plants L{ere left standing and the rosettes were not ploughed in (Figure 4.L2), there were no infected plants nor litere Èhere any aphíds (Figure 4.L3, 4.I4). However, after this mowing, there were plants and flower heads infested (Figure 4.L2) and infected plants by

November, L977 (Figure 4.13, 4-L4) due to the rapid growth

of tlre undamaged rosettes to semi-erect and erect stages.

Because the thistle population in the Southern roadside area is exclusively a new one from September, 1976 on, the marked fall in plant numbers in ,January, L977 which occurred in Top area I (Figure 4.7) did not occur here. Instead' after an initial reduction in numbers of flower heads in January' L977 there vras a recovery and the numbers of flower heads and plants remained fairly similar until March, L977 (FÍgure 4-L2ì.. During this period, from January to March, L977, the numbers of flower heads were generalty higher than the numbers of plants due to the maturation of the favourable plants. Figure 4.I2 shows that the majority of the plants which ger- rninated after the mo'wing in May, 1976 died off due to old age between June, 19'77 and September, 1977. Many replacement plants were destroyed when the area was again mol^ted in OcÈober,

L977 t so that there was a decline in the numbers of flower heads at this time. Figure 4.13 shows that the aphid nrunbers remained high except I2A

in January, 1977 when the temperatures were high (Figure 4.10) and october, L977 af.ter the farmer had sprayed the whole area with aphicide. tr'ígure 4.13 also shows the occurrence of alate 4th instars in the Southern foadside subarea and they can be sumrnarised as follows:

i) Maximum numbers occurred in April and september, L976

and in May and August, 1977 coinciding with peaks in aphid numbers (Figure 4.f3).

ii) None occurred in ifanuary,/Febtuaxy, 1976 and November, L976/January, 1977 when favourable plants Ì¡Iere present but aphid numbers were declining, probably due to the high temperatures (Figure 4.I0). iii)ttone occurred in January/AprlL, L977 when aphid numbers were high (Figure 4.13) and favourable plants present (Figure

4.L2) but the mean weekly temperatures were high (Figure 4.10)

and photoperiods long, inhibitíng alate production.

iv) None occurred in septenber,/November, 1977 when aphid

nurnlcers decreased with the decrease in food supply (Figure 4.I2) and subseguent aphicide treatrnenÈ in Septernber. The increases and decreases on the nu¡nbers of viral infected plants in thòs Southern roadside subarea are shown in Figure

4.I4¡ they can be summarised Èhus¡

i) Decreases cLl January,/February 1976 and December L976/March, 1977 there

\¡rere no marked changes in the nurnber of plants (Figure 4.I2)

but aphid numbers decreased (Figure 4.L3). There is insuf- L2T

ficient data to further explain the January,/February¡ L976 nu¡nbers. The decline in nurnbers of infected plants in the

December L976/March, L977 period can be further explaÍned by

examining the age structure of the plant poputation during ttris ti¡ne. Although the numbers of plants showed no mArked changes, plants which had germinated immediately after the

mowing of the subarea in May, 1976 and had subsequently be-

come favourable and infected by alate aphids present in sept-

ember 1976 (Figure 4.13) \¡rere starting to senesce and die by January, Ig77, as indicated by the decrease in the numbers of flower heads (Figure 4.L2) and nudbers of infected rosettes (Figure 4.L4) at that time. Íhe replacement plants would have

germinated when there \,{ere no alate aphids in this subarea

souÈhern roadside (Figure 4.r3) or the adjacent subarea Top area I (Figure 4.8). b) August,/October 1977 which coincides with a fall in the number of ptants and flower heads (Figure 4"L2) and falling aphid

numbers (Figure 4.13). In October, 1977 most of the remaining senescing favourable planÈs were destroyed by mowingr leaving no viral infected ptants in this subarea. ii) Increases &, August/December, 1976 where there is an increase of favourable ptants (Figure 4.L2) and an increase in the nunbers of aphids and alate 4th instars (Figure 4-f3). sen- b ) March,/June, 1977 when new favourable plants replaced escing plants (Figure 4.L21 and there were high aphid numbers present (Figure 4.I3). Although there Idere no alates in this L22

area until May, :-977 (Fj-gure 4.13) 4th instar alates were present in the adjacent subarea, Top area I in March of that year (Figure 4.8). As described previously (section 4.I.3(c)) apterous aphids walking off aged plants could also

have spread virus to the new plants.

c) ocÈober/November, Ig77 when there t{as an increase in that

nu*r^e.î of flower heads (Figure 4.L2) and an increase in the

numbers of aphids (Figure 4.13). Some of the rosettes infected during tl.e peak in alate numbers in August, L977 would have grown andproduced detectable virus by this time (Section 4.1.3(c)). Figure 4.14 also shows that as with subarea Top I (Figure 4.g and 4.9) the peaks in infected rosettes which occur in

November, 1976 and June, 1977 foLLow peaks in the incidence of alate 4th instars j.n that area (Figure 4.13). No detecÈ- able virus occurs in rosettes when there are no alaÈes in the area (Figure 4.13) or when temperatures are low (Figure 4.r0).

4.L .4 Discussion The 30 successive monthly samples were broken on only one occasion when collection of field data was interrupted for one month (March, Lg76) as a result of a motor accident to the researcher'

They represent a field situation considered typical of lettuce growing areas around Adelaide. Four facts important in understanding the spread óf LD{YV come from this study' i) There are always plants favourable Èo aphids associated with recently cleared land, but because the age distribution of plants L23

withinthepopulatior¡changescontinually'thereareconsequenÈ fluctuations in the nr¡nber of flower heads and the nunber of plants infested with aPhids' de- ii) ttre numbers of aphids in the field are variable, being plants pendent on the temperature, the nu¡nbers of favourable times when and the numbers of flower heads' As expected' the the highest nr:¡nbers of aphids occur (october' 1975 and 1976 flower and June, Lg77), precede the highest numbers of infested heads, and nu¡nbers of ínfested flower heads decrease as aphid numbersfall.Aphiclswerefoundinsmallnurnbersonnon-favour- ableplantswhenaphidinfestationl¡{ashigh.Theaphidcolonies onthenon-favourableplants$'ereinvariablysmall;ineleven out of twenty one cases only one aphid was found' seven out of twenty were one between two and six aphidsr and on only three occasions theremorethansixaphidsonthistlesattherosettestage. iii) Alate formation j-s primarily dependent on the presence of

Iarge numbers of aphids in the population but can be inhibited by high temPeratures and long photoperiods' These findings are consistent with the general principles outlined and díscussed in Section 2.2.4- RandlesandCrowley(1970)discoveredthatmostH.Iactucae weretrappedwhenmeanweeklytemperaÈureswereintherange in 60-70 degrees F(J5-22 degrees C) ' The presence of alates the field during this study confirms their conclusion' iv) presence of viral ínfected plants is dependent mainly on the

number of favourable ptants and on the rnobility of aphids' Thuswhenalatesarepresent'orwhenlargenu¡nbersofaphids L24

infest senescíng favourable plants, the nurnber of viral infected plants íncrea5es. At these times snall nr¡mbers of aphids were

found on rosettes, and some rosettes became inf.ected t¿ith vírus. llhroughout the whole sanpling period it was obvious that predators pl-ayed an insignificant role. No coccinellids l{ere seen and only 27 brown lacewings (Micromus tasmaniae lValker) and 3 syrphids were found. fhe interaction of the aphid, the Plant and the virus are discussed furttrer ín Section D. L25

4.2 AGE STRUCTURE OF APHID POPULATIONS 4.2.I Introduction An estimate ofreproductive activity in aphids has often been used in an attempt to predict Íncreases and decreases in nurnbers; to ex'

plain why changes in numbers occur; to analyse the effects of

parasites, or to identify biotypes (Dixon, 1963; Frazer, L972ai Elliott, L973). Estimates of reproductive actívity of aphid poPu-

lations have been made in the field (Dixon, L97O¡ 1975), in the laboratory under constant conditions and subsequently generalised to

field conrli t-ions (Perrin, L976), and by application of census data

using Hughes method (Hughes, L962, L963, L972). Many computer simulation models of aphid populations have been devised using

Hughes concepts (Kennedy, Booth and Kershaw, 1959; Hughes and e Gilbert, 1968; Gilbert and Hughes, L97I¡ Carter et al, L978). Tt¡e accuracy of the Hughes (f962) method for the estimation of

population growth parameters such as potential rate of increase and

mortality rate, is dependent on the population having a stable age distribution i.e. the population must be reproducing at a con-

stant rate. Although the Hughes method has been recommended for

use in populaÈion studies of Myzus persicae (Sulz) for the Inter-

national Biological Programme (Blackman, L976¡ Mackauer and Way,

L976) it has been shown to be inaccurate when the reproductive rate of a population changes with time (Carter g! aI, 1978). To determine, for H. lactucae whether or not population parameÈers

calculated by the Hughes method would be good estimates on which to

build a model of the population dynamics of the aphid' field popu-

lations were sampled and their age structure tested for goodness of L26

fit to a stable age distríbution.

4.2.2 Methods

The aphids collected during the subsampling described in Section

4.I.2 r'rere sorted into their four separate instars, alate ny,mphs and alate and apterous adults. 1lt¡e different stages could be identified on the basís of the number of antennal segments and on the size of the cauda (OrTake, 1958; Carver, personal co¡mnunication). First

instar nlmphs have 3 antennal segments; the second have 4; the third and fourth have 5; and adults, 6. The cauda of the 3rd instars are wider at Lire base than they are long whilst those of Èhe 4Èh instars are as long as their basal width.

The potential rate of increase (.À ) calculated for aphids i-n """ the whole area and for each of the two subareas on each sampling date using the formula

l, No. aphids & 2 potential increase rate (e in instars I No. aphids in instars 2 & 3 (Hughes, L962)

The expected numbers of the first 3 instars were then calculated using Hughes (1962) formula

expected no. of 3rd Sum no. ids in instars I 2e3 (term instars l) ("À) 3 - r

lrlhere . 1; eÀ - l and (eÀ) ¡ - l were repraced by 1- .À "À "rrd I - ("À) g respectively. The expected number of 2nd and lst instars were obtained by multiplying term I by eÀ and (eÀ)2. 2 * the expected to the observed series were calcu- ,-U, for the fit of lated and Èhe results are shown in Table 4.1. r27

TABLE 4.I l1tre nr¡mber of H. Iactucae sampled, their potential rate of increase, and

*,r_U, for goodness of fit of expected nurnbers to observed numbers of aphids in instars I - 3. * DenoÈes a signifícant value at P = 0.05.

No. of Whole No. of Top No. of S Roadside date aphids area aphids area aphids counted counted I counted

À À À 2 e x2 e x2 e X

3 5.75 L402 r.49 0. 87 .).l1 30 5.7s L.44 L.12 6 6.75 1520 1.88 27.77* 20 6.75 692 r.54 5.7 4r, 233 L.52 9.8* 6 7.75 76 1.55 2.20 I . 8.75 126 L.77 0.06 29 L.27 6.4t 5 . 9.75 595 1.83 I0.88* 3 . 10. 75 958 r.84 0.93 6 .11. 75 551 L.72 7.Ogt 4L6 I .57 L2.2t t6 .L2.75 34 o.82 o.74 250 .77 o.32 7 L-76 89 1.57 r. 48 12 I .81 0.98 2A L.76 122 r. 73 2.22 96r .76 4.93 26 I 67 0.09 27. 2.76 106 2.06 0. I3 106 2 .09 0. 14 I T 00 23. 4.76 2522 2.05 13.39* 2L9L 2 .02 2.7 4 399 2 .07 26.761, 2L. 5.76 185 3.05 0 652 .2r 8. 36* L20 3 .-,, L.O2 18. 6.76 5I L.20 3.6s -0 18. 7.76 L27 6.75 2.29 r27 6 .72 o.79 r3. 8.76 L25 3. t3 0 982 .33 0. 89 t5 9.76 2888 3.15 4.43t 2667 3 .44 3. 50 ,,, I .39 0.55 t3 . 10. 76 I9 2.22 o.62 976 2 .06 0.03 L44 4 .48 7.75t L2 . 11. 76 878 1.45 4.00* 699 I .49 4.94* r79 I .31 0.03 * t0 .L2.76 | 26L7 o.97 L3.29* 2508 0 .98 44.2 109 0 .81 0.90 L4. I .77 2 1.00 0, 0 11. 2 .77 r08 0.96 3.L2 108 0 96 3.L2 11. 3 .77 25L r.44 0 32 5 .05 L.20 2¡.9 I 23 0.59 15. 4 .7'1 89 2.LL 0 79 1.85 0. 38 10 0 t7 5 .77 4669 1.06 26. 38* 86 L.47 o.75 4583 0 95 57 .97* I4 6 77 500 L.L2 5.87* 236 L.29 36.5 * 264 0 94 12.35 15 7 77 277 1. 33 3.46 L4 o.29 0 263 I 39 3 .29 28 8 77 82 2.O5 0.18 82 2 o4 0 .18 15 9 77 50 o.29 o_rn 43 o.29 o.67 7 0 25 0 L4.LO .77 0 0 0 0 L7.l-l-.71 4 0 4 I 00 L28

Table 4.1 shows that for the whole area and for each of the subareas there were many sampling occasions on which the age distribution of nlzmphs in ttre population differed significantly from that of a stable age distribution, and that at no time during the 30 months of sampling was a stable age distribution achieved J-ong enough to enable calculation of accurate population parameters useful in the construction of a population model for H. lactucae. The table also shows that for the whole area, on most of the occasions on which the age structure of the population differed from a stable-age distribution, l"j:re nu¡nbers of aphids were highest, i.e. when the population was probably increasing or d.ecreasing rapidly. Such was the case in ilune, September and November, L975¡ April, September, Nov- ember and December, 1976 and May and June, L977. A similar pattern, although not as obviousr cárn be seen in the data for each of the subareas. It is not surprising that a stable age distribution, and high or low aphid populatíons do not always occur concurrently in the whole area and in the different subareas. The age structure of the plants

(due to mowing and ploughing at different times), and the use of aphicide at different times in the subareas (Section 4.L), would have resulted in aphid populations of quite different densities and age structures on each sampling occasion, regardless of climate

determinants "

Ttre data of Table 4.I suggested that there was a relationshÍp between the value of X2 and the number of observed aphids. To examine this relationship the number of aphids present \^/ere expressed, for the whole area and each of the subareas, as a mean number per infested flower head on each sampting date. This way of presenting 129

the aphid nr¡¡nbers was the best estimate for the data available on the size and density of the aphid populations in the different

areas. Linear regression lines were then calculated for ¡12 on the log of the mean nu¡riber of aphids per infested flower head, the null hypothesis being that the slope of tJ:e regression is zeto.

TABLE 4.2

Línear regression of the values of X'rUf in Table 4.I on the log mean number of aphids per infested flower head (R = correlation coefficient).

Who1e area Y = 1.931 - 0.005 X R = -0.043

Top area 1 Y = 1.801 - 0.011 X R = -0.244

Southern roadside Y = 1.496 + 0.012 X R = 0.511

The equations for the linear regression lines are shown in Table

4.2 and, show that none of the slopes of the lines were significantty

different from zero, indicating that the mean nu¡nber of aphids per infested flower head was not related to the magnitude of the deviation (as measured by X2) of the aphid populations from a stable age distribution.

Because of the short period of time available, and the variabitity of such factors as reproductive rate and age specific mortalities, it must be virtually impossible for an aphid population in the field to achieve stabiliÈy (Carter et al, 1978). A stable instar distri-

bution is not achieved when an aphid population is either increasing

rapidly, and is therefore a population dominated by young nymphs,

or when it is decreasing rapidly and is therefore dominated by 130

FIGURE 4. 15

head and the Log of the mean number of aphids per infested flower of the year' median value of the aphid population at different times

o"""'o mean no' aphids'/infested flower head o.< median value of aphid population

(a) !,Ihole area

(b) ToP Area I

10 3

10 o o o o o < o 102 o o o o d o.. o o o o

o o o

D F M A M J A o N J A o N F M A M A s o N ot

ç ,Fo to' !) g ct E o Ito è .9 p to3 .Ê, E (!cl .Ë o o :.". 2 o) o o () 3 10 o õ (,2, o lt o o O.O t O¡ ç t .o !.g z r¡, 10 o o

o å o o I D J M A M A o N J A s o N J M A M J A s o N

105 4

lo'

lo" /' 2 o o r' o' t=. o b o o o o

o o.. O o N F M A M J J A s o N s o N J F M A M J J A o

1975 1976 19Tl

Months and years expr€ssed in accumulated daY degrees above 4oc L32

adults and old ntrmphs. In an attempt to find a relationship between the age-structure of the population of H. lactucae and its size, on each sampling occasíon, Manly's (L976) method of estimating the median value of a population was used to calculate the median value of each aphid populatíon in the whole area and in each of the subareas (Top area I and Southern roadside) . lttre median value hlas then graphed against the log of the mean number of aphids per infested flower head. The plots are sho\^ln in Figure 4.I5. As discussed above, the age structure of the aphiil populations was diffei:e¡rt between the two subareas on each of many sampling occasions due to the horticultural practices of the falster and the effect that mowing and spraying had on the host thistles and aphid populations. Hence the possible relatíonships between Èhe median value of the aphid population calculated from data pooled from aII the subareas is likety to be masked. None-the-less Figure

4.15(a) shows that, for the whole area i) the median value of the aphid population decreased at the

same time as the log of the mean nunber of aphids per infested flower head increased in December, 1975 to January, 1976i ifune to JuIy, L976¡ February to March, 1977 and June to August, 1977. ii) the median value of the aphid population increased with a

decrease in the log of the mean number of aphids per infested

flower head in July, 1976 to August, L976¡ october, L976 Eo

December, L976 and August, 1977 to September, 1977.

The data for the subarea Top area I represenÈed in 4.I5(b), however, shows that, except for November to December, 1975, January

197'7 in the median to February, 1976 and July to August, ' increases 133

age of the aphid population coincided with decreases in the lo9

of the mean nurnber of aphids per infested, flower headr and decreases occurred when the population density was increasing' Figure 4.15(c) shows that, for the subarea "southern roadside", as in ,,Top area 1", there hlas a sinilar pattern of increases and

decreases in the median age of the aphids as the population densities changed.. Exceptions to this pattern for this subarea occurred in April - May, 1976 and February - Aprit, L977. The relationship between the median age of the aphid population and the log of the mean

number of aphids per infested flower head for each of the whole area and the two subareas may be seen by fitting the data to regression lines. The equations for these lines are shown in Table 4.3.

TABLE 4.3 Linear regression lines of the median value of the aphid populations on the Ìog rnean number of aphids per infested flower head where x = median value, Y = log no. aphids/infested flower head and R = correlation coefficient - for the whole area' top area I and southern roadside.

Whole area Y = 2.425 - 0.312 X R = -0.257

Top area 1 Y = 2.398 - 0.358 X R = -0.386

Southern roadside y = 1.476 + 0.018 x ft = 0.023

The regression lines suggest that for the whole area and for

Top area I there Ìdas a low negative correlation bet\'reen the median age of the aphids and the population densiÈy. Hol^tever, since the L34

stope of the line and the correlation coefficient for the data for the southern roadside subarea were cl0se to zeto there was no relationship between the two variables tested in this subarea' ThatthereisnotagoodcorrelaÈionbetweenthemedianageof aphids and the mean nu¡ribers on each flower head is noÈ surprising since the method of sampling in the field involved counting aphids oneachflowerhead,notoneachbud.AsdiscussedinSection 2.L.I each flower head may have from 2 - 8 buds and the distribution of aphids could be such that, for an infested flower head, atl the aphids could be crowded onto one bud thus forming a large,

young population, whilst the same number of aphids on another

head could be spread over many buds and could represent the ageing

remnants of one or several Iarger colonieS. If the use of median ages is to be of any value in population studies, more information

is needed than was obtained in the samples of this study. 4.2.3 Discussion

"The theoretical study of population dynamics has greatly clarified the understanding of the interactions of population processes, but more long term field studies are still required"' (Dixon, L977)' carter et al (1978) report that severaJ- aphid workers (Milne, I97L¡ Rendetl, Lg73i Perrin, l1g74l have found that, even by Hughes defini- tion their aphid populations did not achieve a stable instar distribution. The field data of Hughes (L962, 1963) also suggest that aphid populations may seldom achieve a stable instar distributíon

and the test used by Hughes is not sensitive enough (carter 4' Ì978). The relatively slow and predictable influences of crowding, dispersal and predation on population numbers and on instar dis- 135

tribution may be confounded in the field by factors such as horticultural practices and rainfall which may very rapidly change the aphid population. Maelzer (L977',) found that rainfall could cause nearly lOOt mortatity of adults and older nymphs in

Macrosiphum rosae (L). Thus after rain there was a small very young population which would show no increase in numbers until the nymphs matured, and then the population consisted almost entirely of newly moulted adults. For aphids such as M. rosae and H. lactucae the age structure of the poputation changes continually because of horticultural practices and age specific mortality, caused by variables such as rainfall and quality of the buds on which they live. During this study particularly, the use of destructive sampling methods would also have had an effect on the age structure. As a result the population dynamics of H. Iactucae cannot be analysed using the Hughes (L962, 1963) method, and another method must be devised. The use of themedian age of a population may have merit as an index in a new model for the calculation of population parameters if a relationship between the median age and the population density (and hence reproductive capacity) can be established- 136

SECTION D

THE INTERACTION OF THE APHID AND THE PLANT

BUDS PRODUCED 5.r GRO!{TH OF TTIE THISTLE - STEM ELONGATION & NT]MBER OF 5.r.1 Introduction Thepossibleoriginsandspreadofthesowthistle,Sonchus oleraceus Lrits taxonomy, occurrence, habitat' growth habit and ftoral biology are well documented, (Bentham, 1866; Dorph-PeteIson Boulos' 1924¡ Buckli, 1936; Gill, 1938; Lewin, 1948; Black ' 1957; 1960, 1961) and have been summarised earlier (section I.2). unlike manyeconomiccropplants,thegrowthofj-ndividualplantsof S. oleraceus under different temperatures and photoperiods has not been studied. Temperature and photoperiod are important in the

growth and migration of aphidsi so to better understand the interaction of H. lactucae and its host plant, experiments were done with s. oleraceus plants to fínd how they grow at each of 3 temperatures and each of 2 daYlengths.

5.L.2 Methods seeds of s. oleraceus from the field were germinated and the seedlíngs were transplanted to IOcm plastic pots containing urlc mix - as in the aphid experiments (section 2.2.2',) and the plants were placed in plant growth cabinets at 150 lux and at each of 6 combinations of 15, 20 and 25o c by 12 hours photoperiod (short day) or 16 hours photoperiod (long day). There were between 20 and 37 plants in each "treatment". The plants were watered daily and records were kept every 7-8 days of the number of leaves, sLetn elongation, number of flower heads and number of flowers for each plant. r37

FIGURE 5.1

Mean accumulative stem length of S. oleraceus against a physiological time scale above a threshold of 4oc.

o a-< 15 c o- -o zooc À.....4 25oc

(a) 12 hours PhotoPeriod

(b) 16 hours PhotoPeriod 3P

o 500 looo 1500 1@o ^ 70 E E G o Èa E o 60 o E tÐ E E a¡ E 50 !ó E Ð ÐE

¡lo t I I I

{,,1,

o 500 1000 150() 2o{00 Iemperotule ln doy degrees > 4oc L40

5.1.3 Results a) Stem el tion

The data for each of tl¡e cumulative means for stem elongation

and for the nu¡nber of flowers have been plotted against' the Èemp- erature converted to day degrees above a threshold of 4oC - the threshold temperature for development of H. IacÈucae. This has been done so that the effect of the exPerimental conditions on the growth of the plant in timer câr be seen in the context of

the development of an aphid population.

The clata for mear¡ stem elongation at each of the 3 temperatures

and each of the 2 photoperiods are shown in Figure 5'1' As Figure 5.1(a) illustrates, at the short day length, the means

for sÈem elongation of Sonchus at the end of the experiment were

almost the same at 20 and 25oC; However' the mean was significantly ô Iess at 15uC (t66 = 4.333; P < O.0Ol). on a physiological time scale the prants grown at l5oc reach their maximum height much sooner than do those plants gror¡ì/n at 20 or 25oc. (Figure S.l(a)).

For Sonchus grown under long day light regime (Figure 5'1(b)) there was no significant difference in the means for stem elongation at 20o and 25oc (tge = 1.413; P > o'10), and arthough the long day Èreatment was terminated before the plants growing at

15oC reached their maximum height, the data ill-ustrated in Figure 5.1(b) suggest that the mean for stem elongation at I5oC

may not have differed significantly from those means for plants

grown at 20 or 25oC if measurements had been continued. It ís of interest to note that when the weekly measurement of stem elongation was terminated (after 127 days) in 3 out of the 20 L4T

plants at 15oC no stem elongation had occurred. Although the

plants at I5oC were still growing, a comparison of means for

stem elongation between plants çtrown at short and long daylengttr

at each of 15, 20 and 25oC show that the plants grown at long daylength were significanÈty taller (15oC; t55 = 3.456; P < O.Ol; 2ooc¡ t49 = 4.062; P < o.oOtz 25oc¡ t4I = 3.742; P < o.Ool). Because the stem elongation of each group of plants grown at

Iong daylength was significantly greater than for those gro$rn at short daylength, daylength was more important than temperature in determining the height of the plants. Hoh¡ever, the smallest

plants were those grown at 15oC and short daylength. The conclusion from these data on stem elongatÍon confirm

field observations that in the winter months when daylengths are short and temperatures Iow, thistles are smaller than during the spring, sunmer and autumn. UnforÈunately, this experiment was terminated before the death of all plants growing at long day conditions so that comparisons between the longevities of plants under tl¡e different treatments could not be made. However, at

each of 20 and 25oC Sonchus reached its maximum height at each

daylength in approximately equat Èimes (expressed as Do > 4oC) so obviously sowthistles grow more slowly at short daylengths - i.e. in the winter months in the fielcl. b) \s$þ9r-9!-Ilgyers-prgq.us9q-p9r-pIs!! The number of flowers produced by each plant at each of 15,

20 and 25oC at each day length with the means for each treatment are shown in Table 5.1 and have been illustrated in Figure 5.2.

Ttre data in Table 5.I show thatplantsgrown at 25oC produce the L42

FIGURE 5.2

Mean accumulative nudber of flowers produced by S. oleraceus

against a physiological time scale above a threshold of 4oC.

O€ 15oc o- -o - 200c À...... 4 - 25oc (a) 12 hours photoperiod

(b) 16 hours PhotoPeriod A¡onul¡led n¡mber el flowers per plonl

o q o o !o o o o o o

I o ) \o ') o o o o o o oá Eo Ê = ) o -ì t o o t qÈ o o o þ ) o ) Ê- o ) ) go o o i ) o Ð ì '.) ). v o ð ). .È o o o o ) ) o o ì o i t ) ) ) i ò o o¡ o o o o L44

TABLE 5.I

Number of flowers on thistle plants at two daylengths at each of

I5 o 2oo and 25oc

PIant Temperature Mean Replicate dayl Nunber 15 20 25

2 23 L4 11 9 19 8 4 20 159 7 L2 20 6 22 98 I 23 23 I 9 138 L4 l8 27 10 20 13 13 11 8 26 L2 I3 L49 I1 6 19 T4 t6 15 17 I 20 t1 16 I5 L99 18 I8 18 Short day I8 II 11 7 7 27 22 20 16 L78 6 I9 15 22 18 t4 10 I 16 31 24 18 13 10 13 20 11 26 L7 I8 L4 9 28 11 11 L2 9 30 9 10 11 I2 32 18 108 34 16 I1 36 T8 7 38 7 8.43

for 14. 35 10.12 18. 21 aÈure

2 20 13 11 5 L2 32 4 15 I T7 15 22 23 6 24 19 T2 22 22 L2 8 10 26 20 t7 L2 L7 10 2I o L7 23 I9 24 day L2 9 24 L7 9 13 t8 L4 34 24 10 15 2L 23 16 18 32 27 I 22 26 I8 23 15 L4 L7 23 23 20 35 20 22 I4 20 20 18. s8

Mean for 19.95 15.60 20.20 Èemperature

¡Mean for temperature lover both daylengths L6.32 L2.27 19.11

i r45

greatest nunber of flowers hthilst those at I5oC produce the least.

Twice as many flowers are produced by plants growing at the long photoperiod than by those growing at the short photoperiod.

In an effort Èo find a relationshþ between the total nu¡nber

of flowers produced by Sonchus and the treatments an analysis of variance was applied to the data (Appendix 5.1). The analysis

shows that the F ratios for temperature and for day length were significant at P = 0.01 indicating thaÈ the number of flowers

produced by Sonchus was dependenÈ on both the temperature and,

tength of day at which the plants \^rere grohln. The analysis also

shows thaÈ the F ratio for the interaction of temperature and day length was not significant (at P = 0'05)

5.I.4 Discussion

Growíng sowthistles under controlled conditions of light and temperature showed that the shortest plants with the least number of flowers h¡ere those gro\^tn at 15oC and short photoperiod whilst the tallest plants with the most flowers gre!ìr at 25oC and long photoperiod. This is consistent with the field observations of Lewin (1948).

He records that in the British Isles S. oleraceus requires good illumination for full growth, and tends to produce a rosette form under short day conditions. Flower stalks only appeared as the day length increased. The rosette growth habit is seen as being important to the survival of plants which germinate in Autumn, because in the rosette form they can withstand temperatures several degrees below freezj-ng point without injury and produce exuberant growth and early flowers in the following spring (Lewin, 1948). L46

That sowthistles gro\it mo1'e slowly and have less flowers at short day lengths and low temperatures has important implicatíons on ttre favourability of the thistle to H. lactucae and on the availability of LNYV in Spring. Thistle plants which germinate in late autumn and which have possibly been infected with LD{YV would grovr very slowly through the winter months and effectively act as an overwintering phase for the virus. Vüheeler (1969) points out that plant pathogens grovr most rapidly when the host is most healthy and growing rapidly, and Allen (1978) showed that the growth of the virus like particles that cause Bunchy top disease of Bananas is dependent on the growth of the host plant. Hence, \,{hen the slow growing infected thistles mature and flower in spring, when day lengths and temperatures are increasing' not only do they become favourable to aphids (sect.ion 4.r"3) but they may also serve as a reservoir source of LNYV. Lewin (1948) found sowthistles in flower the year round ín the British Isles only r:n the Atlantic seaboard when the winters ulere milc1. However, in south Australia where winter days are longer and temperatures higher than in the British Isles Lhere are always t-histles in flower (Figure 4.4J. These thistles may be infested with E. lactucae (Fiqure 4.5) and will thus be important as foci for the spread of H. lactuçlle as aphid numbers build up with in- creasinq temperatuïes in sprlng (I¡igure 4-4; Figure 4'10) ' fn spring, the increasing photoperioo resuits in the appearance of more flower.' heads (I'j.grrr:e 4.4) to which the aphids may migrate.

Some of the plants whi,<:h had been favourable to H. lactucae during

har¡e been infected *rnut. Èhe winter months may also "r* L47

.gt so that aphids reared on these plants during the winter months would be viruliferous (Boakye, 1973) and on emigrating would thus be able to infect lettuces and uninfected thistles early in the season. 148

5.2 THE LENGTTI OF TIME A BUD RE¡4AINS FAVOURÀBLE AND THE POTENTIAT LIFE OF APHID COLONIES 5.2.I Introduction one of the most puzzling features of aphid ecology which has never been satisfactorily explained is why many aphid species are most abundant in the field at surprisingly Low mean temperatures. Thus, in N.2., Latrb (1961) found that maximal rate of increase of BrevÍcoryne brassicae L occurred at 22oC in the laboratory but at lo.zoc in the fierd. sirnirarly, in Adelaide, south Australia, peak

nu¡nbers of many aphid species occur in September-October when mean temperatures in the field are I5-I7oc, e.g. Macrosiphum rosae L

(Maelzer, L977) | Brevicoryne brassicae (McKenzie, 1977) , ToxopÈera citricídis Kirk (KL¡an , L979) and HyPeromyzus lactucae L (Figure 4.4). the rate of increase of all these species is maximal

at much higher temperatures in the laboratory, provided that aII

other environmental variables are optimal. So in the field some process other than the direct influence of temperature on the aphids, rate of increase constrains population growth later in the "season". The two processes most likely to be responsible are (i) a reduction in plant quality at higher temperatures, and'/or (ii) significantly increased voracity of predators at higher temperatures. There is considerable evidence for the second process e.g. Dunn, lg52¡ Frazer & Gilbert, 1976, Maelzer, 1978, but little evidence for the former, although plant quality is known to be-

come unfavourable - for many aphid species - as the "Season" pro- gresses, €.g. Johnson, L966¡ Dixon & Gl-en, L97I"

From a study of tr{acrosiphum rosae and the changing qualitY of L49 the rose'buds on which it feeds, Maelzer (1977) suggested a specl- fic hypothesis for the restriction of aphid population growth by plant quality at high tenperature. The study had indicated that

Èhe aphid had 3 peaks of abundance on irrigated roses that were correlated with 3 flushes of growth of the plant. The nr¡mbers of availabte buds per plant was the same in each flush of growth but each successive peak of aphid abundance was smaller than the previous one and was further characterized by an increase in (i) the percentage of buds infested by aphids, and (ii) the nr¡mber of small aphid colonies. To explain these changes the hyPothesis was proposed (Maelzer, :tg77), that at high temperatures the relative time for which a bud remains favourable for M. rosae is shorter than at low temperatures so that the chance of a

colony becoming intermediate or large is reduced' This chapter describes the testing of this hypothesís with the aphid H. lactucae (L) on sowthistle, S. oleraceus (t). The aPhid can only breed on species of sonchus, and like M. rosae on roses it feeds preferentially on the flower-buds of Sonchus oleraceus'

Tt¡e favourability of the buds for H. Iactucae have been categor-

ized in the same \¡ray as rose buds have been for M' rosae (section 2.I1. So the length of time for which a bud remains favourable was measured at 3 temperatures x 2 photoperiods, and the intrinsic rate of increase (r^) of the aphid was also

measured at each of the 3 temperatures (section 2.3). The poten- tiaÌ size of an aphid colony at each temperature was then calcu- Iated as a function of the length of time a bud remained favourable. 150

5 .2.2 Methods a) Iþ9-re!9-gI-llgreege-eI-E:-leglsgeg t the intrinsic rate of The methods for the estimation of m', increase have been described in Section 2.3.2.

b) of time for which a bud remains favourable seeds of s. oleraceus from the field were germinated and Èhe

seedlings were transplanted to IOcm plastic pots containing U/C mix - as in the aphid experiments (Section 2.2.21 - and the plants were placed. in plant growth cabinets at 150 lux and at each of the 6 combinations of 15, 20 and z5oc by 12 hours photoperiod (short day) and 16 hours photoperiod (long d"y). There were between 25 and 30 plants in each "treatment".

The planÈs were watered daily and records were kepÈ every

7-8 days of the numbers of buds of each stage of growth on each

plant. The sequence of growth of numerous flower heads had earlíer been followed on other plants (Section 2.I) and the stages of growth of the buds categorized as. illustrated in Eigure 2.2. 5.2.3 Results

a) Favourable buds

The aphid H. lactucee, feeds on buds of only stages J--4 (Section 2.L.2') so it was necessary to determine the duration of only these 4 stages of growth at each temperature and photo-

period. The ntunbers of the other stages of growÈh were accumu-

fated as Stage 5. Since every stage 1 bud survived to form a

stage 5 bud, the total number of buds produced by the plants in each treatment was obtained when alt the buds were in stage 5. 151

TABLE 5.2 llhe observed fequencies of buds of stages 1-5 of Sonchus oleraceus when plants were planted as seed and maintained in a þIant growtlr cabinet at 20oC and t2 hours photoperiod. Some of the details for analysis are also gíven; see text for explanation.

Days since Fl"ower stages For analysis planting h. + (r. I 2 34 5 h h l ) j+r h. +1 7 J l

34 0 0 0 0 0 57 I9 22 3 7 0 2 3 7 30 64 2I 29 10 2I 2 7 L4 7L 29 39 t0 4A 13 7 I 15 78 47 50 L2 51 4I I 7 I5 85 34 49 t3 58 76 7 6 13 92 39 30 t6 55 L25 6 t3 99 28 20 9 57 L94 7 9 16 108 I7 I8 3 18 249 9 6 15 1r6 4 4 3 I2 286 6 7 13 l-23 2 2 0 7 302 7 7 T4 r30 2 2 I 0 309 7 7 I4 137 0 0 0 2 3L2 7 7 I4 L44 0 0 0 0 3L4 7 7 T4

Area under curve (Ai) 1882 206 -l 589 240L Total area = 6935

Duration of Total duration of development 6.0 6"6 r.9 7.6 development of a bud (stages 1-4) = 22.L days L52

Tlpical frequencies of each stage of bud on each sampling aate aie given in Table 5.2 - for 2}oc, short day. V'lhen the freguencies of any one stage are plotted against time in days and tl¡e points joined up, the area under the curve can be determined, and the duration of development of thaÈ stage can be estimated (Southwood, L966¡ Ruesink & Kogan, L9751 as:

area under the curve (= total number of buds) number of buds enterÍng that stage

The area under the curve may be determined by numerous methods, One, the trapezoidal method, is used, by Manly (L976) to analyse similar data for stages in insect populations that have, however, survival rates < 1.0. As an illustration of the trapezoidal rule using Manlyrs terminology; for the ith stage:

I area = I +h f. 2 j=r j+1 j where the sample times are denoted as t1 r t2 trr, the corresponding frequency estimates for the stage by ft , f2... frrt

and, h. - t. - t. . The values for t., h. and h. are also ) i l-i J' I l-l given in Table 5.2, as are the estimated areas (4. ) and dura- tions of development (di). Since, in fact, we are really interested only in the total time for which a bud remains in stages 1-4 we can simply accu- mulate the frequencies of stages 1-4 and calculate the area 4 under the curve. The result is to obtain I o*. The observerJ i=I ' frequencies 1-4 for each treatment are given in Figure 5.3. 153

FIGURE 5.3

Sonchus oleraceus buds at temperatures other than 15oC converted to numbers at 15oC so that the respective areas can be compared. - 15oc - zo?c - 2soc

(a) Short Day

(b) Long Day 300

280

240 o. o

a a a o 200 a a a a a o a 160 a a' a a a a a a 120 a a a a a a a a o 80 a a

a a 40 a a ï a a a Ðo a E a o..^ o o -to o € 20 40 60 100 120 o 200 ¡Ð o 160 o ¡È Ð o - aa aa 140 aa aa aa aa to 120 ta a a

a oa a ao 100 a a a

a a a a o a 80 a a a a a a a a a a o 60 a a a a a a a a a a 40 a a a o a a o a a a a a 20 a o a^ to. a to. o o o 20 40 60 80 100 120 140 160 180 200

Doyr fron rowlng 155

The estimated area, the total nr¡¡nber of buds produced and the estimated duration of development (d) of stages l-4 are given for each treatrìent, in Table 5.3. The estimated dura- tions of development were checked by being used to simulate the changing numbers of buds; the "observed" numbers in the simulation were then used to obtain estimates, in turn, of the duration of development of stages 1-4. Ttre two estimates of tlre duration of development for each stage vlere almost identi- cal. The potential size of an aphid colony that can grow at each combination of temperature and photoperiod was then es-

timated by assuming - for each treatment - that on the first day a bud became favourable for aphids it was colonized by one fernale adult aphid and that the colony then grew exponentially

according to the formula: r_d Nd = No'e "t where

N =, size of aphid population at time zero ( = I aphid) o d = time in days for which a bud remains favourable for aphid increase, i.e. time for which the bud is in stages 1-4.

T = innate capacity for increase,/day, of the aphid- m

N size of aphid population after d days. d =

The estimated value of NU for each treatment is given in

Table 5.3. The null- hypothesis that NU is the same in each treatment must obviously be rejected - but it may be accepted

for the temperatures at J-ong day on1y. The values of NU for short day corroborate the hypothesis originally put forward' that the rate of maturation of the bud relative to the rate 156

of increase of the aphid is such that the aphid can produce Iarger colonies at low temperatures (15oC) than at higher temperatures (20-25oC) even though Èhe rate of increase of the aphid is higher at the higher temPeratures'

TABLE 5.3

Property of Plant Short day Long day or aphid 15oc zooc z'oc r5oc 2ooc zsoc

under curve of bud stages 1-4 57 6935 654r 76L3 4811 5310

of buds 473 3L4 464 37L 312 402

tion of develoPmen (d) for bud stages I- (in days) 35.2 22.r 14.1 20 .5 r5.4 L3 .2 r /day for HyperomYzus m-Iac tucae 0.17 0.23 0.26 o.r7 0.23 0.26

(r ) (d) .984 5.083 3.666 3.485 3.542 3.432 m

;PotentiaL size of icolony assuming expon- jential growth from I 397 16I 39 33 35 31 ,aphid | (t."t*o) 'Éo

5.2.4 Discussion

The data of Table 5.3 help to explain why the nurnbers of H. lactucae in South Australia are highesÈ in Septernber-October,

when field temperatures are 15-I7oC and photoperiod is L2-13 hrs/ day, rather than in November-January when field temperaÈures are

much higher and photoperiod is 14-]!6 hrs/day. fndeed, predators are relatively rare on sonchus plants and the nuribers of aphids are largely dependent on the availability of suitable buds (Section 4.r). L57

It has yet to be shown that thehostplants of other aphid species which sirnilarly have peak numbers in SepÈenber-October in southern Australia, respond to temperature and photoperiod in the same way that Sonchus oleraceus does. 158

OF LETTUCE NECROTIC 5.3 HYPEROMYZUS I,ACTUCAE L. AS THE ONLY VECTOR SOUTH AUSTRALIA YELLOV'TS VIRUS IN LETTUCE ( LACTUCA SATIVA L.) IN

5.3.r Introduction H. lactucae is known to be a vector of LNYV (Stu¡Us & Grogan' 1g63; Randles & crovrley, L}TO) with another related aphid species shown to Hype romvzus carduellinus (Theobald) which has also been feed on S. oleraceus and to transmit LNyV (Randles & Carver, 197I) ' this latter aphid, because of its limited distribution' is pre-

sumed to be of minor importance (Randles & carver,.I9'1L)-

H. Iactucae is common on sowthistle but cannot survive on lettuce (Hil1e Ris Lambers, L94g) and has not been recorded feeding on lettuce in the field, however the ubiquitous Macrosiphum euphorbiae

ftromas is often found feeding and breeding on both thistles and lettuce.Thischapterpresentsnewevidenceollthepotentialfor transmission of the virus by H. Iactucae and M' euphorbiae' and reviews the status of the two aphid species as vectors of the virus in the field. TheconclusionthatH.lactucaeisthemainvectorofLNYVin Iettuce is based on both direct and indirect evidence'

(a) Direct Evidence Using l-aboratory colon-ies of aphids, Stubbs & Grogan (1963)

demonstrated that neither H. lactucae nor M' euphorbiae would

transmit LNYV when techníques to demonstrate non persistent virus transmission were used; but field strains of H. Iactucae were aJ¡Ie to transmit LNYV from sowthistle to lettuce and Iettuce to lettuce following a latent period in the aphid. M. euphorbiae did not transmit under similar conditions' 159

(b) fndirect Evidence

(i) Str:bbs, Guy and Stubbs (1963) observed that the incidence

of LNYV in a lettuce crop was reduced if the thistles within

4OO yards of the crop were destroyed. Similarly Rogers &

Baker (1970) showed that the rate of infection by LNYV in

lettuces was reduced from 758 to 68 following the eradication of thistles within 100-150 yards of the lettuce crops. However, field trials by Boakye (1973) suggesÈed that the long range dispersal of H. Iactucae alatae was important for infections within a crop.

(ii) Randles & Crowley (1970) observed thaÈ peaks in the in-

cidence of LNYV occurred in lettuce crops 4-5 weeks after peak

nrunbers of alate H. lactucae were trapped. Furthermore,

in November/December, 1966 no LNYV was deÈected in surveyed Iettuce crops even though there had been a peak of

M. euphorbiae but not of H. l-acÈucae in traps in September of that year. (iii)sehncken (1973), working with another persistent rhabdovirus, Sowthistle Yellow Vein Virus (SYVV) found that H. Iactucae vlas an efficient vector of this virus whilst

lul. euphorbiae v/as an erratic and inefficient vector despite evidence of viral muttiplication in M. euphorbiae.

Because LNYV and SYW share many properties in that they both infect sowthistle and leÈtuce and are transmitted by H. Iactucae (Francki & Randles , L97Or Peters , L97I), it was considered that the possibility that LNYV may be transmitted, albeit rarely, by M. euphorbiae should be investigated because 160

it may have an effect on the epidemiology of the virus and hence influence the direction this study may take.

ftre new evidence presented here is of 3 types" viz: (i) Further transmission studies with field collected

M. euphorbiae (ii) Further trap catches of both aphid species and the inci-

dence and density of both aphid populations on Èhe sow- tlristle, S. oleraceus, together with evidence of LIIYV in the lettuce crops and in the sowLhist-le. (iii) The lack of "clumping" of infected lettuces.

5.3.2 Further transmission' studies

lltre transmission of LNW by M. euphorbiae from sowthistle to sowthistle.

(a) Method

llvo experiments were carried out, one in late spring, the other in early autumn, times at which previous field sampling

had shown high incidence of LNYV in field thistles together with

high aphid nr¡nlcers.

(i) Late spring. 30 mixed age colonies of M . euphorbiae were collected from fiel-d thisÈIes and transferred to virus free young thistles in the glasshouse by breaking off infest-

ed flower heads with 6 to 20 aphids and placing ttrem on the

young thistles. Perspex cylinders capped with fine gauze

were taped Èo the thistle pots to confine the aphids. Tlvo

leaves were also collected from each fietd thistle sampled 161

and these were subsequently tested for virus by grinding

them in a few drops of iced phosphate buffer (O-lM, pH 8-6)

and mechanically inoculating each onto two 5 leaf stage Nicotiana glutinosa L. ptants (indexing). The a¡rhids were confined on the glasshouse thistles for I weeks after which each thistle was also indexed as described above. (ii) Early autumn. For this experiment 20 field thistles with both M. euphorbiae and H. lactucae on tJ:em were selected

and each of a group of. 2 Lo 6 adults of each species were col- Iected from the field and transfer:red by means of a brush to a different virus free seedling thistle and left for 8 weeks. Indexing of the field thistles and of the experimental thistles was carried out as before. (Section 5.3.3 (a) (i))

(b) Results

(i) Late spring. Seventeen out of 30 of the field thistles coloniesd by M. euphorbiae were determined, by indexing, to

contain LNYV but no virus was detected by indexing of the glasshouse thistles, even though 29 out of 30 of the aphid

coLonies became established on these plants. Since each glass-

house colony was started with an aïerage of 13 apterous aphids

at least 500 aphids had access to LNYV.

(ii) Early autumn. In this experiment 12 out of the 20 field pJ-ants were infected with LNYV. At the conclusion of the experiment 10 out of 20 glasshouse thistles inoculated. with H. Iactucae were infected but no infection was evident

in the glasshouse thisttes seeded with M. euphorbiae. The L62

data from both these experiments suggest that M- euphorbiae

is unabte to transmit LNYV from sowthístle to soÛthistle. As

sÈated earlier, LNYV is structurally similar to SYW which is rarely transmitted by M. euphorbiae although it readily in- vades, and survives in, that aphid (Behncken, 1973). Viral

transmission may be blocked by the outer meÍibrane of the salivary gland (Rochow, 1969) or by the inability of the salivary tissues to support virus multiplication or accumulation

(Shikata & Maramorosh, 1965; Granados et aI, L967). Therefore

if the membrane surrounding the salivary glands of M . euphorbiae

restricts the passage of SY\Ã/ as Behncken (1973) suggests,

the membrane also forms an absolute barrier to the passage of

LNYV so that the aphid is physically incapable of transmitting

LNYV.

5.3.3. Aphid Trap Catches, the numbers of aphilts on'thistles and incidence of LNYV in thistles

(a) Methods Trapping of aphids and a survey of sowthistles and their aphid populations l¡Iere carried out amidst, and adjacent to, lettuce crops at Highbury in the Adelaide foothills. For trapping, five tray traps were used, one on each side and one in the centre of

a rectangular patch of lettuce measuring approximately 100m x 60m.

The traps were cleared weekly between 20 August, 1976 and 31

L977 28 June, 1977 - December, L977, and again from 29 April ' to Numbers of alate H. lactucae and M. euphorbiae v¡ere recorded sep-

arately and the numbers of aII other aphid species were combined. A survey of the sowthistle population was made over 30 con- 163

secutive months (section 4.L.2) during which a subsample of 30 sowthistles was collected monthly from areas adjacent to the above lettuce patch. H. lactucae and M. euphorbiae individuals collected from each of the sowthistle plants !ùere counted and sorted into instars on:the basis of the number of antennal segments (ô rafe, 1958; Carver, personal communication). lltre fourth instars were then sorted into potential alatae or apterae.

Each thistle in the sub-samples of 30 was indexed for LlfllV and the incidence of leÈtuces infected with virus Ìdas scored by counting 10 groups of IO0 lettuces. (b) Results

The numbers of aphids on thistles and the incidence of virus in thistles vrere converted to numbers per lOO m2 of the sampling area. The numbers of aphids per trap the numbers of aphíds on thistles and the incidence of LNYV in both sowthistles and lettuce are shown in Table 5.4. The trap catches show that' except in 3 weeks (I5/LO/76, 29/4/77 and LO/5/77), Èhere \^¡ere more

M. euphorbiae than H. Iactucae trapped every week. They also show that the peak catches of the two aphid species occurred at the same time, viz., early September, 1976; mid November, 1976 and early May, L977. An increase in lettuces infected with virus occurred about 4 weeks after two of these peak catches: viz, early in october, 1976 and in June, 1977. Since the peak catches of both aphid species occurred at the same times, it was not possible to conclrrde that eíther one of the two species was responsible for the increase in LNYV in the lettuce crop. How- eveï, an examination of the numbers of the two aphid species on L64

TABLE 5.4

Date No. aphids No. aphids No. alate Number of Incidence per trap on thistles 4th instars thistles (8) of per tOO m2 on thistles with virus virus in per IOO m2 per 100 m2 lettuce H.I M.E H.I M.e H.l M.e

20. 8. 76 2 4 I9 864938650 48 <1 27. 8. 76 4 18 <1 3. 9. 76 6 3 28 5 <1 r0. 9. 76 L7 7 75 3 <1 L7.9. 76 3 I I 2 2852L 139 256L I 23 <1 24. 9. 76 3 6 5 0

29. 4.77 L4.3 7.3

*No lettuces were counted between 15.I0.76 and IO.12.71 165

sowthistles during the trapping periods reveals that there

were always very many more H. lactucae than M. euphorbiae on sowthistles.

Furthermore there were often large nunibers of alate 4th instar H. Iactucae on the thisÈIes but alate 4th instar

M. euphorbiae on thistles were rare. Indeed, during the 30

months of sampling, the largest number of alate 4th instar

M. euphorbiae recorded was 5 per I0O m2 whilst that of

H. lactucae was nearly 4,OOO per lOO m2 (Figure 4.5). Table

5.4 also shows that LNW was always prr,:sent in the sowthístles and that an increase in the incidence of the disease in the field lettuces was always preceded, by about 4 weeks, not only by large nu¡rbers of H. lactucae on thistles but also be relative-

ly large numbers of their alates on the thistles.

Since relatively large numbers of alate M. euphorbiae were caught in the traps but very few - especially alate 4th instars -

were found on thistles, the great majority of the M . euphorbiae population must be breed.ing on plants other than thistles (Cot-

tier, 1953; Daiber, 1963). And since the sowthistle is the main source of LNYV, only a very small proportion of the alate popu- Iation could have had access to the virus by breeding on virus-

infected S. oleraceus. The increase in incidence of diseased

lettuce after peak aphid trap catches must therefore have been due to the alate production of H. lactucae.

5.3.4 Distribution of infected lettuces within crops

(a) Methods Lettuce crops were inspected irregularly for the incidence of L66

LNYV over a 36 month perÍod. At each inspection the percentage occurrence and presence of groups of, infected lettuces were recorded. This was done by recordíng any adjacent infected plants in tO samples of I0 continuous groups of plants down

10 adjacent rows. (b) Results

Diseased plants generally occurred singly and apparently at random within the lettuce crops although edge effects were occasionalllr noticeable. On no sampling occasion was there any e.;iCence of "clumping" i"e. of groups of infected lettuces. Clurnping can be expected if the focus of infection came from within the lettuce crop, e.g. from viruliferous aphids breeding there.

Macrosiphum euphorbiae both feeds and breeds on lettuces whilst H. lactucae does not. Breeding colonies of M- euphorbiae on lettuce are small but some disperslon of apterae from such colonies would be expectecl. So if M. euphorbiae was a vector of LNYV, the virus should be spread to lettuces adjacent to an aphid infesÈed lettuce as apterous members of the colony disperse.

Clumps of infected plants would result. Ttre observation that no such clumping occurred during the 36 month observation period' - during which lettuces were cultivated continuously, is consistent with the hypothesis of M. euphorbiae not transmitting

LNSV within the l-ettuce crops. The scattered occurrence of diseased plants can be more readily exptained by the probing behaviour of H. lactucae. H. lactucae becomes increasingly pre- disposed to probe lettuce as the period of starvation before ]-67

probing increases, i"e. the threshold for probing behaviour decreases with increasing periods of starvation and increases again after probing has occurred (Boakye & Rändles, L974). Migrating H. Iactucae that land on a lettuce and probe are likely there-

fore to move some distance before landing and probing again' rather than to move to and probe adjacent lettuces. If these aphids were viruliferous, only scattered plants within the crop

would be infected. The evidence of the lack of clumping would

be more convincing, however, if the occurrence of diseased

plants had, been recorded and analysed according to the methods of converse et aI (1979) in their study on the local spread of the aphid borne mild yellow-edge virus in strawberries. 5.3.5 Conclusions

The new evidence presented here on the possibility that t4. euphorbiae acts as a vector of LNYV corroborates the earlier more tenuous evidence and clearly indicates that (i) M . euphorbiae did not transmit LNYV under the conditions of the experiments, and (ii) the trap catches of M. euphorbiae, its incidence on thistles, and its colonising patterns on lettuce are consistent with Èhe hypothesis that it has no role as a vector in the transmission of

LNYV. 168

DISCUSSIOI\T AND COTTCLUSTONS

The spread of LNCV depends on the movements of infectÍve H. Iactucae between sowthistles (S . oleraceus) - the príncipte host plant of the aphid and of the virus (Stubbs and Grogan, f963) - and ínto lettuce crops. No representatives of the alternative vector of LNYV,

Hyperomy zus carduellinus (Rand1es and Carver, 1971) were found during the field survey (Section 4.1) and trappíng progranrune (.Section 5.3). Although Macrosiphum euphorbíae was implicated as a vector because of its prese{ ot thistles and its prevalence in traps, and because of its ability to feed and breed on both sowthistle and lettuce, this aphid was shown to be incapable of transmitting LNYV (Section 5.3).

The sowthistle is an annual, but there is little doubt that in the Highbury (South Australia) market gardening area. the virus is acquired almost exclusively from infected sowthistles and not from ttre perennial alternative hosts, Sonchus hydrophi lus and Elnbergeria megalocarpa, both of which can be infected by the virus and support populations of H. lactucae (Randtes and Carver, 1971) but which do not occur in the area.

Ttre role, generally, of the perennials S- hydrophilus and

E. megalocarpa as reservoir plants for the virus is probably unimport- ant as is the transovarial transmission of LNYV in H. Iactucae recorded by Boakye and Randles (L974), f.or the reasons outlined below. (i) The results of the field survey (section 4.1) showed that the sowthistles occurred as a mixed age population throughout the year. At all times there were plants in flower and thus favourable to H. Iactucae and there were always many plants either infected with L69

virus and/or infested with aphids. Hordever, during the winter

months, although the Èotal nu¡nber of plants did not fall narkedly

there were fewer flower heads and aphids present.

(ii) Growth of the sowthistle under laboratory conditions showed

thaÈ S. oleraceus very slowly and has few flowers when grown

at short photoperiods and low temperatures - as in winter - compared with growth at longer photoperiods and higher temperatures (,Section 5.1). So in winter there are thistle plants with a few flower heads which can support overwintering populations of H. Iactucae

as well as rosette forms which are frost resistant (Lewin, 1948)

and which wiII provide many new flowers for aphids as daylengths and temperatures increase in spring.

(iii)IAany of the plants present during the winter were infected by viruliferous emigrating aphids present in late autrmn (Figure 4.5). These plants could effectively serve as a reservoir for the virus in one of two ways. Firstly, the aphids reared on infected plants during the winter would be capable of transmitting the virus

(Boakye and Randles, L974) and on emigrating would spread Èhe virus to new favourable plants. Secondly, the rapid growth of the rosette forms which occurs with increasing daylength and temperature in spring,would provide flowers and an immediate source of virus to newly developing aphid colonies.

Like most- aphid species emigration of H. lactucae from host plants is influenced by currently adverse condiÈions of the environment (e.g.

food shortage and. Iack of space) and an endogenously controlled mech- anism which results in the production of migrant alatae (.SecÈion 2.2). It is these migrant alates of H. lactucae which are mainly responsible l-70

for the spread of LNYV to sowthistles (section 4.1) and ínto lettuce crops (Randles and Crowley, L97O¡ Boakye, L973r. There are more flower heads in the field in late spring,/early sunìmer when daylengths are long and temperatures high (Figure 4.4) as corroborated by the laboratory experiments on thistle gnowttr (Section

5.1). However, the major peaks of aphid abundance occur several weeks earlier in late winter,/early spring when daylengths are shorter and temperatures lower. Although the intrinsíc rate of increase of the aphid increases with increases in temperature, flower buds at higher temperatures and Iong photoperiods mature at a greater relative rate than at lower temperatures and short photoperiods, so that theoretically the relative time that an aphid has to build up large populations is greater at lower temperatures and shorter photoperiods. This hypothesis explains why the peak numbers of H. lactucae occur in early spring (October ) when temperatures in the field are relatively low.

Boakye (1973) suggested that the control of LNYV in lettuce crops could be achieved in any of three ways namely: (i) the eradicaÈion of the source of the virus, i.e. the sowthistle; (ii) prevention of infection of lettuce by changes in cultural practices or by changing the areas of lettuce production, and (iii) the elimination or reduction of populations of H . lactucae. He proposed and critically discussed some interesting ideas which night prevent infection of the lettuce namely, the use of non-host barrier crops, aluminium foil and sites and times of planting to avoid large vector populations. Elimination of the vector or total eradication of the sowthistle !ì¡ere seen as impractical alternat,ives for control. Certainly the use of herbicides I7L or biological agents such as insects is unlikely to be successful in eradicating the sowthistle because of its widespread distribution, but elimination of the thistles in the vicinity of lettr¡ce crops would markedty reduce the all ímportant close range transmission of the disease (Strùbs et 41, 1963¡ Rogers and Baker, L97O¡ Thresh, 1975) -

Because H. lactucae is the only important vector of INYV, and because the critical times for crop infection are when aphid populations are high and are producing large numbers of alatae, i.e. in early spring and late autumn, some control of the aphid population at these times by a parasite specific to the aphid could provide effective control of the disease in lettuce. Such a parasite would only have to reduce the aphid density on the host plant below the density at which alates are induced to effectively reduce the incidence of LNYV. Stary and Schlinger (1967) list two polyphagous aphid parasites which occur in Far East Asia and which infest H. lactucae Ephedrus plagiator (Stary (1970) (Nees) and Praon orientale and Schlinger) ' whilst Stary lists a further t\^¡o polyphagous species with wider distribution; Praon volucre (Hatiday) and Lysiphlebus fabarum (Itlarshall) together with the host specific Aphidius sonchi (Marshall). For H. lactucae in South Australia a parasite must be effective in early spring and late autrnnn and yet be able to survive the hot summer, so parasites from a country such as California might be ideal. It seems then, that the next logi- cal step toward the control of LNYV in lettuces in South Australia is to import parasites; develop suitable rearing techniques for them, and to then study their effects on experimentaf aphid populations in the laboratory and in Èhe field. r72

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APPENDICES 19r

APPENDIX 2.L

Anatysis of variance of the sizes of aphid colonies started with 4 different densities of adult aphids at each of 3 temperatures.

Source of df Sum of Mean F variation squares square ratio

TotaI 34 1738.07

Temperatures 2 327.L2 163.56 3 .506*

Densities 3 2TL.2L 7A.40 r.509

Interaction 6 116.84 19.47 o.4L7

Residual 23 ro72.90 46.65

*Significant at 5t level. t92

APPENDIX 2.2

FIGURE 2.6 Regression of the log t number of aphids that walked off a bud after one generation from colonies started by each of' 2, 4' 6 and 9 adult H. Iactucae on the time in daYs. o- initial densitY 2 adults H initial densitY 4 adults o...... o initial densitY 6 adults A--A initial densitY 9 adults

Initial no. Regression Lines SEofb (a) l5oc of adults 2 Y = 1.14 + 0.075 X 0.07 4 Y = 0.07 + 0.095 X 0.14 6 Y = 0.79 + 0.099 X 0.09 9 Y = 0.62 + 0.117 X 0.06

oc (b) 20 2 Y = I.16 + 0.094 X 0. 16 4 Y = 0.98 + 0.099 X 0.03 6 Y = 0.78 + 0.120 X 0.05 9 Y = -0.L2 + 0.250 X 0.07

(c) 25oc 2 Y = 0.88 + 0.111 X 0.07 4 Y = -0.2O + 0.315 X 0. r3 6 Y = -0.35 + 0.397 X 0.30 9 Y = -0.22 + 0.369 x 0.34 Log % nymphs that walked off bud

o f9 o o 0 o o o 0 0 o o

o a o

J J- \ O'.a > Ì. 3 o \1 I q . a or'. ) ¿ "t fo "a cl o >\ o o > .'ì'¡.- ¡ "a ut o

E o ô 194

APPENDIX 2.3

Analysis of variance of tlte percentage of alate 4th instar aphidsr ËlP- terous 4ttr instar aphids and lst and 2nd instar aphids walking off a bud at 3 densities and 3 temperatures.

Source of df Sum of Mean F Variation squares square ratio

Total 80 91050.99 1138. t4

Temperatures 2 1012.53 506.27 o.465

Densities 2 3225.98 1612.99 r.482

Aphid types 2 483.92 24L.95 o.222

Densities x Aphid types 4 1147 .08 286.77 o.264

Temperatures x Densities 4 20546.86 5L36.72 4.739*

Temperatures x Aphid types 4 2663.24 665.81 o.612

Temperatures X Densities X Aphid types I 6l-.'7L 7 .7L 0.007

Error 57 62033 .09 1088. 30

* Significant at P = 0.05. 195

APPENDTX 2.4

FIGURE 2.7 cumulative number of progeny produced each day by colonies started by each of 2, 4, 6 and 9 adult H. lactucae, time in days. (¡--< initial density 2 adults o4 initial density 4 adults initial density 6 adults H initial density 9 adults

(a) t5oc

o (b) 20 c

o (c) 2 5 c Accumulated number of progeny

a I 3 o

q) ø

o ç

APPENDTX 2.5

Embryo nunber, fresh weight, mean tibia length, mean antennal length and mean lenghts of the 3rd antennal segments and the cornicles of newly moulted aplgreeq adult H. lactucae

No of Fresh Tibia Antennal No. of Fresh Tibia itntennal Mean lengtlt Mean cornicle Enbryos weight length length Embryos weight length length 3rd antennal Iength (nun) (ms) (mm) (mm) (*s) (mm) (mm) seg. (mm)

28 .6 30 1. 307 2.266 36 .696 L.42L 2.O74 25 .520 L -344 2-4I9 34 .700 r.459 2.573 25 .590 L.344 2.342 25 .496 1. 190 L.997 2I .620 1. 190 2.266 31 .610 L.344 2.227 29 .570 L"344 2.342 4L .660 L.42L 2.227 42 .770 L.498 2.4I9 34 .580 I.42L 2.LL2 29 .600 L.42I 2.304 32 .7I0 L.344 2-38L 32 .610 L.344 2.227 28 .954 t.344 2.304 26 .490 0.883 I.997 43 .920 1. 382 2.227 34 .602 L.267 2.150 35 .550 I "229 2. 304 26 .562 L.229 2.IL2 19 .468 r. 190 r.958 L2 .470 o.922 1.882 26 .6t2 L.L52 L.920 39 .975 L.459 2.304 4T .620 L.427 2.266 26 L'7 2 L.42I 2"O74 35 .542 r.306 2.304 33 .030 1.536 2.4L9 36 .672 L.344 2.L89 22 .434 t.229 L.997 27 .828 L.459 2.266 .576 .384 ts (o 33 .746 1.613 2.342 31 .514 L.344 2.LL2 .576 .346 { No. of Fresh Tibia Antennal No. of Fresh Tibia Antennal Mean length Mean cornicle (run) Elnbryos Vüeight length length Hnbryos Vûeight length length 3rd antennal length (*s) (run) (¡nm) (*s) (run) (mn) (nn)

35 .762 1. 382 2.227 35 .950 1.498 2.573 .69L .46L

16 .568 L.382 2.150 30 .754 L.229 2.227 .576 .46L 29 .352 1. II4 2.227 46 r.050 r.574 2.342 .576 .384 35 .560 1.306 I.958 24 .750 t. 306 2.266 .576 .307 32 .660 L-267 2.IA9 T7 .524 L.T52 r.920 .499 -346 38 .752 L.42L 2. 150 48 .990 r.498 2.496 .6L4 .46L 25 -532 0.883 r.767 4L I.060 1.613 2.304 .576 .46L 2I .576 r. 382 2.O35 47 1.380 L.344 2.3AL .6L4 .46L 40 1.095 L.498 2-38I 32 .658 I.306 2.38L .6L4 .384 30 1.080 r. 498 2.LLz 35 .845 t.42L 2 .381 .576 .422 24 .996 1. 382 2.It2 33 .462 1. 459 2.342 .576 .346 26 .514 o.922 L.997 35 -532 L.42L 2.L89 .538 .346 2L -434 L.229 2.O7 4 3I .644 r.459 2.LL2 .576 .346 29 .734 1.382 2.150 31 .472 I.190 2.TL2 .538 .]-92 .6L4 .384 31 "7L4 1. 382 2.LL2 32 .532 I.42L 2.r89 26 .604 L.L52 2.O7 4 47 .916 L.344 2.573 .653 .230 .6L4 .384 27 "454 L.T52 2.L89 23 .7L6 I. I9O 2.304 24 .760 L.459 2.304 35 .635 1.536 2.266 .538 .269

30 .608 1. 382 2.496 30 .852 r.498 2.496 .576 .422 19 .634 T.l.52 1.882 4L .728 r.267 2.342 .576 -46t H 29 -460 r. 190 2.266 25 .624 r. 382 2 .035 .499 .422 (o @ 27 .67 4 1.190 2. r50 32 .764 1. 382 2.458 .6L4 .384 30 .604 L.344 2.035 46 .924 1.382 2.342 .576 .422 Bnbryo Number, fresh weight, mean tibia length, mean antennal length and mean lengths of cornicle and 3rd antennal segment of newly moulted alate adult H. Iactucae

No. of Fresh Mean Mean No of Fresh Mean Mean Mean length Iv1ean cornicl-e Elnbryos weight tibia Antennal Ênbryos V[eight tibia Anterural 3rd antennal length (mm¡ (ms) length length (*s) length lengttr seg. (nun) (run) (nun) (rrn) (run)

5 .434 I.344 2.LL2 30 .6L4 1.536 2.726 .730 .422 8 .320 0 .960 r.843 37 .800 1.651 2. 381 .691 .422 I5 .410 L.382 2. I89 L6 .560 T.427 2.38I .6L4 .346 19 .538 L.267 2.227 23 .830 L.728 2.458 .653 .384 L4 .550 r.459 2.L50 25 .814 L.498 2.496 .6L4 .422 9 .404 L.229 2.L50 26 .772 1.613 2.496 .6l-4 .384 I8 .490 r.306 2.342 27 -794 1.459 2.650 .576 .384 16 .670 L.574 2.342 18 .868 1. 382 2.6TL .6L4 .346 L7 .598 L.42L 2.573 I3 .780 L.344 2.650 .653 .422 16 .502 1.536 2.496 19 -7A4 1.65I 2.650 .69I .422 18 .570 I.344 2.6LI 29 .764 I"65I 2.458 .6l-4 .422 22 .620 L-574 2.496 25 . 715 1.613 2.4L9 .6L4 .384 26 .602 l_.613 2.496 I5 .544 L.459 2.496 .576 .346 22 .626 1.498 2.458 32 .862 r.728 2.458 .69r -422

I5 .498 L.574 2.573 24 . s84 I. 306 2.381 .576 .307 24 .585 1.498 2.L89 25 .830 1.690 2.650 .69r .346 16 .420 1. II4 2.227 2L .796 I .651 2.458 .691 .384 ts \o \0 28 .5r8 L.498 2.573 22 .630 L.42L 2-4L9 .6L4 .346 No. of Fresh l'lean Mean No of Fresh Mean Mean Mean lengttt Mean cornicle (¡ut) Embryos !{ei9ht Tibia Antennal Enbryos Vùeight Tibia Antennal 3ril antennal length (ts) lengttt length (¡ns) length Iength seg. (sm) (¡run) (run) (nm) (wt)

15 .382 1 -267 2. 381 2L .540 L.427 2.t89 .614 .384 20 .400 L.229 2.34L 22 .730 1.536 2.342 -576 .384 L2 -476 L.229 I. 843 23 .780 I.728 2.227 .576 .384 L6 .410 L.459 2.L49 27 -740 t.728 2.650 .6s3 .384

22 .480 L.42L 2.L89 24 .554 t.459 2-342 .576 .346 t5 .444 L.267 2.LL2 18 .758 1.613 2.688 .69L -422 T4 .460 1.190 L.767 19 .664 L.42L 2.573 .653 .384 29 .824 I.651 2.573 .69I .422 22 .420 I.613 2.342 .653 .384

ot\) O moulted Nr¡¡nber of progeny, number of embryos with eyespots, total number of embryos, and fresh weight of newlY apterous adult H. lactucae Total progeny No. Progeny No. embryos Fresh Tota1 No. No. progeny No. embryos Fresh and embryos after 3 days with eyespots weight progeny and after 3 days with eYesPoÈs weight after 3 days after 3 days (ms) embryos after after 3 daYs (ms) 3 days

42 15 13 .672 2I 8 6 .366 10 8 0 .376 t5 5 7 .392 24 L2 9 .632 11 5 7 .233 t0 8 2 .366 l4 6 2 .262 24 9 0 .5L7 L7 4 I1 .357 24 9 I .302 10 5 2 .332 6 .446 I2 6 4 " 363 20 7 9 2 4 . r55 T2 1I 0 .450

23 11 6 .446 18 9 5 .428 L2 9 3 .275 5 0 5 .677 t5 6 6 .290 I6 I 3 .692 L7 7 4 .310 18 10 5 .666 L4 I 5 .400 23 7 7 .586 8 4 2 .190 22 10 4 .520 t2 6 5 .260 32 t4 7 .808 L2 7 3 .398 1t 7 I .338 II 6 5 .308 16 7 6 .430 18 9 4 .402 30 t2 7 -422

I 3 3 .216 7 4 2 .218 N o H Total progeny No. Progeny No. eurbryos Fresh Tota1 No. No. Progeny No. ernbryos Fresh and embryos after 3 days with eyespots weight progeny and after 3 daYs with eyespots weight after 3 days after 3 days ('ns) enbryos after after 3 days (*g) 3 days

l0 5 3 .22a I5 8 6 .308 I6 I 0 .352 18 7 7 .579

15 I1 1 .405 18 6 I .583 I 4 4 .342 13 7 5 .496 23 9 4 .502 31 10 11 .900 13 9 2 .432 2T 9 7 .577 15 10 0 .39r 24 5 .346 L6 8 4 .392 16 4 .548 18 5 6 .420 T9 4 .54s 24 I 4 .526 15 I .330 23 10 6 .62A L4 0 -440 L2 7 2 .287 9 3 .253 .7 L4 6 4 .262 l5 .346 I5 7 5 .312 15 6 .576 t3 6 3 -279 24 7 .544 L4 6 4 .238 T4 4 .484 I8 8 3 .440

l\) O t\) weight rof newly Number of progeny, number of embryos with eye spots, total number of embryos and fresh moulted êlate adul-t H. lactucae-

No. embrYos Fresh weight Total no. No. ProgenY No. embryos Fresh Total No. No. ProgenY daYs with eyesPots (*g) progeny & after 3 daYs wiÈh eyespots weight progeny & after 3 daYs embryos after 3 days (mS) embrYos after 3 after 3 daYs after 3 daYs

0 .398 7 4 2 -426 9 .504 L2 7 5 .680 I5 7 .502 1 4 3 .334 L4 4 .552 6 6 0 .551 11 4 .406 II 2 6 .382 12 3 .258 9 5 2 .376 6 I .356 9 6 3 .408 I 0 .462 I 7 I .494 7 0 .430 7 6 I .366 9 I .404 11 I 3 .370 I I .394 L2 9 I .639 9 2 .4L2 9 7 2 . 3r0 10 5 .334 I6 10 4 .546 9 3 .650 II 2 .504 10 4 19 2 .618 IO 6 .4L6 .490 7 0 .350 I5 6 o .506 IO 2 .6l-2 2 oN .466 (, 7 0 .420 10 4 Total no. No. progeny No. embryos Fresh Total- No. No. progeny No. eurbryos Fresh weight progeny & after 3 days with eyespots weight progeny & after 3 days with eyespots ('os) enibryos after 3 days (*g) embryos after 3 days after 3 days after 3 days

9 2 .506 10 2 .6L2 7 0 .420 10 4 .466 6 0 .352 7 0 -484 7 0 .3l-2 L2 I .393 20 5 -622

ot\J È 205

APPENDIX 3.I

Mean nu¡nber of lesions produced by SE 3 strain of LNYV in each of the 4 paired treatments described in the text, showing standard deviations

of each mean and student t values between each pair of treatments'

DILUTION I:1 DILUTION I:4

Mean no. Mean no. SD EZA Inoculum Figure Iesionsr/L leaf SD tZe Lesions/L leaf

B 3.2 9.4 ro.76 1.2I1* 2-7 2.64 0.847*

D 6.9 6.92 2.O 1. 81 A 3.3 5.4 4.O7 2.370 '7.4 4.6L 2.LO3 c 2.4 2.75 4.3 3. 37 c 3.4 3.8 2.48 2.382 6.1 4.64 2.920

D 6.4 3.89 2.3 r.98 A 3.5 4.4 4.27 2.658 6.5 4.84 L.268t

B 1r. 7 9.74 4.6 3.20

* not significant at P = 0.05. 206

APPENDIX 5.1

Analysis of varlance of npmber of flowers produced by thistle plants gro!ür at 12 hour and 16 hour day length at each of 3 tenpertures.

Source of Sun of ¡lean F Variation ¿tf sguares square ratío

Total 151 5207

Temperature 2 1r32 566 25. 38*

Day length I 785 785 35.20*

Interaction 2 29 14.5 0.65

Residual L46 326L 22.3

* Significant at P = 0.01.