STUDIES ON THE TAXONOMY AND BIOLOGY OF SS 03
POTATO CYST-NEMATODES Globodera spp. Mu-Ivey—a414—Staa4-1944.
JAVIER FRANCO PONCE
A THESIS PRESENTED FOR
THE DEGREE OF
DOCTOR OF PHILOSOPHY'
IN THE
FACULTY OF SCIENCE, UNIVERSITY OF LONDON
MARCH 1977
Imperial College Field Station, Department of Nematology, Ashurst Lodge, Rothamsted Experimental Station
Sunninghill, Berks. Harpenden, Herts. ABSTRACT
Measurements:of diagnostic characters of potato cyst-nematodes
Globodera rostochiensis and G. pallida can be greatly modified by both intrinsic and extrinsic factors. The characters least modified and therefore more suitable for identifying populations are stylet length and distance from head tip to excretory pore in second stage_ larvae; length of vulval fenestra and number of ridges between the anal pore and the vulval fenestra are the characters of cyst terminal areas least affected. Measurements of areas and perimeters of second stage larvae and males were not useful.
Colour changes in developing females must be used with certain restrictions for identification purposes because mixed populations can be misidentified.
Disc electrophoresis, scanning electron microscopy and mating tests showed only small differences between British and Peruvian populations belonging to the same species.
Effects of daylength, temperature and potato host plants on the biology of both species was investigated. Long days (16 hrs.) favoured development of both species by enhancing the development of potato plants. Temperatures below 18°C favour development of
G. pallida, and G. rostochiensis has its optimal development temperature above 18°C.
The potato variety Record was not as good a host for a British
G. pallida population as other varieties tested, as it stimulated hatching poorly and did not permit good multiplication.
In an attempt to explain the distribution of potato cyst- nematode species in the Andean countries of South America a theory about their speciation and dissemination during co-evolution with species of potato is discussed. Two possible routes by which potato cyst-nematodes may have been introduced to Europe are also discussed.
Physiological changes in susceptible and resistant potato
varieties during G. rostochiensis attack were investigated by
measuring various indices of water stress, rates of photosynthesis
and nutrient uptake.
Studies of the tolerance to G. rostochiensis of ten commercial
potato varieties grown in an infested field showed that Maris
Piper and Pentland Crown were the most tolerant because, in spite of
nematode damage, they were able to produce a reasonable yield
compared with that obtained from the other varieties.
Finally a technique for checking tolerance of potato varieties
on a large scale in pots was investigated by using different pot
sizes and various nematode densities.
r - IV -
ACKNOWLEDGEMENTS
I would like to thank Dr. F. G. W. Jones for providing facilities in the NematOlogy Department and for his helpful criticisms and advice in the present work, as well as for his valuable help to me and my family during our stay in Harpenden. I am deeply indebted to
Dr. Ken Evans for his constant help, advice and valuable criticisms throughout this work and also for reading and correcting this manuscript.
Special thanks are due to all members of the Nematology
Department who helped in various ways, in particular Mr. D. N. Greet and Dr. A. R. Stone; to Mr. J. H. A. Dunwoody and Mr. Colin Banfield for statistical analysis of the data and to Mr. F. Cowland and members of the Photographic Department for the photographs and preparation of the figures. I would also like to thank Dr. A. A. F.
Evans of Imperial College Field Station, my director of studies.
Finally I am sincerely grateful to Dr. R. L. Sawyer ,
Director of the International Potato Center, Lima, Peru, for providing the economic support to carry out my studies in'England; and to
Dr. E. French, also from the International Potato Center for his cons'ant encouragement and friendly support. V
CONTENTS
Page No.
TITLE
ABSTRACT II - III
ACKNOWLEDGEMENTS IV
TABLE OF CONTENTS V - VIII
GENERAL INTRODUCTION 1 - 8
- The potato (Solanum tuberosum L.) 1
- The potato cyst-nematodes (Globodera spp.) n.comb. -6
AIMS OF STLDY 9 - 10 MATERIALS AND METHODS 11 - 15
- Potato plants as host 11
- Potato cyst nematodes as inocula 11
. - Collection of larvae 12
- Extraction of cysts and estimation of their egg content 14
- Counting nematodes in roots 15
- Populations under study 15
1. IDENTIFICATION AND SYSTEMATICS OF POTATO CYST NEilATODES
Globodera spy. (n. rank) 16 - 98
Introduction 16
1* FEMALE COLOUR 25 - 31
(i) Materials and methods 26
A. Colour phases 26
B. Colour change for identification 26
(ii) Results and discussion 27
2. MEASUZ-1.:2;NT OF i..ORPMOLOGICAL CHARACTLRS 31 - 67
(i) Materials and methods 35 Preparation of second stake juveniles 35 Preparation of posterior cyst areas 39 Number of observations 39 - VI -
A. Variation in morphology: 44
a. Athin and between populations 44
b. Density of nematodes in roots 44
c. Effect of temperature 47
d. Effect of daylength 47
B. Identification of populations under study 47
(ii) Results 48
A. Variation in morphology: 48
a. Between and within population variability 48
b. Density of nematodes in roots 54
c. Temperature 55
d. Daylength 56
e. Deviations 56
B. Identification of populations: 58
a. Second stage larvae 58
b. Cyst terminal areas 61
c. Image Analysing Computer 65
(iii) Discussion 66 3. ELECTROPHORESIS 67 - 72 (i) Materials and methods 68
(ii) Results and discussion 69 4. SCANNING ELECT.JON MICROSCOPY 72 - (i) Materials and methods 73
(ii) Results and discussion 74 5. MATING 73 - 85
(i) iaterials and methods 81
(ii) Results and discussion 82
6. PATHOTYPES 85 - 96
(i) Materials and methods 90
(ii) Results and discussion 92
7. GENERAL IDENTIFICATION OF POPULATIONS IN THIS STUDY 97 - 98
II. BIOLOGY OF POTATO CYST NEMATODES 99 - 166
Introduction: a preliminary study 99
1. DAYLEilGTH 106 123 (i) Materials and methods 107
(ii) Results io8
A. Host development io8 B. Nematode development 110 C. Hatching 118 (iii)Discussion 121
2. TEMPZRATURE 12/+ - 11+9 (1) Materials and methods 127 (ii) Results 128 A. Host development 128 B. Ne.natode development 128 C. Hatching 143 (iii)Discussion 147 3. HOSTS 149 - 166 (i) Materials and methods 151 (ii) Results 152 A. Hatching 152 B. Multiplication 153 (iii) Discussion 165 III. GEOGPAPHICAL DISTRIBUTION OF POTATO CYST NEMATODES 167 - 197 Introduction 167 1. ANDEAN POPULATIONS 168 (i) Materials and methods 169 (ii) Results 169 (iii) Discussion 171 2. OTHER POPULATIONS 189 (i) - Materials and methods 190 (ii) Results and discussion 190 IV. EFZECTS OF POTATO CYST NEMATODES ON POTATO PLANTS 198 - 255
Introduction 198 1. PHYSIOLOGICAL BEHAVIOUR OF POTATO PLANTS 202 - 237
(i) Materials and methods 208
(ii) Results 210 A. Water relations 210
B. Nutrient uptake 220 C. Photosynthesis 223
(iii) Discussion __230 2. TOLERANCE TO THE POTATO CYST NEMATODE G. rostochiensis 237 - 255
A. Pot experiment 239 (i) Materials and methods 240
(ii)Results and discussion 240
B. Field experiment 246
(i) Materials and methods 246
(ii)Results and discussion 247 SUi•MMARY OF CONCLUSIONS 256 - 261 APPENDIX 262 - 287 REFERENCES 238 - 325 GENERAL INTRODUCTION
The round cyst nematodes, whose hosts lie almost exclusively
in the family Solanaceae, are believed to have originated in Southern
and Central America undergoing co-evolution with their hosts
(Stone, 1975). The two species of potato cyst-nematodes, recently
nominated by Mulvey and Stone (1976) as Globodera rostochiensis
(Wollenweber, 1923) n. comb. and G. pallida (Stone, 1973 n. comb. (formerly Heterodera rostochiensis and H. pallida respectively)
from the family Heteroderinae Skarbilovich,1947, are major pests -
of potatoes in temperate growing areas. Other crops, tomato,
egg plant, are attacked bt4t weeds of the potato family can act as
hosts. The potato cyst-nematodes have a world-wide distribution
following, quite probably, their dissemination with potato tubers
introduced either as new breeding material or as new varieties.
As they have been the subject of much research a brief review
of both the host and the pest will be given.
The Potato, (Solanum tuberosum L.)
Before the 16th century the potato was completely unknown
outside South America where wild potatoes have been widespread
in the Andes for many centuries and must have represented an
important source of food for the early inhabitants. In some
place or places the potato eventually became a true cultivated
plant, and man became less dependent on wild food sources and
able to lead a more sedentary life. Without an abundant and
dependable food supply, the development of an advanced civilization
in Peru and Bolivia would have been impossible, so the potato
became the most important plant. For how long it has been so
important is not known, but even in the Andes in prehistoric
times it was traded to coastal peoples (Martins, 1976) as has been
shown by archae4ogical findings and in designs on pottery
(Hawkes, 1967; Dodds, 1966). Only after the discovery and conquest 2 of Peru by the Spaniards in 1532 (at which time the cultivation of
potatoes extended from Chile to Colombia) did the potato become known in Europe, being introduced into Spain around 1570 and into
England around 1590. However, it was only first grown as a crop in Ireland in 1663 and in the rest of Europe in the mid 18th
century (Salaman, 1946; Hawkes, 1967; CIP, 1973) and only in the
latter half of this century did the crop really become established
as an important source of food. Conservative prejudices against
unfamiliar food broke down and, by the end of the century, named
varieties had appeared. By the end of the following century
(the 19th) modern varieties, such as Abundance (1886), Epicure (1897),
Champion (1876) and Up to Date (1891) and something very like modern
potato agriculture had become established (Simmonds, 1969). At
present the potato is cultivated not only in the temperate regions of
the world but also in many tropical and sub-tropical zones.
Whether the first potatoes to reach Europe came from the Northern
Andes or from Chile is still in controversy (Grun, 1976). Russian
authorities claim the latter, whereas English argue for the former.
The evidence seems to indicate that the first potatoes came from
the northern Andes and although these were short-day types they became
long-day types through selection under European conditions. The
fact that Chile had not yet been conquered when the potato reached
Europe makes it rather unlikely that it was introduced from this
region. Whether it was looted from the Incas or from the mines of
Potosi in Bolivia it was probably exported from Callao, the harbour
of Lima (Salaman, 1946).
Like most other plants of ancient domestication its early history
as a cultivated plant is obscure. Nevertheless the origins and
evolution of the cultivated potatoes have been extensively studied
since the publication of the results of the Russian plant collecting
expeditions to South and Central America in the years between 1925 -
1932 and the British expeditions in 1938. We can now reconstruct 3 the main outlines of its evolution as a cultivated plant and point with some degree of confidence to the wild species from which it was derived.
We can also propose a centre of origin by identifying the centre of diversity as the Andes of Peru and Bolivia, in particular the Lake Titicaca basin on the border between these two countries.
Possible origins of some cultivated potatoes according to
Hawkes (1976), Cribb (1972) and Huaman (1975):-
WILD sp. CULTIVATED
S.canasense (2x) _ )S.stenotomum (2x)
Mutation and selection
S.megistacrolobum(2x) > S.ajanhuiri(2x) S.phureja (2x)
S.acaule (4x) S.juzepczukii (3 )
S.sparsipilum (2x) S.tuberosum ssp. andigena(4x)
Selection under long daylength conditions. S.tuberosum\ ssp.tuberosum(4x) S.curtilobum (5x)
The breeding potential of the South American cultivated potatoes had been grossly neglected in Europe until recently, and even now only a few commercial varieties, such as those resistant to
Globodera rostochiensis, have been produced utilising Solanum tuberosuM ssp. andip-ena germ-plasm (Simmonds, 1969). 4
Salzman (1946, 1949) and Hawkes (1967) have shown that all
European S. tuberosum potatoes have been derived from one or two introductions of S. andigena potatoes into Spain and England in the 16th century and a few others mainly in the 19th and
20th centuries. Thus the S. tuberosum potatoes have a narrow genetic base, and there is immense potential for the use of
S. andigena potatoes (Simmonds, 1976) and the even more promising wild species as breeding material (Hawkes, 1972). Unfortunately, the reservoir of genetic variability is diminishing at an alarming rate, either because of rapid urban development or rapid replacement of native cultivars by improved varieties (CIP, 1973; Ochoa, 1975).
The taxonomic position of cultivated species according to
Hawkes (1963) is as follows:
Genus: Solanum
Section: Tuberarium
Sub-section: Hyperbasarthrum
Series: Tuberosa
Species: ( 2x ) S. ajanhuiri, S. phureja and S. stenotomum with
ssp. stenotomum and ssp. goniocalyx.
( 3x ) S. chaucha and S. juzepczukii.
( 4x ) S. tuberosum with ssp. tuberosum and ssp.
andigena.
( 5x ) S. curtilobum.
Finally, little stress needs to be laid on the importance of
the potato as a staple crop in the past (hence the Irish famine
of the 18401 s) and in the present world agricultural economy, where
the potato is the major vegetable in much of the world, ranking first
among the 10 major food crops in terms of yield of calories per unit
area per day and second in protein production per unit area (CIP, 1974).
(Sec table overleaf) -- . World Potauo Cultivation and Production (PAO 1975)
Ar6a Harvested (1000Ha) Yield (Kg./Ha) WORLD SECTION 1961-65 1973 1974 1975 1961-65 1973 1974 1975
Africa 290 433 439 453 7219 8127 7952 7939 North America 670 634 678 616 20059 23046 24667 24008 Central America (Mexico included) 65 75 79 79 7692 10368 10302 10627 South America 977 951 946 955 7184 8678 9975 8463 Asia 4407 5107 5195 5319 8727 9871 10096 10554 Europe 8626 6716 6684 6400 16027 19174 19451 18247 Oceania 52 46 44 49 15565 21025 19925 18818 USSR 8638 8017 7983 7912 9449 13496 10149 11183
Developed Countries 4952 3492 3505 3298 18459 22021 22815 21087 Developing Countries 2117 2453 2503 2590 7493 8833 9315 9129 6
The Potato Cyst-Nematodes. (Globodera n. comb.
The potato cyst-nematodes, Globodera spp., werefirst described by Kuhn (1881) as a potato-atticing strain of the plant parasitic nematode Heterodera schachtii Schmidt. Although Massee (1913) reported the potato-root eelworm as a commonly occurring parasite in Scotland it was not until the following year that Zimmerman (1914) definitely declared that the potato plant could be severely damaged by the potato strain of H. schachtii. After this year, many reports of its widespread occurrence in Europe began to accumulate, from England (Taylor, 1917), Sweden (Kemner, 1929), Ireland (Carroll, 1933 and other ones (Spears, 1968). Wollenweber (1923) with'specimens from
Rostock, Germany was the first worker to recognise morphological differences between the sugar-beet and potato-root eelworms i4 respect of cyst shape and the lengths of larvae and their stylets.
On the basis of these differences he proposed a new species,
Heterodera rostochiensis. This specific name wap not immediately accepted. Goffart (1928), for example, claimed that the potato eelworm could be induced to attack sugar-beet, and that on this latter host 'lemon-shaped' cysts would be produced. Later Franklin (1940) was unable to reproduce the findings of Goffart by transferring sugar-beet nematodes to potatoes. She then carried out a series of studies and described further morphological differences, so confirming the specific status of H. rostochiensis. The next year,
1941, the potato root eelworm was found for the first time in the
American continent, Long Island, N.Y., and it was called the
'golden nematode' by Chitwood.
It was not until 1952, when the potato cyst-nematode was found widely distributed in Peru (Willi and Bazan de Segura, 1952) that the idea of a European origin was discarded and replaced by the idea of an 'andean origin' in parallel with an andean origin of its hosts.
Further support came from the findings in Bolivia by Bell (1955) and
Argentina by Brucher (1958) who further suggests the centre of origin of the potato cyst-nematode to be the andean regions between Bolivia and Argentina, due to the presence of numerous sources of resistance in this area e.g. Solanum vernei, S. sanctae-rosae,
S. spegazzinii, S. Kurtzianum, S. oplocense (Anderson, 1972;
Huijsman and Lamberts, 1972; Ross, 1972).
So, with these factors in mind, it seems likely that potato cyst-nematodes were introduced to Europe with the tubers introduced as breeding material in the search for resistance to potato blight in the middle of the 19th century. The nematodes' presence was first noted about 40 years later - about the time that would be required for noticeable field infestations to build up. Nevertheless earlier introductions of this nematode cannot be ruled out since the potato had become the staple food crop for the peasants in Ireland by the middle of the 17th century. This is in contrast to the rest of Europe where the crop became established only in the latter half of the 18th century, modern varieties being bred at the end of the 19th century. Thus, the lack of findings may be attributed either to the late establishment of the potato in Europe - perhaps still in transition from andigena to tuberosum type (Simmonds, 1964)- or to a failure to detect the nematodes (Southey, 1965; Peters, 1951), when it must be supposed that few populations were high enough to attract attention by producing symptoms and when the scale of dissemination of cysts from one field to another would be minimal.
Besides the introductions made in the 19th and early 20th centuries, others may have occurred when different European expeditions to the
Andes collected potato material as breeding sources in the search for resistance to different pests and diseases and improved growth quality. The potato cyst nematodes, Globodera rostochiensis and
G. pallida are by far the most important pest of potatoes in temperate regions - either high altitudes or islands or coastlands where the climate is moderated by the sea - of the world and also one of the most difficult to control. This is due to the difficulty of detection during the early stages of colonisation. Spread is slow at first, speeding up as the area of each focus increases, so, providing better opportunities for dissemination by means of any process that involves movement of tubers and soil. The resistant cyst wall means that they can survive under unfavourable conditions.
Some countries have acted vigorously to contain and eliminate this pest by means of severe quarantine regulations but they cannot entirely prevent cysts passing their defences by guarded or unguarded routes, so further spread to remaining uninfested areas is likely.
(Jones, 1968; 1970).
For these reasons it is virtually impossible to eliminate from land and farmers have to learn to live with it (Simon, 1954).
It causes substantial reductions in crop yields when susceptible potato varieties are grown in infested soil. Jones (1966) showed that the maximum pre-planting levels of infestation that potatoes will tolerate without loss of yield is about 10 eggs/g. on a sandy loam soil but 10-20 eggs/g on a black fen soil. However, when susceptible varieties are grown continuously the population reaches between 100 and 500 eggs/g. of soil, at which level the potato crop virtually fails. Brown (1969) found that, on average, 3250 kg. of tubers were lost per hectare for every increase of 10 full cysts per
100 g. of soil, or 2500 kg. per hectare for every increase of 20 eggs per gram of soil before planting, whatever the potential yi€ld.
Estimates of the economic losses caused by this pest are scarce but
Jones (1970) suggests that these may total some 7-10 million pounds sterling annually in England and Wales, whilst in Peru annual losses have been estimated at 5-7 million pounds sterling. (Dias, gl.al, 1974). Recent inflation, however, and scarcity of potatoes because of poor growing conditions mean that these figures under-estimate the losses by factors of possibly up to five. Aims of Study.
A great deal of research has been done in Europe and America on different aspects of potato cyst-nematodes, but most of them considered only one species, Globodera rostochiensis and its resistant-breaking pathotypes. Therefore, when Stone (1973) described a second species of potato cyst nematodes, G. pallida, certain doubts arose about the results of early workers since they did not know that they were working with different species.
Only recently have studies been made to compare European representatives of the two species with their relatives from
South America, collected from countries located along the Andes.
Therefore, it was decided to investigate certain aspects of the distribution and biology of different potato cyst-nematode populations collected from different parts of the world, with special emphasis on those from South America. The following lines of investigation were adopted.
A. Identification of collected potato cyst-nematode populations by:-
i. Measurements of 2nd stage larvae and terminal areas of cysts
(dead mature females).
ii. Female colour change during development.
B. Studies on the variability of body measurements of selected
populations:-
i. Between and within populations.
ii. Factors of variability.
C. Comparative study of British and Peruvian species by means of:-
i. Protein gel electrophoresis.
ii. Scanning electron microscopy of head features.
iii. Controlled attraction and mating.
iv. Host-parasite relationships with differential clones.
v. Image analyzing computer.
D. Response in hatching, invasion and development of selected
populations to:- 10
i. Daylength.
ii. Temperature.
iii. Host variety.
E. Effects of potato cyst nematodes in the most connon commercial
potato varieties in:-
i. Pot experiments.
ii. Field experiments. 11.
MATERIALSand METHODS.
As different stages of the potato cyst-nematodes were used throughout this study, this section deals with the well-established techniques which were used. Special or modified techniques will be described in the relevant sections.
Potato plants as host.
Potato tuber pieces with a 1-2 cm. sprout were cut with a sterile vegetable scoop from the tubers. One day was allowed for callus to develop on the cut surface before the tuber pieces were planted in sterile loam in 9cm. diam. pots. Then the soil - was thoroughly wetted by soaking the pots in a tray with water.
The plants were grown either inaglasshouse or outdoors. In the glasshouse summer temperatures were kept below 25°C by a thermostatically operated fan and winter temperatures were kept o above 18 C by hot water pipes. Plants were watered every day.
When plants were grown outdoors the pots were pinged in a sandy bed until maturity.
Potato cyst nematodes as inocula.
When cysts were used as inoculum they were mixed at planting time with the soil just beneath the tuber piece. When larvae were used they were inoculated by means of a hypodermic syringe into the soil in which 7-10 day old potato plants were growing. They were inoculated in 3.5 ml. of water containing a known concentration of larvae. Pots were watered regularly before and after inoculation in order to keep the soil moisture near to field capacity, as this resulted in the best invasion rate.
This was checked by growing potato plants from Arran Banner potato pieces weighing 10 g. in 9cm. plastic pots containing
400 g. of soil. Unifor7—ly sized potato plants were chosen and inoculated with 4000 larvae of G.rostochiensis or G.pallida.
Two days before inoculation five regimes of soil moisture were 12 established. These percentages of water were 5, 7.5, 10, 15 and 20% W/W. The soil moisture levels were maintained by days watering to a constant weight twice daily until fourteen after inoculation, when three pots per treatment were used to estimate the rate of invasion.
Tee moisture characteristic of the sandy-loam soil was determined by the method described by Wallace (1954). Moisture content was plotted against suction and in Fig 1 the suction pressures at the five points corresponding to the moisture contents at which invasion was investigated are given.
The greatest invasion rates for G. rostochiensis and G. pallida species of potato cyst-nematode were obtained at the
10% (95cm. suction pressure) water content. However,best growth of the potato plants represented by root and top weights and top length
(Table 1)3oesnot correspond to the point of maximum invasion.
Table 1 :- Growth of potato plants under different levels of
soil water content.
Moisture content Top length Top weight Root weight (%) (cm) (g) (g)
5.0 14.5 2.17 1.18 7.5 21.5 3.53 1.38 10.0 25.3 5.10 1.80 15.0 25.5 5.02 1.68 20.0 28.1 6.61 2.02
Collection of larvae.
Larvae were collected by placing cysts on a nylon sieve and soaking them first in water fora week and then in potato root diffusate at 18°-20°C. The larvae were collected after one
Fig. 1. Soil moisture characteristic curve and the effects of soil-moisture regimes on rates of invasion of G. pallida (UK-2) and G. rostochiensis (UK-3) populations. e-■ a) E
0
34 `c- 2) 0 a. 4 x Globodera rosrochiensis 0 0 Globodero pa 'lido 30.0 -t" 8 0 0 77 12 W 25.0 0 0 E 3 lb E (T) 20.0 20
0 t) 24 0 E 15'0 > 28 0 2 32 1 0. 0
35
1 11 1 S.0 40 I • I -1 10 30 50 70 90 110 130 ISO 170 190 210 230 250 Suction pressure (cm. of water) week and when necessary kept for no longer than a week at - 5°C.
The number of larvae available was estimated by counting the number in 3 one ml. aliquot samples from the suspension. This was done in a Fenwick Multichamber perspex slide. After this count the concentration of larvae was readjusted so that approximately
1000-1500 larvae per ml. could be introduced to the plant, according to the inoculation density required.
Extraction of cysts and estimation of their egg content.
When large numbers of cysts were necessary they were obtained by passing the soil from large pots which had been filled with infested field soil, plunged in the field and planted with a potato seed tuber through the apparatus described by Green and Parrott (1967).
When the number of new cysts formed on a plant had to be counted the potato plants were left until maturity and the watering stopped.
After this the tops were cut off the plants and-after thorough drying the soil was passed through a A" sieve. Then the cysts were extracted from 200-300 g. soil samples using a Fenwick can, as described by Goodey (1963). After drying the "float" the cysts were separated from other debris by either the 'hand rolling' technique (Goodey, 1963) or the "acetone floating" technique
(Anonymous, 1969). The cysts were counted under the microscope using Shepherd's modification of Fenwick's couating tray.
Egg contents of cysts were estimated by picking 50 cysts at random from each sample and transferring them to water. After soaking overnight the 50 cysts were squashed using the metal channel and glass rod as described by Goodey (1963). The squashed cysts were washed into a boiling tube and,after agitating the suspension for about 15 seconds by means of an electric stirrer in order to break all the eggs free of the cyst walls, thelolume of the suspension was made up to 25 or 50 ml. The number of full eggs and larvae in one ml. aliquots were counted for each sample in a 15
Fenwick Multichamber Counting Slide.
Counting nematodes in roots.
To estimate the number of larvae invading potato roots the plants were removed from their pots about two weeks after inoculation and the roots gently washed with a jet of tap water.
The clean roots were immediately placed in F.A.A. (Goodey, 1963) and stored for one week. The roots were then weighed again, cut into small pieces about 1 cm. long, mixed thoroughly, and a 2 g. sub-sample taken and wrapped in a small square piece of muslin to which a wire marker was attached. The sub-sample was then plunged for 2 minutes in a boiling 1.1% solution of cotton blue in lactophenol, after which it was rinsed in hot running water. It was then macerated by placing the roots in a food blender, which was run at half speed for minute and full speed for another -
minute. The homogenate was carefully washed into a plastic beaker and made up to a volume of 200 ml. To count the stained nematode stages, a 10 or 20 ml. sub-sample was taken with a pipette whilst the nematodes were kept in suspension with a "Vibro-mixer", and the deeply-stained nematodes were counted.
Populations under study.
One hundred and sixty one populations from 21 countries were increased on Arran Banner (S. tuberosum ssp. tuberosum) potato plants. To facilitate the description of each population, they are identified by a code. This code contains two capital letters to represent the name of the country of origin and a number, which represents the population within that country
(identified below by a locality or race name). The countries are listed
(in Appendix Table 1) according to their latitudes from North to south and the second letter with the Peruvian populations corresponds to the department. 16
I. IDENTIFICATION AND SYSTEMATICSOF POTATO CYST NEMATODES, Globodera spp. (n-rank)
The systematics of the family Heteroderidae (Fidipjev and
Schuurmans Stekhoven 1941) Skarbilovich 1947, have been reviewed by
Wouts and Sher (1971) and Wouts (1973a, 1973b, 1974). The subfamily
Heteroderinae Skarbilovich, 1947 was emended by Wouts (1973a) Skarbilovich
(1959) placed the spherical and pear-shaped species of Heterodera
in the subgenus Globodera, and the lemon-shaped species in the subgenus
Heterodera. Mulvey and Stone (1976) considered that round cyst-
nematodes form a distinct group among the cyst forming family Heteroderidae
and raised the subgenus Globodera to generic rank. They considered
as type species Heterodera schachtii Schmidt, 1871 and Globodera
rostochiensis Wollenweber, 1923 respectively.
Therefore, in the present study, both species of potato cyst-
nematode will be referred to under the new combination.
The potato cyst-nematodes Globodera rostochiensis (Wollenweber
1923) n-comb. and G. pallida (Stone, 1973) n. comb. are the most easily
recognised species in the genus being only widespread on potato and
tomato crops. The first sign that more than one species was involved
were obtained when potato clones were found which were resistant to
some nematode populations. After Ellenby (1945, 1948, 1952, 1954)
screened the Empire Potato Collection (now the Commonwealth Potato
Collection) and found nematode resistance in clones of Solanum
tuberosum ssp. andigena and S vernei (Ellenby, 1948), breeding
programmes designed to incorporate this character were begun. (Ellenby,
1952; Toxopeous & Huijsman, 1952, 1953; Mai & Peterson, 1952; Jones, 1954).
Since then, "resistance-breaking" (Jones, 1957, 1958), "aggressive
populations" (Dunnett, 1957) or "pathotypes" (Cole & Howard, 1966)
have been found on resistant lines from the clone CPC-1673 containing
gene H from S. tuberosum ssp. andigena (Quevedo, 1956; Dunnett, 1957; 1 17
1960; Van der Laan, 1957; Goffart, 1957; Howard, 1959; Schick, 1959;
Gooris et al, 1962). Howard (1959) proposed the first pathotype scheme for British populations and designated the populations which
developed only on susceptible potatoes with the symbol "A" and
those which developed on ex andigena resistant potatoes as "B".
Dunnett (1961) found that the wild potato S. multidissectum, had a
gene H which gave resistance to pathotype 'B' but not against that 2 which H gene Dunnett 1 material was resistant to. With this new H2 (1962) referred to the existing pathotypes by arabic figures 1(=_1131 ),
2(=A) and 1,2(=a pathotype able to multiply on both genes H1 and H2).
Cole and Howard (1966) by using the two genes for resistance (H1 and
) recognised three pathotypes: 'A' could produce many cysts on H2 potato clones with gene H but none or few on those with genes H or 2 1 H1H2•; 'B' could produce many cysts on H clones but not on those with 1 H or H H and 'C' could produce many cysts on all three types of 2 1 2 resistant clones, i.e. those with H11112 and 111112. Later Guile (1967)
adopted Dunnett's term pathotype 'E' (proposed in 1967) in place of 'C'
to avoid confusion with the Dutch pathotype nomenclature.
With the incorporation in different countries, e.g. Great Britain,
Holland and Germany, of new sources of resistance, which permitted the
recognition of more than three pathotypes, schemes were independently
developed (Huijsman, 1960a, 1962; Kort, 1962; Dunnett, 1962, 1964;
Jones and Parrott, 1965; Noheni, 1969; Ross and Huijsman, 1969;
Howard et al, 1970; Stelter, 1971). Potato species found resistant to potato cyst-nematodes are
listed in Table 2 according ...to their ploidy, place of distribution,
and author's name. This list does not mean that every sample of the
mentioned species is resistant or that each resistant species has
genes of resistance distinct from those of other species. Table 2 Ploidy and distribution of potato species found to be resistant to Globodera spp.
WILD SPECIES
SERIES SPECIES PLOIDY COUNTRY OF DISTRIBUTION AUTHOR Commersoniana S.chacoense 2n=24 Argentina Huijsman & Lamberts,1972 S.tarijense 2n=24 Bolivia Rothacker & Stelter,1961 Circaeifolia S.capsicibaccatum 2n=24 Bolivia Dunnett,1960 A caulia S.acaule 2n=48 Peru, Bolivia, Argentina Rothacker11957 Polyadenia S.polyadenium 2n=24 Mexico Dunnett,1960 Cuneolata S.infundiliforme 2n=24 Bolivia, Argentina Dunnett,1957 Megistacroloba S.megistacrolobum 2n=24 Peru, Bolivia, Argentina Dunnett,1957 S.sancta-rosae 2n=24 Argentina Dunnett,1960 S.boliviense 2n=24 Bolivia Ross & Rowe,1965 S.raphanifolium 2n=24 Peru Dunnett,1960 Tuberosa S.canasense 2n=24 Peru Ross & Huijsman,1969 S.bukasovii 2n=24 Peru Ross & Huijsman,1969 S.cajamarquense 2n=24 Peru Mai & Peterson,1952 S.chiquidenum 2n=24 Peru Huijsman & Lamberts,1972 S.sandemanti 2n=24 Peru Ross,1972 S.neohavkesii 2n=24 Peru Dunnett,1960 Table 2 (Contd) WILD SPECIES SERIES SPECIES PLOIDY COUNTRY OP DISTRIBUTION AUTHOR
S.multidissectum 2n=24 Peru,Bolivia Dunnett,1960 S.sparsipilum 2n=24 Peru, Bolivia Ross & Huijsman,1969 S.leptohyes 2n=24 Bolivia, Peru Ross & Huijsman,1969 S.ganderillasii 2n=24 Bolivia Ross & Huijsman,1969 S.sucrense 2n=48 Bolivia Mai & Peterson1 1952 S.microdontum 2n=24 Argentina, Bolivia Dunnett,1960 S.oplocense 2n=24 Argentina, Bolivia Dunnett,1965-1963 S.rechei 2n=24 Argentina Ross,1972 S.kurtzianum 2n=24 Argentina Huijsman,1956 S.gourlayi 2n=24 Argentina Ditnnett,1957 - S.vernei 2n=24 Argentina Ellenby,1948 S.venturii 2n=24 Argentina Goffart & Ross,1954 S.spegazzinii 2n=24 Argentina Goffart & Ross,1954 S.maglia 2n=24, 36 Chile, Argentina Dunnett,1960 CULTIVATED SPECIES Tuberosa S.andigenum 2n=48 Peru,Boliva,Ecuador,Colombia, Venezuela,Argentina Ellenby,1948 S.juzepczukii 2n=36 Peru,Bolivia Howard1 1961 S.curtilobum 2n=60 Peru,Bolivia Scurrah,1973 20
The different sources of resistance used in different countries
made it difficult to compare pathotyping results from.those countries and
when Stone (1973) described the second species of potato cyst-nematode,
all the schemes had to be reviewed and there are now pathotypes within
the two species (Trudgill and Parrott, 1972; Parrott, 1972, 1974).
TABLE 3 British and Dutch pathotyping schemes (Stone, 1975)
l 2 2 BRITISH A B E
Solanum tuberosum ssp. tuberosum + 4, + S. tuberosum ssp. andigena CPC 1673 - + +
S. multidissectum + - +
l l l 2 2 l DUTCH A B C D E F
S. tuberosum ssp. tuberosum + + + + + + S. tuberosum ssp. andigena CPC 1673 - + + +
S. kurtzianum KTT 60-21-19 - - + + + +
S.vernei G-LKS 58-1642/4 - - - + + +
S.vernei (VTn)2 62-33-3 - - - -
1) G. rostochiensis
'2) G. pallida
Although a considerable amount of work had been done on pathotype
identification by 1975 there was still a good deal of confusion on their nomenclature. To solve this problem, work began simultaneously
but separately in Europe (Kort, 1974) and Peru (Canto, 1975). In
order to rationalise this situation, the two proposed schemes are
being compared in order to establish a new, common and international 21
scheme on pathotypes of the potato cyst-nematodes (Stone 1976,
pers. comm.).
In spite of all the work done on the biology of potato cyst-
nematodes, further studies and observations were made to try and
differentiate populations. Thus, although Shepherd (1965) thought
that British populations were undistinguishable on morphological
criteria, Guile (1966) observed that the length of the yellow phase
of maturing females varied. He observed that a bright yellow phase
was characteristic of certain populations, i.e. pathotype A (Globodera
rostochiensis), and that these justified the name of 'golden nematode'
given by Chitwood (1948, 1951). Guile also (1967, 1970) observed
that with resistance-breaking populations either a long cream phase
was followed by a short yellow one or a long white phase was followed
by short pale cream phase. The results can be summarised as follows:-
Pathotype A (=G. rostochiensis), short white-cream phase, long yellow
phase, becoming bright yellow to golden colour.
Pathotype B (=G. pallida ), long cream phase with short. yellow phase,
not becoming bright yellow.
Pathotype E (=G. pallida), long white phase, short pale cream phase,
No yellow phase.
These distinctions can lead to some confusion as yellow females are
sometimes found on plants with gene H1 (Trudgill et al, 1970), but
further studies on morphological and biochemical characters showed
more differences between pathotypes.
Webster & Hooper (1968) found serological differences between
pathotypes. Larvae from populations with yellow females are shorter and have shorter stylets with differently shaped knobs than those
with cream or white females (Evans and Webley, 1970; Webley, 1970).
Although pathotypes B and E interbreed freely, fewer fertilizations occurred when they were crossed with males or females of pathotype A
(in Jones 1967, 1968, 1969, 1970). 22
After Jones' et al (1970) suggestion that there were two species of potato cyst-nematodes further evidence supported this hypothesis.
Trudgill (1970) found the males had different measurements and
Trudgill & Carpenter (1971) were able to distinguish populations with yellow and cream or white females by electrophoresis of their proteins in polyacrylamide gels. Green (1971) found distinguishing characters in the vulval region of mature females. Stone (1972) found differences in the configuration of the lips and oral disks of second stage larvae.
Eventually Stone (1973) with all this evidence and more (Bouwman
& Ross, 1972; Behrens, 1972) supporting Jones' hypothesis, described the second species of potato cyst-nematode. He considered the two species to be sibling species and very similar but the differences between populations with yellow and cream or white females were as
great or greater than differences between some species now in the
genus Heterodera Schmidt 1871, e.g. Heterodera carotae Jones, 1950 and H. cruciferae Franklin 1945. Latterly, more studies of the differences between species of the potato cyst-nematodes with
populations from different world areas have been done on the reliability and usefulness of each of the characters used to separate these two
species. (Scurrah, 1972, 1973; Mulvey, 1972, 1973; Hesling, 1973;
Ellis & Hesling, 1974; Hesling & Ellis, 1974; Oydvin, 1974; Stone, 1975;
Canto, 1975; Wouts, 1976). Table 4 Some characters used for differentiating G. rostochiensis and G. pallida
Character G.rostochiensis G.pallida Author FEMALE:
Colour Golden—yellow White or creamy Guile (1966,1967,1970)
Stylet length 22.9 ± 1.2 um 27.4 ± 1.1;26.0 ± 1.6 um Stone (1973)
CYSTS:
Mean fenestral diameter 18.8 ± 2.2 um 24.5 ± 5.0;20.7 ± 3.2 um Stone (1973)
Fenestral length usually < 19 um usually > 19 um Ellis & Hesling (1974)
Fenestral shape Circular Oval Ellis & Hesling (1974) Distance from anus to fenestral edge 66.5 ± 10.3 um 49.9 ± 13.4;50.2 ± 9.8 um Stone (1973)
I I 55 ± 1.4 um 46 ± 1.2 um Ellis & Hesling (1974) - Number of ridges on anus to fenestra axis 21.6 ± 3.5 12.5 ± 3.1;11.8 ± 2.4 Stone (1973) usually > 14 usually < 14 Hesling & Ellis (1974) usually > 11 usually <11 Green (1971) Granek's ratio 3.6 ± 0.8 2.1 ± 0.9;2.5 ± 0.8 Stone (1973) usually ).3 usually <3 Ellis & Hesling (1974 "V" shaped arms from anus straight Bent outwards Behrens (1972) Table 4 (contd) Character G.rostochiensis G.pallida Author SECOND STAGE JUVENILES: Stylet length 19.3 ± 0.69 um 21.2 ± 0.68;21.1 ± 0.61 um Webley (1970) 20.9 ± 0.19 um 23.3 ± 0.21 um Bouwman & Ross (1972) 21.8 ± 0.7 um 23.8 ± 1.0;23.4 ± 0.6 um Stone (1973) 21.0 ± 0.7 um 22.8 ± 0.7 um Oydvin (1974) It 22.9 ± 0.6 um 24.3 ± 0.62 um Canto (1975) Distance from head tip to stylet. base 23.1 ± 0.16 um 25.1 ± 0.29 um Bouwman & Ross (1972)
tI 24.3± 0.7 um 25.7 ± 0.7 um Oydvin (1974) Lip length 4.73 ± 0.2 um 5.22 ± 0.4 um Canto (1975) Distance from median bulb valve to excretory pore 31.0 ± 2.4 um 36.8 ± 2.9;36.3 ± 3.4 um Webley (1970) 3].3 ± 2.3 um 39.9 ± 3.3;36.7 ± 3.0 um Stone (1973) 36.2 ± 4.0 um 43.0 ± 4.1 um Oydvin (1974) 33.81 ± 2.8 um 33.5 ± 2.1 um Canto (1975) Stylet knobs shape Forked backward Large with forward prof. Webley (1970) Lip region contourns Rounded Angular Stone (1972) MALES: Spicule length 36 ± 2 um 40 ± 2 um Behrens (1972) 25
I.1 FEMALE COLOUR
The term female is used throughout to denote live individuals seen or
collected on living potato roots.
When females of potato cyst-nematodes begin to protrude through the
root cortex to be fertilized they are always white or pale cream in
colour. After fertilization, that white colour either continues or
changes to an intermediate yellow phase, of varying duration and
intensity. If a yellow phase occurs, it is not due to a colour change
in the cyst wall, but in the female content (Franklin, 1951). Although
the presence of lipo-fuscin pigments has been shown, it is considered
that the pigmentation of the developing females content is largely due to
a quinonoid substance (Smith and Ellenby, 196 7). Later colour changes
to brown take place in the cyst wall. Proteins in the wall undergo
a gradual tanning process involving polyphenol oxidase 1946)ana
the deepening of the brown colour continues for some time after cyst
formation.
Although developing females of Globodera rostochiensis and
G. pallida, British pathotype E, can be readily distinguished by the
yellow phase in the first and white phase in the latter, the identifi-
cation of G.pallida, pathotype B, is more difficult because of possible
confusion with the creamy-yellow phase of G. rostoc'liensis. However,
these differences in colour occurring during female development have
been shown to be correlated with potato cyst-nematode species (Stone, 1973, 1975), although populations from Panama, thought to be introductions of G. rostochiensis from Europe, contrary to expectation
exhibited white females when they were tested in Peru (CIP, 1974). As
mentioned previously Guile (1970) defined three distinct sequences of
colour change for British pathotypes, but although Canto (1975, 1976) • 26
found similar colour phases, he found that the duration of these phases
sometimes differed from that suggested by Guile.
The experiments described below used Guile's criteria to identify the
populations under study.
(i) Materials and Methods.
A. Colour phases.
Selected populations, PP-8, UK-1, UK-2 and UK-3, were studied to
follow how the colour varies during the development of potato cyst-
nematode females. Five transparent pots per population were pladted with
Arran Banner (Solanum tuberosum ssp. tuberosum) and inoculated with
3,000 larvae each. Larvae were used to avoid the prolonged period
of invasion which occurs when a cyst inoculum is used. Two extra
transparent pots per population were also planted. When young
females appeared on the root ball surface of the extra pots, young
females were removed from one of the five, and from the remaining
four at weekly intervals thereafter. The colours of the females
collected at each sample date were compared by means of a Colour Chart
(Royal Horticulture Society) and colour photographs were taken.
B. Colour change for identification.
Sprouts of susceptible Arran Banner were planted in two transparent
plastic pots inoculated with 25-30 cysts/pot ..of each population.
Ten to twelve weeks after planting young females started to appear
on the root balls, and 10 of them per pot were marked. The colour
sequence was followed until the dead females became brown in colour
(i.e. reached the cyst stage). The potato plants were grown in a
greenhouse during the summer season. The colours which were
considered during this study were: white, cream, yellow, golden and
brown, because it is not possible to use intermediate colours for 2? 1 such subjective observations, and more categories simply complicate ,, the ...... t heme for identification. The duration of each colour phase was recorded, but not considered to be important for identification purposes.
(ii) Results and discussion.
The colour sequences of selected populations of G. rostochiensis (UK-3 and PP-8) and G.pallida (UK-1 and UK-2) are shown in Figure 2
(a, b, c, d, respectively). No differences were noted between British and Peruvian populations of the same species.
The colour sequences for the two species were matched with the colour chart and can be described as follows:
Observed colour Colour chart
Globodera rostochiensis White White (155A)
Cream Greyed-Yellow (160D)
Yellow Yellow (7A)
Golden Greyed-Orange (164C)
Brownish Greyed-Orange (167A)
Brown Greyed-Orange (175C)
Globodera pallida White White (155A)
Cream Greyed-Yellow (161A)
Cream (dirty) Greyed-Orange (164B)
Brown Greyed-Orange (165B)
When these British and Peruvian populations of G. rostochiensis and G. pallida were tested in growth cabinets under different daylength conditions (12 and 16 hrs.) no changes from the colours character- istic of each species were found.
The study of colour change for identification purposes has aimed only at recognition to species of potato cyst-nematodes. Little Fig. 2. Colour changes of females of G. rostochiensis.
(a) Colour changes of a British population (UK-3) during female develop- ment from their first appearance on roots to the cyst stage.
(b) Colour changes of a Peruvian population (PP-8) during female de - velopment.
... , ' ', ' '' • -' A%.,t.1,1 .; •)4 a .' 1■(' • .4 • ' ..` ' A % . No; 1%,' ' ‘‘.; z.•.‘ • , ,„‘ . N• AO . 4 •- ei ,.,. , •,-• * .; ,,"%-. ..„.,4,,,,si..,-.,,.1 • ;..,-..• L., tn.., A '“A: 4 '. ' • 4 • ../ • 1(.4. , 1. • - - •. 1 . • • 10 ' iv . • $ 1 A' • , . • - -' - .• • •, t. '. ..' SI .., 15 , • Ito • ' ' ' , • : \ . • • '(; '....'t • • . ;,■;,!..-.. • . 4...... ■ ' .- • .%. • 41 •..‘. %, • ,,;■.• ...• , • ., ... . ,
4 ' •
•
•
• .
, • • . •• Fig. 2. Colour changes of females of G. pallida.
(c) Colour changes of a "white" British population (UK-2),showing fewer intermediate colours than G. rostochiensis.A Peruvian "white" popu- lation (PL-4) showed similar colour phases.
(d) Colour changes of a "cream" British population.(UK-1) ..
30
TABLE 5 Species present in South American countries (identified by fe%iale colour)
VENEZUELA COLCflBIA ECUADOR PERU BOLIVIA CHILE TOTAL -
G.pallida (,,hite) 9 54 10 73
G.pallida (Cream) 1 2 17 0 '20
G.rostochiensis (Yellow) 2* 12 7 21
G.pallida + G.rostochiensis - 1 - 7 8 1 17 TOTAL 131
* These populations may have been introduced from Europe.
TABLE 6 Species present in countries other than those of South America
(identified by female colour).
IC DE UK GE CA NF JA IT SP GR MA'CY IN. SA NZ
G.pallida (halite) 1 1 1 1 1 1 6
G.pallida (Crean) 3 - - - - 4
G.rostochiensis (Yellow) 3 - 1 1 1 3 2 1 3 1 1 1 ,18
G.pallida + G.rostochiensis 1 - - 1 ------2 30 31
effort was spent on trying to recognize pathotypes, particularly and Appna Ta5les 233 as some populations seem to be mixtures of species. (Tables 5 and Appenaix From the results shown in the„Table 2 for Andean populations
different distributions were noted for the two species, as also found
by Evans et al. (1975). North of 15.6°S mostly G. pallida was found, but
south of this latitude most populations were G. rostochiensis or a
mixture of the two species. Possible reasons for this distribution will
be discussed later with additional experimental results.
Results in Table 6, for populations considered as introduced in the
Old World show a clear predominance of G. rostochiensis.
1.2. MEASUREMENT OF MORPHOLOGICAL CHARACTERS.
Morphological features are useful characters for reference to
by taxonomists as they can be preserved for study over many years.
They are therefore more useful than attributes that are lost once the
organism is dead e.g. physiological and genetic characters (Franklin,
1967). Nevertheless as comparisons between populations from different
localities have been made, biologists have found that within a species
there is often marked morphological variability (Tarjan, 1967).
Variability within a population can be extrinsic, due for example
to host plant (Trudgill et al, 1970), density (Fisher, 1965), anaFisfier, anaEvanS) environment (EvansA 1970 , 1970a, 1970b 074) or it may be intrinsic,
due to genetic variability, (Tarjan, 1974) and this can cause deviations
from the mean of as much as 10-35% (Thorne & Allen, 1959).
In different nematode genera different characters have proved
useful for species differentiation. Brzeski & Szczygiel (1963)
studied 15 morphological characters of 5 species in the genus
Paratylenchus and found that only the length of the stylet shaft was
consistent and different between species because differences in other
characters were small and the ranges overlapped. Many other workers 32
have studied the usefulness of characters for differentiating between species, e.g. Geraert, 1965; Goodey & Hooper, 1965; Sanwall, 1965, etc.
In the genera Heterodera (and Globodera) the cyst often possesses reliable long-lasting morphometric and anatomical features which can
be used in species identification or species grouping (Hesling, 1965).
Differences in cyst shape have been used both by Skarbilovich (1959) and Mulvey & Stone (1976) to classify round-cyst species and lemon-
shaped species in either separate sub-genera or genera. Morphology
of larvae and males also provide useful means for identifying species.
The different stages of the potato cyst-nematodes Globodera
rostochiensis and G. pallida are not easily distinguished at any
stage of their life cycle, and these dimensions can be influenced
by Certain factors (Muller, 1958; Cooper, 1955; Rasinya, 1962; Kerstan,
1971; Netscher, 1973; Behrens, 1974; Sigar'ova, 1974). Webley (1970)
and Evans & Webley (1970) found that G. rostochiensis larvae had different
measurements from those of G. pallida, e.g. body length, stylet length,
distance between the median bulb valve andexcretory pore and that the
shapes of the stylet knobs were different. Trudgill, Parrott & Stone
(1970) found the males also had different measurements, but that these
were not reliable differential characters. Green (1971) examined the
terminal areas of females, using the form of the cysts' papillated
vulval crescents (= vulval fenestral Hesling, 1974) as
distinguishing features and also the number and form of the ridges on
the external cuticle-pattern of the perineum, i.e. more than 11 ridges
in G. rostochiensis and less than 11 in G. pallida. Greet (1972),
working with larvae and males of the round cyst-nematode group found
differences between species in body and stylet length, but not in body
breadth, distance between median bulb and excretory pore, tail length
and length of the hyaline portion of tail. Behrens (1972) comparing 33
populations from England, Scotland and Germany did not find clear differences in terminal cyst areas because of great variability within populations. The form of the V-shaped mark (Granek, 1968) on the cuticle at the anus differed in that the two arms of the "V" were straight in
G. rostochiensis but at least one arm was bent outwards in G. pallida.
The distance between the arms at a point 25 um from the anus was also characteristic but the ranges of values overlapped. With males,
the two species could be separated by spicule length, measured dorsally
from the inner lateral viewpoint. Bouwman & Ross (1972), studying
populations from Europe, India and Peru, found that the two species could to some extent be differentiated by larval morphometrics. Scurrah
(1972) found that three Peruvian populations had great variation, but
were closer to G. pallida than G. rostochiensis in body and stylet length and distance between median bulb valve to excretory pore. Stone
(1972) describing G. pallida indicates that this differs from
G. rostochiensis in having a larger second stage larva with a stylet
on average 23 um long (21 um on G. rostochiensis), basal stylet knobs
pointed anteriorly (rounded in G. rostochiensis) and 2 to 8 refractive
bodies in the larvae tail (1 or 2 in G.r.); in having cysts with
Granek's ratio less than 3.0 (greater than 3.0 in G.r.) and only 8-20
cuticular ridges between anus and fenestra (16 to 31 in G.r.). He also
describes differences between males and females of the two species.
Hesling (1974) and Ellis (1974) showed that potato cyst nematode
species could be placed in two groups on the basis of cyst fenestral
length, width, area and shape and on the basis of Granek's ratio, number
of perineal ridges and "B" (= distance between anal pore and the
nearest fenestral margin). However, cyst characters such as pigmentation,
punctation, perineal pattern, thornlets and microbullae were no help
in distinguishing them. Oydvin (1974), working with Norwegian populations, 34 - proposed as sole characters to differentiate the two species stylet length, distance from head tip to stylet base or distance
between median bulb valve and excretory pore of second stage larvae.
Canto (1975) found much variability in most characters measured in
second stage larvae from South American populations. This made it
difficult to use most of the characters for identification purposes.
The length of the stylet and length of the lip region were the two characters that were most constant within populations and they were sufficiently different to allow differentiation of the species.
However, measurements of larvae from Peruvian populations are generally
quite different from those given for the type species. Wouts (1976)
measuring 25 characters from G. rostochiensis and G. pallida populations
from Germany found that the total length is of no diagnostic value and that the total body length is reliable only for G. pallida
populations with larvae more than 475um long. However, stylet
length, the size of the stylet knobs and the width of the lip region are distinct and consistent characters useful for separating the species.
So, in practice, cysts and second stage larvae are the stages in the life cycle of potato cyst-nematodes which can be used in
diagnosis. Because measurements of all morphological features are variable, and have overlapping ranges for the two species, diagnosis based on both stylet length and stylet knob shape of second stage larvae are the most reliable (Stone, 1975).
As identification of second stage larvae depends on small
differences in measurements it is important to be accurate, use a
standard method of preparation which does not cause dimensional changes (Stone, 1971) and place the specimens in the correct position.
In addition, sufficient specimens must be measured, though the number 55
may sometimes be small. For instance, Oydvin (1974) claimed that there is little advantage in measuring the stylet length of more than
2 specimens in a pure population.
(i) Materials and methods
The populations in this study were first multiplied on the susceptible potato variety Arran Banner (Solanum tuberosum ssp. tuberosum), although some cysts from original samples were also used. Measurements were made on both larvae and cysts, but more emphasis was placed, on larval measurements. The techniques used_ to process specimens for microscopic observation and the statistical analysis used to determine the minimum number of specimens to be examined are described first.
Preparation of second stage juveniles
From each population multiplied on Arran Banner 30 cysts were soaked first in water (one week) and then in potato root diffusate.
The hatched larvae were killed and fixed according to Stone's technique (1971) to avoid changes in the measurements. When possible they were measured immediately after fixation but, in most cases they were stored as fixed material for up to 12-15 months. This meant that the clarity of some internal structures such as the stylet tip was impaired. Accuracy of measurement was therefore reduced and the time taken increased. To solve this problem, the effect of adding dyes to stored material was investigated. The dyes which were selected have been used before by other workers but with different purposes (Hollis, 1961; Shepherd, 1962; Ogiga & Estey, 1974; Jatala,
1975). New Blue R, Meldola Blue, Nile Blue A, Cotton Blue Lactophenol,
Potassium permanganate and potassium dichromate were tested at different concentrations and temperatures (room temperature and 50°C). The best results were obtained when a drop of 0.5% aqueous solution of 36
KMn04 was added to the sample and left overnight at room temperature
(Figure 3). Good results were also obtained when samples
treated either with KMnO4 or K dichromate were kept for 30 to 60
minutes at 50°C. However, the treatment at room temperature was chosen
and this markedly improved the definition of stylets, giving the
most successful results with material stored in 4% formalin.
The characters measured in second stage juveniles were the
following:-
Characters Symbol Magnification
Total length (by camera lucida) TL 120x
Stylet length (and knob shape) SL 760x
Head tip to median bulb valve MB 760x
Median bulb valve to excretory pore MEP 760x
Head tip to excretory pore EP 760x
Tail length TL 760x
Some way through this study a new technique was developed by
Hooper (1976). Freshly hatched larvae were treated in a drop of
water on a cavity slide with one or two drops of 2% ammonia for 1
minute and then were relaxed by adding &ri excess of iodine in
potassium iodide solution. Using this technique, It was possible to
prepare larvae for examination 80% of which had protruded mouth
stylets and were therefore very easy to measure. However, as changes
in the shape of the median bulb in ammonia-treated larvae were observed,
("shrinkage") a comparative study with the standard technique was made
(Figure 3)- Fig. 3. Effects of different processing techniques on second stage larvae of potato cyst-nematodes.
Upper photographs:show effects of ammonia treatment on live 2nd. stage larvae: stylet protrusion and "shrinkage" of mediam bulb valve.
Lower photographs:show effects of potassium permanganate on 2nd. stage larvae stored in formalin for one year: good definition of the stylet tip and other features. S. Table 7 Analysis ofIariance of second stage larvae measurements of two G. pallida populations treated with two different techniques (n=30)
Total Stylet Distance Between Distance Tail 1 2 TECHNIQUES Length Length to MBV MBV-EP to EP Length
KM 0 436.4 24.0 106.7 n 4 71.7 35.0 55.3
Ammonia 444.0 23.8 64.5 42.5 107.0 54.3
S.E.D. 5.2 0.2 0.7 0.7 0.9 1.1 L.S.D. 5% N.S3 N.S3 1.4 1.4 N.S N.S
1% 1.9 1.9
1. Median Bulb Valve
2. Excretory Pore
3. No Significance
The analysis of variance of second stage larvae measurements of two populations of G. pallida (PJ-16 and PJ-40) is shown in Table 7. the Significant differences atA l% level between measurements of headtip to medium bulb valve (MBV) and MBV to excretory pore (EP) in larvae prepared by the 2 methods were found. However, as no significant differences in the measurements of headtip to excretory pore between ,thepopulations were found, the two first measurements were replaced for identification by the latter one (headtip to excretory pore) and the rest of the populations were measured by the new technique. 39
Preparation of Posterior cyst areas (vulval fenestra, fenestralia)
To examine cyst fenestralia the cyst wall was cleaned and cleared by soaking whole cysts in 5% hydrogen peroxide for a week at room temperature (+ 20°C) or 30 min at 65°C. Afterwards the posterior end of each cyst was cut, cleaned and mounted in warm clear lactophenol to show the vulval, perineal and anal regions. The features which were considered and / or measured are shown in 1'J Figure 4 and are the following:-
Characters Symbol Magnification
Vulval fenestral length L 320x
Vulval fenestral width W 320x
Anal pore to nearest edge of vulval fenestra B 320x
Number of ridges on sides of triangle located between anal pore and fenestra lateral 1 projections n,n 320x
Number of observations
To establish the minimum number of measurements and the most useful features to separate the species of potato cyst-nematodes, two populations were compared. Measurements from sixty larvae and fifty cyst areas from the populations UK-3 and EC-1 (Globodera rostochiensis and G. nallida respectively) were analysed and the number required to give 95% confidence established . (Table 8) 40
W
Vulval slit Vulva! Hyaline parapet fenesrra Margin Shelf Basin
Distances: W-Vulval fenesrra width L -Vulval fenestra length B -Anal pore ro nearest edge of vulva! fenesrra
Fig. 4. Fenestralia of potato cyst-nematodes showing the morphological features measured. 41.
Table 8. C+ulation table of 'n' values at 95% of probability.
"n" t2(n- 1) /
1
2 3.04
3 1.6o
4 1.22
5 1.03
6 0.91
7 0.82 8 0.76
9 0.71 10 0.66
Large 2/ lir 16 0.50 25 o.4o
36 0.33 49 0.29
Tables 9 and 10 show the statistical analysis used to compare the measurements of second stage larvae and cyst terminal areas, respectively.
In the last column of each table the usefulness of the features to be used is given by the minimum sample size ('n') required to discriminatethe'two species.
Thus, to discriminate one species from the 'other • , using second stage larvae measurements the best features are: stylet length (2 samples are enough to recognize each species), distance from headtip to excretory pore (4) and tail length (4). To discriminate species using cyst terminal area measurements the best straight features to be used are 42 Table 9. Discriminatory values of second stage larvae measurements of G. rostochiensis and G. pallida populations.
Difference Minimum MEASUREMENTS Between Common Common DBM/ /2-XSD Sample Means Variance S.D. ' Size (n)
Total length 8.82 392.26 19.81 0.31 4o Stylet length 2.65 0.19 0.44 4.26 2 Dist.to Median Bulb Valve 2.47 9.99 3.16 0.55 14 Dist.between MBV to excretory pore 4.86 5.15 2.27 1.51 4 Dist. to excretory pore 7.34 13.24 3.64 1.43 4
Tail length 5.83 7.37 2.71 1.52 4
Table 10. Discriminatory values of cyst terminal area measurements of G. rostochiensis and G. pallida populations.
Difference Minimum MEASUREMENTS Between Common Common DBM// .7xSD Sample Means Variance S.D. Size
Fenestral length 5.3o 5.52 2.35 1.6o 3 (L) Fenestral width 1.80 2.26 1.50 0.85 7 (W) Number of ridges 5.4o 8.49 2.92 1.31 4 (n) Number of ridges 5.3o 8.70 2.95 1.27 4 (n1 )
Dist.between Fenestra to Anus 14.60 301.30 17.36 0.59 12 (B) Graneles ratio 1.25 0.51 0.72 1.23 4 Fenestra area 122.00 4012.64 63.35 1.36 3 43 fenestra length (3) and number of ridges (4). Useful derived features are : fenestra area (3) and
Granek's ratio (4). Table 11 shows the actual means of second stage larvae and cyst
terminal area measurements of the populations studied.
Table 11 Means of actual measurements used to discriminate two species of potato cyst-nematodes.
UK-3 EC-1 SECOND STAGE LARVAE Measurements (G.rostochiensis) (G.pallida) Total length 457.34 466.16
Stylet length 21.02 23.67
Dist.to Median 64.51 66.98 Bulb Valve
Dist.between MVB to EP 31.02 35.88
Distance to Excretory Pore 95.53 102.87 Tail length 46.37 52.20
CYST TERMINAL AREA
Fenestral length 20.7 26.0 Fenestral width 20.6 22.4 Number of ridges 17.2 11.9 Dist.between Fenestra to Anus 70.2 55.6 GraneK 's ratio 3.4 2.1
Fenestra area 337.o 459.0 44
A. Variation in morphology
The effect of some factors on the variation or change that they can cause in some characters has been studied:- a) Variation within and between populations
This was done with 6 populations. To study the variability in measurements "between" and "within" populations cysts and'larvae were. compared in the F1 generation. This first generation was obtained by growing pieces of Arran Banner tubers in small pots inoculated with single cysts (10 cysts per population). When mature the newly formed cysts were extracted from each pot. Cysts from selected sub-population were-placed either singly or in batches into root diffusate to obtain active larvae.
The posterior areas of cysts in batches were then mounted on slides and
15 fenestra and larvae per sub-population were measured. With the single on cysts similar measurements were made and,15 larvae from each of them
(see Figures 5 and 6).
Measurements were made as above on cyst terminal area and larvae.
All these measurements were compared by a "Hierarchical Analysis of
Variance". Some F cysts were also used to produce an F generation. 1 2 The cyst.,measurements have been compared between populations, within populations (=between sub-populations) and within sub-populations
(=between cysts). Larvae measurements have been compared between, populations,within populations (=between sub-populations) and within sub-populations (=between larvae).
The cyst and larvae measurements for single cysts have been compared
..:_ between populations, within populations(=between cysts) and within cysts(=between larvae). b) Effects of density of nematodes in the roots
Potato plants grown in 9 cm. pots(400 g. of soil) were inoculated with 500, 5000 or 50,000 Globodera rostochiensis larvae. Four replicates were used. When the plants were mature the new cysts were extracted and placed in potato root diffusate to obtain active larvae. Where
45
Figure 5
4 populations
(EC-1;CO-1;PL-4030-18)
1 10 cysts/popn
PARENT
Single cyst cultures
se 40 F 1cysts/sub-pop.(4 sub-pops. from each popn.) / / / F1 10 cysts / 30 cysts / / /
Single cyst cultures Bulk hatched in P.R.D.
MeasurementsI made on: (i) 15 cyst terminal areas
(ii)15 larvae
'(WITHIN POPS.and WITHIN SUB-POPS.) 1 i 1 F cysts F 2 2
46 Figure 6
2 populations (UK-2, UK-3)
10 cysts/popn PARENT
Single cyst cultures
4 40F cysts/sub-pop. 10 cyst/sub-pop. 1 (from one sub-pop. (4 sub-pops.from in each popn) each popn).
F 10 cysts 30 cysts 1
Single cyst Bulk hatched cultures in P.R.D.
1 1 Measurements made on: Measurements made on:
(i) 15 cyst terminal (i) Cyst terminal area area
(ii) 15 larvae (ii) 15 larvae
(WITHIN POPS. and (WITHIN POPS. and WITHIN SUB-POPS.) WITHIN CYSTS) ---- 1 1
F2 cysts 47
possible twenty five larvae from each density were measured.
c) Effects of Temperature
Potato plants were inoculated with Globodera rostochiensis or
G. pallida and grown at 10°, 140or 18°C. Larvae were collected
from the cysts that formed and processed and measured as above.
d) Effects of Day length
Larvae for measurement were hatched from cysts reared on a susceptible
potato variety. The plants were grown under controlled conditions
in growth cabinets at 12 or 16 hours day length. Further details-
will be given in section II.1.
B. Identification of populations under study
Fifteen second stage juveniles from each population were measured
and their means and standard deviationscompared. Four of the
measurements made were chosen to compare populations by means of
Canonical Variate Analysis. This combines the information to show
which isolates are most closely related and which are their nearest
"neighbours". Fifteen vulval fenestra characters of 25 selected populations
were also measured and analysed in the same way. In addition to
simple morphological measurements the usefulness of an image analyzing
computer (quantimet 720) to identify species of potato cyst-nematodes
was investigated. The perimeters and areas of second stage juveniles
and males were compared by analyzing their optically derived images.
The apparatus consists of a microscope to produce the image which
is formed directly from specimens mounted on a slide. The optically
formed image is scanned by a 720-line plumbicon scanner 10.5 times
per second and the output is passed to a detector where features
are selected for measurements according to their common grey level
characteristics. The accuracy of measurements depends greatly on 43 obtaining sufficient contrast between the specimen being measured and all other material. The signal from the detector is passed directly to a series of modules which make the measurements. An integral display monitor shows which features are being measured and displays the digital results on the monitor screen. Results are passed into a teletype printer or punched on computer tape.
The second stage larvae and males to be measured were killed and fixed as described before. To get good contrast both were stained in hot cotton blue-lactophenol and mounted in warm clear lactophenol. For each of the eight populations investigated, fifty larvae and males respectively were measured. The populations used were the following:- UK-3, PA-2, (G.rostochiensis)
UK-2, CO-1, PL-4 (G. pallida) and B0-22 (G.E. +
(ii) Results
The results on how measurable morphological characters of potato cyst-nematodes can be modified by different factors and the use of these features as a tool to identify populations will be described separately.
A. Variation in morphology
Measurable morphological characters of different developmental stages of potato cyst-nematodes have been widely used to identify them as either G. rostochiensis or G. pallida. However, most of these measurements are not clear cut between species, with overlapping ranges of values which have led to misidentification. Some of the factors which may influence this variability, such as characters of the population itself (intrinsic), or conditions during development of the nematodes (extrinsic), are presented in this study. a) Between and within population variability
Results of a " Hierarchical Analysis of Variance" of measurements made on features of cyst terminal areas and larvae of the 6 49 selected populations (G.rostochiensis: UK-3; G. pallida: UK-2,
EC-1; CO-1 and PL-4; G.r. + G.E.; B0-18) are presented in Tables
12, 13 and 14.
All values of Sums of Squares (ss) and Mean Square (MS) have been 4 divided by 10 . Table 12 shows the results of the hierarchical analysis on the 5 cyst-measurements made on 4 sub-populations of the F1 generation
(see Figs. 5 and 6, pagesIt5 7 4b ) of the 6 populations studied. The dimensions of vulval fenestral length and width, distance from anal pore to nearest edge of vulval fenestra and number of ridges between the anus and either side of the fenestra are represented by C (1), C (2), C (3), C (4) and C (5) respectively.
The character C (1), will be used as an example to explain the results obtained. To test whether the variation between populations is no larger than that within populations ( = between sub_populations) against the alternative that is larger, the appropiate F statistic value (F = 13.51) is compared with the tabular F value at the 5% level (F 5% = 2.77). In this case the observed F is significant and it can be said that there is evidence at the 5% level that the variation between populations is greater than the variation within the populations. Similarly, when testing whether the variation within populations is no larger than that within sub-populations
( = between cysts), the observed F value (5.36) is significant at the 5% level (F = 1.70.) So, the evidence is that in C (1), the 5% variation within populations 39 greater than the variation within sub-populations. In addition the variance component (last column) indicates that variation between populations is 0.0387 in excess of the variation within populations, and that the latter is 0.0101 in excess of the variation within sub-populations. Considering the
Table 12. Hierarchical analysis of variance of measurements on cyst . generation from single cyst cultures- terminal areas of the T1 of six populations. 1 Measurement Source of variation DF SS MS F F5% Variance component
C(1) Between populations 5 12.19 2.439 13.51 * 0.0387 Within populations 18 3.25 0.180 5.36 * 0.0101 4Between sub-pops)
Within sub-populations 326 10.98 0.034 0.0337 (=Between cysts)
Total 349 26.42
C(2) Between populations 5 3.10 0.620 5.96 • 0.0088 Within populations 18 1.87 0.104 5.30 * 0.0058 (=Between sub-pops)
Within sub-populations 326 6.41 0.020 0.0196 (=Between cysts)
Total 349 11.38
C(3) Between populations 5 155.55 31.111 5.43 • 0.4345 Within populations 18 103.22 5.735 3.41 • 0.2787 (=Between sub-pops) Within sub-populations 326 547.97 1.681 1.6809 (=Between cysts) Total 349 806.74
C(4) Between populations 5 0.32 0.064 63.74 * 0.0011 Within populations 18 0.02 0.001 1.55 NS 0.0010 (=Between sub-pops) Within sub-populations 326 0.21 0.001 0.0006 (=Between cysts)
Total 349 0.55
C(5) Between populations 5 0.30 0.602 74.35 * 0.0010 Within populations 18 0.01 0.001 1.32 NS 0.0000 (=Between sub-pops) Within sub-populations 326 0.20 0.001 0.0006 (=Between cysts) Total 349 0.52 1 Tabular F value at the 5% level with Degrees of Freedom 5 = 2.77 18 = 1.70 51
C (2) and C (3) measurements in the same way, there is evidence
(at the 5% level) that the variation between populations is greater than variation within populations and that the latter is greater than within sub-populations. With the C (4) and C (5) measurements only the variation between populations is great; variation within populations and within sub-populations are small and similar, indicating that these characters are consistently and markedly different between populations.
In Table 13 the results of hierarchical analysis of 5 measurements the made on hatched larvae from 4 sub-populations ofA F1 cyst-generation
(see Fig. 5) are presented. The characters measured and represented by L (1), L (2), L (3), L (4) and L (5) are body length, stylet length, distance from head tip to median bulb valve, distance from median bulb valve to excretory pore and tail length, respectively.
For L (1) and L (3) the variations between and within populations are similar and the variation within populations (= between sub-populations) is significantly greater than that within sub-populations (= between larvae from a batch of cysts). This indicates that body length and distance from head tip to median bulb valve of larvae hatched from a singlecysesprogeny are less variable than those of larvae hatched from different cysts of the same population. Thus it is established that these characters are not significantly different between populations because the variation within populations is too great. For L (2), L (4) and L (5) the variation between populations is significantly greater than within populations, which in turn is greater than that within sub-populations. However, although variation of these larval measurements decreases from ana between to within populations,4t he excess of variation between populations is greater than the excess within populations, the variations shown by the observed F values between and within 5a
Table 13. Hierarchical analysis of variance of measurements on hatched generation-cysts of six populations. larvae from F1
Measurement Source of variation DF SS MS F F5%' Variance:, component
L(1) Between populations 5 239.14 47.828 1.18 NS 0.1360 Within populations 16 646.00 40.375 12.04 * 2.4680 (=Between sub-pops)
Within sub-population308 1033.20 3.355 3.3545 (=Between larvae)
Total 329 1918.30
L(2) Between populations 5 3.70 0.740 32.17 * 0.0131 Within populations 16 0.37 0.023 11.76 • 0.0014 (=Between sub-pops) Within sub-population308 0.60 0.002 0.0020 (=Between larvae)
Total 329 4.67
L(3) Between population 5 2.73 0.547 1.34 NS 0.0025 Within populations 16' 6.52 0.408 5.60 * 0.0223 (=Between sub-pops)
Within sub-population308 22.43 0.073 0.0728 Total 329 31.68
L(4) Between populations 5 14.33 2.866 6.80 * 0.0446
Within populations 16 6.74 0.421 6.69 * 0.0239 (=Between sub-pops) Within sub-population308 19.39 0.063 0.0629 (=Between larvae)
Total 329 40.46
L(5) Between populations 5 8.23 1.645 4.22 * 0.0229 Within populations 16 6.25 0.390 5.33 • 0.0221 (=Between sub-pops)
Within sub-population308 22.57 0.073 0.0733 (=Between larvae)
Total 329 37.04
' Tabular F value at the 5% level with degrees of freedom 5 = 2.85 16 = 1.70
53
Table 14. Hierarchical analysis of variance of measurements on hatched larvae from single cysts of the F1 generation of two populations. 1 Measurement Source of variation DF SS MS F F5% Variance component L(1) Between populations 1 7.88 7.876 0.29 NS -0.1289 Within populations 18 489.80 27.211 8.02 * 1.5880 (=Between cysts)
Within cysts 280 949.49 3.391 3.3910 (=Between larvae) Total 299 1447.20 L(2) Between populations 1 5.03 5.029 632.52 * 0.0335 Within populations 18 0.14 0.008 3.72 * 0.0004 (=Between cysts)
Within cysts 280 0.60 0.002 0.0021 (=Between larvae) Total 299 5.77 L(3) Between populations 1 0.01 0.009 0.03 NS -0.0020 Within populations 18 5.58 0.310 5.58 * 0.0170 (=Between cysts) Within cysts 280 15.54 0.055 0.0550 (=Between larvae) Total 299 21.13 L(1%) Between populations 1 23.89 23.885 107.06 * 0.1573 Within populations 18 4.02 0.223 3.94 * 0.0111 (=Between cysts) Within cysts 280 15.88 0.057 0.0567 (=Between larvae) Total • 299 43.78 L(5) Between populations 1 2.04 2.042 10.27 * 0.0123 Within populations 18 3.58 0.199 2.76 * 0.0084 (=Between cysts) Within cysts 280 20.16 0.072 0.0720 (=Between larvae) Total 299 25.78 1 Tabular F value at the 5% level with Degrees of Freedom 1 = 4.41 18 = 1.70 54 populations in characters L (4) and L (5) are nearly the same, indicating that they vary as much within populations as between populations. The observed F values for L (2) indicate that variation between populations (32.17) is greater than that within populations
(11.76) and that therefore stylet lengths are quite different between populations.
The results of the analysis of measurements of larvae hatched from
generation of two single cysts of 1 sub-population of the F1 populations (UK-2 and UK-3) are presented in Table 14. As in
Table 13, the variations of L (1) and L (3) are similar between and within populations, and the variation within populations (= between cysts) is greater than within cysts (= between larvae) indicating great • variability. In L (2) and L (4) the variation between pop- ulations is greater than within populations (which in turn is greater than within cysts, indicating that variability decreases as the degree of inbreeding is stronger) and the variation between populations is by far much greater than in character L (5).
To summarize it can be said that variation in cyst measurements of the
populations studied is greater between populations than within and that it decreases under the imposed conditions of inbreeding. Variations in larval measurements decrease in the same way but L (1) and L (3) showed similar variation between and within populations.
b) Density of nematodes in the roots.
Measurements of second stage larvae from G. rostochiensis population
(UK-3), reared at different inoculation densities are shown in
Table 15. 55
Table 15. Effect of different nematode densities on measurements (um) of second stage larvae (Means of 25 -observations)
DENSITIES Total length Stylet length Dist.to Dist.M.B.V. Dist to Tail M.B.V. to E.P. E.P. length
Low 452.9 21.9 67.9 33.4 101.0 48.3
Medium 447.0 21.8 66.5 32.6 98.0 48.0
High 433.1 21.6 65.2 31.7 98.0 47.1
S.E.D. 4.5 0.1 0.7 0.7 1.0 0.6
L.S.D. 5% 9.0 0.2 1.4 N.S. 2.0 N.S. 1% 12.1 0.3 1.9 2.7
The results indicate that most of the measurements of larvae from low and medium densities (1.25 and 12.5 larvae/g of soil respectively) were significantly different (at the 1% level) from those of larvae from the high density (125 larvae/g of soil). Distance between median bulb muds as valve and excretory pore and tail length were not affected asA the other measurements, although they also were shortened as the density increased. c) Temperature
The means and standard deviations of measurements of second stage larvae of a G. rostochiensis (UK-3) and a G. pallida (UK-2) populations are presented in Table 16.
Larvae to be measured were hatched from cysts formed on potato plants grown at different temperatures. Stylet lengths of G. rostochiensis- larvae from the 10°C treatment were not measured because the larvae failed to hatch. These results indicate that although stylet length of G. pallida larvae was only affected slightly and only by the
10°C treatment, body length measurements of both populations were greatly affected. In both populations body length increased as the temperature rose. 56
Table 16. Means and standard deviations of second-stage larval measurements of G. rostochiensis and G. pallida populations reared under different temperatures.
TEMPERATURES n UK-3 (G. rostochiensis) n UK-2 (G. pallida) (°C) Stylet length Body length Stylet length Body length
10 29 417 I 38.9 25 22.4 I 0.49 469 -1 47.4
14 11 22.1 ± 0.79 488 35.0 25 23.8 ± 0.67 516 ± 24.2
18 25 22.1 ± 0.49 472 I 16.1 25 24.1 ± 0.63 522 f 30.3
•Not measured
d) Daylength
The means and standard deviations of stylet and body length measurements of second stage larvae hatched from cysts of G. rostochiensis (UK-3) and G. pallida (UK-2) formed on potato plants grown under different daylengths are presented in Table 17. These results indicate that as daylight duration was increased the dimensions of stylet and body length were also increased.
Table 17. Means and standard deviations of second-stage larval measurements of G. rostochiensis and G. pallida populations reared under different daylengths.
DAYLENGTH n UK-3 (G. rostochiensis) n UK-2 (G. pallida) (hrs) Stylet length Body length Stylet length Body length
12 20 21.5 1 1.01 477 + 30.8 25 23.3 -1 0.32 478 f 38.1
16 23 22.3 1. 1.03 501 1. 19.6 24 24.0 I 0.27 494 ± 27.6
e) Deviations
During the various studies on the use and measurement of morphological characters to identify populations of potato cyst-nematodes, unusual factors have been observed. Fig. 7 shows some of these deviations which Fig. 7. Photographs showing some "deviations" from normal features in some potato cyst-nematode populations.
"Giant" and normal size second stage larvae of a Peruvian G. rostochiensis population.
Cyst ,:erminal pattern Meloidogyne-like (left);cyst fenestralia with double fenestration.(right)
Lateral position of inverted "V" arms (left); reticulate pattern of a G. pallida population.(right) 57
R. 58 were found in either second stage larvae ('giant larvae') or cyst terminal areas (lateral position of inverted "Ni" feature;
Meloidogyne- like pattern and bifenestra).
B Identification of populations under study
As mentioned earlier, most if not all of the measurable characters of second-stage larvae and cyst terminal areas are variable and overlap forA two species of potato cyst-nematodes. However, the earlier results (pA2 ) on the most useful characters and the minimum number of observations to be made to separate the species-, enable diagnoses to be made with some degree of confidence. a) Second-stage larvae
The means and standard deviations of four sets of measurements made on second-stage larvae of populations from different countries are presented in Tables 4Imi.51114TemWWhe populations in this table have been divided into two groups according to their places of origin
(Andean and Others) and arranged according to the latitudes of their places of origin - from north to south. The last column indicates the species present in each population based on the dimensions of . the features measured and their respective standard deviations.
Populations' measurements which do not seem to fit either species and have large standard deviations have been considered as mixtures of the two species. (e.g. PJ-24; PJ-29; PJ-36; PJ-39; PP-1;B0-1;B0-5;
PP-22; B0-13; PA-2; PA-3; BO-19; BO-20; BO-21; BO-22; BO-24; BO-25;
IT-1; IN-1).
The features used above to classify the populations of potato cyst-nematodes were also used to show the relative similarity of some of those populations by canonical variate analysis. With four sets of variates each population can be represented by a point in four dimensional space, and four axes were derived on which to 59
plot the means (Table 18). The first of these axes is the vector
which accounted for most between-population variation, the second .
is the vector at right angles to the first which accounted for
most of the remaining between-population variation, and so on.
The first two axes accounted for 76.6% of the total variation and
are represented in Fig 8 with the contribution (loading) of each
character to each of the axes. On axis 1 and 2 the characters
which contributed most are stylet length (SL) and distance of head
tip to excretory pore respectively(EP).
Table 18. Contribution of each character mean, on the canonical axes as a loading factor and the percentage of variance of each of the axes. AXES Character 1 2 3 4 BL 0.0240 0.0507 0.0399 -0.0012
SL -1.0403 0.8329 - 0.6204 0.3959
EP -0.0981 -0.2628 0.0968 0.1129
TL -0.0818 -0.1103 -0.0743 -0.3607 % Variance 46.9576 29.6041 14.1103 9.3280
Although in Figure 8 axis 1 separates the G. rostochiensis
from the G. pallida populations, there are some populations which
fall in between (NZ-1; IC-2; IC-3; PC-1; PP-7; B0-2; B0-13; PP-22
and EC-1) and may be considered as mixed populations. However,these
populations of uncertain identity lie in .between either because they
are truly mixed populations or because characters contributing to
this axis misclassified them. According to the measurements in Appendix
Tables A- and .5 the populations EC-1, PC-1, PP-7, B0-2, IC-2, IC-3
and NZ-1 have been misclassified because all of them belong to
G. pallida; B0-13 and PP-22 are mixtures but G. pallida predominates
over G. rostochiensis.
Axis 2 O G.pollido 4-000 • G.rostochiensis O Both oUK I oPJ 38 3200
oPK2 eCY1 2.400 oNZ I olC3 0B022 C01 0 Phdopni 0IC 2 1.600 o(VA' PL 3 0 0 oEC I 8018 EC2 oPC I PL4 0PP 24 0.800- PP2I oPP7 ft I T2 190g114•• 4: A A oPP9 "'SP2 pj5 J23 .1 0 o pj 2.8i3vig 0B013 api33opp i o jyzo 802 0000- PJ 20 0 00 0805 OPP8 pj18,,, .114 P,J8 toPJ 29 r-1 '''PJ3 K3 Ft oPP22 B021o9PJAP223 oB023 oUK3 PJ 25o P-14pLui °p PPI2 0A300 N3(508025 -0-800 PJ2b0 PL2o0B012 602400PA2 BORt) JAI (01T I 0PJ Ib oSPI B0200 CHI oPH I -1-600 08015 0SAi oPn °GE! G R1 ooPJ I ooGR 3 PA4 &NI o B09 -2.400
,957. confidence radius -3.200 -2.40 460 -080 0 0.80 1.60 2.40 3.20 400 Axis I
Fig. 8. Spatial relationships of ninety-five populations of potato cyst-nematodes on the first two axes of the canonical va - riate analysis of 2nd. ge larvae.The 95% confidence radius corresponds to X /h 61
Other populations which lie within either the G. rostochiensis or the G. pallida groups are mixtures where
either G. rostochiensis (PJ-29, B0-5, B0-24) or G. pallida (PJ-8
PJ-24, PJ-36, PP-1, B0-l,etc.) predominates.
Nevertheless, as this figure is presented to show the relative similarity of populations rather than to facilitate their identification, it can be seen that most populations coming from any one geographical, area are grouped in the figure and overlap each other; EC, PJ, PH,
IC, GR, etc. Although most of the BO populations are generally quite scattered, indicating a wide range of diversity, some of them show similarity with populations JA, SA, GE and GR. PP populations are also scattered but less so than BO populations.
The population CY-1 (G. rostochiensis) occupies a quite different possition, having no similarity with the rest of the populations.
POpulation IN-1 also occupies a unique position and seems to be a G. pallida population possessing G. rostochiensis characters. b) Cyst terminal areas (Fenestralia)
The means and standard deviations of four direct measurements and two derived characters of cyst terminal areas of 25 populations are presented in Table6(A'p).The last column indicates the species apparently present in each population after comparison of the
:lean of each character with those used to discriminate species (p.4.3)
Populations with large standard deviations and/ or contradictory values in some character have been considered as mixed populations e.g. PP-9 (number of ridges 11.6 3.3 and distance B, 44.8 3.2) B0-18 (number of ridges 12.7 ± 2.5 and B = 49.2 ± 2.9) IC-2 and
IC-3 (also with similar values for these characters) 62
Table 19. Contribution of each character mean to the canonical axes as a loading factor.
AXES
Character 1 2 3 4 L 0.2156 -0.2524 -0.0776 0.4906 -0.1053 -0.1605 0.3774 -0.5595 B 0.0227 0.1016 0.0449 -0.0025
n -0.3615 -0.3374 -0.0321 0.1578
For the canonical variate analysis four axes have been devised and the contribution of each character on each of the axes is
presented in table 19. Table 20 presents the coordinates of the
populations on each axis.
As the first two axes account for 82.2% of the total variation,
they are represented in Fig. 9. On axis 1 and 2 the characters which
contributed most are number of ridges (n) and fenestral length (L).
Although in this figure axis 1 separates the G. rostochiensis
from the G. pallida populations, there are some populations (PL-3,
NZ-1, IC-3 and IC-5) which fall in between. Possible reasons for
this are that they are true mixtures of species or that characters contributing to this axis misclassify them. In this group of uncertain identity, IC-3 is the only one which seems to be a mixture in Table5l.ApIA) suggesting that IC-5, NZ-1 and PL-3 have been misclassified. The
population IC-4 (G. rostochiensis) occupies a different position
from the rest, due to the high number of ridges. Nevertheless,
the validity of these results and the real identify of these populations will be confirmed later on when results from other sources (e.g.
female colour, second stage larvae, differential plants) are tabulated 63
Table 20 Co-ordinates of populations on four canonical axes.
POPULATIONS AXES
1 2 3 4 co-1 0.8792 -0.3453 0.2260 0.0140 EC-1 1.7883 0.4135 0.1512 0.1625
EC-2 1.7905 0.6241 0.4891 -0.0850 PC-1 0.5903 -0.1476 -0.1218 -0.6122 PL-3 -0.0237 0.4042 0.0159 -0.5818 PL-4 1.3284 0.5016 -0.1210 0.1300
PJ-32 1.7119 0.1713 -0.2286 0.8698
PJ-38 1.1890 -0.7529 -0.1232 -0.6940 PK-2 1.5633 0.5183 0.4776 -0.0534
PP-8 -1.2478 1.4591 -0.1454 -0.0926 PP-9 -0.6965 0.4452 -1.9005 0.0891
B0-18 -09741 0.2964 -1.4554 0.0502 B0-22 0.6262 0.0215 -0.5312 -0.2806 IC-1 -1.4042 0.9139 0.1985 0.1521 IC-2 -0.4902 -1.1659 1.0438 -0.3781 IC-3 -0.2714 -1.1622 -0.1458 -0.1072
IC-4 -3.4601 -1.3393 -0.0089 0.6346 IC-5 0.1693 -0.9475 -0.6636 -0.5382
UK-1 1.7247 -0.7078 -0.1607 0.6284
UK-2 0.9081 -0.7335 0.3405 0.7437
UK-3 -2.2154 -0.4681 0.1800 -0.1034
IT-2 -1.4948 1.1345 1.0228 0.0413
SP-2 -1.3229 0.3992 0.1805 -0.2011
CY-1 -2.2274 0.3449 0.5993 0.2802 NZ-1 0.1733 0.0425 0.4727 -0.1544
% Variance 65.2522 16.8971 12.7100 5.1407
Fig. 9. Spatial relationshii)s of 4..wenty-five populations of potato cyst-nematodes on the first two axes of the canonical variate analysis of cyst terminal areas.The 95% confidence radius corresponds
tofXT /n Axis 2 4000 o G.pailida • G. rostochiensis 3200. o Both
2400
1.600 OPP8 • IT 2 IC I 0.800 EC 2 0CY I GISP 2 epp9GPL3 PL400Pcp 0EC I °8018 NZ I 0000 co ()BO 22 PJ 32 opc OUK3 ()COI O UK I -0-800 UK20 IC2 c/IC5 PJ38 ©01C3 •1C4 -I.b00
-2.400
-3.200
95% confidence radius -4.000 3.20 2.40 I.50 0.80 0 0.80 1.60 2.40 3.20 400 Axis I 65
and compared. c) Image analysing computer The means and standard deviations of area and perimeter of second stage larvae and males of six populations of potato cyst-nematodes (2 G. rostochiensis, 3 G. pallida and 1 mixed) determined by the
image analysing computer (Quantimet, - ) are presented in Tables 21 and 2Z. None of the measurements considered are useful for differentiating the species, since their values overlap .inseparably. Table 21. Means and standard deviations of measurements of second stage larvae of some populations by the Image Analysing Computer.
POPULATIONS n AREA (sq.um) PERIMETER (um) Species' CO - 1 51 20842 ± 1703 1545 t 204 G. pallida
PL - 4 26 16370 I 1810 1445 I 78 G. pallida UK - 2 28 21439 ± 1494 1664 ± 72 G. pallida
BO - 22 27 19396 t 1703 1587 I 91 G.p/G.r. PA - 2 29 17380 ± 1807 1485 ± 61 G. rostochiensis UK - 3 26 20330 t 1481 1615 t 80 G. rostochiensis
Identified by female colour and / or measurements of 2nd stage larvae and cysts.
Table 2Z. Means and standard deviations of measurements of males of some populations by the Image Analysing Computer.
POPULATIONS n AREA (sq.um) PERIMETER (um)
CO - 1 51 16367 ± 1944 1668 ± 120
PL - 4 54 12236 ± 1678 1466 ± 126
UK - 2 50 14626 ± 1459 1580 t 105
BO - 22 49 13390 ± 1047 1545 t 84
PA - 2 53 12310 ± 1349 1441 ± 90 UK - 3 52 14807 t 1144 1643 ± 121 66
iii) Discussion
Diagnostic characters of cyst terminal areas and second-stage - larvae of potato cyst-nematodes vary due to intrinsic factors
(e.g. genetic) and extrinsic factors (e.g. density, temperature and daylength)
The genetic variability of cyst terminal areas characters should be unchanged or decreased by inbreeding. As the level of inbreeding becomes greater so the variation between populations increases and the variation within populations decreases, although this is only true for those characters of high diagnostic value e.g., number of ridges between vulval fenestra and anal pore and fenestral length. However, in characters such as vulval fenestral width and distance between anal pore and nearest edge of fenestra, the variation within populations is as large as that between populations, and variation within a population may be as large as that found between populations belonging to different species.
The variability of diagnostic characters in second-stage larvae is also affected by intrinsic and extrinsic factors. Body length, distance from headtip to median bulb valve (MBV) and tail length (TL) are characters with greater variation than those such as distance between MBV to excretory pore (EP) and stylet length (SL). This means that these first three characters are of little value as diagnostic characters because the variability within populations is as great as that between populations. They seem also to be affected by density of nematodes in the roots, temperature and daylength, body length being shorter when numbers of nematodes in roots are great, at low temperatures (10°C) and in short daylengths (12 hours).
Although SL is also affected by the factors mentioned above, it seems to be the character with the smallest variation within populations; and its variation between populations is the largest, 67 indicating its great value as a diagnostic character for identifying populations and separating the species of potato cyst-nematodes.
With the above knowledge and that obtained from the measurements of morphological characters in second-stage larvae (95 populations) and cyst terminal areas (25 populations) it is clear that 15 observations per population are sufficient to enable diagnosis of species to be made with confidence.
The most reliable character in second-stage larvae is stylet length because it permits clear recognition of species, even when populations are mixed.
Measurements of cyst terminal areas made on 25 populations indicate that numbersof ridges, Granek's ratio and vulval fenestral area are the best diagnostic characters.
When relative similarity of these populations was mapped by canonical variate analysis using cyst characters, the positions occupied by populations of G. rostochiensis and G. pallida were more widely separated than when larval characters were used.
The use of the Image Analysing Computer to measure perimeters and areas of second stage larvae and males of 8 populations was of no use for identifying species because values overlapped and were very variable.
1.3. ELECTROPHORESIS
Analysis of tissue proteins by gel-electrophoresis has been used as a taxonomic tool in many groups of animals (Sibley, 1962;
Leone, 1964; Dessauer & Fox, 1964) and plants. (Danielson, 1956;
Ibrahim, 1975; Stegemann, 1976). Electrophoretic separation of proteins has also been useful in recognising species of nematodes
(Benton & Myers, 1966; Dickson et al 1970; Gysels, 1968, 1970;
Eriksson & Granberg 1969; Trudgill & Parrott, 1972; Trudgill, 1972;
Greet, 1972). 68
Disc electrophoresis gives a characteristic pattern of protein
bands but the pattern depends on the age and condition of the nematodes
(Ishibashi, 1970; Evans, 1971; Trudgill & Carpenter, 1971).
Nevertheless, Dickson, et al. (1970) found that species of Meloidogyne
could be differentiated. Similarly Trudgill and Carpenter (1971)
showed that Globodera species were different; G. rostochiensis was
distinguishable from G. nallida by the absence from G. rostochiensis
of two bands and by the presence of another rarely found in G. pallida.
Differences between pathotypes were not found.
The experiment described below compares the protein patterns
produced by disc electrophoresis from British and Peruvian Globodera spp.
and studies the effect that daylength may have on them.
(i) Materials and methods.
Females of populations to be studied were produced by inoculating
14 day-old potato plants (var. Arran Banner), growing in 9 cm diameter
plastic pots, with 6000 freshly hatched larvae. The potato plants were
grown under controlled conditions in SAXCIL growth cabinets at 12
and 16 hours daylength. At the same time, three nematode-free potato
plants were also grown. The populations studied were, UK-3, PP-8
(British and Peruvian G. rostochiensis respectively) and UK-2, UK-1
and PL-4 (formerly British pathotype E and B and Peruvian G. pallida
respectively). Forty-two days after inoculation the soil was washed
from the plant roots and the clean roots transferred to a one litre
glass beaker. By shaking the roots females were dislodged from them
and separated from root debris by an adaptation of the prototype for
the fluidising column (Trudgill, Evans & Faulkner, 1972). A small
sieve containing females and debris was fitted in the top of the column.
After leaving the sieve and its contents exposed to the air for one
minute the water supply was turned on at low speed. The females 69
floated, leaving most of the debris behind, and were collected from the collar of the column in a small beaker.
A solution of soluble nematode proteins was prepared by grinding approximately 200 females (45 n1) with 45 pl of extraction buffer (Staples
& Stahmann, 1964). This suspension was then centrifuged for 5 minutes at 3,000g to prevent breakdown or degradation of proteins. The separated liquid was layered on to 7.5% acrylamide gels. The gels were cast in 65x5mm (i.d.) tubes using acrylamide containing 5% NN- methylene bisacrylamide and TRIS buffer, as described by Ornstein-&
Davis (1964) except that spacer gels were photopolymerised on top of the separating gel. Ten or 15 ul of the protein extract were layered onto each gel, and four drops of 0.1% bromophenol blue in ethanol added to the tuffer solution in the top cathode compartment. Electrophoresis was started with a current supply of lmA per gel until the marker band appeared at the interface of the large and small pore gels, when the current was increased to 4mA per gel. Electrophoresis continued for about 75 minutes at 5°C until the bromophenol blue marker reached the lower calibration mark on the gel tube. As rapidly as possible the gels were removed from their tubes and stained by immersion in 1% naphthalene black in 7% acetic acid for a minimum of one hour. The gels were then destained and stored in 7% acetic acid. A standard population was included in each run, and soluble proteins of potato plant roots were also prepared for electrophoresis by macerating and squeezing frozen root pieces enclosed in muslin bags.
(ii) Results and Discussion
The protein patterns in Figure 10 shows that the populations studied fall in two groups. UK-3 and PP-8 are members of the same species G. rostochiensis and UK-2, UK-1 and PL-4 are populations of G. pallida. However, the band patterns obtained for the two species Fig. 10. Gels showing the protein patterns of immature females of British (UK-1,UK-2 and UK-3) and Peruvian (PP-8 and PL-4) populations of G. rostochiensis and G. pallida collected from potato plants grown under 12 and 16 hours daylength.The lines on the right side of each pair of gels indicate the bands present.Bands 2,3,12 and 14 are common for both species. G.rostochiensis G.pallida UK 3 PP 8 PL 4 UK 2 U K1
12 13
14
15 71
of potato cyst-nematodes differ from those of Trudgill and Carpenter
(1971), as will be discussed.
The dotted line in Fig. 11 indicates the position of Trudgill
& Carpenter's band N o. 17 (band No. 12 on Figs. 10 and 11), common
for both species, The next band down is No. 18, found only in
G. pallida. However, band No. 19 (band No. 14 in the present study)
considered by Trudgill & Carpenter as characteristic for G. rostochiensis
also occurs in band patterns of G. pallida (PL-4, UK-2 and UK-1)
suggesting that this band is common to both species. 0
Figure 11 Protein band patterns obtained for the two species of potato cyst-nematodes in the present study (right side) and by Trudgill & Carpenter (1971, left side).
ftrropmge" 2 3 5 7
orfrearesA 9 16 - 1? 12 19 14
G.rostochiensis G.pallida G.rostochiensis G.pallida G.pallida (UK-3 and PP-8) (PL-4) (UK-2) 72
Although UK-1, UK-2 and PL-4 (members of G. pallida) all have bands
12 and 13,differences between UK-2 and PL-4 occur (British and Peruvian populations respectively). Band patterns in Fig. 10 show that in spite of presence in both of them of bands 12 and 13, characteristic
of G. pallida, their positions are not quite the same. Further differences are the presence of bands 5, 7 and 9 in the British population but bands 6,8, 10 and 11 in the Peruvian. Both populations lack band 1 found by me in G. rostochiensis and band 15. Band 5, found in British G. rostochiensis and British G. pallida, does not occur in
Peruvian G. pallida. The bands 2,3,12 and 14 are common to both species.
Furthermore, although only poor resolution•of bands was obtained for Uk-1 (formerly British pathotype B), the positions of bands 12 and
13 suggest a closer relation with population PL-4 than with UK-2
(formerly British E).
In the study of soluble protein in potato plant roots no clear band patterns were obtained, probably due to either the small amount of roots used or their poor protein content.
1.4 SCANNING ELECTRON MICROSCOPY
Scanning electron microscopy makes it possible to view features which are difficult to discern with the light microscope and morphological characters have been revealed in biological specimens, which bring better understanding in taxonomic and evolutionary studies.
In nematology, it has proved useful in the development of both of these fields. (Lippens & De Grisse, 1974; De Grisse et al. 1974; Stone 1975).
Different sorts of tissues and structures have been studied, e.g. hard surfaces, such terminal area of cysts of Heterodera spp. and
Globodera spp. (Lamberti, 1971; Green, 1971; Mulvey 1973, 1974); heavily sclerotised structures ej.spicules (Clark, et al. 1973); and softer areas such as labial regions (De Grisse, 1969a; De Grisse and
Loof 1970; Smart, et al. 1972; Stone, 1972). 73
Many techniques for the preparation of specimens have been
described but most require much time and careful handling, due to the
problems of distortion of shrinkage of surface structures during the
removal of volatile materials (chiefly water) from the nematode tissues.
(Green, 1967; Pasternak, 1970; Stone & Green, 1971; De Grisse, 1973;
Turner & Green, 1973; Turner & Smith, 1974; Green et al. 1975).
Green (1967) working with Meloidogyne females, males and females of
Heterodera species, showed how sharply the cuticular structures are
defined under the stereoscan microscope. The same author (Green, 1971),
comparing the morphology of terminal area of the round-cyst nematodes
(Globodera spp.) found distinctive characters (just visible by
transmitted light) which showed that differences between G.
rostochiensis and the then undescribed G. pallida were as great
as those between described species.
Stone (1972), examining the configuration of the oral region
of second-stage larvae found unsuspected morphological differences
between and within the genera Heterodera and Globodera. In the
former, the sub-median lips of the larva are fused with the 'oral'
disk, while in the latter group these sub-median lips are distinct.
although completely reliable differences in broad head characters
were not found between species of potato cyst-nematodes and related
species in the same genus, G. rostochiensis can be distinguished from
G. pallida. The first has 'oval' shaped lips and oral disk contours,
but G. pallida has 'rectangular' lips and disk contours and sometimes
the median division between the lips of one or both of the sub-median
pairs is absent.
(i) Materials and methods.
Second-stage larvae and cysts of five potato-cyst nematode
populations were examined in a Cambridge Instruments Stereoscan MK IIA at 5 kv accelerating voltage. The populations studied were UK-1,
UK-2, UK-3, PL-4 and PP-8. 74-
The specimens were prepared by following a slight modification of the technique described by Stone & Green (1971). -Fixed larvae in 4% formaldehyde were transferred to 3 cm. diameter glass cavity blocks and washed in two changes of distilled water. The blocks were placed in a dessicator containing dry acetone and left at room temperature for at least 24 hrs and then transferred to acetone by vapour exchange.
After acetone had replaced the water, the specimens were placed in a
50/5C solution of Spurr's low viscosity epoxy resin (Spurr, 1969) and acetonet and were kept in an acetone-filled desiccator for approximately
21/2 hours. Then the blocks were removed from the desiccator and fresh Spurr's resin was added. The blocks were covered with glass tops and after 6 hours the specimens were transferred to fresh resin, and placed in a dry desiccator again and kept at 4°C for 2 days.
After infiltration the excess of resin was washed off by holding the specimen on a needle and dipping it briefly in 96% alcohol. The specimen was then immediately placed onto an adhesive-coated stub.
Specimens positioned on stubs were kept overnight in an oven at 70°C to polymerise the resin. Next day specimens were coated with a layer of gold, 200 R thick, on a rotating turntable to give even deposition.
As the cuticles of cysts are tough and their surface patterns are not easily degraded (Green, 1971), dry cysts were stuck onto adhesive coated stubs and plated in vacuum with gold/palladium.
(ii) Results and discussion.
The lip regions of populations examined are shown in Fig. 12 (a,b,c,d, e) Fig.12 shows that lips and oral disks of second-stage larvae of PP-8 (a) and UK-3 (b) populations (British and Peruvian respectively) are similar to those described by Stone for G. rostochiensis. In the same Figure, c, d and e are UK-1, UK-2 and PL-4 respectively and show that their features are similar to those described by Stone for G. pallida.
However, the differences in lip region and oral disk contours, described a) b)
c)
d) C)
Fig. 12. Scanning electron micrographs of heads of second-stage larvae. a) and b) Peruvian and British G. Rostochiensis (PP-8 and UK-3, respectively); c) and d) British G. Pallida (UK-1, UK-2); e) _Peruvian G. Pallida (PL-4). All approximately x13,000. 76
Fig. 13. Scanning electron micrographs of the terminal area of British and Peruvian G. Rostochiensis populations. a) and b) Vulva and anus far apart on populations UK-3 and PP-8, respectively (x700); c) and d) Vulval shape of same populations (x2,400); e) Anal pore (x2,400). 77
a) b )
c) d)
e) f)
Fig. 14. Scanning electron micrograph of the terminal area of British and Peruvian G. Pallida populations. a), c) and e) Vulva and anus close and few ridges on populations UK-2, UK-1 and PL-4 (x900); b) and d) Vulva shape of populations UK-2 and UK-1 where the vulva has fenestrated (x2,000); f) Vulval shape of population PL-4 (x1,800 78 as "oval" for G. rostochiensis or "rectangular" for G. pallida, are not completely distinct. However, the length of the oral disk (across the stoma) in G. pallida tends to be greater than in G. rostochiensis but the width seems to be more or less the same for both species. Also, fused sub-medium lips were frequently observed in specimens of
G. pallida, both British and Peruvian populations.
Figures 13 and 14, show the presence of distinctive characters in the vulval region of cysts from populations PP-8 and PL-4, resembling those found by Green (1971) in populations of the potato cyst- nematode that became G. rostochiensis and G. pallida. The fenestra shape in population PP-8 (Fig. 13) is 'rounded' (length CND bredth), but in PL-4 (Fig. 14) it is oval and the anus is located at one fenestra length from the fenestra edge while in PP-8 it is located at two or more fenestra lengths. The number of ridges between fenestra and anus is higher in PP-8 than in PL-4. The differences indicate that populations PP-8 and PL-4 belong to the species G. rostochiensis and G. pallida respectively.
1.5. MATING.
Morphological criteria form the basis of species differentiation but solaetimes morphological and ecological data conflict and difficulty arises in d,ciding the relative importance of the different criteria. Biological and physiological affinities also have to be taken into account.
Ecological factors have two-fold effects upon the individual: the direct effects upon the phenotype, and their indirect effects upon the genotype, since the genetic structure of populations is the outcome of longterm interactions between genetic processes which generate and maintain genetic variation, and environmental factors, which mould that genetic variation. 79
Speciation is ultimately an adaptive process that involves establishment of intrinsic barriers to gene flow between closely related populations by development of reproductive isolating mechanisms, which is fundamentally different from the process involved in local adaptation (Carson, 1971). The latter entails only minor genetic adjustments, whereas speciation frequently involves a reorganisation of some crucial component in the genetic system that results in a quantum step towards the origin of interspecific differences (White, 1973).
The species concept is defined as breeding communities, capable of producing viable and fertile offspring (Loof, 1967) or as groups of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups (Mayr,
1969). A failure of individuals to interbreed indicates genetic incompatibility and leads to the establishment of new species.
These new species of sexually reproducing individuals arise only after a period of adaptation (Bush, 1975).
Variation in nematodes is recognised to be largely genetically controlled (Sturham, 1973). Bovien (1955) discusses interbreeding experiments as a means of determining biological relations of the various stem nematode races. Mayr (1964) considered the possibility that host races among parasite nematodes might be regarded as sibling species, defined as sympatric forms, morphologically similar and closely related, but with specific biological features and reproductively isolated. Sturham (1971) elaborated Mayr's sibling species concept with regard to plant parasitic nematodes, and maintains that this category of species is well applicable among nematodes.
When cyst-nematodes were thought to be one species, there was much talk of the danger of populations adapting to new hosts being grown frequently. These fears proved groundless and speculation 8o ceased when the species were adequately described and their host ranges determined (Jones, 1972).
The potato cyst-nematodes have a host range clearly separated from that of the other species in the genus, but differences between populations were first observed when some of them were able to reproduce on potato plants containing genes for resistance
(Dunnett, 1957; Jones, 1957).
Parrott (in Jones 1967) studying the inheritance of ability to reproduce on Maris Piper, made single reciprocal crosses between populations. Matings within population groups were equally as successful as selfings, but populations from different groups mated much less successfully. Globodera pallida pathotypes (formerly if and E) interbred freely but fewer fertilisations occurred when they were crossed with males or females of G. rostochiensis (formerly A).
The production of fertilised eggs averaged 63% for crosses between
G. rostochiensis ( A x A ) and 64% for crosses between G. pallida
(E x E; B x B and E x B), but only 20 and 27% between species
(A x B and A x E respectively). The new females produced by culturing larvae from successful crosses on a susceptible potato variety were counted. Not only did fewer females from crosses between species produce eggs but fewer larvae hatched from them and many fewer of these produced F1 females (Parrott 1969, 1972).
Bedi (1968) without evidence of infertility between species of potato cyst-nematodes, made controlled single larva matings between
G. rostochiensis and G. pallida (B) and obtained F1 eggs from 14% of the females.
The species of potato cyst-nematodes are presently considered as sibling species with very similar morphology (Sturhan,1970;
Kuhn, 1971; Stone, 1975). Therefore, the experiment below describes single reciprocal crosses between British and Peruvian populations 81 of potato cyst-nematodes.
(i) Materials and methods.
Potato plants were grown in pots, and larvae added to the soil.
Sixteen days after inoculation the roots were washed free of soil and suspended in water, so that the females remained on them but the males fell away after emerging. The plants were supported on slotted wooden lids placed over buckets containing aerated water.
Beginning four days later, males were collected every day be straining the water through a 150-mesh sieve. The contents of the sieve were transferred to a 100a aperture nylon sieve just submerged in water and only those males passing through the nylon sieve within 2 hours were collected for use. Females were collected forty-two days after inoculation using the technique described in section I. Two sets of plants, inoculated at different times, were used in order to collect fresh males and females at the same time.
The apparatus used by Green (1966) for attraction tests was used for mating single males and females. Agar plates were made by pouring 5 ml of 0.85 water agar into 5 cm plastic petri dishes.
White females were placed in the centre of these plates. A single newly-emerged male was placed next to each female. Twenty dishes were used for each cross. Control females were unmated. The dishes we,•e kept in darkness at 20°C and the females examined for eggs after 4 weeks. If a female contained eggs it was assumed
that it had been fertilised, as sufficient evidence exists to show
that unfertilised females do not contain eggs (abnormal, non-
embryonated eggs may occasionally be found - Ellenby, 1957).
The populations studied were UK-3 and PP-8 (British and Peruvian
G. rostochiensis respectively) and UK-2 and PL-4 (British and
Peruvian G. pallida respectively.)
82
(ii) Results and discussion.
The results in Table 23 show that the populations studied can
be divided into two groups.
Table 23. Total number of females and eggs per female
WITHIN GROUP BETWEEN GROUPS CROSSES CROSSES (6G.r.xG.r.dG.p.xG4.) (dG.r.xG.p.dG.p.xG.r.)
Total No. of females:
Mated 160 160
With eggs 46 15
Mean eggs per cyst: 179.5 109.5
Percentage females with eggs 21.25 36.25 13.75 5.4) Mean 28.75 9.37
Crosses within species gave a higher percentage of successful
matings (29%) and a higher number of eggs per cyst (179) than those between
species (9% and 109). The percentage of fertilised females in the
latter group was higher when G. rostochiensis were used as the males
(13.75%) than G. pallida (5.00%). The results of the tests within
and between groups and populations are shown in Table 24-. From this
table, the condition of males or females due to external factors such
as handling, poor development in host, etc., can be inferred, although
different behaviour may also be involved, such as:-
a) Ability and preference of males to fertilise females.
b) Ability of females to be fertilised.
We can see that the condition of males was similar in all crosses
(20, 15, 20 and 21%), and that G. rostochiensis males were able to
fertilise either group of females (27, 12, 15 and 15%). G. pallida Table 24. Results of crosses within and between British and Peruvian populations of G. rostochiensis and G. pallida. (n = 20)
n Condition and compatibility de condition and capability to to be fertilised (%) fertilise (%) Crosses n with Successful Crosses in with Successful ns x des eggs mating means des x ns eggs % mating means Within Between Within Between
UK-3xUK-1 1 20.00 15.00 UK-3xUK-3 20.00 27.50 PP-8 10.00 PP-8 35.00 UK-2 2 0.00 2.50 UK-2 20.00 12.50 PL-4 5.00 PL-4 5.00 Mean 8.75 20.00 condition
PP-8xUK-3 35.00 27.50 PP-8xUK-3 10.00 15.00 PP-8 20.00 PP-8 20.00 UK-2 15.00 7.50 UK-2 25.00 15.00 PL-4 0.00 PL-4 5.00 Mean 17.50 15.00 condition Table 24 (Contd.)
?9 Condition and compatibility as Condition and capability to to be fertilised (%) fertilise (%)
Crosses ?9 with Successful Crosses ?? with Successful ??s x ads eggs % mating means dois x eggs mating means Within Between Within Between
UK-2xUK-3 20.00 22.50 UK-2xUK-3 0.00 7.50 PP-8 25.00 PP-8 15.00 UK-2 40.00 52.50 UK-2 40.00 32.50 PL-4 65.00 PL-4 25.00
Mean 37.50 20.00 condition
PL-4xUK-3 5.00 5.00 PL-4xUK-3 5.00 2.50 PP-8 5.00 PP-8 0.00 UK-2 65.00 UK-2 25.00 20.00 40.00 PL-4 15.00 PL-4 15.00 Mean 12.50 21.25 condition
1 Globodera rostochiensis populations. 2 Globodera pallida populations. 85
males prefer to fertilise their own females (32 and 40%) and not
G. rostochiensis females (7 and 2%). The condition of some females
seems to have been poor, the mean success rate for fertilisation of
UK-3 being 8% and for PL-4, 12%. The success rate for all females
was higher with males from their own group, (15,.27, 52 and 20%)
although 22.5% of UK-2 females (G.pallida) were fertilised by G.
rostochiensis males.
The results also show that British or Peruvian populations
can interbreed but that intra-specific crosses are always the
most successful and produce greater numbers of embryonated eggs.
It also seems that G. pallida males prefer to mate their own females
though the females may not prefer their own males. G. rostochiensis
males seem more able to fertilise the other species, but G. rostochiensis
females seem to prefer their own males. (Perhaps this means that
G. rostochiensis would compete less well than G. pallida in a
situation where both species were present).
1.6 PATHOTYPES
The discovery of some potato cultivars resistant to potato cyst-
nematodes (Ellenby, 1952, 1954) was followed soon afterwards by the
discovery of nematode populations able to overcome this resistance
(Quevedo, 1956; Dunnett, 1957; Jones 1957, 1958). These resistance-
breaking populations were called pathotypes rather than biotypes by
Cole and Howard (1966), since they may be either homozygous or
heterozygous and also describe interspecific units: ecotypes. The
.Federation of British Plant Pathologists (1973) considers pathotypes
a subdivision of a species distinguished by common characters of
pathogenicity, particularly in relation to host range. Bedi (1968)
defined a pathotype of potato cyst-nematode as a group of biotypes
or individuals possessing a certain resistance-breaking property or
specificity. The term specificity is derived from the fact that the 86
major resistance genes used to differentiate pathotypes confer patho-
type-specific resistance. As far as is known pathotype-specific
resistance inhibits female development, virtually supressing cyst
formation in some cases, but does not prevent root invasion or the
development of males (Jones, 1954). Initially, the availability of
only one resistance gene meant that only two pathotypes could be identified; one reproducing on Solanum tuberosum ssp. tuberosum
but not on S. tuberosum ssp. andigena, and one reproducing on both
plants. In most countries these pathotypes were given the code-
letters A and B and it was assumed that all of them were identical.
However, it is now known that there are two species of potato cyst-
nematodes, both of which have pathotypes differing in their ability
to reproduce on potatoes with genes for resistance. For instance,
pathotypes of G. rostochiensis able to reproduce on plants containing
the H gene for resistance have been found in populations from 1 Holland (Kort, 1974) and Bolivia (Trudgill et al., 1970) but no
British G. rostochiensis population able to overcome gene H1 has
yet been found. There are now known to be sources of resistance
other than H and pathotype identification in different countries 1 has developed independently according to the sources of resistance
found by breeders. In the United Kingdom:breeders have found
resistance in lines of S. multidissectum (gene H2) and from another
S. tuberosum ssp. andigena line (gene H ) (Dunnett, 1961 and Howard 3 et al., 1970, respectively). British pathotypes are identified using
these three genes in a series of different hosts.
In Holland, the identification of pathotypes uses a series of
differential plants which includes one with the H1 gene, but also
others from S. kurtzianum and S. vernei. A total of six pathotypes
(A to F) are recognised. Pathotypes in Germany (West) are
identified by using hybrids containing genes for resistance from 87
S. snegazzinii, S. vernei and S. oplocense (Ross, 1972).
Thus, although various schemes for pathotype nomenclature and identification have been proposed (Howard, 1959; Schick & Stelter, 1959; Dunnett, 1964; Huijsman, 1962) there is still a need for a unified system which is both easy to use and readily adaptable to all countries' needs.
The first attempt to resolve this situation appeared in a circular issued as the result of a meeting between British, Dutch and German potato breeders at Gross Lilsewitz, East Germany in 1967.
Pathotypes were denoted by a letter code, with each letter in the code referring to a particular differential host. Capital letters
(A, B, C, D, E) indicated on which plants the pathotype reproduced and a small letter (a, b, c, d, e) placed in brackets, indicated
that no (or insufficient) information was available for that host.
Test plants on which the pathotype did not reproduce were left out
of the code. Trudgill and Parrott (1972) proposed a new scheme
derived from this. Ability to reproduce on a test plant was
indicated by a capital letter, inability by a small one, and untested
by a dash. Further plants containing a new factor for resistance
could be added to the list as required.
The same year, during a meeting of the International Organization
for Biological Control (IOBC) in England, attention was paid to ttis
problem and the major difficulties were indicated. As a result, a
co-operative effort to provide a pathotyping scheme was initiated at
Rothamsted Experimental Station (England),the Institut fur Nemato-
denforschung (West Germany) and the Plant Protection Service (Holland).
Test plants and pathotypes from the different countries were used
by workers in all the countries in parallel tests. After three
years of joint work Stone (1976) writes: "The following was proposed
at a meeting held in November, 1975 in Munster (West Germany) and will 38
be fully documented in Nematologica and in Potato Research.
RO R0 Pathotypes of G. rostochiensis are to be designated 1, R02' 3 and those of G. pallida PA1, PA2, PA3 , according to ability to
multiply on chosen differential Solanum hosts. The resistance in
the hosts is also designated in the same way". He also indicates that
the proposed scheme can be extended if new pathotypes of existing species are found and that six from eleven resistant clones tested distinguished clearly between the G. rostochiensis and G. pallida
populations used (Table 25). The scheme proposed is shown in Table 26.
Table 25. Resistant clones of Solanum ssp. used to distinguish
between G. rostochiensis and G. pallida.
G. rostochiensis G. pallida
S. tuberosum ssp. andigena with gene H3
S. vernei hybr. 65.346/19
S. vernei hybr. C.8099
S. vernei hybr. C.8087
S. spegazzinii hybr. 66.1044/112
S. oplocense hybr. J.7886
Although the first steps to obtain an internationally- recognised scheme have now been taken it seems that there is still not full agreement. Canto (1975) in Peru, testing native and foreign populations of potato cyst-nematode on European differential
plants, found that some "races" could not be fitted into the current classification and proposed a new scheme with the two species and
5 races, of which 2 were not previously recorded. He gave his ▪ Table 26. Designation of pathotrs by their ability to multipin specified potato hybrids.
n G. rostochiensis * Ro indicates G. rostochi- G. pallida ensis, Pa indicates tio 0 G. pallida a P n British .0 -c4 P A 0 ■-,3 rzi 40 0 ig .1-1 a) P s Dutch 4 -4 P4 XI a) 0 N cq A PI .4 ** able to reproduce co Z -P Pc m H m + .1-1 41 Pc ,s1 M ,C 4 0 vi 4 m o 4 •ri o -P P 0 -i-) de German -1-1 0 +D o H o o •ri 4-) ri +2 0 -1-D -I-3 2 0 d 4.) 'ri - unable to reproduce •ri 0 .CI 0 0 d H F-I 4 A N 0 A l'-i Old SAP:1 CIM M A 0 A A M M * * Differential host New - designation Re Ro Ro Ro Ro Pa Pa Pa 1 2 3 4 5 1 2 3 Resistance Code
** S. tuberosum tuberosum S. tuberosum andigena - + + - + + + + CPC 1673 Rol, 4 S. kurtzianum Hybr. KTT 60-21-19 Rol, 2 S. vernei Hybr. Ro G-LKS 58.1642/4 l, 2, 3 S. vernei Hybr. Rol, 2, 3, 4 (VT )2 62.33./3 Pa1,2 S. vernei Hybr. 65.346/19 R01,2, 3, 4, 5
S. multidissectum Pa1 P 55/7
Ro S. Vernei Hybr. l, 2, 3, 4, 5 IMO MID OMI• WM' 69.1377/94 Pal, 2, 3 90
populations numbers for races and letters for subraces.
Eventually, full understanding between the proponents of these
two schemes would result in an international pathotyping scheme
acceptable to all the workers involved either in breeding or
identifying populations of potato cyst-nematodes in different countries,
as Howard (1972) suggested.
The tests described in the present study were done to identify
pathotypes in selected populations, by recording the development of
females (cysts) on potato plants containing some genes for resistance
against the potato cyst-nematodes. Plant material containing all the
genes for resistance known to be useful in pathotype identification
was, unfortunately, not available.
(i) Materials and methods.
This work was done with fifty-three populations (i.e. those
from which sufficient cysts were available to carry out such a large
test) and stem cuttings from 4 potato clones: the susceptible variety
Arran Banner (S. tuberosum ssp. tuberosum) was the control; Maris
Piper (ex S. tuberosum ssp. andigena, CPC 1673) contains gene H1,
(formerly British A); clone P55/7 resistant to G. rostochiensis Rol (ex S. multidissectum) with gene H2 gives resistance to cream
G. pallida Pal (formerly British B); and the hybrid E45/65 (ex
S. andigena CPC2775) with gene H gives resistance to white G. pallida 3 Pa (formerly British E) (Howard et al. 1970, 1971). As it was 3 necessary to use stem cuttings,a preliminary test to assess the
reliability of this method was carried out. Sixteen 9 cm pots
planted with either tuber pieces or stem cuttings of the variety
Arran Banner were inoculated with fifty cysts of G. pallida.
After 10 weeks root-ball counts in both treatments were made,
but a complete lack of correlation was observed. Therefore the 91 final populations were compared. (Table 27). Although about twice as many cysts were recovered from plants raised from tuber pieces as from plants raised from cuttings it was decided that the results
from cuttings were sufficiently consistent for the purpose of this
study.
Table V. Multiplication of a G. Dallida population on Arran Banner's tubers and cuttings.
Reps. Number of cyst from Ratio Tubers Cuttings 746ens/CuRirT,s
1 1014 544 1.86
2 577 378 1.53
3 1227 552 2.22 4 1334 365 3.65
5 1364 300 4.55 6 1380 804 1.72
7 1613 805 2.00
8 980 344 2.85
Mean 1186 512 2.32
Therefore the available tubers from each differential clone, including Arran Banner, were planted in large pots. When good development of stems was observed they were cut and transplanted for rooting in trays containing organic soil. After 12 days single stem cuttings were transferred to 9 cm pots to allow good establishment. A week later, 3 plants of each differential clone were inoculated with 50 cysts (enclosed in a muslin bag) from each of the 53 populations. At maturity the tops were cut off the plants and the pots left to dry. The newly-formed cysts were extracted from each pot and counted. 92
(ii) Results and discussion.
The numbers of new cysts (expressed as a percentage of the nu.zber recovered from Arran Banner) found on plants with genes H1, H2 or 113 are
given in Table 28 for the 53 poialations tested. 2he place of collection
and its latitude are also given and the last column in the table indicates
the soecies by colour of the developing females (i.e. a separate test).
Results were not always clear cut, but using 40,; of the reproduction
on Arran Banner as a threshold, Table 29 shows that from the 44 South
American populations tested most but not all of the 35 G. pallida
populations were able to multiply on Hi or H2 and 18 (more than 50) were
able to multiply on H3. Of the 6 G. rostochiensis populations 3 were able
to multiply on Hi, but only one on H3, and of the 3 populations suspected
to be mixtures, two on H2 and H3. One mixture was predominatly a cream
type and the other two were mainly white types.
The right hand side of Table 29 is derived from the left hand side
rather than from hybrids with the different gene combinations. However,
it shows that the 17 G. pallida populations able to reproduce on H1H3 are
not the same 17 that reproduce on H2H3. If they were, the figure under
H1112H3 would be the same i.e. 17 not 15. In other words the behaviour •
of the populations towards these 3enes is complex, leading to the n definition of many pathotypes (n differential plants allows 2 pathotypes
to be distinguished). For instance, the two populations from Ecuador belong
to the white G. pallida sroup (for female colour and measurements) but
EC-1 behaves differently on the hybrid with gene H1. (See Table 28).
In the gr-up of G. rostochiensis populations the situation is also complex,
even considering only the two Peruvian populations. With the same criteria
as above, a few European populations were tested on the same differential
plants. The results in Table 30 show that the situation is less complex
than for the South American populations, with all of them behaving in
the same way as pathotypes already recognised from Europe. 93
TABLE 29. Ability of South American populations to multiply on resistant
1:otato hybrids.
Species* Country No. popI.1 Hybrids with resistance gene(s)** tested H1 H2 H3 111112 H1H3 H2H3 H1H2H3 G.pallida Colombia 3 3 3 2 3 2 2 2
Ecuador 2 1 2 1 1 0 1 0
Peru 30 26 25 15 20 15 14 13
TOTAL: 35 30 30 18 24 17 17 15
G.rostoch- iensis Peru 2 1 2 0 1 0 0 0
Bolivia 4 2 4 1 2 1 1 1
TOTAL: 6 3 6 1 3 1 1
G.r./G.u. Peru 3 3 2 2 2 2 2 1
* The species was determined from the colour of developing females.
**H1 = Gene from S. tuberosum ssp. andigena (CPC1673) resistant to G. rostochiensis.
H3 = Gene from S. multidiss':ctum (P55/7) resistant to cream G. Dallida.
H3 = Gene from S. tuberosum ssp. andigena (E45/65) resistant to white
G. pallida. 94 Table 28. Numbers of cysts found on plants with genes for resistance expressed as a percentage of the number found on Arran Banner. Resistance gene H2 H3 Latitude Species Country Code H1
Colombia CO-1 70 204 55 1.5°N G. pallida CO-2 279 557 183 1.5°N G.p,
CO-3 142 228 36 1.2°N G.p.
Ecuador EC-1 20 41 41 3.0°S G.p. EC-2 103 153 18 3.0°S G.p.
Peru PC-1 445 242 218 6.5°S G.p. PL-1 74 127 47 7.8°S G.p. PL-2 47 46 28 7.8°S G.p. PL-3 102 87 82 7.8°S G.p. 7.9os IDL-4 55 65 53 G.p. PH-1 42 27 92 9.8°S G.p.3 PJ-1 429 382 49 11.2°S G.p. PJ-2 176 91 57 11.2°S G.p. PJ-3 155 78 23 11.2°S G.p. PJ-4 83 57 19 11.2° S G.p. PJ-5 91- 66 71 11.2°S G.p. PJ-6 156 30 6o 11.2°S G.p. PJ-8 37 83 25 11.2°S G.p. PJ-9 87 57 168 11.2°S G.p. PJ-14 118 66 31 11.2°S G.p. PJ-16 82 133 38 11.5°S G.p. PJ-18 60 131 11 11.7°S G.p. PJ-19 118 25 13 11.7° S G.p. PJ-23 146 290 69 11.7°S G.p.
PJ-24 39 311 4o 11.7°s G.p. PJ-25 54 169 72 11.8°S G.p. PJ-26 95 97 8 11.8° S G.p.
PJ-31 9 94 29 11.8°S G.p. PJ-33 39 90 25 11.8°S G.p. 95
Table 28.(contd) Resistance gene H Latitude Species 1 Country Code H1 II'2 3
PJ-37 165 13 88 11.8°S G.p. PJ-38 137 143 46 11.8°s G.p. PJ-40 51 64 57 11.9°S G.p. PJ-44 63 129 29 12.1°S G.p. PK-3 77 99 109 13.5°S G.P. PP-7 50 10 14 15.6°S G.p.3 PP-8 8 97 56 15.9°S G.r. / G.p. PP-21 135 71 15 16.2°S G.p. / G.r. PP-23 132 92 34 16.2°S G.r.2
PP-24 95 5 14 16.2°s G.p.3 PA-1 4 72 19 16.4°s G.r. Bolivia B0-1 39 311 40 15.6°S G.r. 2 Bo-18 57 76 75 17.3°S G.r. 2 B0-21 43 238 32 17.4°S G.r. B0-23 14 133 18 17.5°S G.r. Iceland IC-2 102 17 5 65.0°N G.p. IC-3 82 lo 3 65.0°N G.p. IC-5 71 22 5 65.0°N G.p. United UK-1 166 3 8 56.0°N G.p. Kingdom UK-2 134 121 6 53.4°N G.r. UK-3 5 198 22 52.3°N G.p.
Italy IT-1 2 42 12 41.0°N G.r. IT-2 14 25 6 41.0°N G.r.
Spain SP-2 1 19 10 37.0°N G.r. 1 Determined by developing female colour - see above. 2 Overcome resistance gene H1. 3 Cream G. pallida. 96
Table 30. Ability of some European populations to multiply on resistant potatoes.
Species Country No.Pop. Resistant gene(s) Tested H H H H H H H H H H1H-2H 1 2 3 1 2 1 3 2 3 3 G. pallida Iceland 3 3 0 0 0 0 0 0 United Kingdom 2 2 1 0 1 0 0 0
TOTAL: 5 5 1 0 1 0 0 0
G.rostochiensis Italy 2 0 1 0 0 0 0 0
Spain 1 0 0 0 0 0 0 6
United Kingdom 1 0 1 0 0 0 0
TOTAL 4 0 2 0 0 0 0 0
It seems likely, then, that European populations of potato cyst- nematodes contain only a fraction of the variability found in South
America, where they evidently evolved with their host plants, the
Solanum species. This 'old' association has lead to a gradual co- evolution, with speciation of the host and the parasite. Between host and parasite there is an intimate relationship (Hogenboon, 1975;
Person & Ebba, 1975), regulated by matching gene systems, and changes in the host may lead to adaptive changes in the parasite.
Conversely, changes in the parasite may induce changes in the host.
This mutual influence is likely to be frequent in regions where different populations of both host and paraite occur (Braverman and Leppik, 1972). Further discussion on the evolution and distribution of the potato cyst-nematodes and their hosts will be covered in Section III. 97
I. 7. GENERAL IDENTIFICATION OF POPULATIONS IN THIS STUDY.
The populations studied come from many of the areas of the world
where potato cyst-nematodes are found.
In Table?(Allx)all the populations of South American origin (131
populations) are listed by a code corresponding to their country of
origin viz. Venezuela (VE), Colombia (CO), Ecuador (EC), Peru (P-),
Bolivia (BO) and Chile (CH). Although all of them were identified-
by female colour during female development some, which gave uncertain
results due to the difficulty of differentiating the "cream" colour
from the yellow one, were also identified by measurements made on second
stage juveniles (74 populations) or cyst terminal areas (13 populations).
In this way it was found that three populations, (B0-2, B0-21 and PA-4)
previously considered to be mixtures of species or G. rostochiensis, were
actually G. pallida. On the other hand, populations considered to be pure
on the basis of female colour, (PJ-24, PP-1, B0-1, B0-5, PA-3, B0-22 and
B0-24) were found to be mixtures of species from measurements made on
second stage juveniles. Results of measurements on cyst terminal areas
agreed well with those obtained by the other two methods of identification. When some of these populations (44) were tested, using differential
clones, for the presence of pathotypes, three populations identified
as G. rostochiensis overcame the resistance gene H1 contained in Maris
Piper. (PP-8, PP-23 and B0-23).
After evaluating and comparing results obtained by the different
methods and from the differential hosts, the actual status of each
of the 131 Andean populations is indicated in the last column of
Table? ftaWhenever populations were suspected to be mixed, the final
identification was from measurements of second stage larvae. When
one species was only just dominant, its name is first (PJ-24, PJ-29,
PJ-36, PJ-39, B0-1, B0-5, PP-22, B0-13, PA-2, B0-18, B0-19, B0-20,
B0-21, B0-22, B0-24 and B0-25) but when one species is almost totally 98
dominant the population is considered as pure for practical purposes
(PJ-8, PP-1 and PA-3).
Finally, it is worth mentioning that a good correlation was found
between identification by female colour and identification from
measurements on larvae, as 14 populations were considered to be mixtures
using the first method and 16 using the second.
In Table8(Affithe populations coming from geographical areas outside
South America are listed.
Although two populations (IC-7 and GE-1) were found to be mixtures
of the two species on the basis of female colour, measurements
showed that only IC-7 was a mixture and GE-1 was probably a "cream"
type of G. pallida (as was NF-1). The population IT-2 was found to be a
pathotype of G. rostochiensis because multiplication ocurred on potato
plants with the H1 - resistance gene. In population JA-2 considered in
the last column as G. rostochiensis, the presence of few G. pallida -
like second stage larvae suggests the presence of a small proportion of
this species. A unique case was that of IN-1, which from female
colour was identified as G. pallida but which had measurements typical
of G. rostochiensis. 99
II BIOLOGY OF POTATO CYST-NEMATODES
The potato cyst-nematodes are obligate parasites of -roots of wild and cultivated potato plants. The life cycle described by O'Brien and Prentice (1931) for Scottish conditions and by
Chitwood and Buhrer (1946) for Long Island, U.S.A. conditions was only for the species described at that time, Globodera rostochiensis (Wollenweber, 1923) l . The potato cyst-nematodes Ro - are characterised by the transformation of the body wall of the dead adult female into a tough resistant cyst containing the eggs. Within the cyst, the quiescent larvae contained within eggs can remain viable in the soil for several years. In the absence of a host plant only a small proportion of larvae (30-33%) hatch each year. When potatoes are grown most of the larvae (60-80%) are stimulated to hatch by a specific chemical "hatching factor" diffusing from the potato roots (Triffitt, 1930). Second stage juveniles invade the young roots just behind the root tip, penetrating the root cortex.
At first they move through the cells, leaving a trail of damaged tissue.
Soon, however, they settle to take up a position near the endodermis and reaching the stele where they induce formation of specialised transfer cells (syncytia) which are multinucleate with a densely granular content on which the now sedentary larvae feed. The syncytia are formed mainly from phloem parenchyma cells by the breakdown of cell walls and enlargement of the cells. Special wall ingrowths develop on the walls adjacent to vessels to increase the surface area of the plasmalemma and so increase the rate of transport of solutes (Jones and Northcote, 1972). The larvae thicken, shorten a little and moult three times before becoming adult. The sex of larvae becomes obvious in the third stage and from then on males and females diverge. The fourth-stage male re-elongates within the cuticle of the third-stage which by now has either ruptured the cortex EMBRYONATED EGG
j3 a
j4
MATURE y
IMMATURE y
Fig. 15. Life cycle of potato cyst-nematodes Globodera spp. 101 of the root or lies just beneath the epidermis. After the fourth moult, the fully formed male, a little over 1 mm long, escapes into the soil.
Fourth-stage females remain saccate and enlarge further until they rupture the cortex completely, remaining attached with the head buried in the root and held in place by a sticky secretion produced in the neck region. The female, now a large sac-like body protruding from the root surface at about 28-38 days after invasion, produces a secretion which is highly specific attractant for males (Green, 1966, 1967) which begin to emerge at about 20 days after invasion. The females are fertilised within about 10 days of the start of male emergence.
After this time the males become inactive, in contrast to the females which remain receptive to fertilisation for long periods (Evans, 1970).
After fertilisation the female body is spherical, and the body wall eventually becomes the tanned so-called "cyst" which is the toughened integument of the dead female. At this stage, they are either easily dislodged into the soil or remain attached to the roots or the surface of tubers and stolons on which they also sometimes develop. The dead female contains 200-600 eggs, each of which encloses a coiled larva.
The interval from embryonated eggs to embryonated eggs is not less than
38 days and rarely exceeds 48 days (Chitwood & Buhrer, 1946; Feldemesser
& Fassuliotis, 1950). However, Morris (1971) in Canada found cycles between 50-58 days, depending on disruptive influences occurring either within the host plant (resistant varieties) or in the soil.
Jones (1970) used data of Cooper (1953 ) and Winfield - (1965) to show when a crop other than potatoes is grown or the land is left fallow, the nematode population decreases regularly by about one- third every year regardless of population density. When a susceptible potato variety (a suitable host) is grown in lightly infested soil, the nematodes that hatch multiply greatly, sometimes by as many as fifty 102
times, but usually by twenty-five to thirty times. In heavily infested soil, increase is less because larvae comp ete for root space and root systems are stunted so that there are fewer sites to produce females.
Although the most obvious factor influencing development is host specificity, different parts of the potato cyst-nematode life cycle have different environmental requirements for an optimum and full development. Thus we need to know how environmental factors influence their behaviour such as hatching, invasion of the host and reproduction. Some of these influences have been studied in -
G. rostochiensis, but little work has been done on the biology of the second species G. pallida. For instance, it has been shown that the soil type affects water content and the degree of aeration in the soil and consequently the amount of oxygen necessary for hatching
(Triffitt, 1930). Changes of temperature increase the total hatch
(Bishop, 1953, 1955; Winslow, 1956). FenwicK (1951) found that 25°C and darkness were the best conditions for hatching. Feldemesser and
Fassuliotis (1950) found that larvae did not hatch at temperatures of o 40 C. Oostenbrink (1967) states that cyclic influences or diapause cause depression or' delay of hatching and appear to be inherent in the seasonal emergence cycle of mature cysts. Other workers have also studied the effect of temperature on the life cycle of G. rostochiensis
(Fenwick, 1951; Ferris, 1957; Ellenby, 1975). Trudgill (1967) found that the conditions to which larvae of G. rostochiensis were exposed in host-plant roots determined the sex ratio of the adults.
Therefore in this section the effect of some factors on the biology of British and Peruvian populations of both species of potato cyst-nematode will be examined. However, due to scarcity of information on comparative rates of development of G. rostochiensis and G. pallida, a preliminary experiment will be described. 103
(i) Materials and Methods
In late August 9cm plastic pots were planted with Arran Banner
(S. tuberosum ssp. tuberosum) tuber pieces and grown in greenhouse
conditions. Similarly sized plants were chosen and inoculated
with 4,500 larvae from each of the following populations: UK-3
PP-8 (G. rostochiensis) and UK-1, UK-2, PL-4 (G. pallida). Seven
days after inoculation, and every week thereafter for three
w-eks, three plants from each treatment were taken to determine
invasion and stage of development as described earlier. After
ten weeks the tops of the remaining plants were cut off and the
soil in the pots was allowed a further two weeks to dry thoroughly.
The cysts formed were extracted, counted and the number of eggs
per cyst was also estimated. The multiplication rate was
calculated by dividing the number of eggs per pot by the number
of larvae in the inoculum (4,500).
(ii) Results and Discussion
Under the experimental conditions for this time of year, the
results in Fig. 16 show that invasion at seven days after inoculation
was greater for the white G. pallida populations, UK-2 and PL-4
than for the G. rostochiensis populations UK-3 and PP-8, which
reached their invasion peak at 14 days. However, as the greater
invasion observed with those populations is not reflected in
the numbers of cysts formed, it may indicate only a temporary
invasion as reported for larvae of Meloidogvne incognita (Reynolds,
et al, 1970; McClure et al, 1974) and Heterodera avenae (Davies,
et al, 1976). On the other hand, the life cycle of the white
population PL-4 seems to be shorter, since between 14 and 21 days
( a time at which males are only just beginning to leave the
roots, Evans 1970) the number of larvae found in roots goes down
sharply to reach almost the number of new cysts formed, suggesting
Fig. 16. Invasion and multiplication rate of British and Peruvian populations of G. rostochiensis and G. pallida under glasshouse conditions. 2800 b0-0 G.rostochien sus : o UK-3 • PP-8 g 2400 G. pa II ido : 6 UK-2 • PL-4 50.0 O UK-I 2000 -0 40-0 0 O 1500 c') 30.0 • 1200 -0 0 ..0 20.0 E E 800 C
-6 400 10'0 0
7 14 21 28 Harvest Days after inoculorion 105
that after 28 days, no more males left the roots. With the other populations the numbers of larvae in roots continue to fall right up to 28 days, suggesting that males were still leaving the roots up to this time. The pattern of invasion observed for the cream population (UK-1) of G. pallida suggests that it is slower to develop than white populations of this species.
The results in Table 31 show the differences in the numbers of cysts formed and the multiplication rates for each population.
G. pallida (UK-2, PL-4) produced more cysts and therefore has a greater multiplication rate than G. rostochiensis (UK-3, PP-8), and the Peruvian population of each species produced more cysts than British populations of the same species but the differences in numbers of eggs per cyst were small. After finding these differences in invasion and multiplication between populations and species further experiments were done to study their biology and the influence of a number of factors on it.
Table 31. Multiplication rate under glasshouse conditions
(Means of three replicates).
SPECIES POPULATION NUMBER OF No.EGGS/ MULTIPLICATION CYSTS CYST RATE
Globodera rostochiensis UK-3 399 36o 31.5 PP-8 603 369 49.5
Globodera pallida UK-2 709 343 54.8
PL-4 988 310 68.1
UK-1 344 225 17.2
S.E.D. 140.2 22.8 9.19
L.S.D 5% 323.0 53 21.2 1% 44.4 72.3 29.1 X06
II. 1. DAYLENGTH
Although life on earth has evolved in the presence of a daily cycle of daylight and darkness, the depth to which light penetrates into the soil is small (Beck, 1968). Many true soil animals never expose themselves to full daylight or to sunlight (Kuhnelt,
1976). Of course, plant-parastic nematodes depend ultimately on light as a source of energy, which they obtain through the plant, but light may affect directly only those nematodes species which occur above ground level, e.g. Ditylenchus and Anhelenchoides. _
It is doubtful whether it has any influence on nematodes in the soil, except indirectly via the host plant. The closer the association nematodes have with their hosts (e.g. cyst-nematodes and root-knot nematodes) the greater must be the chance of this effect occurring.
In the complex relationship a plant has with its environment, many interdependent factors may affect the various functions of the plant either directly or indirectly, perhaps by altering morphology or modifying enzyme systems (Thomas, 1955). Mokronosov etal (1959) found that the sap of a European potato variety contained a larger variety and amount of aminoacids in daytime than in night-time; with plants 15-20 days old the aminoacid content was much smaller under short day conditions than long day conditions.
Quantitative changes in ribonucleic acids of potato in response to photo-period and temperature have also been found (Oslund et al,
1971). A direct effect of light on potato cyst-nematodes has only been found during hatching of G. rostochiensis. The emergence of larvae was unaffected in the dark or by exposure to diffuse light but in bright light there was no emergence (Lownsberry, 1950; 107
Fenwick, 1951). Long days have already been shown to influence the reproductive rate and pathogenicity of the root-knot nematode,
Meloidogyne incognita (Tarjan et al, 1955; Gillard, 1961). Also,
Ellenby (1958) found that G. rostochiensis produced fewer females on potatoes grown under short days than on those grown under long days. Evans et al,(1975) studying the distribution of
Andean populations of potato cyst-nematodes, suggested among the possible reasons for the distinct distributions of the two species, the indirect influence of daylength on the hosts.
Therefore, the experiment below describes the effect that different daylength conditions may have on the biology of different populations of potato cyst-nematodes through effects on the host.
(i) Materials and Methods
Potato plants of two varieties of Solanum tuberosum ssp. tuberosum were planted in 9 cm plastic pots and inoculated with larvae hatched from cysts produced in a greenhouse. Potato plants,var
Arran Banner (main crop) and Arran Pilot (early)l were grown in o 2700 Saxcil cabinets at 18 C, 70-80% RHmlumens and daylengths of 12 and 16 hours. Fifteen days after planting (March, 1975), 3000 larvae from each of the following five populations: UK-3 and
PP-8 (G. rostochiensis) and UK-1, UK-2 and PL-4 (G. pallida) were added to each pot. Seventeen days after inocu_ation the rate of invasion and sex differentiation was determined. The roots of three plants from each treatment were washed and weighed and stored in fixative until the larvae they contained were counted.
Ten weeks after inoculation watering was stopped and the soil in the pots allowed to dry. The new cysts were then extracted and counted. A further experiment was then done to find out if growing conditions during cyst-formation or root diffusate collection 108 affected hatching. To study the hatching rate of the newly formed cysts, root diffusate was collected from uninOculated
Arran Banner potato plants grown in the cabinets in the different daylengths and tested on cysts produced on both varieties grown in both daylengths. There were four replicates of each treat- ment combination. For each replicate 25 cysts were placed on a 5 mm diameter sieve in a glass tube partly filled with root diffusate and kept in an incubator at 20°C. At weekly intervals for 10 weeks the hatched larvae were collected and counted.
Each time new root diffusate was added.
(ii) Results a. Host development
The first noticeable effect of daylength on both varieties was observed in the tops. Arran Banner and Arran Pilot in 12 hour days had large soft leaves and light-green leaflets. Internodes were short and stems easily broken. No flowers were produced.
In 16 hour days both varieties had small hard leaves and grey- green leaflets. Internodes and stems were longer. However,
Arran Pilot was shorter, produced more stems and stolons, and flowered and matured earlier than Arran Banner. Figure 17 shows the effects of different daylengths in plants 32 days old. Both
varieties produced more roots and tops in 16 hour days, but
fewer tubers. By ten weeks these effects were greater and
differences between the varieties obvious (Table 32). The height
of both varieties increased in 16 hour days but in both daylengths
Arran Banner was taller than Arran Pilot, presumably a varietal
difference (Bodlaender, 1963). Arran Pilot had heavier tops.
Both varieties had more roots in longer days, Arran Pilot having
the heavier root system in both daylengths. The number and weight
of tubers decreased in longer days (see Driver and Fawkes, 1943;
Burton, 1966). Fig. 17. Weights of roots, tops and tubers of Arran Banner (main crop) and Arran Pilot (early) potato varieties at 32 days after planting.
A. Bonner A.Pilor -) C ) 0 o Roo rs a Tops 0 Tubers
SD LD (12h) (I 6h) Daylight length (hours) 110
Table 32 Effects of daylength on two potato varieties 10 weeks after planting (Mean of three replications)
Daylength Variety Stems Height Top wt Root wt Tubers (hrs) (Nos) (cm) (g) (g) No wt
12 Arran Banner 2 22 26.1 11.5 4 40.9
Arran Pilot 2 15 30.9 14.3 4 48.7
16 Arran Banner 1 30 26.7 24.2 2 16.2 Arran Pilot 3 18 47.0 26.9 4 27.1
b. Nematode Development
An analysis of variance of the number of larvae that had invaded the root at 17 days is in Table 33. Only variety and the interaction between populations and daylengths had significant effects. Results in Table 34 show that the total number of larvae invading in 12 hour days was the same for all populations but slightly greater on Arran
Pilot than on Arran Banner. In 16 hour days invasion was greater on both varieties, but as before greater on Arran Pilot than Arran
Banner. The numbers of the different larvae stages found in the roots are shown in Fig.18, and the greater invasion found in Arran
Pilot is also shown. Table 35 shows the percentages of males and females and the sex ratios in the roots of Arran Banner 17 days after inoculation. Development of Globodera rostochiensis populations
(UK-3 and PP-8) in 16 hour days seemed to be slower then in 12 hour days, in which the percentage of sexually differentiated females was greater (3.0%) and the male/female sex ratio smaller (4.1 compared with 10.6). G. pallida populations behaved the same in both 12 and I. '
'" 't
2000 Arra n Ban'ner (ma i ncrop) Arran Pilot (early crop)
1600 ~ c o ~ 1200
800 o E o UK-3 PP-8 PL-4 UK-2 UK-I UK-3 PP-8 PL-4 UK-2 UK ~ I 2nd 12 hour days G.rostochiensis: UK- , PP-8 3 rd 16 hou r days c? G,pallida: PL-4 ,UK- ,UK-I ~ Fig. 18 . Total numbers of different l arval stages in the roots of potato plants at 17 days aft er inoculation. - - ..- - 112 Table 33. Analysis of variance of logaritmic transformation of total nematode number in roots per plant, 17 days after innoculation. SOURCE OF VARIATION DF MS VR Level of significance REPLICATIONS 2 0.02178 0.374 DAYLENGTH 2 0.01924 0.330 POPULATIONS 4 0.03376 0.580 VARIETIES 1 0.32367 5.559 5% DL x POP. 8 0.12967 2.227 5% DL x VAR. 2 0.04760 0.818 POP. x VAR, 4 0.03295 0.566 RESIDUAL 118 0.05823 113 Table 34 Effect of daylength on invasion of G. rostochiensis and G. pallida populations 17 days after innoculation. (Means of three replications) Transformed results (log total number + 1) Arran Banner Arran Pilot POPULTS. 12 hour 16 hour 12 hour 16 hour UK-3 2.903 3.014 3.131 3.247 G../. PP-8 2.969 3.040 3.000 3.133 PL-4 2.840 3.080 3.004 3.089 G.? UK-2 2.972 2.788 2.878 2.977 UK-1 2.981 3.047 3.092 3.047 Means: 2.933 2.994 3.021 3.099 S.E.D. 0.114 L.S.D. 5% 0.225 10% 0.298 De-transformed results (UK-3 800 1033 1352 1766 G. r. PP-8 931 1096 1000 1358 PL-4 '692 1202 1009 1227 .1). UK-2 938 614 755 948 UK-1 957 1114 1236 1114 Means: 864 1012 1070 1070 938 1177 114 16 hour days - the percentages of females (3.4% and 3.3%) and the sex ratios (2.6 and 2.3 ) were similar. The smallest sex ratio recorded was 1.2 for G. pallida PL-4 in 16 hour days perhaps indicating good adaptation by the nematode to the host. Table 34 Sex ratio and percentages of male and female, larvae found in roots of Arran Banner 17 days after innoculation (Means of three reps.) Daylength G. rostochiensis G. pallida UK-3 PP-8 Mean PL-4 UK-2 UK-1 Mean a 10.1 14.3 12.2 9.8 6.5 9.9 8.7 12 hours T 1.4 4.6 3.0 4.6 2.7 2.9 3.4 a /? 7.2 3.1 4.1 2.1 2.4 3.4 2.6 a 9.0 9.9 9.5 8.3 3.5 11.3 7.7 16 hours T 1.0 0.8 0.9 6.8 1.5 1.7 3.3 a /? 9.0 12.4 10.6 1.2 2.3 6.6 2.3 The multiplication rates of the five populations were eseaated by comparing the numbers of cysts produced per plant. The analysis of a logarithmic transformation of cyst numbers is in Table 41. Daylength, populations and varieties all had effects significant at the 1% level. Two-factor interactions were also significant, at the 5% level, with the exception of that between daylength and variety. The treatment means in Tables 36 and 37 show that more cysts were produced in 16 hour days than in 12 hour days, and that more cysts were produced on Arran Banner than Arran Pilot. With Arran Pilot as a host, differences in numbers of new cysts between populations and daylength treatments were not significant. The de-transformed results of Table 37 are presented as histograms in 115 Table 36 Analysis of variance of daylength effect on multiplication of potato cyst-nematode populations (Means of three replicates) SOURCE OF VARIATION DF MS VR Level of significance REPLICATIONS 2 0.03155 1.994 DAYLENGTH 2 0.12631 7.981 1% POPULATIONS If 0.25959 16.404 15 VARIETIES 1 0.40160 25.378 1% DL x POP. 8 0.03639 2.300 5%_ DL x VAR. 2 0.02740 1.732 POP. x VAR. 4 0.0446 2.809 5% DL x POP. x VAR. 8 0.03459 2.186 5% RESIDUAL 58 0.01582 Treatment Means Daylength Populations Varieties 12 hours 2.391 UK-3 2.449 Arran Banner 2.508 G.r. 16 hours 2.514 pp-8 2.468 Arran Pilot 2.374 PL-4 2.632 G.p• UK-2 2.395 UK-1 2.320 S.E.D. 0.033 0.042 0.027 L.S.D. 5/0 0.066 0.084 0.054 1% 0.087 0.111 0.143 116 per plant Table 37 Effect of daylength interactions on number of cysts/(Means of three replications) Transformed results (Log Number cyst + 1) Populats. Arran Banner Arran Pilot 12 hours 16 hours 12 hours 16 hours UK - 3 2.473 2.641 2.350 2.331 PP - 8 2.590 2.472 2.437 2.372 PL - 4 2.653 2.853 2.467 2.553 UK - 2 2.379 2.625 2.224 2.352 UK - 1, 2.027 2.441 2.310 2.503 Means: 2.424 2.606 2.358 2.422 2.508 2.374 S.E.D. 0.103 L.S.D. 5% 0.205 1% 0.273 De - transformed results UK - 3 297 438 240 214 G. r. PP - 8 389 297 274 236 PL - 4 450 713 293 357 UK - 2 G.p. 239 422 168 225 UK - 1 106 276 204 318 Means: 296 429 236 270 363 253 Arran Banner (rnain crop) Arran Pilor (early crop) 800 700 1E 500 0 -a. 500 400 E 300 200 6 100 0 UK-3 PP-8 PL-4 UK-2 UK-1 UK-3 PP-8 PL-4 UK-2 UK-1 G.r G. p. p EI 12 hour days 10 lb hour days Fig. 19. Multiplication rates of British (UK-1,UK-2 and UK-3) and Peruvian (PP-8 and PL-4) populations of potato cyst-nematodes estimated by the number of cysts produced per plant. 118 Fig. 19 and these clearly show the differences in multiplication on the two hosts and the large multiplication rate of the Peruvian population PL-4 (G. pallida) on Arran Banner in 16 hour days. The percentages of larvae invading Arran Banner plants which formed cysts are shown in Table 38 which also compares the multiplication rates of the two species of nematode. Similar proportions of the G. rostochiensis larvae formed cysts in 12 hour (11.5%) and 16 hour (12.3%) days but a greater proportion of larva,. of G. pallial formed cysts in 16 hour days than in 12 hour days (15.7% compared with 8.8%). Table 38 Percentages of larvae forming cysts on Arran Banner Daylength G. rostochiensis G. pallida 12 hours 11.5 8.8 16 hours 12.3 15.7 c. Hatching Test The testing of root diffusates produced in different daylengths as hatching agents for cysts produced in different daylengths were done because cysts disseminated over long distances may find themselves in daylengths different from those in the native land. Inefficient hatching might lead to failure to establish or compete successfully in the new environment. The ability to hatch promptly is estimated by comparing the number of larvae hatching during the three first weeks. From Fig. 20 it is clear that larvae from PL-4 (G. pallida), followed closely by UK-3 (G. rostochiensis) hatched faster from cysts produced in 16 hour days, but did so better from diffusate 5 1— V) l) _ Lu 0 0 0 LL —I 1.-- cc LL I LLQ 2 0 ° 0 ec LL ¢ LLJ — Cl- —1 J Fig. 20.Cumulativehatchedlarvaepercystforpopulationandrootdiffusates collectedfromArran D Z I Lu cc Z 12 hour days ' _c u 150 46 o E 0 = u (1) > Banner plantsgrownunder12and16hoursdaylength. 50 200 200 100 50 O 0 CYSTS PRODUCEDONPOTATOPLANTSUNDER: 1 12 hourdoys Weeks incubated inporaro rootdiffusore 3 45 7 1 234 57 lb hourdays & o o O . UK-I UK-2 PL -4 UK-3 pp 1 _ 8 G.rosrochiensis G.pallida 120 Table 39. Hatching from cysts produced in different daylengths on response to root diffusates collected from plants growing in different daylengths. Analysis of Variance SOURCES OF VARIATION D.F. M.S. V.R. Level of Significance Population 4 37540.2 53.909 1% Diffusate source 2 2045.3 2.937 Cyst source 2 12666.9 18.190 1% Pop.x Diffusate 8 694.2 0.997 Pop.x cyst source 8 7408.7 10.639 1% Diffusate x cyst source 4 3086.4 4.432 19'x - Pop. x Diff. x cyst source 16 431.3 0.619 Residual 135 696.4 Treatment Means Populations Cyst source - 3 117.3 12 hours 64.4 /PP - 8 59.7 16 hours 104.5 (P1 - 4 134.6 spluic - 2 70.5 (UK - 1 56.7 S.E.D. 6.2 4.8 L.S.D. 5% 12.3 9.5 1% 16.2 12.6 121 collected from plants growing in 12 hour days. The other populations hatched more slowly and slowest of all when both cyst and root diffusate were produced in 16 hour days (e.g. G. pallida population UK-2). The analysis of variance of total larvae hatched per cyst after seven weeks of inoculation is presented in Table 39.-. Populations and cyst source had effects significant at the 1% level. The interactions between populations and cyst sourcel and diffusate and cyst source were also significant at the 1% level. The table of treatment means shows that most larvae hatched from populations PL-4 and UK-3 (135 and 117 larvae/cyst respectively) and that more larvae hatched from cysts produced in 16 hour days than 12 hour days (105 and 64 larvae/cyst respectively). (iii) Discussion The results obtained, using the European potato varieties Arran Banner and Arran Pilot (both Solanum tuberosum ssp. tuberosum) as hosts, on the biology of British and Peruvian populations of Globodera rostochiensis (UK-3 and PP-8) and G. pallida (UK-1, UK-2 and PL-4) clearly show an effect of daylength through the host. The rate of invasion seventeen days after inoculation was greater in plants grown in 16 hour days, in which both varieties produced a large increase in root weight (Fig.17). Although both varieties reacted similarly, Arran Pilot (early) developed more roots than Arran Banner in both 12 and 16 hour days and was invaded by more nematodes (Fig.l8). The differences in root weight between the varieties is a reflection of their different physiological development rates, which determines their earliness (Bodlaender, 1963). The greatest invasion of Arran Pilot roots was by the British population UK-3 (G. rostochiensis). 122 of The sexA developing potato cyst-nematodes seems to be determined by environmental conditions in the host roots (Ellenby, 1954; Trudgill, 1967). The ratio of male larvae to female larvae 17 days after inoculation on Arran Banner showed little change with daylength for the G. pallida populations (PL-4, UK-1 and UK-2) but was greater in 16 and 12 hour days for the G. rostochiensis populations (PP-8 and UK-3) (Table 35). Population PL-4 had the smallest sex ratio in either daylength. The numbers of new cysts formed only partially reflected the numbers of larvae invading. More cysts were produced on plants grown in 16 hour days but Arran Banner supported the largest numbers (Table 36, Fig. 19) even though more larvae were found in Arran Pilot roots. The explanation of this may be in the slower development of Arran Banner in which developing larvae were possibly less affected by early maturation than when in Arran Pilot was the host. The G. rostochiensis populations UK-3 and PP-8 multiplied differently under different daylengths, indicating an influence of place of origin. UK-3 multiplied better in 16 hour days and PP-8 better in 12 hour days. G. pallida populations all did better in 16 hour days (Fig. 19). Thus, the inter-relationships between nematode and host as measured by invasion, sex determination and multiplication were affected in different ways. Invasion was related to the amount of root development but sex determination and multiplication were more affected by the inherent characteristics of the potato variety. However, the populations tested behaved differently: the Peruvian G. pallida population PL-4, being best adapted to the host 123 in all conditions. So far, only effects inside the host roots have been discussed. However, larvae that hatch quickly gain an advantage in competition for root space. The results from hatching test suggest that day length may be important during both hatching and cyst production. Table 39 shows the superior hatching from cysts produced in 16 hour days and Fig. 20 that larvae from populations PL-4 (G. pallida) and UK-3 (G. rostochiensis) hatched faster than the rest. The more rapid and complete hatching of the G. rostochiensis population UK-3 than all others except G. pallida PL-4, in all conditions of cyst and root diffusate production suggest that this and similar populations may be very well adapted to spread into new environments. Perhaps this is why many populations around the world originating from Europe are G. rostochiensis, and perhaps population PL-4 from Peru (and others like it) would rapidly colonise new areas in to which they were introduced. Since this population is unaffected by many of the resistance genes identified by European workers, such an introduction could have dire consequences. 121+ IL Z. TEMPERATURE Soil temperature is determined by the amount of radiation falling, the amount reflected and by the amount of cooling. Warming of the soil by radiation depends on its water content and the vegetation cover. In general the heating is inversely proportional to the water content (Schubert, 1930). Water content may also affect heat losses as cooling due to evaporation lowers soil temperature if the capillary water reaches the surface. Temperature fluctuations are much greater on the surface than in the deeper layers of soil. A moist soil tends to retain heat, because when the temperature falls, the water vapour within the soil air becomes partially condensed and the liberated latent heat delays fall of the soil temperature. In northern areas snow cover inhibits cooling of the soil so that when the snow melts in spring the temperature gradient runs from above downwards and causes a general temperature increase. In the autumn, the temperature gradient runs in the opposite direction. However, heat loss in the autumn from the deeper layer and the surface tends to be delayed by fallen leaves or by vegetation cover. In contrast, heavy rain can cause temperature equalisation between all upper soil layers. As a rule, low wint.lr temperatures penetrate only very shallowly into the soil and the soil fauna is not harmed, particularly as soil animals are able to survive low temperatures (Kuhnelt, 1976). Besides determining the rate of development an important effect of temperature for plant parasitic nematodes is the indirect regulation of the abundance of food. When the food supply is adequate, temperature affects such functions as movement, rate of growth, generation time (Jones, 1975) and determination of sex (Evans, 1971+). Studies on the temperature relations of nematodes and plants are complicated because the plant itself is often affected by temperature. 125 The average soil temperatures in potato growing areas of countries with a moderate climate, such as the north western part of Europe and the northern part of the United States etc., are about 15-18°C, similar to those found in potato growing areas of South America. Jones et al (1922) found such soil temperaturQ;were optimal for tuber development. Gregory (1954) observed that high soil temperature increased stem length and top weight, especially at low air temperatures, am decreased tuber weight, especially at high air temperature (30°C). The optimal temperatures for tuberisation vary with the potato variety (Bodlaender et al, 1964). Wallace (1963) divided the influence of temperature on the activity of plant nematodes into five arbitrary phases: (1) non-lethal low temperatures at which activity is inhibited, (2) optimum temperatures, (3) non-lethal high temperatures at which activity is inhibited, (4) lethal low temperatures, and (5) lethal high temperatures. Different nematode activities may have different temperature requirements and different populations of the same nematode species may have different temperature requirements, as found with the potato cyst-nematode Globodera rostochiensiS(Fenwick, 1949, 1950; Ellenby and Smith, 1975). The development of G. rostochiensis within the host plant is inhibited ( at 29 to 32°C (Fenwick, 1951; Ferris, 1957; Mai and Harrison, 1959) but emergence of larvae from the cyst may continue until 36 to 37°C (Mai, 1952; Slack and Hamblen, 1959). The optimum temperature for invasion of this species is 15 to 16°C (Chitwood and Buhrer, 1946), for emergence from the cyst 21 to 25°C (Lownsberry, 1950; Fenwick, 1951a) and for development within the host 18 to 24°C (Ferris, 1957). Bishop (1953, 1955) and Winslow (1956) showed that the rate of emergence from cysts increased with alternating temperatures. Feldmesser (1950) found that larvae hatch at temperatures as low 126 as 4.5°C. Calam et al (1949) report larvae hatching at 6 to 15°C and Oostenbrink (1967) found an insignificant larval hatch at 10°C • with best hatch at 21.5°C. The above refer almost exclusively to G. rostochiensis Rol, the wide ranges of temperature requirements suggest that some of them may have been working with the second species of potato cyst- nematode, G. pallida. However, it was not until 1967 (Guile) and 1972 (McKenna and Winslow; Kuhn) that differences found in the hatching behaviour of species of potato cyst-nematodes (formerly pathotypes) were thought perhaps to play some part in changes in the relative numbers of the two species in mixed populations. Guile (1967) and McKenna (1972) showed that at 25°C cysts of G. rostochiensis hatched more readily than those of G. pallida. Kuhn (1972) found that hatching occurs at a minimum temperature of 10°C for G. rostochiensis and 9°C for 'another' species, and that the rate of hatching at 20.5°C was faster with G. rostochiensis. Oydvin (1974) found that some larvae of G. pallida hatched after dry storage for a week at - 30°C, but only a few larvae of G. rostochiensis hatched after one day or one week at - 25 or - 30°C. Ellis (1975) tested the hatching of 12 isolates of G. rostochiensis and G. pallida at 18°C and found that emergence from isolates in which G. pallida predominated was more rapid than that from isolates in which G. rostochiensis predominated. The rate of development of potato cyst-nematodes has been less studied. McKenna and Winslow (1972, 1972) in a pot experiment outdoors found that G. rostochiensis developed faster than G. pallida and in a heated glasshouse its multiplication rate over a year (and therefore several generations) was greater. When a mixture of species was present, G. pallida was unable to compete with 127 G. rostochiensis. Lane and Holliday (1974) found that G. pallida increased at the expense of G. rostochiensis and that it increased even more on plants with a northerly aspect when compared to multiplication on plants with a southerly aspect. Kuhn (1974) found that emergence of males from potato roots varied according to the time of the year but that it was earlier for G. rostochiensis than G. pallida. The experiments described below were done to clarify the effects that temperature has on the biology of the two species of potato cyst-nematodes. (i) Materials and methods To study the development and multiplication rate of populations of Globodera rostochiensis (UK-3 and PP-8) and G. pallida (UK-1, UK-2.and PL-4), pots containing uniform Arran Banner potato plants were placed in water-baths maintained at constant temperatures (10, 14 and 18°C). Twelve days after planting they were inoculated with 3000 larvae per pot. Each pot contained 300 g of soil and was made waterproof by placing it inside a plastic cup of the same size. Sixteen days after inoculation four pots from each population from each water-bath were taken to estimate the rates of invasion and development. Eight weeks later the plant tops were cut off and a week after that the pots were taken out of the water-baths and the soil allowed to dry. The following week (10th) the soil was emptied from the pots and the roots and tubers weighed. When the soil had dried completely cysts were extracted and counted and their egg contents determined. The hatching test was conducted with cysts reared on Arran Banner and kept as stock in a cold room (5°C) until use. Twenty- seven batches of 50 cysts each were picked from each population. 128 Three of them were used to determine the egg content and the other twenty-four were randomised amongst six temperature treatments: 5, 10, 15, 20, 25 and 30°C. The hatching tests were carried out during eight weeks in incubators using equipment already described (page 108). Root diffusate collected from Arran Banner potato plants and kept in a refrigerator was used for this study. (ii) Results A. Host development • The potato plants at first grew faster at 18°C than at 10°C. Later on this was reversed, but no other differences were noticed in the morphology of above ground parts of the plants. Analysis of variance of root weight of potato plants 28 days old (Table 40) did not show significant differences. The weights at 10, 14 and o 18 C were 4.3, 4.6 and 4.7 grams respectively. Tuberisation was similar at 10 and 14°C but lower at 18°C. Table 40. Analysis of variance of root weight (g) of potato plants 28 days old, grown at 10, 14 and 18°C. SOURCE OF VARIATION DF NS VR REPLICATION 3 0.0295 0.045 TEMPERATURE 2 0.9727 1.472 POPULATION 4 1.1752 1.779 TEMP. x POR 8 0.5058 0.766 RESIDUAL 42 0.6607 B. Nematode development An analysis of variance of total invasion counts 16 days after inoculation is presented in Table 41. Both temperature and population had significant effects. Treatment means show that total numbers of 129 nematodes found in the roots of Arran Banner were higher at 10 and 14°C than at 18°C and that the invasion rate for population UK-2 (G. pallida) was greater than the rest. The results for interaction between populations and temperatures (Table 42) also show a greater invasion rate at the different temperatures for UK-2 than the rest, and that as the temperature increased the numbers of nematodes found in the roots were generally lower. Populations UK-1 and UK-2 are exceptions, with best invasion at 14°C. G. pallida showed a higher invasion rate than G. rostochiensis. Table 43 shows the analysis of variance of number of males found in the roots. Only temperature had an effect significant at the 1% level. The results in Tables 43 and 44- show that as temperature increased the number of males also increased. 1.30 Table 41. Analysis of variance of total number of nematodes per plant at 16 days after inoculation. SOURCE OF VARIATION DF MS VR Levels of significance REPLICATIONS 3 379297 1.489 TEMPERATURE 2 843232 3.309 5. POPULATION 4 701256 2.752 5% TEMR x POP. 8 255310 1.002 RESIDUAL 42 254798 Treatment Means Temperatures Populations Species(obtained from population means) 10°C 1722 UK-3 1488 G.rostochiensis 1442 an 14°C 1610 PP-8 1396 G.pallida 1632 18°c 1336 PL-4 1448 GT- UK-2 1927 UK-1 1521 S.E.D. 206 160 L.S.D. 5% 417 323 1% 557 432 Table 42- Total nematode number per plant at 16 days after inoculation for each population at different temperatures (mean of four replications) Populations Temperatures (°C) 10 14 18 UK-3 1808 1499 1156 PP-8 1528 1292 1368 PL-4 1739 1260 1344 UK-2 G•P• 2095 2205 1481 UK-1 1440 1793 1330 131 Table 43. Analysis of variance of number of males per plant, 16 days after inoculation and means of different treatments. SOURCE OF VARIATION DF MS VR Level of significance REPLICATIONS 3 4841 0.192 TEMPERATURES 2 1121291 44.465 1% POPULATIONS 4 12573 0.499 TEMP. x POP. 8 41323 1.639 RESIDUAL 42 25217 Treatment Means Temperatures Populations Species 10 C 44 UK-3 232 G.rostochiensis 212 G.r. 14°C 139 PP-8 192 G.pallida 229 18°c 484 PL-4 193 G.. UK-2 234 UK-1 261 S.E.D. 50 65 L.S.D. 5% 100 130 1% 135 176 Table 44. Number of males per plant at 16 days after inoculation for each population at different temperatures (Means of four replications) Populations Temperatures (0C) 10 14 18 UK-3 52 85 557 PP-8 23 112 648 PL-4 70 197 312 UK-2 53 141 507 UK-1 23 160 393 S.E.D. 112 L.S.D. 5% 224 1% 303 132 Species Temperatures (°C) 10 14 18 G.rostochiensis 38 99 603 G.pallida 49 166 404 The table above, obtained from the results on interaction between temperatures and populations (Table 44) shows that lower number of males were found with G. rostochiensis at 10 and 14°C than with G. pallida, suggesting a slower development. At 18°C the situation is reversed. The analysis of variance of number of early stage females found in the roots is presented in Table 45. Again, temperature had an effect significant at the 1% level and with the interaction between temperatures and populations significant at the 5% level. Treatment means show the highest number of females at 18°C. Although differences between populations were not significant, the values for species in Table 45 show more females developed in the G. pallida populations; the numbers of females for the two species at the different temperatures are given below. Species Temperatures (°C) 10 14 18 G.rostochiensis 0 54 26o G.pallida 4 36 507 133 Tablert5 Analysis of variance of number of females per plant, 16 days after inoculation and means of different treatments. SOURCE OF VARIATION DF MS VR Level of sip;nificance REPLICATIONS 3 23032 1.844 TEMPERATURES 2 1014595 81.251 1% POPULATIONS 4 21352 1.710 Tam x POP. 8 34592 2.770 5% RESIDUAL 42 12487 Treatment Means Temperatures Populations Species 10°C 3 UK-3 98 G. rostochiensis 104 G.r. 14°C 43 pp-8 110 G. pallida 182 18°C 4o8 IDL-4 184 G.p. UK-2 203 UK-1 160 S.E.D. 35 46 L.S.D. 5% 70 92 1% 95 124 Table 4-6— Number of females per plant for each population at different temperatures (Means of four replications) Populations Temperatures (°C) 10 14 18 UK-3 0 86 209 G. PP-8 0 21 310 PL-4 13 23 505 UK-2 GI' 0 39 570 UK-1 0 33 447 S.E.D. 79 I.S.D. % 178 1% 214 134 The results in Table 46 show faster development of females in populations PL-4, UK-1, UK-2 (G. pallida) at 18°C than populations UK-3 and PP-8 (G. rostochiensis). The sex ratio (6: ?) for all the populations at different temperatures is presented in Table 47. Populations of G. pallida at 18°C had a lower sex ratio than G. rostochiensis populations, suggesting that this temperature is more favourable for production of G. pallida females. Table 47. Sex ratio (Males:Females) for each population at different temperatures (Means of four replications) Populations Temneratures (°C) 10 14 18 UK-3 52.0 1.0 2.7 PP-8 23.0 5.3 2.1 PL-4 3.4 5.6 0.7 G.p UK-2 53.0 3.6 0.9 UK-1 23.0 4.9 0.9 Fig. 21 shows the development of the different larval stages of each population at the three temperatures. Although the interaction between temperatures and populations did not have a Eignificant effect on invasion, the G. pallida populations (PL-4, UK-2 and UK-1) developed faster than the G. rostochiensis populations (UK-3 and PP-8). Table 48 shows the analysis of a logarithmic transformation of the numbers of new cysts formed on each plant, which may be used as an estimate of multiplication rate. The effects of temperatures, populations and the interaction between them were all significant at the 1% level. At 18°C the numbers of cysts were larger (mean 507) than at 10 and 14°C (means 207 and 218 respectively). Populations PL-4 and UK-2 135 10°C 14 °C I8°C o b cdo b cd a b c • d_ so - 25 UK-3 [:i> 0h 25 -50 PP-8 PL-4 UK-2 Fig. 21. Percentages of 2nd. stage larvae (a),3rd. stage larvae(b), males(c) and females(d) found in the roots of plants grown at different temperatures 16 days after inoculation. 156- Table 48. Analysis of variance of logarithmic transformation of number of cysts per plant and means for different treatments. SOURCE OF VARIATION DF MS VR Level of significance REPLICATIONS 3 0.00233 0.074 TEMPERATURE 2 0.95298 30.411 1% POPULATION 4 0.26783 8.547 1% TEMP. x POP. 8 0.10694 3.413 1% RESIDUAL 42 0.03134 Treatment Means Transformed Results Temperatures Populations Species 10°C 2.316 [UK-3 2.227 G.rostochiensis 2.335 Cr 1 4°C 2.339 PP-8 2.443 G.pallida 2.532 18°C 2.705 PL-4 2.522 GP• UK-2 2.635 UK-1 2.440 S.E.D. 0.056 0.072 L.S.D. 5% 0.112 0.144 1% 0.149 0.192 Detransformed Results Temperatures Populations Species 10°C 207 UK-3 169 G.rostochiensis 223 a.r. 14°C 218 PP-8 277 G.pallida 347 18°C 507 PL-4 333 al- UK-2 432 UK-1 275 137 produced the greatest numbers of cysts overall (means 333 and 432 respectively). The results for the interactions (Table 49.) show that almost all populations (except UK-1) produced most cysts at 18°C, and that the increase in number over that at 10°C was greatest for UK-3. The numbers of new cysts for the two species at different temperatures (for de-transformed results)wereas follows: Species Temperatures (°C) 10 14 18 G. rostochiensis 144 181 486 G. nallida 278 283 557 133 Table h9. Transformed and de-transformed number of cysts per plant, for each population at different temperatures (Mean of four replications) Transformed Results Populations Temperatures (°C) 10 14 18 UK-3 2.049 1.900 2.732 S.E.D. 0.125 G r PP-8 2.244 2.450 2.635 L.S.D. 5% 0.250 PL-4 2.389 2.381 2.796 1% 0.333 G.P. UK-2 2.579 2.462 2.864 IUK-1 2.320 2.502 2.498 De-transformed Results Populations Temperatures (°C) 10 14 18 {UK-3 112 79 540 PP-8 175 282 432 PL-4 245 240 . 625 G-P• UK-2 379 290 731 UK-1 209 318 315 139 The numbers of eggs produced per plant were calculated by multiplying the numbers of cysts produced by the numbers of eggs they contained and an analysis of the log transformation is presented in Table 50. Populations and the interaction of the two had effects significant at the 1% level. The transformed and de-transformed treatment means showed the same pattern of multiplication found for cysts, either for single effects or the interaction of them (Table 51). The numbers of eggs per plant for each species presented below shows a lower value at 18°C for G. pallida than for G. rostochiensis, due mainly to the poor multiplication rate of the population UK-1 at 18°C. Species Temperatures (°C) 10 14 18 G.rostochiensis 18160 36643 107745 G.pallida 37373 47293 92497 The efficiency of multiplication estimated by the number of eggs formed for each larva inoculated is shown diagrammatically in Fig. 22. From this figure it is obvious that all the populations behave differently, even within species. G. pallida populations (PL-4, UK-2 and UK-1) and the Peruvian G. rostochiensis (PP-8) multiplied better at lower temperatures (10 and 14°C) than the British G. rostochiensis (UK-3), which only multiplied well at 140 Table 50. Analysis of variance of logarithmic transformation of number of eggs per plant and means for different treatments. SOURCE OF VARIATION DF MS VR Level of significance REPLICATION 3 0.01967 0.332 TEMPERATURE 2 1.58184 26.682 1% POPULATION 4 0.42921 7.240 1% TEMP, x POP. 8 0.28419 4.794 RESIDUAL 42 0.05928 Treatment Means Transformed Results Temperatures Populations Species 10°C 4.407 r-3 4.366 G. rostochiensis 4.540 • Gs. 14°C 4.556 Pp-8 4.713 G. pallida 4.704 18°C 4.952 PL-4 4.763 GI' UK-2 4.825 UK-1 4.524 S.E.D. 0.077 0.099 L.S.D. 5% 0.154 0.198 1% 0.205 0.263 De-Transformed Results Temperatures Populations Species 10°C 25530 r-3 23230 G. rostochiensis 37435 G.r. 140c 35970 PP-8 51640 G. pallida 52730 18°C 89540 r-4 57940 G-13- UK-2 6683o UK-1 33420 141 Table 5J. Transformed and de-transformed numbersof -cysts per plant for each population at different temperatures (Means of four replicates) Transformed Results Populations Temperatures (°C) 10 14 18 UK-3 4.021 3.955 5.123 cr. PP-8 4.412 4.808 4.918 S.E.D. 0.172 PL-4 4.571 4.606 5.113 L.S.D. Gr UK-2 4.7/-3 4.684 5.049 5% 0.344 1% 0.458 UK-1 4.291 4.725 4.555 De-transformed Results Populations Temperatures (0C) 10 14 18 r-3 10500 9016 132700 C.r. PP-8 25820 64270 82790 37240 40360 129700 a-p- UUK-2K-2 55340 48310 111900 UK-1 19540 53210 35890 SO E 10°C o u400 14 °C so 30 8 °C v, >0 CP ,_ _c o u E 10 WM& z 0 UK-3 PP-8 PL-4 UK-2 UK-1 POPULATIONS Fig. 22. Number of new eggs formed from each larva used as inoculum of British (UK-1,UK-2 and UK-3) and Peruvian (PP-8 and PL-4) populations of potato cyst-nematodes. 143 18°C. Furthermore, the British G. pallida (UK-1) was the only one which showed a lower efficiency of multiplication at 18°C than at 14°C, perhaps suggesting that this population develops best in temperatures between 14 and 18°C. C. Hatching The temperatures used during the hatching tests were 5, 10, 15 20, 25 and 30°C. However, in the presentation of results neither of the two extremes have been included. At 5°C no larvae hatched from any population during five weeks. After this time they were transferred to 20°C and hatching occurred, with best hatching from the UK-3 population (G. rostochiensis). No larvae hatched at 30°C from cysts kept for five weeks even when they were subsequently transferred to 20°C, indicating the lethal effect of keeping - at 30°C in root diffusate. The numbers of larvae emerging per cyst during the first three weeks from each population at 10, 15 and 20°C is presented in Fig. 23. The most striking observation to be made is the faster and greater hatching from the Peruvian population PL-4 (G. pallida). The British population UK-2 (G. pallida) hatched faster than UK-3 (G. rostochiensis) at 10 and 15°C and its optimum hatching temperature probably lies between 15 and 20°C, whereas the optimum temperature for UK-3 may be slightly higher than 20°C. The populations PP-8 (G. rostochiensis) and UK-1 (G. pallida) did not hatch properly, presumably due to poor condition of the cysts. ▪ 100 ° UK-3 a) 0 ° PP-8 80 ° UK-2 — Lre -0 L...) PL— 4 uc 9 50 ° UK — QJ ca. 40 > 20 2 2 2 3 10°C 15°C 20°C TEMPERATURES Fig. 23. Hatching of potato cyst-nematode populations at different temperatures dul.ing the three first weeks. 11+5 Table 52. Analysis of variance of total number of hatched larvae / cyst during 8 weeks. SOURCE OF VARIATION DF MS VR Level of significance BLOCK 3 561.1 0.696 POPULTN 4 10967.0 13.609 1% TEMP. 3 16597.4 20.595 1% POPULTN. x TEMP. 12 2131.2 2.645 1% RESIDUAL 57 805.9 The analysis of variance of total number of hatched larvae per cyst in Table 52, shows that temperature, populations and the interaction between the two all had effects significant at the 1% level. As this total hatching is dependent on the number of viable eggs contained in cysts. Table 53 presents the analysis of variance (angular transformation) of numbers of hatched larvae per cyst as a percentage of the estimated egg content for each population. Temperatures, populations and their interaction had effects significant at the 1% level. The table of treatment means shows no great differences between populations with the exception of PP-8 and UK-1, which showed low total hatch (8.4 and 23.3% respectively) Although very lcw hatching occurred at 25°C (3.3%) there were no significant differences between the totals of larvae hatched at the other temperatures (30.0, 29.1 and 33.3%). The results for interactions, however, (Table 54) showed that the populations UK-3 and PP-8 increased as the temperature increased up to 20°C and hatched more than other populations at 25°C, perhaps indicating a preference for temperatures slightly above 20°C. The populations PL-4 and UK-2 (G.pallida) behaved similarly at all temperatures 146 Table 53. Analysis of variance of angular transformation of percentage of larvae hatched per cyst. SOURCE OF VARIATION DF MS VR -Level of significance BLOCK 3 62.21 0.964 POPULATION 4 1253.16 19.423 1% TEMPERATURE 3 3729.82 57.811 1% POPULTN.x TEMP. 12 262.81 4.073 1% RESIDUAL 57 64.52 Treatment Means Populations Temperatures o UK-3 26.0 10° cC 30.0 C.r. PP-8 8.4 15°C 29.1 PL-4 32.1 20°c 33.3 GI UK-2 29.8 25°C 3.3 UK-1 23.3 S.E.D. 2.8 2.5 L.S.D. 5% 5.7 5.1 1% 7.6 6.8 Table 51t. Total hatching as percentage of egg content for interaction between populations and temperatures (Means of four replications) Populations Temperatures (0°C) 10 15 20 25 rK-3 21.9 32.2 39.9 lo.o G.T. PP-8 7.2 9.0 13.9 3.6 PL-4 44.6 39.0 43.1 1.8 G.p. UK-2 41.3 36.6 40.1 1.2 IUK-1 34.9 28.9 29.6 0.0 S.E.D. 5.7 L.S.D. 5% 11.4 1% 15.1 11+7 with little variation in hatch between 10 and 20°C, but at 25°C hatching was negligible. Most larvae hatched from cysts of population UK-1 at 10°C, and none at all at 25°C. The table for species presented below shows that, although G. rostochiensis hatches less well than G. pallida, it hatches over a wider range of temperatures. Species Temperatures (0°C) 10 15 20 25 G. rostochiensis 14.6 20.6 26.9 5.7 G. pallida 40.3 34.8 37.6 1.0 (iii) Discussion The results obtained on the effect of temperature on the biology of potato cyst-nematode species represented by five populations have shown a direct effect on their rate of development, since the potato plants were not much affected by the range of temperatures tested. The rate of development of populations of G. rostochiensis (UK-3 and PP-8) and G. pallida (UK-1, UK-2 and PL-4) shown by the results for invasion, sex determination and cyst and egg production suggest that the temperatures tested (10, 14 and 18°C) were more favourable to G. pallida populations. Total invasion (Table 41) was higher for G. pallida than G. rostochiensis and although it seems generally to be lower at 18°C, this is probably due to faster maturation of the larvae at this temperature and males leaving the roots. This was more evident with G. pallida populations in Fig. 21 (narrowing at the males value), since the number of males found in the roots (Table 43 ) was lower than that for G. rostochiensis populations at 18°C. The development of females at 18°C also was faster for G. nallida populations (Table 47) and this temperature seems to be quite close 148 ! to the optimum for this species, since the sex ratio (males/females) was less than 1.0. The total numbers of cysts produced per plant reinforce this point on optimal requirements (Tables 46 and 149-). At 18°C the numbers of cysts for the populations PL-4 and UK-2 (G. pallida) were higher than those for UK-3 and PP-8 (G. rostochiensis). However, the cream British population UK-1 (G. Dallida, ex-pathotype B) seems to have the lowest temperature requirements. The numbers of eggs per plant (Tables 50 and 51) show the same trends but also show that the UK-3 population multiplies best at 18°C. Fig. 22 shows the number of new eggs formed for each larva inoculated and indicates that G. pallida populations developed better than G. rostochiensis populations at temperatures of 10 and 14°C.. A temperature of 18°C may be close to the optimum temperature for this species and this would agree well with findings by Martin (1965) who worked with a Peruvian population and found an optimum development temperature of 17.7°C. G. rostochiensis seems to require a slightly higher temperature (Ferris, 1957; Oostenbrink, 1967) The temperature requirements for optimum development suggested above correspond fairly well with the conclusions reached by Guile (1967) and McKenna and Winslow (1972) regarding optimum temperatures for hatching. My results for hatching also show the same trends. Although population PL-4 (G. pallida) hatched best at 20°C. all G. pallida populations hatched faster and more at temperatures below 20°C than G. rostochiensis populations (e.g. UK-3). At 20°C, UK-3 hatched as well as UK-2, and initially hatched slightly faster (Fig. 23). These results agree with those of Kuhn (1972) who found a faster hatching of G. rostochiensis at 20.5°C than G.nallida, and with those of Ellis and Hesling (1975) who found a more rapid hatching of G. pallida populations at 18°C. 149 The percentages of larvae which had hatched by 8 weeks (Table 59) indicate that G. rostochiensis populations hatch better than o G. pallida at temperatures above 20 C, but that G. pallida hatches o much better at 10 C. II.LHOSTS Different species and races of plant nematodes have different host ranges. These ranges may include many species in many different genera for such polyphagous species as Ditylenchus dipsaci; Meloidogyne incognita, H. schachtiil etc., or be very restricted for nematodes like G. rostochiensis and G. pallida. The balance of favourable and unfavourable factors determines the degree of success or failure of the parasite. Root secretions from growing plants are an important feature of the environment of nematodes. With endoparasites, at least, the host provides shelter, nutrition and physical environment, and possibly stimuli necessary for hatching, host finding,feeding, moulting and other functions. Failure to provide any of these requirements of the parasite could result in the plant being unsuitable as a host (Rohde, 1972). Crop-nematode relationships vary with cultivar, nematode species and environment. Plant-parasitic nematodes develop and reproduce in close association with their hosts and this can be modified by plant variety and age (Barker and Olthof, 1976). Growing non-host crops or fallowing sometimes results in a dramatic decline of population densities of nematodes and a poor host may result in an even greater decline than will fallow (Jones, 1956; Seinhorst, 1965). As the rate of reproduction of a nematode depends largely on the suitability as a host of the plant it is parasitizing, any mechanisms or intrinsic functions which disrupt the normal development of the nematode may be considered as resistance mechanisms. In resistant varieties and non-host 1.50 species there may be no reproduction, so that the population density falls, and in some host-plants the reproductive rate may be insufficient to compensate for mortality losses, so that population again declines. Thus, there are what might be termed "efficient" and "inefficient" hosts (Seinhorst, 1967). The hatching of the eggs of many plant-parasitic nematodes is stimulated by exudates from the roots of host plants. For instance hatching of G. rostochiensis eggs is greatly enhanced by exudations from the roots of solanaceous plants (Bird, 1975). Winslow (1955) suggested that emergence of larvae from cysts of Globodera spp. tends to occur in response to root exudates from their own host but not to the exudates from non-hosts. Infective larvae of many species of the Heteroderidae enter the roots of resistant and susceptible plants eaually well. Those larvae invading resistant plants may fail to develop and die within the tissue, develop into males, or leave the plant as undeveloped larvae (Webster, 1975). In general, resistance shows up after infection and is most often based on failure of the host to respond to nematode secretions in a manner favourable to nematode development (Rohde, 1965). Resistance to G. rostochiensis larvae seems to be a property of plant cells since resistant potato plants (ex-andigena clones) stimulate the hatch of G. rostochiensis eggs aid many larvae invade their roots (Williams, 1958). Gemmell (19L3) has shown that the potato cyst-nematode G. rostochiensis responds differently to different varieties of potato. He found that there were fewer and smaller cysts on Epicure and Doon Star than on Golden Wonder and Majestic, and when stimulated by potato-root excretions, cysts from the two former produced fewer larvae. Ellenby (1946) found significant differences in responses to the root excretions of different 151 potato varieties. Trudgill (1968), working with a G. rostochiensis population, suggested that even susceptible commercial varieties of potato may possess a degree of resistance. These examples of potato varieties not possessing genes for resistance and considered as suitable hosts, but which in some instances behaved as unsuitable or inefficient hosts in failing to produceAffly active root exudate or in inhibiting the formation of females or eggs, lead to a study of the effects of some commercial potato varieties on the biology of G. rostochiensis and G. nallida populations. (i) Materials and Methods To investigate the responses of the two species of potato cyst- nematodes to root diffusates collected from different potato - varieties, cysts of G. rostochiensis (UK-3) and G. pallida (UK-2), which had been kept as stock at 5°C, were used. Three batches of fifty cysts for each treatment were soaked in water for a week. The water was then replaced by root diffusate and this was subsequently changed at weekly intervals. Batches of cyst were kept in water as controls. The root diffusates were collected from potato plants of the varieties Arran Banner, D^siree, Pentland Ivory, Record (all Solanum tuberosum ssp. tuberosum) and Maris Piper (ex-andigena) grown under similar conditions in pots containing sterile soil. The root diffusates were kept at 5°C after collection, but before use were left for 3-6 hours at room temperature. Each batch of cysts was placed in a small sieve (see page 108 ) and the numbers of larvae emerging each week were counted. The total numbers of larvae that hatched over a period of five weeks were used to estimate the root diffusate activity. The hatching tests were carried out at a constant temperature of 20°C and the 152 of egg contents of cystsA both populations estimated from three extra batches of fifty cysts. To investigate the rate of emergence and multiplication in presence of the host plants, a second experiment was carried out with the same populations. Eight tuber pieces of Arran Banner, . Pentland Ivory, Record and Maris Piper were planted in 9 cm pots inoculated with cysts enclosed in nylon bags, each bag containing the equivalent of 10000 viable eggs. Seventy days after planting the tops were cut off the plants and watering stopped. Four weeks later the dry soil was removed from pots to extract the newly .. formed cysts. At the same time the nylon'ipags containing the original cysts were recovered in order to check their egg contents. The egg contents of the new cysts were also determined. (ii) Results A. Hatching The results of the hatching test are presented in Fig. 24. Hatching of G. rostochiensis was faster than that of G. pallida with more or less equal response to all root diffusates. G. pallida, however, hatched less well with root diffusate of the variety Record. The analysis of variance of transformed results (Table 55) shows that population, root diffusates and the interaction between them all had effects significant at the 1% level. The treatment means show that hatching of G. rostochiensis was significantly greater than G. pallida and that hatching of G. pallida was significantly decreased in the presence of root diffusate from Record. The difference in hatching between the species is partly due to differences in egg content. Table56.. shows that the populations contained significantly different numbers of :eggs, but Fig. 24 shows that slightly greater percentages of G. rostochiensis eggs hatched. 153 Table 55. Analysis of variance of logarithmic transformations of number of hatched larvae per cyst with different potato root diffusates. SOURCE OF VARIATION DF MS VR Level of significance BLOCK 2 0.004699 0.758 POPULATION 1 0.140404 22.638 1% ROOT DIFFUSATE 5 0.946565 152.620 1% POR x RD 5 0.033782 5.447 1% RESIDUAL 22 0.006202 TRANSFORMED RESULTS (Logs) Treatment Means Populations Root diffusates Populations x Root diffusates G.rostochiensis G.pallida G. rostochiensis 2.122 Arran Banner 2.315 AB 2.279 2.252 Record 2.019 R 2.221 1.817 G. pallida 1.997 Maris Piper 2.260 MP 2.338 2.182 Pentland Ivory 2.276 PI 2.296 2.257 Desiree 2.210 D • 2.228 2.192 Distilled water 1.277 DW 1.271 1.284 S.E.D. 0.026 0.046 0.064 L.S.D. 5% 0.054 0.095 0.133 1% 0.073 0.130 0.180 UNTRANSFORMED RESULTS Populations Root diffusates Populations x Root diffusates G.rostochiensis G.pallida G. rostochiensis 169 Arran Banner 211 AB 240 181 Record 118 R 170 66 G. pallida 127 Maris Piper 187 MP 219 154 Pentland Ivory 190 PI 198 182 Desiree 163 D 169 157 Distilled water 19 DW 19 20 154 Table 56'_. Analysis of variance of number of eggs per cyst. SOURCE OF VARIANCE DF MS VR Level of significance. BLOCK 2 0.003737 0.997 POPULATION 1 0.055059 14.691 1% ROOT DIFFUSATE 5 0.001795 0.479 POR x RD 5 0.000653 0.174 RESIDUAL 22 0.003748 Treatment means TRANSFORMED RESULTS UNTRANSFORMED RESULTS Populations Populations G. rostochiensis 2.485 G. rostochiensis 307 G. pallida 2.1+07 G. pallida 258 S.E.D. 0.020 L.S.D. 5% 0.041 1% 0.056 Percentage of egg content 100 U Globodera rostochiensis Globodera pallid° (i) 0 250 0 0 200 _c(1) e U 150 a) 1 00 50 2 3 4 1 2 3 4 5 °Arran Bonner oMoris Piper o P. Ivory Record oDesiree CZ Distilled water Fig. 24. Hatching of British G. rostochiensis (UK-3) and G. pallida (UK-2) larvae with root diffusates from different potato varieties. 156 Table 57. Analysis of variance of number of larvae hatched as percentage of egg content per cyst. SOURCE OF VARIATION DF MS VR Level of significance BLOCK 2 25.99 1.209 POPULATION 1 296.22 13.780 1% ROOT DIFFUSATE 5 3695.38 171.910 1% POR x RD 5 383.24 17.828 1% RESIDUAL 22 21.50 Treatment Means Populations Populations x Root diffusates G. rostochiensis G. pallida G. rostochiensis 55.2 Arran Banner 73.7 AB 79.6 67.9 Record 42.1 R 57.7 26.4 G. pallida 49.4 Maris Piper 61.2 MP 67.6 54.8 Pentland Ivory 70.3 PI 65.3 75.3 Desiree 59.5 D 54.7 64.4 Distilled water 6.9 DW 6.1 7.7 S.E.D. 1.6 2.7 3.8 L.S.D. 5% 3.3 5.6 7.9 lip 4.5 7.6 10.7 100 LSD 90 0 5% 1% 80 75-2 I I 0 70- b0 u arosrochiensis c 50 Fl o o u 40 G.pallido g: 30 ai 20 L-) 10- OL LSO Distilled Desiree Pentland Arran Maris Record Ivory Banner Piper Water Fig. 25. Total hatched larvae per cyst with different potato root diffusates as a percentage of egg content for G. rostochiensis and G. pallida. 158 Analysis of variance of total numbers of hatched larvae as percentage of egg content (Table 57) shows that population, root diffusate and their interaction had effects significant at the 1% level and treatment means for populations show a significantly greater hatch for G. rostochiensis than G. pallida. Fig. 25 shows that the two responded differently to different root diffusates. G. rostochiensis larvae hatched better with Arran Banner root diffusate (79.6%) than with Record or Desiree root diffusates (57.7 and 54.7% respectively). G. pallida hatched better with Pentland Ivory root diffusate (75.3%) than with Record root diffusate (26.4%). B. Multiplication The numbers of new cysts formed and the numbers of unhatched larvae remaining in the nylon bags are shown in Fig 26. Although the greatest numbers of unhatched larvae of G. pallida were found with plants of the variety Record, the differences in the numbers of larvae were not significant. The analysis of variance of numbers of new cysts formed per plant (Table 58) shows that variety and its interaction with population had effects significant at the 1 and 5% levels respectively. Treatment means by variety show fewest cysts with the G. rostochiensis resistant variety Maris Piper and Record (means 139 and 179 cysts/ pot respectively compared to 367 and 521 for Arran Banner and Pentland Ivory respectively). This variety effect is even clearer in the table for interactions where, as expected, Maris Piper had very few cysts of G. rostochiensis, compared with Arran Banner and Pentland Ivory (means 359 and 475 cysts/pot respectively) and even Record (mean 295 cysts/pot). Although Arran Banner and Pentland Ivory were also very good hosts for G. pallida (means 374 and 567 Globodera rosrochiensis Globodera pallid() 0 200 a 400 L) b00 800 g 1000-4t. 1200 1400 lu 17 1500 (800 !al- 2000 -g AB R MP PI AB R MP PI Fig. 26. Hatching of G. rostochiensis and G. pallida larvae and their ability to form new cysts on four different potato varieties. 160 Table 58. Analysis of variance of numbers of new cysts formed per plant. SOURCE OF VARIATION DF MS VR Level of significance POPULATION 1 8922 0.295 VARIETY" 3 249657 8.261 1% POR x VAR. 3 83422 2.761 5% RESIDUAL 23 30220 Populations Varieties Varieties x Populations G. rostochiensis G. pallida G. rostochiensis 285 Arran Banner 367 AB 359 374 Record 179 R 295 63 G. pallida 318 Maris Piper 139 MP 10 269 Pentland Ivory 521 PI 475 ' 567 S.E.D. 62 87 123 L.S.D. 5% 130 180 254 1% 173 243 344 161 cysts/pot respectively), this was not true for Record (mean 63 cysts/pot). Analysis of variance (log transformation) of numbers of eggs per new cyst (Table 54) also show significant effects for population, variety and their interaction (1.% level). G. rostochiensis cysts found on Maris Piper contained almost no eggs, and although the egg content of G. pallida cysts on Record was not decreased as much as was the numbers of cysts that formed, the egg content of cysts of this species on this variety was less than that of all other cysts except G. rostochiensis on Maris Piper. Table 60 is the analysis of numbers of new eggs produced by each egg in the inoculum and it shows that variety had an effect significant at the 1% level. The table of means for variety shows significantly lower multiplication rates on Maris Piper and Record potato varieties (means 2.1 and 3.1 respectively), but there was no significant difference between the overall means for reproduction of the two species of nematode. Somewhat surprisingly the population x variety interaction did not have a significant effect. Multiplication rates for G. rostochiensis varied from 0 on Maris Piper to 8.3 x on Pentland Ivory, and for G. pallida it varied from 0.8 x on Record to 11.5 times on Pentland Ivory. Table 61 is an analysis of the numbers of hatched larvae divided by the numbers of new cysts formed and shows effects significant at the 1% level for population, variety and the interaction between them. Treatment means show that G. rostochiensis required a larger number of hatched larvae to produce a new cyst (mean 277 larvae) than G. pallida, obviously due to Maris Piper (resistant to G. rostochiensis). The table for interactions shows 162 ) Table 59. Analysis of variance of logarithmic transformation of number of eggs per new cyst formed. SOURCE OF VARIATION DP MS VR Level of significance POPULATION 1 1.64801 137.855 1% VARIETY 3 1.84136 154.028 1% POPULTN.x VARIETY 3 1.72516 144.308 1% RESIDUAL 23 0.01195 TRANSFORMED RESULTS (Log) Transformed means Populations Varieties Varieties x Populations G. rostochiensis G. pallida G. rostochiensis 1.768 Arran Banner 2.261 AB 2.248 2.274 Record 2.170 R 2.227 2.113 G. pallida Maris Piper 1.279 MP 0.358 2.200 Pentland Ivory 2.272 PI 2.241 2.302 S.E.D. 0.039 0.055 0.077 L.S.D. 5% 0.080 0.114 0.159 1% 0.109 0.154 0.215 UNTRANSFORMED RESULTS Populations Varieties Varieties x Populations G. rostochiensis G. pallida G. rostochiensis 131 Arran Banner 186 AB 178 193 Record 149 R 169 130 G. pallida 170 Maris Piper 80 MP 2 158 Pentland Ivory 187 PI 173 200 163 Table 60. Analysis of variance of multiplication rates. SOURCE OF VARIATION DF MS VR Level of significance POPULATION 1 5.65 0.406 VARIETY 3 1o4.66 7.545 1% POPULTN.x VARIETY 3 31.57 2.276 RESIDUAL 23 13.87 Treatment Means Populations Varieties Varieties x Populations G. rostochiensis G. pallida G. rostochiensis 5.1 Arran Banner 7.1 AB 6.8 7.4 Record 3.1 R 5.4 0.8 G. pallida 6.0 Maris Piper 2.1 MP 0.0 4.2 Pentland Ivory 9.9 PI 8.3 11.5 S.E.D. 1.4 1.9 2.6 L.S.D. 5% 2.9 3.9 5.4 1% 3.9 5.3 7.3 164 Table 61. Analysis of variance of hatched larvae to produce a new cyst. SOURCE OF VARIATION DF MS VR Level of significance POPULATION 1 355754 18.749 1% VARIETY 3 445388 23.473 1% POPULTN.x VARIETY 3 525496 27.695 1% RESIDUAL 23 18974 Treatment Means Populations Varieties Varieties x Populations G. rostochiensis G. pallida G. rostochiensis 277 Arran Banner 36 AB 39 34 Record 109 R 42 177 G. pallida 66 Maris Piper 521 MP 1008 34 Pentland Ivory 21 PL 21 21 S.E.D. 49 69 97 L.S.D. 5% 101 142 200 1% 137 193 271 165 how poor a host Maria Piper is for G. rostochiensis (1008 larvae required to produce one new cyst). naris Piper was an efficient host for G. pallida, as were Pentland Ivory and Arran Banner. Although Record was not significantly different from these three for G. pallida, it did not behave as a very efficient host (177 larvae to produce a new cyst). (iii) Discussion The results show clear differences between commercial potato varieties with regard to the biology of potato cyst-nematodes, as suggested earlier by other workers (Gemmel, 1943; Ellenby, 1946; Kort, 1966). The hatching results showed that G. rostochiensis hatched faster than G. pallida and this was presumably due to the temperature (20°C) at which the tests were conducted (see page143) They also showed a poor response by G. pallida to root diffusate from the variety Record, suggesting a poorer stimulating effect (Fig. 25), which did not occur with G. rostochiensis. Growing this variety might therefore favour multiplication of G. rostochiensis in a population consisting of a mixture of the two species. Results of the second experiment described in this study, confirm the status of Record as a poor host. Hatching was estimated from the number of unhatched larvae remaining in the inoculum and showed poor hatching of G. pallida in the presence of the variety Record. The number of new cysts of G. pallida produced on this variety was also lower than with the other varieties (Fig. 26), and this was not only due to the poor hatching. Table 61 shows that a greater number of hatched larvae was required to Produce each new cyst. Furthermore, these new cysts on Record contained smaller numbers of eggs (mean 130 eggs/cyst) than those on the other varieties (Table 59-). 166 Huijsman (1974) suggests that all potato varieties, including resistant ones, are hosts for both species of the nematodes, as larvae will invade even the most resistant and establish giant cell feeding sites: it is after this stage that the resistance mechanisms operate. However, the variety Record, with no major genes for resistance but of uncertain pedigree has recently been shown to possess a measure of resistance to G. pallida in that multiplication in the field was less than expected (Dunnett et a:I, 1976). It may also have played some role in unexpected experimental results (Ambrogioni, 1966) and may even be important in the competition and distribution of species of potato cyst-nematode in areas where this variety is cultivated. The behaviour of this variety in some way may be similar to that shown by resistant lines of Solanum vernei: low root diffusate activity and poor host condition (Williams, 1958; Dunnett, 1960). This uncommon host-parasite relationship shown by the potato variety Record (Solanum tuberosum ssp. tuberosum) and the population UK-2 of potato cyst-nematodes (G. pallida), could be only part of the great variation of interaction between individual pathogen isolates with host cultivars (Raski, 1952; Oostenbrink, 1955; Meeuse, 1973; Johnson and Taylor, 1976; parker and Olthof, 1976). 167 III. GEOGRAPHICAL DISTRIBUTION OF POTATO CYST-NENITOD,2_:S The potato cyst-nematodes (Globodera rostochiensis and G. pallida) where occur in most countries potatoesare grown intensively. Recent records in some countries do not necessarily indicate recent introduction. The main infested areas include most of temperate Europe, Iceland, Newfoundland, New York State (including Long Island) U.S.A. and Vancouver Island, Canada, Israel, New Zealand, India, the Mediterranean basin and South America. They are thought to be indigenous only in S. America, from where they have spread _ to other countries. Much long distance spread has been with seed potatoes exported from Northern Europe. Near the equator potato cyst-nematodes occur either at high altitudes, or on islands or coastlands where the climate is moderated by the sea: in Finland their distribution extends to just north of the Arctic Circle (Sarakoski, 1976). Spread in parts of the world other than those mentioned above is continuing and there is no reason to suppose that potato cyst- nematodes will not eventually be found wherever potatoes are grown except perhaps in the lowland tropics. It is doubtful whether they can flourish in hot soils where most species of root-knot nematodes (MeloidoRyne sp.) are abundant and where the soil temperatures exceed the tolerance of potato cyst-nematode populations for part or the whole of the year. In hot Moroccan soils, populations decreased by 95% within seven months of harvest and so withholding potatoes for two years results in virtual elimination of the pest (ScHhter, 1976). The distrihution of these nematodes is compiled from many different sources and in such as the annual reports of the European and Mediterranean Plant Protection Organization (EPPO) (Anon.1955,1966, 1974). 168 The national and world distribution patterns of potato cyst- nematodes have been studied more thoroughly than those of any other species. The distribution of these nematodes (formerly regarded as one species, G. rostochiensis) was mapped by Oostenbrink in 1950, who reported its from 14 countries. Since then additional records have been listed or mapped as well as the possible routes by which the potato cyst-nematodes have spread from the Andes (Jones, 1964; Southey, 1965; Spears, 1968, 1969, 1970; Stelter, 1973). The most recent map . of the distribution of G. rostochiensis and G. pallida separately is that in Evans & Trudgill (1976), who report . occurrence. in New Zealand, Japan, Morocco, South Africa, Cyprus (Jones, 1976) and some Latin American countries e.g. Venezuela, Colombia, Ecuador, Mexico, El Salvador and Costa Rica. III.1 ANDEAN POPULATIONS Willi et al (1952) first reported the wide distribution of "golden nematode" in Peru, and Bell et al (1955) its occurrence in Bolivia. Martin et al (1963), sampling potato growing areas in the Peruvian Andes, found the "Golden nematode" widely distributed, with populations varying from 1 to 700 cysts per 100 g. of soil. Infestation were mostly between 2000 and 4000 m. above sea level with the heaviest infestations between 2900 and 3800 m.a.s.l. From these records and the Indian practice of fallowing land where potatoes are grown (Mishkin, 1946; Simon, 1954) which demonstrates their knowledge of the deleterious effect of growing potatoes continuously. Jones (1951) and Willi et.al.,(1952) suggested that potato cyst-nematodes originated from the Andes. Potato cyst-nematodes have been found in South American countries lying along the Andes Cordillera usually under the name 169 "golden nematode" which is strictly G. rostochiensis, i.e. Peru (Willi et al, 1952), Bolivia (Bell et al, 1955), Argentina (Brucher,' 1961); Chile (Spears, 1968); Venezuela (Dao & Gonzalez, 1971; Osoris, 1971); Colombia (Nieto et al, 1973); Ecuador (Baeza, 1972). However Evans etal (1975) mapped the distribution of G. rostochiensis and G. pallida in some of these countries, and showed that most Andean populations north of Lake Titicaca are G. pallida, which is not the golden nematode. The distribution of G.roaocKensis is mainly south of Lake Titicaca. _ (i) Materials and methods The identification of many of the one hundred and thirty one populations from Six Andean countries based on female colour (T. ZApps.) has been confirmed by measurements of cysts (R6Appx-) , and second- stage larvae (114.4r..) and by tests of ability to multiply on differential clones bearing genes for resistance (p. 92.). Addition- ally, the measurements of second-stage larvae of some populations have been submitted to canonical variate analysis. (ii) Results Figure 27 gives the location of potato cyst-nematode populations studied. It confirms Evans et al's (1975) view, that the two species occupy different zones in the Andes. The demarcation line between the two species is near 15.60S. With few exceptions populations north of this line are mainly G. pallida. Those from areas around Lake Titicaca and further south are predominatly G. rostochiensis with few G. pallida or mixtures of both species. Population B0-25 , the most southerly from the east side of the Andes is a mixture of G. rostochiensis and G. pallida. Populations VE-1, VE-2 and CH-1 may represent recent introductions. Canonical variate analysis plotted on the first two axes (Fig. 8 p. 60 ) which combines the measurements of stylet length, 170 • • G. rostochiensis 0 G.pallida o Grast/ G. pall. • Fig. 27. Distribution of potato cyst-nematode species in some South American countries. 171 distance from tip of the head to the excretory pore, tail length and total body length, showed that most populations from Colombia, Ecuador and North and Central areas of Peru identified as G. pallida vary considerably, with the exception of those coming from the same valley (C0-1, C0-2, etc. and PJ, PI etc.). Populations from the south of Peru and Bolivia are very different. Although G. rostochiensis populations are clearly separated from G. pallida, mixed population are dispersed and occupy intermediate positions. Populations of G. pallida from southern areas also appear to be - different from those coming from northern areas. (iii) Discussion The two species of potato cyst-nematode are differently distributed in the Andes and factors which may be responsible include daylength, temperature, altitude, rainfall or the interaction of any of them with the host potato. Human activities over centuaries may also have influenced distribution. Although results (Section II) on effects of daylength and temperature on the biology of the two species apply only to Solanum tuberosum ssp. tuberosum varieties, they showed the beneficial effect of long days (16 hrs.) on both species. However, we know nothing of the effect of these conditions on the sub- species andiFena as host. Therefore it is impossible to say what interactions between daylength or temperature and this host occur though it may be little different from effects on tuberosum varieties. Nevertheless small differences may be important as the nematodes and their hosts have evolved together for many years. Both species had more or less the same optimum temperature for development, but with different ranges. Altitude, mean annual temperature (°C), mean annual rainfall (mm.), latitude and daylength of different 172 places along the Andes are in Table 62. More detailed information (i.e. daily or monthly maximum and minimum temperatures)are excluded because records are unavailable for many localities. Nevertheless, daylength fluctuations are larger as latitude increases but where the two species of potato cyst-nematodes overlap, daylength fluctuates little and As. the same for both. However, at the same or similar latitudes, changes in temperature and rainfall are large, depending mainly on the altitude and whether the site is on the eastern or the western slopes of the Andes. Table 62. Meteorological information of some localities along South American Andes. Daylight(hrs.min)/ 2 3 Country Locality Latitude Max Min Altitude oC mm Longitude Venezuela Merida 8.6°N 12-38 1f-37 1640 18.7 1779 71.2 Colombia Pasto 1.2°N 12-12 12-03 2594 14.1 816 77.4 Ecuador Quito 0.2°S 12-08 12-07 2850 12.5 1120 78.5 Peru Cajamarca 7.2°S 12-33 11-42 2810 14.6 1144 78.5 Cerro de Pasco 10.7°s 12-46 11-28 4350 5.7 885 76.3 Jauja 11.8°S 12-50 11-25 3450 10.9 497 75.4 Cuzco 13.4°S 12-55 11-20 3380 10.7 8o4 71.9 Puno 15.8°S 13-04 11-11 3822 8.3 963 70.0 Arequipa 16.3°S 13-06 11-09 2451 13.8 106 71.6 Bolivia La Paz 16.5°S 13-07 11-09 3658 9.3 562 67.9 Cochabamba 17.4°S 13-11 11-05 2575 17.3 462 66.0 Sucre 19.0°S 13-17 11-00 2850 12.4 665 65.2 011ague 21.2°S 13-26 10-51 2695 6.8 62 68.0 Argentina La Quiaca 22.1°S 13-30 10-45 3458 9.6 296 65.7 Ledesma 23.8°S 13-37 10-40 458 21.2 782 64.8 S.A.Cobres 24.1°S 13-38 10-39 3777 8.6 104 66.3 173 Table 62 (Contd.) Daylight(hrs.min)1 2. 3 Country Locality Latitude Max Min Altitude °C mm Longitude Argentina Guemes 24.6°S 13-40 10-35 • 19.9 513 65.0 Salta 24.7°S 13-41 10-36 1220 17.5 712 65.6 Tucuman 26.8°S 13-50 10-27 481 19.1 974 65.5 Andalgala 27.6°S 13-54 10-23 1070 18.6 272 66.5 Catamarca 28.5°S 13-53 10-19 547 20.5 357 66.0 La Rioja 29.4°S 14-02 10-15 429 19.6 321 66.9 Cordova 31.4°S 14-12 10-06 482 17.0 714 64.2 San Juan 31.6°S 14-13 10-04 630 17.2 94 68.6 Mendoza 32.8°S 14-20 9-59 755 15.8 194 68.8 San Luis 33.2°S 14-22 9-57 708 16.6 566 66.4 Chile Coquimbo 30.0°S 14-05 10-12 27 14.6 114 71.0 For Northern latitudes : maximum in June and minimum in December- January Southern latitudes : maximum in December and minimum in June- July 2 Lean temperature 3 Rainfall Although ecological factors influence the genetic variability of individuals and / or populations (Alice et al, 1969) it cannot be assumed that the factors listed above had an effect on the speciation of the potato cyst-nematodes, since this event must have occurred of many thousand/years ago, when the climatic conditions in the Andes, as will be seen later - were different, and speciation of the potato and other oolanum hosts was also occurring. Divergent evolution depends upon reproductive isolation, which includes any factor that prevents gene flow from one population to another. Then, under different conditions of selection, heritable variations accumulate. So, biological (reproductive) isolation is 174 a necessary condition for species formation. Reproductive isolation results after other types of isolation occur, such as geographical (physical barriers, mountains, lakes, deserts, etc.), ecological (behaviour etc. modified by the environment) or genetic (sterility between groups of individuals from sudden changes in the gene and chromosome mechanisms, due to mutation or polyploidy). Most species with mutually exclusive geographic ranges (allopatric distribution) are isolated by topographic barriers which lead to the formation of new species in separated geographic areas. However, speciation also occurs in closely related species living together (sympatric species) but occupying different habitats. Furthermore, if a selection pressure (such as a host plant with resistance genes)is greater than dispersal and cross breeding between partially isolated populations, divergence of populations may also occur. Adjustments to variation in seasonal factors such as temperature or light may also bring reproductive isolation between closely related populations. So we may suggest that in the conditions of the mountain plateaux of South America, where potatoes and their "followers" i.e. round-cyst nematodes co-evolved, there were spatially isolated, inbreeding populations in which genetic change was enough to cause partial reproductive isolation, but from a common ancestor. Nematodes themselves are relatively immobile, so each local habitat (field)is isolated and isolation is strengthened by deep valleys and other topographical features. This isolation probably played an important role in speciation. In these habitats the Solanum sp. and the potato cyst-nematodes evidently evolved together, the nematodes diverging along separate paths provided by diverging species of Solanum, separating first into races and ultimately into 175 species as also occurredwith the round cyst-nematodes group as a whole and the family Solanaceae. From the distribution pattern of • the two species of potato cyst-nematodes and their races, it seems likely that different modes of speciation have occurred. In the first place, sympatric speciation probably occurred on the eastern slopes of the Central Andes. This is assumed because this mode of speciation occurs at the centre of a 'species' range rather than at the periphery, (which would be parapatric) and because previous reproductive isolation before populations shift to new areas they were no longer able to mate successfully. Under the conditions of the geographical area under study, a shift to a new area (including new hosts) could have a profound effect and provide a strong barrier to gene exchange, resulting in the development of sufficient genetic divergence to guarantee the speciation. Final development of reproductive isolation by allopatric speciation may have occurred due to the presence of a geographical barrier such as the Altiplano between the southern edge of Peru and north western and central areas of Bolivia,' which lies between two parallel mountain chains, the Cordillera Occidental (western range) and the Cordillera Oriental (eastern range). On this plateau, which averages more than 4000 m. above sea level, the lowest parts are occupied by Lake Titicaca (3180 m.a.s.l.) in Peru and Lake Poopo (3688 m.a.s.l.) in Bolivia (Carpenter, 1976). Allopatric sneciation is very common in almost all groups of sexually reproducing animals (Dobzhansky, 1970; Grant, 1963, 1971; Mayr, 1963) since gene flow is interrupted and genetic differences begin to accumulate as each population responds to its own array of selective forces and tracks its everchanging environment,including its host plants,which themselves evolve resistance genes. These populations will not fuse once they 176 reestablish contact if enough genetic differences have accumulated during isolation to ensure that hybrids between them are of such poor fitness that they are strongly selected against or eliminated. Such patterns of isolation along with the geographic or ecological ones probably occurred during and after the Pleistocene, when a series of glaciations occurred. It is known that both biological and geological investigations in situ have increasingly revealed that Quaternary climatic events actually played a significant role in the evolution and adaptations of the biotas of South America (Vuilleumier, 1971). The evidence supporting this conclusion is derived from two sources: analysis of speciation patterns of the flora and fauna throughout the continent, and geological and paleobotanical studies that document the requisite climatic events. It has been established that, in using speciation patterns as clues to the evolutionary history of a group of organisms, it is necessary to locate areas of contact between distinguishable forms. These areas are recognised as stepped clines, hybridization belts, or areas with narrow sympatry of closely related taxa. Consequently we can assume that an area in which we now find overlapping of differentiated forms and indicate where the two forms had previously been separated. Such a distribution is even more evident if it coincides with a visible physical or ecological feature, i.e. the Altiplano or Lake Titicaca. Speciation patterns in South America have already been divided into geographical regions, each of which circumscribes a more or less coherent biota and appears to be (and, therefore, was also in the past) under a distinct climatic regime; the southern Andes, Patagonia, the Central and Northern Andes, the lower mountain slopes and the tropical lowlands (Vuilleumier, 1971). However, for our purposes, only some of them 177 will be considered from the point of view of speciation pattern during the Pleistocene when, after the uplifting of the Andes, an alpine-like vegetation developed and angiosperms were already evolving. The Central Andes consist of several parallel mountain chains that begin in Northern Peru and the broad, flat Altiplano into which they merge. This plateau, flanked by high ranges, covers southern Peru, western Bolivia, north western Argentina and adjacent parts of Chile. The Altiplano itself lacks ecological barriers but there may have been geographical ones. In Bolivia zones of contact between species occur along climatic and vegetational gradients. During the Pleistocene a great ice expansion occurred and mountain glaciers in Peru, Bolivia and Argentina were more extensive and reached lower down the slopes, with the snow line coming lower in the east. Moreover, the interandean region (Altiplano) became flooded by huge lakes, now represented by small remnants (Titicaca and Poopo). The combination of extended areas of glaciers or tongues of ice created enough of a gap in the puna vegetation to have caused divergence of populations on either side of it (Vuilleumier, 1969). Also the increase in glacial lake surface area would have restricted distribution of animal and plant taxa living there to ice-free and non-flooded refuges (Fig. 28). If these glaciers and expanded lakes restricting dispersal and gene flow were removed during an interglacial period, secondary contacts of previously isolated populations would be possible. If this is correct, glacial maxima in the Central Andes would have corresponded to periods of genetic differentiation in relatively small, geographically isolated populations living in refuges in the interandean region. During an interglacial, removal of most of 178 80 70 60 50 40 90 80 70 60 50 40 30 20 Fig. 28. Features of the Wurm glaciation and an interglacial episode. Dotted areas show the location of interglacial sea trans - gression and the large,inland,freshwater lake in Western Argentina. 179 the ice and lakes would promote range expansions. It is also known that mountain glaciers, now above 4500 m may have reached down as low as 3000m locally in the northern Andes and that the simultaneous temperature depression during the Pleistocene caused a lower and narrower belt of vegetation than exists today (Fig. 29). An upward extension of forest during an interglacial (as now) would restrict the non-forest vegetation to higher and more isolated populations. It seems therefore that geographically isolated populations (plants and/or animals) may have shifted latitudinally from the Central Andes to the Northern Andes during glacial times, becoming geographically re-isolated during interglacial times as the vegetation line moved upwards. Opportunities for speciation in the Andes during the Pleistocene were repeated because there were three or four glacial-interglacial episodes. Although the presence of glacial advances and their positive influence in the modification of plant and animal distributions in the southern and Central Andes is known, the effects of glaciation on the flora and fauna of the northern Andes is only beginning to develop. From Venezuela to Ecuador the area above the timberline and below the zone of permanent snow is now covered by paramo (humid grassland). These high paramos are isolated from one another by expanses of lowland or montane forests but an analysis of these paramo "islands" has shown that it is primarily derived from the high central Andes and not from Central America, and this could have been caused by shifts in the paramos as a consequence of glacial events. It is, in fact, known that glaciations did occur in the northern Andes and organisms from high altitudes were thus allowed to disperse comparatively freely when vegetation zones were lowered, but were restricted in interglacial times, when warm climates Elevation (1000 meters) — 0.- o 2-* 1171-.V. r2, cr Co San Valentin 4058m H P• 0 0 O e■ t7 f7 • t7 CoTronador 3410m O Gzi CD 0 o 0-• =4r. c 9 CD 0 el- Cn 0" C°Aconcagua 702Im c-t- 0 , 0 H CD --t G., 5 ft> \ 0 C OD Vns Ojos del Salado 5885if • CD IV o N O 0 C•1 O 0 • 0 Nv Illimani 6457m ti d- I o 0 Cord. Vilcabamba Rs. I: / • Nv Huascaran 6768m O R. c+ H P- Vn Chimborazo 6272 m 0 I Cotopaxi 5896m / I Cr4 . . Nv de Tolima 5620m • o ta co . Sierra Nevada (Merida) 5002m ct- CD ct L___---)Santa Marta 5775m CD .••••••-- o z z 0 -181 prevailed. Undoubtedly, these events have also played a part in the evolution of the wild tuber-bearing Solanum species. Hawkes et al (1965) suggests continental drift as a more acceptable theory than that of land bridges, in the early dissemination of the Solanaceae. This continental drift occurred during the Mid-Jurassic (150 million years ago) to the Mid-Cretaceous (90 million years ago), when the present outlines of the continents were defined and the genus Solanum was disseminated as sub-genera in the Southern hemisphere (in South America, Australia, New Zealand and Africa). At this dating of continental origin and drift, the Angiosperms are thought to have been actively evolving and the genus Solanum was already in existence. This may be the reason why this genus is one of the largest in the plant kingdom. . The same author (Hawkes, 1958, 1966) postulated that the tuber- bearing Solanums might have originated somewhere in the region of Mexico to the southern United States during late Cretaceous to Tertiary times. Before the mid-Eocene certain species had migrated to South America where further evolutionary development took place. The Central American land bridge was destroyed from mid Eocene to Pliocene times (45 to 7 million years ago) but was then restored. A return migration of diploid species might then have taken place, with the restoration of this bridge, producing tetraploid species by hybridization with the original Mexican diploids. A second, and probably very recent, return migration would have brought in a second diploid species, S. verrucosum. Hawkes (1958) supports these theories of evolutionary pathways in Solanum species by serology and crossability studies, which show that Mexican diploid groups stand out sharply from polyploids and South American diploids, 182 but the polyploids contain an indigenous Mexican genome and it is perhaps significant that S. polyadenium seems to form a bridge between the two groups (Fig. 30). Although there are present-day climatic belts in Central America over which S. velfucosum would not migrate it may be assumed with some certainty that changing conditions during the Pleistocene (since 2 million years ago) made such a move possible. Thus, once the tuber-bearing Solanum species were in South America, the evolutionary events during Pleistocene explain their• great variability largely due to the wide range of climatic and other selection pressures which acted and isolated one from the other by eco-geographical barriers in well-defined altitudinal belts (Fig. 31). These events must have occurred more intensively on the Altiplano around Lake Titicaca, where the wild diploids from which the cultivars arose are native and the greatest variability of potato is to be found. To the West (Central Andes), the Cordillera is the watershed between the Pacific and the Altiplano and the tropical lowlands. The north-eastern section of the Eastern Cordillera drops precipitously to the Yungas (Amazonian forests) but in the scuthern section the descent to the tropical lowlands is more gradual. These slopes are dissected by long valleys in which large basins provide favourable areas for cultivation and settlement. This area, where the suspected ancestor to all the modern cultivars of the potato, the primitive group of Stenotomum cultivar is found, has regularly occupied the attention of potato specialists as the place of domestication of the potato. The botanical origins or the wild species from which the diploid cultivars might have derived are not well established. Hawkes (1967) contends that these may have arisen from the wild diploids native to the Titicaca Lake, 133 Morel ii form to Bulbocastuna Pinnatisocta LongIpedicellata DEMISSA Conicibaccata and South Polyadenia American series FIG. 30. Crossability groups In Mexican potato series. 111111 till Eruberosa S. brevidens — S. calvescens S. commersonil subsp. commersonli Commersoniano S. commersonil subsp. malmeanum S. chacoense subsp. ,nacoense I S. chacoense subsp. muelleri I I S. torljense I 1 I I S. acaule subsp. acaule _ _ 1 I Acaulia I I I S. acaule subsp. aemulans I I I I I I S. infundauliforme --.— ------Cuneoalaro I I 1 I 1 S. megistacrolobum 1 I Meosracroloba I S. boliviense ----- 1 I I I S. sanctae-rosse I I I 1 I -- 1 S microdontum suosp. microdontnm I I — I S. microdontum subsp. gigantophyllum I I S. venturit I I S. vernal subsp. vernal 1-- I I S. vernal subsp. balleii 1 1 S. gourlayi — Tuberosa S. oplocense (based also on Bolivian material) 1 I I I S. vidaurrel I S. 1 egazzinil I 1 S. kurtrianum 811) I I — S. maglia 1 I I LJIIIIIIII f I t I L I I I 0 t.p -- NJ r....1 t.^I 41 7 0 lf, t./1 O 0 (61 8 `16' o 0 O 0 8 0 0 0 0 0 0 o 0 0 0 0 0 0 Metres FIG. 31. Altitude ranges of wild potato species. 184 S. leptophyes,S. canasense and S. soukunii, while Bukasov (1941) agrees with the last two species but adds two more, S. multiinterruptum (from Peru) and S. sparsipilum (from Bolivia and Peru). The date of domestication of the potatoes is difficult to decide with any accuracy, but like wheats, corn and many other crops, the common potato did not come into existence until well after man's arrival (10 to 15000 years ago). This arrival of man in the South American Andes, according to archeologists, is assumed to have been either down the Andes from the north via the Isthmus of Panama, or from eastern parts of South America over the Andes quite early in the history of the South American agriculture (Correll, 1962). The latter supposition is supported by the observation that traces of groundnut, a plant which originated east of the Andes (probably in north western Argentina) occur in archeological sites on the Peruvian coast (1000B.C.) (Engel, 1960). In addition, some authors agree that the initial impetus of Central Andean agriculture may have been within the highlands or to the east of the Andean crest (Lathrap, 1969; Willey, 1971; Patterson, 1971a,b). The early man brought with him an economy based soley upon hunting, fishing and plant gathering (Towle, 1961). The tubers of the various wild potato species might well have been included among the very first plant species that were gathered and utilized (7 to 5,000 B.C.; Jensen & Kautz, 1974). The crucial first step acquired at this stage was undoubtedly the knowledge of variation in alkaloid contents (bitterness) of the tubers by the first potato selectors. Then, the accidental or deliberate introduction of the tubers of two or more wild species of potato to the campsites of early man may have been the next step toward the development of a primitive 135 cultigen, favouring the hybridization of two or more formerly isolated species (Ugent, 1970). However, as settled populations became larger .(4 to 2000 B.C.), man learned to exercise a higher degree of clonal selection upon his crop, planting those cultivars which showed favourable yield, desirable flower, or other good qualities. This hybrid-clonal-heterozygosity cycle terminates or diminishes with the introduction of the spontaneously formed tetraploids, which were selected quite rapidly into their present Andean form when their superior qualities became known. Evidence so far accumulated by studies of this tetraploid complex (s.tuberosum) indicates that both S. stenotomum and S. sparsipilum may have been involved in the origin of the tetraploid group in the Central Andes (Cribb, 1972). Then, as when tetraploids were disseminated by Man (over long distances because of their quality) to the north and south, the potato cyst-nematodes which had already evolved as separate species and were co-existing with wild potato species in different ecological sites in the Altiplano and eastern slopes of Central Andes, may have also been spread further. The above suggestion is made on the basis that plant pathogens in their most diverse and primitive forms are found where the ancestors of cultivated crops still exist as wild species (Zhukovsky, 1965; Braverman, 1972). Furthermore, as Leppik (1965, 1966) indicates, it is possible to trace the evolution of host and parasite as consequences of reciprocal selective factors when the actual centres of both have long been associated with each other. Examples of these reciprocal adaptations are those such as the gene center of wheat and the greatest number of physiological races of Puccinia graminis; the gene center of wild species of tomato contains genes for resistance to common diseases and insects;similary for cucumis center, lettuce center, etc. (Leppik, 1970; Dinoor, 1975). 186 It is also known that interference by man invariably disturbs this balance and organisms of minor significance in one situation may become involved in epiphytotics when transferred elsewhere (Watson, 1970). With the potato both situations are known. Firstly, the great number of wild potato species from Mexico and the Andes found to be resistant against fungi, bacteria, virus, insects and nematodes and secondly the potato blight pandemic in Ireland. An interesting point and one which may be significant is that Argentinian and Bolivian potato species have been widely acknowledged as outstanding sources of resistance genes for potato breeders (Rowe, 1969; Hawkes, 1970; Okada, 1976). Resistance against the potato cyst-nematodes (Hawkes and Hjerting, 1969) has also been found in many wild potato species (p.24 ) of the north western area of Argentina and in the cultivated sub-specie andigena in Bolivia (Fig. 32). This situation leads to common assumption, as Brucher(1966) had suggested, that the center of variability and origin of the potato cyst-nematodes is located in that area. This is in disagreement with Dunnett (1960), who considered that the resistant species S. vernei and S. sancta.Tosae owe their resistance to occupying a position at the fringe of the potato cyst-nematodes' host range rather than to natural selection for resistance during the process of coexistence over long periods of time. He reached this conclusion because of their poor ability to stimulate hatching and the poor invasion of their roots by larvae of potato cyst-nematodes. The presence of G. rostochiensis (may be G. pallida) in Argentina (Jujuy and Salta provinces) reported by Brucher and the sympatric distribution of both species found in Bolivia (on the Altiplano) between latitudes 16 and 25°S may mean that the resistance of S. vernei and S. sanctae-rosae is a result of long co-existence of host and parasite, which has led to evolution of resistance in form of "field" or "horizontal" (polygenic) resistance • 137 80 70 60 - 50 a — --10 10 I _ _ — — ••••.....••• VENEZUELA \. -•1 ...: t...... • !G UI A NAS / ( \ i i COLOMBIA ( \ ) ‘ . f • -s • ... • - -• . ••••.. . -"" • r.. • •■• 0 CUA DOW'. •-••. i • f • ■. BRAZIL 10 ...•. BOLIVIA .'•‘. I- -•1 x x i .'x.x" •-• ` ( x •• I• x I : ‘. i x x • 20 • x..., ..•. : • x •NPARxAxGx xtixAxy W i -. . x .. ix :...-I .'". x 1 i X ( x •-■ x . ) j X x l;..._._, _. x X .\ x I x / x 1-0 x I • • %. . x 7 .. x •• i Q i . . x 30 • • -13 x :• l la x ,. U R iiiGUAY ..: t‘.. ▪ 1 : ,. x x ,i x x 30 1 : x •. .. . 4.7 L • :: 0 I - /-40 80 60 40 188 (Bennett, 1970; Van der Plank, 1975). More recent contact between host and parasite would lead to the type of "vertical" (single gene, monogenic) resistance found in S. tuberosum ssp. andigena and a gene for gene relation drip as suggested by Jones (1974). Similar relationships appear to exist in non-tuberous solanums e.g. S. sarrachoides (Jones & Pawelska,1963). If resistance against potato cyst-nematodes has developed in the manner suggested above, it would seem that G. rostochiensis originated first on the eastern (warmer) slopes of the Andes on wild species of potato, and due to the geological events during the Pleistocene, G. pallida arose as a second species in cold niches, showing sporadic distribution on this side of the Andes. This may explain why G. pallida is better adapted to lower temperatures than G. rostochiensis and why the potato species with the most promising resistance to G. pallida are also frost resistant. Later on, with the appearance of the first diploid cultivated potatoes (S. stenotomum, frost resistant), G. pallida probably moved through the Altiplano and beyond the barriers represented by Lake Titicaca and the chain of high mountains to the north of it. Eventually, G. pallida became widely distributed to the north of the Altiplano with the widespread cultivation of S. tuberosum ssp. andigena in the Central Andes. This theoretical suggestion may fit with Cribb's conclusions (1972) that within the area of greatest potato diversity, certain areas such as the higher valleys of the southern Central Andes where the fields are predominantly free of symptomcausing diseases would seem to be the most likely places where potato cultivation could have arisen. Furthermore, the lack of wild and weed potatoes closely related to the cultivated species on the Altiplano of North Bolivia 189 indicates that potato domestication in its early phases may have occurred at lower altitudes, in the warmer valleys that lead up to the Altiplano. This would also explain the lack of resistance against the potato cyst-nematodes in potato species of Central and Northern areas of Peru, Ecuador and Colombia due to the relatively recent contact between host and parasite. 111.2. OTHER POPULATIONS. Although parasite and host can move together with cysts adhering in soil to seed tubers or actually embedded in their tissues and- the first transportations might have been by this means, much of the earlier spread of the pest in Europe was probably casual. The year of the earliest introduction to Europe in unknon and was possibly after 1850 but could have been earlier. Cysts on potato roots were first recorded in 1881 in Germany. In Great Britain the earliest records of symptoms suggestive of serious damage to potato crops date from around 1900 and the pest was not positively identified until 1913 (Southey, 1965). From small beginnings before 1900 to the present, potato cyst-nematodes have spread in most countries of Europe and their distribution is increasingly coincident with the cultivation of potatoes. Later on, undoubtedly with the development of improved varieties in Europe and their trade, potato cyst-nematodes were carried to virtually all the potato-growing areas of the world. There may be a few exceptions such as Australia, Hungary and countries located in the low and wet lowland tropical areas. Some traffic in cysts may have been in produce other than seed potatoes, e.g. daffodil bulbs. The infestation in Vancouver Island, Canada may have arisen in this way as Dutch daffodils were grown there. 190 (i) Materials and methods. Thirty populations, of G. rostochiensis, G. pallida or a mixture of the two previously identified from measurements of second stage larvae and cyst terminal areas and from tests on differential clones containing resistance genes, were compared and added to existing records of potato cyst-nematode in countries outside South America. (ii) Results and discussion. The known World distribution of Globodera rostochiensis and G. pallida (Fig. 33) shows that the first predominates. However, as identifications are improved, new records of G. pallida are frequently reported e.g. New Zealand (Wouts, 1976), Norway (Oydvin, 1974), U.S.S.R. (Makovskaya, 1975) etc. The recognition of G. pallida in populations from Spain and Denmark in this study is the first indication that it occurs in these countries. Jones (in litt) informs me that his report of G. rostochiensis in India was in fact G. pallida. Although G. rostochiensis seems to predominate in the Old World, the recent incorporation of resistant varieties in agricultural schemes of many European countries has increased the numbers of G. pallida often demonstrating that this species was already present in small numbers in many fields. In areas predominently G. rostochiensis a few fields are almost wholy G. pallida. The situation seems to vary from country to country. Among countries where surveys have shown G. pallida is present, England has the greatest incidence around 50% (Anon 1957, 1960, 1961; Jones, 1958) Holland has 20 - 24% (Kort, 1962, 1974; Huijsman, 1964) and Germany (Stelter, 1971) The world map shows possible routes of spread by potato cyst- nematodes from the Andes where these nematodes are thought to be indigenous and occupy different geographical areas. N 0 N 0 / N N .;)13/ N ):5? • , ---* Globodera rostochiensis G. pallida HIM ----* Fig. 33. World distribution of potato cyst-nematodes Globodera spp. and possible routes of dissemination. 192 The spread of potato cyst-nematodes throughout the World may be explained by various historic-geographical events which occured during and after the discovery and conquest of the New World, and from knowledge of their present distribution in the South American Andes. The potato was probably first introduced into Europe around 1570 when a Spanish ship brought it from the northern Andes (Salaman, 1946). From Spain potatoes were widely spread around Europe by the end of the century. Evidence shows that ther-e were few initial introductions but that the first introduction to Britain (about 1590) was probably independent of the earlier Spanish one (Hawkes, 1967). If as Salaman suggests, the first introductions of potatoes came from the Colombian area the potato cyst-nematode introduced would have been G. pallida but if, as Lechnovicz (1961) suggests, the potatoes were introduced from the Southern Andes, G. rostochiensis would probably have been introduced. However although the potato spread widely in Europe during the 17th century, it was then only a botanical curiosity or of medicinal potential. Except in Ireland, where the potato was quickly taken up as a staple food, it had no agricultural impact. Acceptance of the potato in England was slow (Cobbett, 1823). In Sweden, the potato was introduced in 1725 but was not widely cultivated until 1764 (Corre11,1962) and in Scotland potatoes were first cultivated as a field c..-op in 1739. In 1744 Frederik II of Germany, forced the peasants to cultivate potatoes which were established as a crop in that part of Europe during the Bavarian War (1778 - 79). During the latter part of the 18th century the potato also became a part of the agriculture of France. Therefore whether potato cyst-nematodes were introduced to Europe with the first potatoes during this time is impossible to tell, but both species might have been introducedthenAn alternative which would have favoured G. rostochiensis, is that when the potato 193 reached Europe the Spanish conquerors were exploiting the silver mines in Potosi (Bolivia) and tubers and "chuno" (dry potato) were transported to feed the 'Indians' working in the mines. Hence containers used to transport silver or potatoes might have carried soil with cysts as far as Spain. At the beginning of the 19th century, from a remarkably narrow genetical base provided by the first introductions of potatoes and a few later additions such as those of Daber to Germany, a few named varieties were extent. Later on, the disastrous famines in Ireland during the 1840s. due to blight fungus Phytophthora infestans, was a significant episode in the history of the potato. Besides the tremendous social and historical impact of the disease it imposed a major new selective factor on the potatoes of the time. Some resistance became available in the 1870s in the variety Champion. However the modern potatoes which emerged around the end of the 19th century were still all founded genetically upon the original introductions as mentioned above. It was about this time that cyst-nematodes were first noticed on potatoes by Kuhn in Germany (1881) and by others from 1890 onwards, suggresting that they were introduced to Europe with breedins. materials imported in the mid-1800's to improve resistance to late blight. The pathways of introduction of the potato cyst-nematodes must remain a matter of speculation although the above suggestions fit the known facts fairly closely. yet However there isAanother possible route which seems possible. Inagaki and Kegasawa (1973) found viable cysts of G. rostochiensis in Peruvian guano, imported into Japan and suggested that this organic fertilizer appeared to be a means by which potato cyst- nematodes reached Japan. The word guano is derived from the Indian (from Peru) word "huanu" meaning excrement. This material is one of the oldest fertilizer known to man, for the excrements of 194 birds and animals were used in agriculture at least 200 years B.C. Whilst there appears to have been a period during which the fertilizer merits of guano were unappreciated or forgotten in certain European countries, it again came into use in the 1840's when a firm from Peru sent a cargo of Peruvian guano to, London (Waggaman and Easterwood, 1927). The most famous deposits of guano are those occuring on certain groups of islands off the coast of Peru between 13 to 21°S latitude, and on shores where it has been deposited over periods thousands of years. (Johnston, 1841). It is estimated that more than 10 million tons of Peruvian guano were shipped from the Chinchas islands alone (13 - 14°S) between 1851 and 1872 (Voss, 1890). In addition, guano was also shipped from other islands such as Mejillones, etc. (Bolivian territory before 1879) and .of from Iquique (20°S). Considering that Inagaki found cysts in 100 g. of guano, the introductions of cysts into Europe (mainly England and Germany) may have been as great as 26,000 viable cysts for each ton, and this occurred before Kuhn first noticed the potato cyst-nematode in Germany in 1881. Although this means of introduction and dissem- ination seems possible, neither the means of contamination of the guano nor the species of potato cyst-nematode which would have been introduced is clear. Inagaki and Kegasawa suggest that guano may be contaminated after the birds visit areas where potato cyst-nematodes occur and carry the nematodes either in their digestive tracts or on their feet, beaks etc. to the islands. However, theyrecomend further investigation since according to Krusberg and Hirsch:mann (1958) the potato cyst-nematodes are scarce in the coastal areas of Peru. th e r-Qfo Such a methodA seems most unlikely. 195 Brodie (1974, 1976), studying the effects of birds ingesting G. rostochiensis cysts on the viability of eggs and larvae, found that, depending on the species of bird concerned the cysts were completly voided in from 0.5 and 6 hrs. This was enough to destroy all larvae;viability of cyst contents was inversaly proportional to exposure in birds' excreta. So, even accepting a large flight range of the guano producing birds from the high mountain valleys to the coast or off-shore island, the flight time required to carry cysts of G. rostochiensis would be too long. Ingested cysts would have been voided before arrival or their contents Killed. In these circumstances guano would be more likely to be contaminated by the species occurring closest to the guano islands i.e. G. pallida but this appears not to have been so. Since the birds would have to cross the western Cordillera to pick up cysts in Central Andean valleys it seems unlikely that nematodes of either species were moved by this route, and that if the guano deposits were ever contaminated by cyst-nematodes, then they were probably picked up from the scarce coastal infestations. The coastal potato plantations of Peru use seed from the central Andean highlands, mainly Junin in central Peru. If, then, guano were contaminated via the birds in some way there arises the question of whether long exposure to the birds' excreta would allow any cysts to remain viable and in the light of Brodie's work this seems unlikely. Another hypothesis is proposed. Bags and sacks were and still are filled indiscriminately with fertilizer or potatoes for transport. Large quantities of guano were used on fields in southern areas of Peru during the 1840's when cargos of Peruvian and Bolivian guano were shipped from Iquique (20.0°S), Mollendo (17.0°S) and Callao (11.7°S) to Europe. If guano was packed and shipped in contaminated bags 196 potato cyst-nematodes may have been introduced to Europe (England and Germany) from the 1840's onwards. This agrees with the presence. in England of almost equal proportions of the two species and with the timing of the first discovery of potato cyst-nematodes in Germany in 1881. It may also explain why G. pallida infestations centre on the Humber port of Hull. It would also explain the movement to Europe of the two species of potato cyst-nematodes particularly as England was by far the biggest customer for guano during the mid-ninetenth century, and permit the suggestion that there may have been two different routes of introduction to Japan. One with imported guano from Peru, which could have been either species, and another with seed potato tubers imported from Europe which was mainly G. rostochiensis. This latter is assumed because only G. rostochiensis has been reported from Japan, However the dimensions of stylet length of hatched second stage larva from cysts extracted from Peruvian guano samples given by Inagaki and Kegasawa (1973) are closer (23 pm) to G. pallida than G. rostochiensis. This is also true of larval from population JA - 2 (from Japan) which appear to be G. pallida. The next stage of potato cyst-nematode dissemination begins in the 20th century during the first quarter of which breeding methods were still empirical, being improved by more scientific approach when introductions of wild potatoes from South America began. Although the contribution of the wild species and primitive cultivars brought from South America by expeditions has had significant impacts on potato breeding (resistance against potato cyst-nematodes, virus, disease) and the botanical concepts of the potato and its relatives, it is also possible that many pests and diseases were introduced with potato tubers (and true seeds) brought by early 197 expeditions to European countries. Quarantine regulations were not so strict as now. Expeditions started with the Russian ones under the leadership of Bukasov in 1925 and 1932, but before this there were several others e.g. that of Wight in 1913 who travelled through Chile and Peru. In 1930, Baur and his associates made a trip through South America for the Max-Planck Institute of Germany and at about the same time Sweden sent an expedition too. In 1930 and 1931 Russell and others explored first Mexico and then South America for the United States Department of Agriculture. In 1938 Balls collected wild and cultivated potatoes from Mexico and in 1939, joined by Hawkes,collected from Peru, Colombia, Ecuador, Bolivia and northwest Argentina. Correll explored Mexico in 1947 - 8 and South America in 1958. Hawkes, in 1949 and then in 1969, with Hjerting and Lester collected potatoes from the United States to Nicaragua. The same year Ross and Rimpao of Germany explored South America. Since then many more collecting expeditions have been made. These expeditions show the world-wide interest in the potato but especially of Europe, where potatoes are a substantial part of the human diet and a source of income from the export of improved varieties, either as seed tubers or for consumption. About 89% of Venezuelan potato crops are raised from seed imported from Canada and Europe. Chile,Ecuador, Panama, Mexico and other countries all over the world also plant seed from Europe. This traffic in potato tubers and indeed the opportunities of spreading cysts in soil adhering to many types of produce must have provided opportunities for further extensive dissemination of potato cyst-nematodes, so that they now seem to occur wherever potatoes are grown. 198 IV. EFFECTS OF POTATO CYST-NEMATODES ON POTATO PLANTS Plant parasitic nematodes injure their hosts in several ways: by direct tissue destruction resulting from penetration and movement into the host and by damaging centres of differentiation. These injuries result in crop damage by altering host physiology (Bergeson, 1968; Dropkin, 1972; Wallace, 1974; Wang and Bergeson, 1974) and this may also be responsible for tissues becoming more susceptible to associated fungi and bacteria (Powell, 1971; Bergeson, 1972). The general symptoms in crops infested with root-feeding nematodes are patches of stunted plants which are often overlooked because similar symptoms can arise from many other causes. Furthermore, the syndrome of an infested crop may be affected by factors such as rainfall, temperature and varietal differences. The role of nutrition in the host-parasite relationship is related to the nutritional requirements of the parasite as they may or may not be satisfied by the host environment. The parasite gets all of its nutritional requirements from the host at the site of development. Thus, provided a larva of Globodera sp. induces a syncytium of sufficient size in the root stele, it receives all the food necessary to develop into an adult female, but when the syncytium is inadequate for this purpose the larva either dies or becomes male. In this syncytium, cytoplasm is dense, outer walls are thicker than normal, but walls of cells which comprise the unit area are partly broken, nuclei are enlarged and there is no central vacuole. The walls of this syncytium next to vascular tissue have prominent ingrowths or protuberances especially where contiguous with xylem vessels (Dropkin, 1976). Jones and Northcote (1972a, b) considered these units as examples of transfer cells, specialized for the flow of nutrients from source (xylem) to sink (nematode), as Bird (1975) also found with Meloidogyne sp. The 199 Globodera-induced syncytia contain enlarged nuclei of incorporated cells. There is no evidence of nuclear division, and ample evidence of cell-wall dissolution. Jones et al (1975) found that syncytia display many of the characteristics of normal plant transfer cells and suggest that some of the observed pathology result from the gradual withdrawal of cell contents by the nematode. The wall ingrowths may develop in response to the high nematode demand for nutrients, since at this time the nematode is actively depositing glycogen throughout its intestine and hypodermis (Dropkin and Acedo, 1974). These types of changes, resulting from highly specific associations between the host and parasite, eventually reflect the fundamental quantitative relationship between the plant-parasite nematodes and growth and yield of crops, depending primarily on preplant densities. Several workers have represented changes in nematode densities by mathematical models (Seinhorst, 1965, 1966, 1970; Jones et al, 1967; Seinhorst & den Ouden, 1971) and have developed the concepts of tolerance to plant diseases, (Schafer, 1971), host efficiency or suitability and susceptibility to damage (Jones, 1956). Other workers have attempted to characterize all possible nematode-host relationships. Hollis (1963) separated parasitic and pathogenic action and suggested that 90% of phytophagous nematodes are simply parasites and about 10% are pathogens. Dropkin and Nelson (1960) developed a scheme by which parasite and host growth were related on the basis of tolerance, susceptibility, resistance and intolerance. Cook (1974) diagrammatically characterized relationships in terms of the concepts of resistance and susceptibility in reference to host efficiency, and in terms of 200 tolerance and intolerance on reference to host sensitivity. Jones (1976) suggested different terms for types of resistance according to the activity of the pest, tolerance to damage and host preference. Other schemes for quantitative purposes involve the concepts of a tolerance limit to characterize host sensitivity and 'maximum rate of reproduction and equilibrium density (Seinhorst 1965, 1967) to characterize host efficiency. Tolerance limit refers to the density below which no loss in yield occurs, but multiplication of parasite does. This limit is a dynamic rather than a stable phenomenon because it varies with the host and several environmental factors. Huijsman, et al (1969) studying the tolerance range of 118 potato varieties to G. rostochiensis found that the varieties Multa and Panther gave the highest yield and showed most tolerance. Differences in physiological reactions between more and less lolerant varieties were also shown. Seinhorst, et al (1971), found that the tolerance limit of Multa was 6 eggs per gram of soil in comparison with 1.5 eggs per gram of soil for the intolerant variety Libertas. Jones (1966) noted that the maximum preplanting population of potato cyst-nematodes that potatoes will tolerate without loss of yield is about 10 eggs/g on a sandy loam soil but 10 - 20 eggs/g on a black fen ft il. Brown (1969) with the same nematodes found that there was an average loss of 2.13 tomes/ha for each 20 eggs/g of soil before planting. Stelter and Meinl (1974) found that 3 larvae/cm3 of soil resulted in yield decline. Chitwood and Buhrer (1946) suggested that reparative growth may be involved in the improved growth sometimes found in systems parasitized by G. rostochiensis. Thus, the economic damage to crop growth, yield, and quality by plant-parasitic nematodes is known and recognized. However, 201 relatively recently the interrelationships between nematode populations and the mineral and nutrient composition of plant tissues and their water relations and metabolic processes have been studied. Kirkpatrick, et al (1959) working with Xiphinema americanum on cherry trees, found that while leaf phosphorous concentrations increased, the potassium concentration was reduced, without significant changes in leaf calcium, magnesium and sodium concentrations. Meinl and Stelter (1963) measured the effect of potato cyst-nematodes (G. rostochiensis) on growth, time of flowering, CO 2 uptake, and water requirements as well as tuber production. Hollis (1963) on the other hand suggests that poor haulm growth resulting from nematode damage could be caused by the withdrawal of nutrients, the injection of toxins or the disturbance of plant hormone systems. Loveys and Bird (1973) showed that the photosynthetic rate of tomato plants was significantly decreased by large numbers of Meloidogyne javanica within one to two days of infection. It has also been shown that Meloidogyne sp. can cause a decrease in cytokinins and gibberellins in root tissues and xylem exudates of tomato plants, (Brueske & Bergeson, 1972). There is more information on the mechanisms of damage for various species of plant-parasitic nematodes but more information on the mechanisms of damage by potato cyst-nematodes has recently been obtained by Trudgill, Evans and Parrott (1975a, b) who studied the effects of different numbers of potato cyst-nematodes on the growth, nutrient uptake and yield of resistant and susceptible potato varieties in experimental plots. It was found that invasion of the roots by larvae of potato cyst-nematodes decreased root and haulm growth and concentrations in both fresh haulm and haulm dry 202 . matter of K, P and to a lesser extent Mg. Decreased tuber yield of infested potato plants was correlated to decreased plant growth_ caused by the limited K uptake, which induced chronic K difficiency in the haulms. Evans, Parkinson and Trudgill (1975) showed that yield was also decreased by water stress which caused infested plants to senesce prematurely. So, in an attempt to understand better the effects of these limiting factors on the growth of infested potato cyst-nematode plants and to investigate the degree of tolerance of some commercial potato varieties to G. rostochiensis as well as techniques for assessing tolerance, results of field and pot experiments with different varieties are reported in this section. IV. 1 PHYSIOLOGICAL BEHAVIOUR OF POTATO PLANTS The economic yield of a crop is a function of the total dry matter produced and the proportion passing into the yield-bearing organs - in potatoes generally about 75-85 per cent. Watson (1956) has suggested that variation in total dry matter production is associated more closely with differences in the size of the photosynthetic system (leaf area) than with differences in its efficiency (net assimilation rate). Specifically, he considered dry ( matter yield to be a function of the leaf area integrated over the growth period. Ivins and Bremner (1965) indicate that dry matter distribution within the potato plant, particularly early in its life, exercises the dominant role in determining both total dry matter production and yield. Growth and development in the potato crop, and their relationships with yield, lie in a balance struck between the conflicting demands of foliage and tuber growth. Hence, any factor that affects the young plant may modify the course of growth. The majority of tubers are initiated within 203 a period of about two weeks, after which tuber, numbers remain relatively stable. Final yield depends on the rate of tuber bulking and the length of time over which it takes place. After initiation, yield increases exponentially for a time and then the rate falls off, tuber growth ceasing completely with the death of foliage. Tuber bulking is limited by the capacity of the plant to supply assimilates. Both the rate of tuber growth and the time of foliage senescence are related to the amount of leaf growth made by the time of tuber initiation. If tuber initiation occurs before the basis of a large leaf area is established, the leaf surface will be restricted in size and tuber bulking will be correspondingly limited in rate. Early senescence of the leaves naturally or as the result of a disease, reduces the period of tuber bulking and hence the final yield, (Lupton, 1974). However, plants do not only grow above the ground they also occupy a large volume below the soil surface, a fact which has been often neglected. The root system plays a very important role in the uptake of both water and nutrients, and anything that limits its growth is important. Thus, the photosynthetic system of a plant cannot be built nor can it function unless sufficient amounts of nutrients and water are taken up from the soil but there will, on the other hand, be no formation of roots to absorb these nutrients if there is no steady flow of carbon assimilates from the photosynthesizing organs. The net effects of a reduced supply of assimilate are a slowing of root extension and a smaller root system. These considerations lead inevitably to the source-sink problem. Organic assimilates are formed in specialized tissues, which are referred to as sources, and sooner or later transferred to other parts of the plants (sinks). Developing organs (roots, tubers, stems, etc.) 201+ represent strong sinks and in most cases their ability to attract and accumulate assimilates are of particular value. However, the mechanism which regulates this source-sink relationship is still unknown and variation in the amounts or activities of various endogenous growth hormones such as auxins, gibberellins, cytokinins and abscisic acid have been considered as potential means of inducing and controlling sink activity (Stoy, 1974). Another critical factor in plant growth and dry matter production is the resistance and susceptibility of plants towards adverse conditions (e.g. pests and diseases), since the sink capacity of different plant organs may be influenced by the incidence of pests or diseases, which induce tissue change in the host (e.g. potato cyst-nematodes, Jones and Northcote, 1972a, b) and may constitute a sink acting in competition with other plant parts (Lupton & Sutherland, 1973). So, it can be concluded that although yield is necessarily dependent on the production and distribution of carbon assimilates, there are many other factors which may interact with these processes and with the capacity of the crop to utilize the carbohydrate formed (Lupton, 1974). The damaging effect of plant nematodes is more than just an act of parasitism, rather, it is a deleterious change in the biochemical and physiological processes required for normal cellular function, differentiation and reproduction. Nematodes slow root extension, induce branching and remove nutrients. The chemical composition of the material shunted to the exterior through their bodies is largely unknown. It contains sugar and nitrogenous products, some Of them excretory, and possibly also cations which otherwise would have found their way in the sap stream to the overground parts of 205 the plant. Work in pots suggests that the mechanical damage caused by root-invading larvae is more important that that from their feeding (Seinhorst & den Ouden, 1971). Trudgill (1968) found that potato cyst-nematodes affect root growth since it was virtually stopped when nematode attack was severe. Evans, etal (1975) found that roots of potato cultivars Pentland Dell and Maris Piper were smaller and did not penetrate deeply in the soil when many nematodes were present. The functioning of plants with infested roots has been measured by many workers who have examined water absorption and nutrient uptake but their conclusions are often contradictory. O'Bannon and Reynolds (1965) concluded that the water consumption of Meloidogyne incognita-infested cotton plants was as great as or greater than that of uninfested plants, when water was not limiting, but that under water deficits, infected roots are not able to transport sufficient water to maintain normal growth of cotton plants. Meinl and Stelter (1967) found that the water requirement of potato plants infested with potato cyst-nematodes was greater than that of uninfested plants to produce the same amount of dry matter. Webber, et al (1970) found that under water stress conditions, transpiration rates from M. incognita-infested tomato plants were greater than from uninfested and were disproportionally higher in relation to the amount of leaf tissue. Odihirin (1971) found that the same nematode greatly increased the rate of transpiration of infected tobacco plants but that eventually the plants wilted even when abundant water was available, apparently because the damaged roots were unable to meet the demand for water. Wallace (1974) reported that the most important effect of root-knot nematodes on tomato plants was probably on water uptake and thereby on photosynthesis. Evans et al, (1975) in field conditions and with potato plants subjected to intermittent water supply in the form of natural rainfall found that plants infested 206 with many potato cyst-nematodes had greater stomatal resistances and more negative water potentials than lightly infested plants and suggested that the early senescence of infested potato plants is associated with the increased internal water deficit caused by water stress. Hunter (1958), Jenkins & Malek (1966) and Bergeson (1966, 1968) all found that Meloidogyne sp. did not affect translocation of nutrients to the top of plants, although they sometimes caused nutrients to accumulate in roots. Shafice and Jenkins (1963) also found that M. incognita and Pratylenchus penetrans increased concentrations of K, Na, P and N in infected plant roots without changing the mineral content of the leaves, and suggested that stunted haulm growth was caused by toxins secreted by nematodes. Stelter and Meinl (1969) found no changes in ash content of potato plants infested with potato-cyst nematodes and concluded that uptake of water and plant nutrients was unaffected. However most evidence suggests that nematodes affect mineral nutrient uptake and translocation to the tops and the elements whose uptake is most affected are P and the cations K, Mg and Ca (Parris and Jehle, 194'; Lownsberry, 1956; Sher, 1957; Kirkpatrick et al, 1959; Oteifa and Elgindi, 1962). Widdowson, et al, (1973) found that Heterodera trifolii and M. hapla greatly reduced the efficiency of P utilization in white clover plants. Dasgupta and Deb (1972) found that absorption of N, P, K, Ca, Fe, Mn and Cu in tomato plants inoculated with M. javanica and M. arenaria was lower than in uninfested and that considerable loss of labelled P from root to shoot occurred in infected plants 21 days after inoculation. Results from Trudgill, Evans and Parrott (1975a, b) showed that concentrations of K, P and to a lesser extent Mg and N decreased in potato plants infested by potato cyst- nematodes. In most instances it is not clear whether these differences 207 in uptake and translocation are caused by the modification of the root form which usually accompanies infestation, or whether the ability of the root to absorb nutrients is impaired. Studies on the influence of the cereal cyst-nematode, Heterodera avenae on root development and function in oat plants showed that the effects on absorption of nutrients appear to be primarily a result of the reduced size of the infested root system (Price & Clarkson, 1975; Price, et al, 1976). So, the physiological disturbance caused by nematodes in the root system as a result of their invasion or establishment elicits a series of interacting events throughout the whole plant system that ultimately influence top growth. One way of determining the extent of these disturbances is to examine the physiological activity of infested plants by measuring photosynthetic rates. Meinl and Stelter (1965) studying the assimilative capacity of different potato varieties of similar "physiological age" found that the maximum level of assimilation varied according to the potato variety but it was always before flowering. They also found that nematode attack limited dry matter production by increasing the nightly respiration losses in spite of a high net photosynthesis. Loveys and Bird (1973) found a decrease in photosynthesis of tomato seedlings two days after inoculation with M. javanica, and this was maintained throughout subsequent growth. During latter stages at least the reduced rate of photosynthesis was due to the smaller size of infested plants. Wallace (1974) with the same host and different infestation levels of M. javanica also found 14 that both photosynthetic rate and 00 incorporation into the 2 roots decreased as infestation level increased. The experiments described below, on water relations, nutrient uptake and photo- synthesis, constitute an attempt to understand better the factors limiting haulm growth and eventually yield of potato cyst-nematode 208 infested plants. (i) Materials and Methods Three potato varieties were used, Maris Piper (MP) a variety resistant to G. rostochiensis (pathotype Rol) and two susceptible ones, Pentland Dell (PD, mid-season) and Maris Peer (MR, early). . Tuber pieces were planted in 9 cm pots filled with 350 g of sterilized sandy-loam soil, containing a standard commercial fertilizer. Before planting cysts were added to some of the pots to give nematode densities of 0, 160 and 800 eggs of G. rostochiensis per gram of soil. The plants were grown in a glasshouse in similar conditions to those described earlier. Three plants from each treatment were used to study water relations and another three were used to measure rates of photosynthesis and the concentration of nutrients in the haulm. Ten weeks after planting potato plants of the varieties Maris. Piper (MP) and Pentland Dell (PD) were removed from the glasshouse and placed in a growth room. Maris Peer potato plants were discarded because of premature senescence. Conditions in the growth room were: 20°C (day) and 15°C (night), with 16 hours photoperiod, 1000 u Einsteins m-2s-11ight intensity measured at the top of the plants, and a water vapour content of 9.30 gm-3 (day) and 6.94 g m-3 (night). The temperature and water vapour content of the air were monitored continuously with an aspirated wet and dry bulb psyc1ometer. Two soil moisture conditions were considered: wet and dry (pot capacity and about -2.0 bars respectively). The dry condition was reached by withholding water supply for two days. All treatments were replicated three times. After acclimatizing the plants, the evaporative flux density -2 -1 E(gm s ) was estimated over a period of 4 hrs. by measuring the leaf area and the change in weight of the pot and plant. (It was 209 assumed that evaporation from soil surface was negligible because the soil was covered by polyethylene granules.) The temperature difference between leaf canopy and ambient air was measured with four pairs of 40 s.w.g. constantan-chromel P differential thermocouples connected in parallel; one junction of each pair was in air and the other inserted into a leaf lamina at different parts of the canopy. Canopy diffusive resistance (rs) was calculated from: aX E - r r a + where(g cm-3) iss the difference in vapour content between the ambient air (DC ) and air at the evaporating surfaces within the a leaves (X 1,which was assumed to be the saturation vapour pressure s -1 and r (scm ) are the at the temperature of the canopy); ra s resistances to the diffusion of water vapour offered by the canopy boundary layer and the stomata plus intercellular spaces can be neglected in of leaves within the canopy respectively. ra comparison to rs, if the air close to the plants is stirred vigorously (Lawlor & Lake, 1976). The leaf water potential (Y) was measured on four leaves per plant with a pressure bomb (Scholander, et al, 1965). Th._ same leaves were used to evaluate relative water content (PWC) (Barra, 1968). The osmotic potential (1r) of leaf tissue was measured from leaf discs after freezing and thawing. The pressure potential (P) is the difference between Iff andlT. The concentration of nutrients in haulm tissues was determined from 0.5 g of dry matter. The haulms were dried in an oven for at least 2 hrs. at 1100C, milled and the concentration of P, K, Ca and Mg determined. K content was measured by flame photometer after extraction with hydrochloric acid. The concentration of the 210 other minerals was estimated after ashing at 480°C for 2)-3 hours. The rate of photosynthesis was measured at 3, 4, 5, 6, 8 and 9 weeks after planting on the same set of plants used in the analysis of nutrients. The day before measurement the plants were placed in a growth room in order to acclimatize. Conditions were similar to those described above. Two terminal leaflets of the youngest fully expanded leaves on each plant were used to measure the rate of photosynthesis (Frier, 1975). The gas exchange chamber, in which the attached leaf was placed, was a clamp type made of perspex and a gasket of non-intercellular sponge rubber, concentration of air with an exposed area of 2 x 2 cm. The CO2 •passing through the chamber was measured on a infra-red gas analyser, which was connected to a pen recorder. Gas flow rate was adjusted to 18 1 hr-1 by a flow meter placed between the air supply (compressed air cylinder) and the leaf chamber. CO2 exchange was estimated by comparing CO2 concentration in the original air from the cylinder (315 ul CO2 1-1) with that of air which had passed over the leaf surfaces in the chamber. The degree of opening of stomata was assessed by measuring the amount of water transpired from the leaf in the perspex chamber: e.g. no transpiration, no open stomata. After the last measurement (9th week) of photosynthesis the leaf areas of the plants were measured using an electronic "Leaf Area Meter" (Patton). (ii) Results In all the results given below, the potato variety names will be presented as MP (Paris Piper), PD (Pentland Dell) and MR (Maris Peer). A. Water relations Treatment means of single factors or two factor interactions which had significant effects in an analysis of variance of various plant-water relationship indices are presented in Table 63 along TABLE 63. Treatment means of leaf water potential, relative water content, transpiration rate and stomatal resistance in Norio Piper (resistant) and Pentland Dell (susceptible). Measurements Varieties Soil moisture Nem. density Soil moisture x Nem. density Water potential Wet Dry - bars Maris Piper . 5.7 Wet 4.4 0 6.6 0 4.1 9.1 Pentland Dell 6.5 Dry 7.9 160 5.3 160 3.9 6.8 800 6.4 800 5.2 7.7 S.E.D 0.4 0.4 0.5 0.7 L.S.D 5% 0.8 5% 0.8 5;) 1.0 55 1.4 Relative water content, 5 N.S. Wet 89.5 0 84.8 0 89.2 80.3 Dry 83.3 160 88.0 160 89.3 86.7 800 36.3 800 89.8 82.8 S.E.D. 0.9 1.1 1.6 L.S.D. 1.8 5% 2.3 5% 3.2 Transpiration rate (mgs-lm-2 ) Maris Piper 11.0 Wet 21.8 0 15.3 0 22.8 7.9 Pentland Dell 21.5 Dry 10.7 160 16.5 160 21.7 11.3 800 17.0 800 20.9 13.0 S.E.D. 1.3 1.3 1.7, 2.4 L.S.D. 1',',() 3.6 1`:, 3.6 5% 3.5 5% 4.9 Table 63 (contd) Measurements Varieties Soil moisture Nem:density Soil moisture x Nem.density. Wet Dry :stomatal resistance Maris Piper 8.80 Wet 4.57 0 7.93 0 4.15 11.70 (scm-1) Pentland Dell 4.98 Dry '9.25 160 6.78 160 4.75 8.80 800 6.03 800 4.8o 7.25 S.E.D. 0.62 0.62 0.76 1.08 L.S.D. 1% 1.73 1% 1.73 5% 1.57 5% 2.23 213 with their standard errors and least significant differences. Results for which no significant differences were measured are omitted (e.g. osmotic potential and turgor). Leaf water potential ( ) was lower for PD (-6.5 bars) than MP (-5.7 bars). Although there was no clear effect of nematode density alone, the interaction of nematode density with soil moisture indicates that potato plants in wet soil had decreasing leaf water potentials as nematode density increased but in dry soil was more negative than in wet soil and increased with increasing nematode density. Relative water content (PWC) was not significantly different between varieties, but was greater in wet soil than in dry. The effect of nematode density showed no clear trend. The transpiration rate (E) per unit area was higher for PD (21.5 mg s 1m-2) than MP (11.0 mg s-im-2) and for wet soil than dry soil (21.8 and 10.7 mg s-lm-2 respectively). The rate of transpiration increased as nematode density increased but when the interaction with soil moisture is considered this effect is only observed in dry soil because healthy plants use-water reserves of pots faster. Diffusive water resistance or stomatal resistance (r ) was sc significantly greater in MP than in PD (8.80 and 4.98 scm.1 respectively) -1 and it was greater in dry soil than in wet soil (9.25 and 4.57 scm respectively). Although stomatal resistance decreased as nematode density increased, the treatment means for the interaction with soil moisture show a slight increase in wet soil as nematode density increases but with values much lower than those for dry conditions. In dry soil stomatal resistance decreased as nematode density increased. 214 The treatment means for the multiple interactions of potato variety, nematode density and soil moisture; are presented - in Table 64. Some of these are also shown in graphs. The relationship between Y and RWC, and ir and RWC (Fig. 34) were similar for both varieties, except that Tr and Y decreased slightly more in MP per unit decrease of RWC. Also turgor (P) was a little larger in MP than in PD throughout the range of RWC studied, and this may allow MP to grow better than PD at lower RWC, if turgor pressure is an important factor in elongation (Hsiao, 1973) and in any case a larger decrease of Y per unit decrease of RWC confers better drought resistance (Jarvis and Jarvis, 1963). The leaf diffusive resistance (r ) of both varieties sr, increased when leaf water potential ( T ) decreased (Fig. 35), but rsc increased slightly faster in MP than in PD and showed greater values at a given Y , again suggesting better drought tolerance. The combinati6n of slightly greater values of turgor pressure (P) and large rsc for NP compared to PD indicates that MP has a greater ability to minimise water loss and maintain growth. Figure 36 shows that NP had a lower transpiration rate than PD, throughout the range of Y tested, and that MP is therefore more resistant to water loss than PD. When infested by nematodes both varieties behaved similarly in wet soil, but in dry soil Y of PD was significantly lower (Fig. 37a) even without nematodes (-8.6 and -9.6 bars of MP and PD respectively, Table 64). These results are differentfromthose of Evans, et al (1975), but we have to consider the different growing conditions. Measurements were taken just at one age and small pots were used, whereas Evans et al, made field observations. , Table 64,.. Treutw,ent means for multiple interactions of potato variety, - nematode density and 11 oisture. 2 -1 Soil i:loisture iiriety Nematode Y(-bars) ir(-bars) P(bars) RWON E(mgsm ) r(ocm ) density sc Wet L,:.,ris Piper 0 4.0 8. 4.3 86 15.1 5.4 -) 160 3.8 (.,c ,_, 4, 5 90 13.9 6.6 Q. L,800 „iu 3.8 9.3 5.5 89 1 3.5 6.3 .rentland Dell 0 4.2 8.2 4.0 92 30.5 2.9 160 4.0 9.3 5.3 85 29.5 2.9 -) , 800 4.3 9.1 4.8 90 28.0 J ....) Dry Laris Piper 0 5.6.0,- 11.3 2,7 80 5.2 15.7 160 7.3 9.4 2. 4 87 8.0 10.9 800 7.3 10.5 3.3 82 10.3 8.1 Pentland Dell 0 _.... 11.3 1.7 80 10.5 7.7 160 7.6 9.0 1.0 85 14.5 6.7 800 6.2 10.9 2.7 83 15.6 6.4 216 Pentland Maris Piper(---) o n" .33.0-0-27 RWC o**. 44-8-0-41 RWC o w** =40.3 -0-39 RWC .**= 51.6-0.53 RWC 0 2 N I° 0 0 JD 8 6 0 CL 4 Z13 0 2 (19 95 . 90 85 80 75 °10 Relative water content (RWC) Fig. 31 Relations between relative water content (RWC) and leaf water potential (y) and leaf osmotic potential (W) of resistant and susceptible potato v&rieties. 2 1 7 (3) Maas Piper rs**= 0- 97 +1- 38y (0) Penrland Dell rs*I-0- 49 +0.844, ai 20 V • • . . • 12 0 . 8 4 O 50 0 0 40 0 ,...-.... LLJ %...... 30 20 0 10 Woe • • i 2 4 6 8 10 12 14 (-bars) Leaf water potential (') 218 In small pots the plants without nematodes (and therefore with larger root systems and tops) use water more quickly than those with nematodes (Fig. 37b). As the plants were left for two days without watering, the plants in pots without nematodes used more water and developed greater water stress than those with nematodes, a situation which would not occur in field conditions, where water in deeper soil layers could be used by plants with well-developed root systems. The stomatal resistances (r ) of both varieties in wet sc conditions followed a similar pattern but in dry conditions the plants without nematodes had greater resistances (Fig. 37c) than those with nematodes. This also differs from the results of Evans et al, but agrees with those of Odihirin (1971): a nematode- infested plant may transpire as much as a healthy plant depending on the stage of infection, plant age and soil water content. This excessive water loss causes temporary wilting of nematode infested plants which is observed in field conditions, particularly in hot afternoons (O'Bannon & Reynolds, 1965). Initial infection by nematodes may promote a mechanism of defence, so obliging infested plants to use more water to maintain its metabolic equilibrium. Stelter and Meinl (1962) reported that consumption of water for production of one gram of dry matter was mush greater in plants grown in nematode-infested soil. This means that the efficiency of water use is lower in susceptible nematode infested plants than in healthy or resistant plants. Odihirin (1971) found that nematodes may stimulate growth in tobacco plants as long as water supply is plentiful: the stomata seem to open oftener during early stages of infection. However, susceptible 219 I 0 a (a) 4-- 8 e_, N o 0 6 a) _co, 4 8 ----- 8- - - - 0 cu —J 2 Maas Piper Pentland Dell Wet 0 0 0 0 .•••••-■ 40 0 Dry • ---0 41110■■■■•■■• OJ 0,7 30 - (b) c E ID IN 20 0 0 o- e-■ N L., I ,11.)- 0 a) (c) 0 I2 (1) 111 8 o E 4 0 (r) 0 0 160 800 Nematode density (eggs q-' soil) Fig. 37. Effects of different nematode densities cn (a) leaf water potential,(b) transpiration rate and (c) stomatal resistance of resistant and susceptible potato varieties under wet and dry soil moisture conditions. 220 plants infested by many nematodes have much smaller root systems than resistant plants, and this would affect exploitation of water reserves in the soil rather than the rate of movement through the plant. B. Nutrient uptake The results for amounts of nutrients taken up by Maris Peer (MR), Pentland Dell (PD) and Maris Piper (MP) are expressed as the concentrations of phosphorous (P), potassium (K), calcium (Ca) and magnesium (Mg) in their haulm dry matter. The treatment means for percentages of P, K, Ca and Mg for single factors (potato variety and nematode density) are presented in Table 65. Results for varieties show that K, Ca and Mg are found in significantly greater amounts in PD than MR and MP. The concentration of P in PD was similar to that in MR but significantly higher than in MP, which in general shows a low concentration of the four elements. This may be related to differences in plant development as will be discussed later. The amounts of nutrients found in plants grown at different nematode densities show clear trends but are not significantly different. Concentrations of P, K and Mg in haulm dry matter the decrease as nematode density increases whereas concentration of Ca increases. The effects of the interactions between variety and nematode density on the concentrations of the four elements are shown in Figure 38. Where significant differences were found, the least significant difference (LSD) and level of significance are indicated. The percentages of P and K in haulm dry matter of MR and PD decrease as nematode density increases but increase in MP, especially P. 221 0-5 0.4 P 0.3 0 0-2 .1; :a. 5.0 E I< 3.0 E 7 _c 3-0 „, 2-0 . • • - " cr Ca ...... 4E 1.0 Maris Peer Pentland Dell — 12_Z1-.) 0.6 Maris Piper •••• 0-5 •••••• Mq 0.4 0.3 0 160 800 Infestation density (eqqs 09' soil ) Fig. 33. Effects of different nematode densities on percentages of P, K,Ca anu Mg in haulm dry matter of susceptible (Pentland Dell and Maris Peer) and resistant (Maris Piper) potato varieties. 222 Table 65. Percentages of P, K, Ca and Mg in haulm dry matter for potato varieties and nematode density of krostochiensis (Means of nine plants 9 weeks after planting). % in Dry matter Varieties Nematode density (eggs g isoil) P Maris Peer 0.4o Nil 0.39 Pentland Dell 0.40 160 0.37 Maris Piper 0.31 800 0.35 S.E.D. 0.03 0.03 L.S.D. 5% 0.06 K Maris Peer 3.22 Nil 3.50 Pentland Dell 3.63 160 3.36 Maris Piper 3.18 800 3.16 S.E.D. 0.18 L.S.D. 5% 0.38 Ca Maris Peer 1.75 Nil 1.68 Pentland Dell 2.22 160 1.79 Maris Piper 1.52 800 2.02 S.E.D. 0.14 0.14 L.S.D. 1% 0.40 Mg Maris Peer 0.38 Nil 0.46 Pentland Dell 0.51 160 0.39 Maris Piper 0.39 800 0.43 S.E.D. 0.03 0.03 L.S.D. 1% 0.09 223 The percentage of Mg seemed unaffected by nematodes in MR and PD, but declined in MP as nematode density increased. Percentage of Ca increased in haulm dry matter of all potato varieties as nematode density increased. C. Photosynthesis Figure 39 shows the effects of the interactions between potato varieties and nematode densities on the rate of photosynthesis per unit leaf area measured from the 3rd to the 9th week after planting (no measurements were made in the 7th week). When differences were significant in any one week the LSD and level of significance are shown. The resistant variety MP showed a steady increase in rates of photosynthesis at all three nematode levels during the first three weeks of measurements, followed by a gradual decline as the plants began to mature. The rate of photosynthesis was lowest at the high nematode density and highest in the control. Uninfested PD plants photosynthesised at a steady rate up to 6 weeks, and then the rate declined. Nematode infestation at the low level increased photosynthesis up to 6 weeks (possibly a physiological reaction to nematode damage) but photosynthesis was reduced by the high level of infestation throughout the experimental period. The rate of photosynthesis of uniljested MR plants increased up to 6 weeks and then declined, but it increased more and earlier with both low and high levels of nematode infestation. Again this may have been a reaction to nematode damage, and the difference in pattern of reactions between the two varieties due to intrinsic varietal differences. Treatment means for single factors are summarized in Table 66 and the analyses of variance for leaf area, root weight and tuber weight of plants at 9 weeks are presented in Tables 67, 68, 64, and 70. Photosynthesis per 224 4 5 6 8 9 Weeks after planting Eggs /9 of soil 0 160 000 Maris Piper Pentland Dea moris Peer Fig. 39. Rate of photosynthesis per unit leaf area at weekly intervals of susceptible (Pentland Dell and Earls Peer) and resistant (naris Piper) potato varieties inoculated with different naTa- tode densities. Table 66 , 2 Rates of p mtosynthesis per unit 19.,1 area at weekly intervals (mg c02 am, - hr -1 ). , Net photosynthesis, leuf area kdm ), root weight (g) and tuber weight per plant at thi-: 9th week. OB=VATION INonrumf 1;!?,NSITTI;2 (ecrr's soil Photosynthesis at: Peer P.72ell 1'.Piper 8.-2.5. L.S. 0 160 800 3.E.D. L.j.* 3rd week (mg 00., dm-2hr-1 ) 5.10 5.68 5.40 0.58 N.S. 5.77 5.52 4.89 0.58 U.S. 4th week 2.96 6.06 5.27 0.52 lir- 4.75 5.25 4.29 0.52 N. J. 5th week 6.70 4.83 7.57 0.80 15: 5.73 7.1', 6.21 0.80 N.S. 6th week 5.18 6.35 4.06 0.71 5;'- 5.12 6.50 3.96 0.74 l'il vi 8th week 2.47 2.37 1,66 0.44 N.S. 2.28 2.46 1.96 0.41- N..), 9th week 1.66 2.58 1.95 0.32 5c/1 2.15 2.47 1.57 0.32 5 , Per plant at 9th ) 0 week .4 3.55 6.76 0.62 15, 5.29 5.15 2.27 0.62 l; Leaf area (dm`) 1.38 1.35 3.16 0.11 lip 2,41 2.28 1.49 0.11 12 Root weight (g) 0.96 1.19 0.93 0.08 17, 1.38 1.06 0,64 0.08 17- Tuber weight (g) 3.80 9.20 10.10 1,00 15- 11.60 6.30 5.22 1.00 1;- Level of significance ** G. rostochicnsis 226 Table67 . Analysis of variance of results on total photosynthesis rate per plant at 9 weeks after planting SOURCE OF VARIATION DF MS VR Level of significance POT 2 5.523 1.596 POT x LEAF 3 1.490 0.430 VARIETY 2 92.130 26.618 1% INFEST. 2 52.308 15.112 1% VARIETYxINFEST. 4 5.702 2.647 RESIDUAL 40 3.461 2 Table 68. Analysis of variance of results on leaf area (dm ) determin- ation on potato plants 9 weeks old and infested with different nematode densities SOURCE OF VARIATION DF MS VR Level of significance POT 2 2782 2.468 POT x LEAF 3 0 0.000 VARIETY 2 262956 233.270 1% INFEST. 2 45017 39.935 1% VARIETYxINFEST. 4 3591 3.186 5% RESIDUAL 40 1127 227 Table 69. Analysis of variance of results in root weight (g) of 9 weeks old potato plants inoculated with different nematode densities SOURCE OF VARIATION DF MS VR Level of Significance POT 2 0.09407 1.661 POT x LEAF 3 0.0000 0.000 VARIETY 2 0.36074 6.370 1% INFEST. 2 2.43185 42.943 1% VARIETYXINFEST. 4 0.58630 10.353 1% RESIDUAL 4o 0.05663 Table 70. Analysis of variance of results on yield (g) of 9 weeks old potato plants inoculated with different nematode densities SOURCE OF VARIATION DF MS VR Level of significance POT 2 13.212 1.468 POT x LEAF 3 0.000 0.000 VARIETY 2 209.645 23.291 1% INFEST. 2 211.560 23.504 1% VARIETYXINFEST. 4 54.531 6.058 1. RESIDUAL 40 9.001 223 plant at the 9th week was greater for MP than PD as a result of its huge leaf area. MR having a similar leaf area to PD (1.38 and 1.35 dm2, respectively) showed a slightly lower rate of photosynthesis (2.4 mg CO2 dm 2h-1) than PD (3.6 mg CO2 dm-2h-1) which does not reflect the differences in yield (3.8 and 9.2 g respectively). The results for effect of nematode density on the rate of photosynthesis show an increase in photosynthesis of uninfested plants at the 5th and 66th week, probably due to tuber development. ROwever, the corresponding increase in photosynthesis in infested plants with 160 eggs/g of soil is greater and persists longer (6th week) suggesting that it may be a mechanism of recuperation which is further prolonged by a delay in tuberization. The situation with heavily infested plants (800 eggs/g of soil) is quite different since the photosynthesis rate is lower and begins to decline earlier. The total photosynthesis per plant at the 9th week after planting showed no significant differences between uninfested plants and those infested with few nematodes, in spite of significant differences (at the 1% level) in root weight and tuber production. The more heavily infested plants had very low rates of photosynthesis and root weights, but this was not reflected in tuber weights when compared with plants infested with 160 eggs/g soil,- suggesting that different proportions of photosynthate went to the tops and roots at the two nematode levels. Treatment means for the interaction between variety and nematode density are presented in Figure 40 (a and b). At the highest nematode density, although MP suffers a reduction in leaf area and photosynthesis rate, it is still able to divert photosynthate to the tubers (Fig. 40b). On the other hand MR and PD ••• Peer ' - Maris . • • • • ••• . • • •.■ •.■ --- Pentland Deli . • • •'I- Mark Piper (b) 18 0 14 4 E N 10 a_ ***** . -1•• . • • ..41• 2 •••• O. • • • ■ 1 O 0 0 160 800 eggs /q of soil Fig. 40. Effects of numbers of nematodes on (a) total photosynthesis per plant;(b) weight of tubers and roots and leaf area of 9 weeks old susceptible and resistant potato varieties. 27)0 have poorer root systems, smaller leaf areas and lower yields, indicating that they cannot support and tolerate the damage caused' by the nematodes. Figure 40a shows photosynthesis per plant at the 9th week and, although PD has a lower rate of photosynthesis at all nematode levels at this time, it produced as many tubers as MP at the low nematode level and in the control. This may mean that PD is more efficient at producing tubers than MP. Figure 40 b shows that although root weight of the resistant variety MP was not much affected by nematodes, root quality may have been as leaf area and tuber production decreased as nematode density increased. Nevertheless, MP seems to tolerate high levels of infestation better than PD and MR which were more severely damaged. (iii) Discussion The damage observed on susceptible potato plants (Pentland Dell and Maris Peer) grown in soil infested with cyst-nematodes (G. rostochiensis) consisted of poor root and top development compared with uninfested plants. When root damage is not intensive a compensatory development occurs by an increase in both length and diameter of lateral roots, the ability of the root system to exploit the soil may be much modified (Geisler & Maarufi, 1976; Russell, 1971). This seems to have occurred with Maris Peer (MR) which showed a similar root weight to the resistant Maris Piper (MP) when both were grown in infested soil although yields were quite different. (Fig. 40b) Although the suitability of the soil as an environment for root growth and function depends on the availability of water, nutrients, oxygen, etc, the rate of absorption by the whole root system is dependant on the total amount of root, on its absorbing 231 capacity and on its rate of expansion (Eavis & Payne, 1969), abilities which perhaps diminished in heavily infested potato plants (800 eggs/g soil) as uptake of K and P was impaired. An exception was the absorption of Ca, which increased in infested plants (Fig. 38). Alterations in metabolic processes and translocation patterns of damaged non-photosynthetic tissues, such as roots may seriously disturb the normal events in non-parasitized photosynthetic tissues. The succesful parasite much necessarily create a carbon economy favourable for its own growth because large pots of organic reserves are often required for subsequent development (Daly, 1976; Jones, 1976). The supply of carbohydrates and other metabolites from shoots and the uptake of water and nutrients are some of the most obvious physiological factors which influence the development of root systems. Water absorption in potato plants watered regularly (wet soil) .was slightly affected by the presence of nematodes, since the transpiration rate decreased as nematode infestation increased. However, it was higher in the susceptible PD than in the resistant MP (Fig. 37b). When plants were water stressed (dry soil) the situation was changed: transpiration in both varieties increased with nematode density but was lower in MP than PD at all nematode densities because MP had greater stomatal resistances (Figs. 37b and 37c) This may allow MP to grow better than PD, because it uses water more efficiently and because turgor pressure is an important factor in enlargement and elongation of new cells (Treshow, 1970; Hsiao, 1973). The relationship between the shoot and root in the developing plant might be described as a "push" and "pull" system, for the movement of photosynthates, nutrients and growth substances in which the direction and rates of flow of the substrates are partly determined by the sizes of the sinks for their utilization. The 232 most active sinks are meristems which have a continued demand for a assimilate. In potato plants, before tuber bulking,the root system is an example of an ideal carbohydrate sink which potentially is of unlimited growth (Humphries, 1963). This may explain the early increase observed in the rate of photosynthesis of PD plants infested with160 eggs/g-of soil. This increase"Might represent compensatory action to repair the damaged roots by a greater flow of photosynthates to the root system, because although the rate of photosynthesis increases after tuber initiation most of the increased assimilates exported from leaves go to the tubers (Moorby, 1968)-. This latter and indispensable increase in photosynthesis explains a basic physiological feature: the smaller the leaf area at the time of tuber initiation then the slower is the rate of bulking and the lower the final yield (Moorby & Milthorpe, 1975). Furthermore, maximum rates of dry matter production by potatoes depend more on the maintenance of a certain rate of leaf production than on the maintenance of a certain leaf area, because potato leaves become functionally impaired as they age ( Ivins& Bremner, 1965; Frier, 1975). This also explains the poor yield of the susceptible potato plants (PD and MR) infested with many nematodes (Fig. 40b) whose new leaves were produced more slowly and senesced earlier than those of uninfested plants (Trudgill, et al, 1975). One of the factors controlling the rate of leaf production is the efficient use of nutrients available in the soil (Benepal, 1967). The value of nutrients in soil depends on their accessibility to roots which in turn is related to their availability in terms of chemical form and their mobility in the soil, but also depends of the area of absorbing root surface of the whole plant. Mobile nutrients (e.g.N) are taken from the total volume of soil containing 233 roots whereas immobile nutrients (e.g.K) are absorbed from a thin layer around the roots. Availability of immobile nutrients therefore decreases with the distance from the root surfaces and shortage readily occurs when roots develop poorly (Bald, 1946; Cornforth, 1968.). In ion absorption, plant roots have two main functions: at the surface of the root mainly in the first few centimeters behind the growing tip and therefore in the region of elongation (region of most active metabolism and growth extension) ions are absorbed from the soil or solution; and from within the roots, ions are _ initially transported to other parts of the plant, mainly in the xylem, with secondary redistribution occuring in the phloem. Water movement across the root cortex affects ion fluxes through both the apoplastic (extra cellular space) and symplastic (cell to cell) pathways. In general the parenchyma cells of the stele are interconnected by plasmodesmata in much the same manner as are the cortical cells; the cortical and stelar symplasm are continuous through the endodermis (Pitman, et a111976). However the formation of the casparian bands (endodermis) and their firm attachment to the plasmalemma prevents direct access to the stele through the apoplast, because the enaodermis is the inner limit of the free space and has a capability for ion concentration and discrimination. Stout and Hoagland (1939) indicated that upward movement from the roots into the shoot seems to depend on the transpiration stream and occurs mainly if not entirely in the xylem, but the lateral exchange of ions between xylem and phloem is possible where the two tissues are in contact. Later on, Sutclife (1976) indicates that the amounts of solutes reaching the individual leaves via the xylem depend on the volume flow of the xylem sap to the leaf and its concentration. The first is usually closely related to the transpiration rate. Sap concentration is 234 influenced by the rate of which solutes are delivered to the xylem sap in the roots and by the extent to which ions are removed during passage through the xylem. Particular points are that transfer of most ions from the external solution to xylem across the roots involves metabolic transport, but that certain ions (P,K) seem more dependant on metabolism than others (Ca and Mg). Transport of the first group is strongly reduced by inhibition of energy metabolism, but little affected by transpiration (Sutclife, 1962). Therefore the lower percentages of K and P in the dry matter of heavily infested susceptible potato plants (Fig. 38) may be due to the plants having a lower level of metabolic activity caused by the reduction in the rate of photosynthesis. On the other hand where transport across the root is dependant on metabolism, there is a possibility that transfer to the shoot can be regulated by feedback of signals from the shoot. Klepper and Kaufam (1966) showed that during passage through the plant the concentration of xylem sap may decrease considerably, because solutes are absorbed from the transpiration stream by neighbouring cells, (especially by the actively-growing cambium and xylem parenchyma) and because some are transferred from xylem to phloem, perhaps via specialized "transfers cells"(Gunning & Pate,1968,1974). The presence of these transfer cells, where cyst-nematodes induce formation of syncytia on which the sedentary larvae must feed in order to develop further, hate been reported by Jones & Northcote (1972). Another reason for the low concentrations of P and K may be the differences in endodermis activity. Clarkson & Robards (1975) suggested that suberization of the endodermis effectively different- iates those ions reaching the stele mainly from the apoplast, e.g. Ca from those which enter via the symplast, such as K and P. Suberization of the endodermis occurs with aging and inversely affects water uptake and Ca absorption, but translocation of P and K is not altered because the alternative pathway into the endodermis, 235 via the plasmodesrcsata is not affected (Clarkson & Sanderson 1971). This would suggest that K and P gain access to the .vascular tissue. from the symplast which is not disturbed by formation of Casparian bands, but in damaged potato plants the plasmodesmata may be modified by the formation of syncytia near the endodermis and the translocation of K and P impaired. Ferguson (1974) also suggests that suberization of the hypodermis may be of great significance in preventing translocation of P along to root axis. The situation with Ca is quite different, since it moves only in the xylem via the apoplast and it has been found to be at least partly dependant on the rate of water transpiration (Leopold, 1964; Lazaroff and Pitman, 1966). Although many studies have been made on this last point and it has been found that ion uptake increases with increase in transpiration, most investigators have failed to establish any quantitative relationship between the two processes. Russell and Shorrocks (1959) found that uptake of P was greater at low than at high rates of transpiration. Uptake of K and Na was found to be independant of transpiration rate by Pitman (1965, 1966). Growth regulators have also been found to have effects on ion transport as the distribution of materials in plants is profoundly affected by the pattern of growth. (Sutclife, 1976). For instance the fact th&t cytokinins have the capacity to delay senescence of tissues and stimulate movement of materials towards active tissues (Mothes, 1961), may explain the early senescence found in root-knot nematode infested tomato plants having lower concentrations of root cytokinins than uninfested plants by Brueske and Bergeson (1972). So far the effects of potato cyst-nematodes on uptake of nutrients have been mentioned but it is also necessary to explain the effects that may produce these differences in uptake and these are closely related to plant growth. 236 Potassium is present in particular abundance in all actively growing tissues such as stems, root apices and reproductive bodies. There is a close connection between abundance of K and active growth, but there is no evidence that K precedes and provokes the growth (James & Penston, 1933). Potassium plays an important role in translocation, since its deficiency decreases the translocation of photosynthates from leaves to other parts of plants. This is due to a decrease in efficiency of the assimilatory organs and slow translocation of photosynthates into tubers, which is reflected in production of - small tubers. (Hartt, 1969; Haeder, et al, 1973). In stems and leaves K aids the conversion of photosynthates to protein and it is also essential in the synthesis of simple sugars and starch and in the translocation of carbohydrates (Smith, 1968). In particular, K is necessary to initiate and maintain the opening of stomata in light (Gauch, 1972) and the low concentration found in haulm dry matter of heavily infested plants may be reponsible for the reduction in rate of photosynthesis per unit leaf area (Fig. 39). Another factor contributing to the poor development and yield of heavily infested susceptible potato plants may be the low percentage of P found in their haulms, since P is critical during early stages of potato plant growth, when meristem development and rapid vine growth are necessary for a high yield. P also increases growth and assimilation and apparently leads to greater out put of carbohydrates, which in turn increase weight and size of tubers (Smith, 1968; Benepal, 1967). Although the uptake of Mg was not affected as much as that of K and P, it may also be involved in determining the rate of photosynthesis because it occurs in chlorophyll and therefore is involved in photosynthesis. The role of Ca is not understood but in a study of its effect on growth and elasticity of sunflower 237 hypocotyls, Uhr strom (1969) concluded that Ca imparts rigidity to the cell walls, and although it is necessary for growth, at higher. concentrations the cell wall becomes too rigid and cell elongation is inhibited. IV. 2 TOLERANCE TO POTATO CYST-PlEi•IATODES. When a nematode pest is present, its numbers can be decreased to levels that the crop will tolerate by fallows non-host crops, chemicals or resistant varieties, by combinations of these means and sometimes by changes in the management of the crops. The use and practice of these control measures depends on many factors. Firstly, those related to educational, social and political structures in countries where it is necessary to deal with the pest, and secondly the biology of the pest itself - the potato cyst-nematodes - including the dynamics of populations, species, pathotypes etc. Solme of the European countries enforce strict regulations governing importation of produce, Seed quarantine, movement of farm machinery and,potatoes, frequency of growing potatoes in either free or infested soils planting of resistant varieties and chemical application. However, measures directed at decreasing losses by controlling or avoiding the pest are not always applicable in countries outside Europe(e.g. Andean countries in Latin America) due to difficulties ofageographical, educational or economic character. In such conditions, with nearly all the land suitable for growing potatoes infested by potato-cyst nematodes, it is necessary to look for alternative ways to overcome the problem by adjusting to the situation and growing reasonable potato crops in spite of the pest. Computer models (Jones and Parrott, 1968) suggested that resistant varieties alternated with susceptible varieties in a 236 three or four-course rotation would be of some benefit even when unwanted pathotypes were 10% of the population. With such a sequence, nematode numbers when the susceptible variety is planted resemble those after 7 to 9 years without potatoes. In England potatoes are . grown one year in four or five on heavy soils, but only one in six or seven years on light soil or peats (Cooper, 1953; Winfield, 1965). However, in practice rotations of 4 or 5 years are used and the farmers accept slight losses because they find it unprofitable to have longer rotations. No single method of controlling potato cyst-nematodes so far developed is ideal. Control by rotating potatoes with non-host crops means that potatoes cannot be grown as frequently on a field as many farmers would wish because there is not always a profitable cash crop to substitute for the potato crop; chemical control is expensive and requires special application equipment due to the great toxicity of the chemicals used; resistant varieties are not yet available for all the pathotypes of G. rostochiensis or for G. pallida, and their uncontrolled use leads to a rapid increase of resistance-breaking populations. Nevertheless, of the control measures listed above the use of appropriate resistant varieties (where they are available) is so far the best method of control as there is little extra cost to the farmer. This is why plant breaders continue to try to find resistarce genes and then incorporate them in good commercial potato cultivars. However, a thorough study of the various responses of potato plants to nematodes may lead to development of tolerant cultivars, although such cultivars would probably allow large nematode populations to develop and therefore endanger susceptible crops of desirable or special qu ...—lities. However they would not exert selection pressures on the pathotype or species composition of the nematode populations 239 and would suffer smaller reductions in yield at high nematode densities. Huijsman, et al (1969) found that when the potato variety Multa was grown in an infested soil it outyielded many other commercial varieties. Degree of tolerance is assessed by comparing yields In infested and uninfested conditions and it is difficult to establish a technique suitable for screening large numbers of seedlings or cultivars for tolerance to nematode attack when only a limited range of densities may be used. The study described below was to investigate the suitability of a method for testing tolerance in pots, a method which could be used either for recently bred cultivars (when only a few tubers are available) or for commercial cultivars in large scale use. The second study describes a field experiment designed to estimate the level of tolerance of some popular commercial varieties. A Pot experiment Experiments in pots, although necessary to test ideas and establish principles, are difficult to translate into field practice. This is especially so when standard size pots are not used. Small pots are usually choosen in order to facilitate handling or to economise on space. In small pots the roots are confined to a limited volume, and. nutrient deficiency will only show in a particular treatment if there are large differences in ability to absorb nutrients. (Newman 8, Andrews, 1973). On the other hand, healthy plants grow rapidly and many exhaust the supply of nutrients, particularly nitrogen, so leading to earlier senescence than that of attacked plants (Jones, 1976). A lack of continuous water and nutrient supply has also been considered to be the factor limiting plant performance in small containers (Stevenson & Fisher,1975)• 2/40 Slight changes in size of pots in greenhouse experiments, also alter the effects of nematodes on plant growth (Ouden den, 1965). For all the reasons indicated above, in this experiment different pot sizes were used to find the minimum pot size that could be used to grow potatoes without altering their growth patterns in infested soil. The work was done because large pots could not be used to screen for tolerance in large breeding programmes. (i) Materials and methods Four equal sized tuber pieces (10g) of the potato varieties Pentland Crown (PC) and Pentland Dell (PD) were planted in 9, 12 and 16 cm diameter clay pots (350, 1000 and 1900 ml in volume respectively) previously filled with uninfested or G. rostochiensis infested sandy-loam soil. The potato cyst-nematode densities were 10, 100 and 1,000 eggs/g of soil. Four replications for each treatment were used and after planting pots were kept in a sand plungeoutdoors. Thirty five days after planting,height, number of leaflets and leaves and leaf area per plant were determined. At 80 days height was measured again. When potato plants reached maturity the tubers of each plant were counted and weighed and the soil was allowed to dry. Cysts of G. rostochiensis were then extracted from 300 g of soil and counted. The egg content per cyst was determined. (ii) Results and discussion Results of analysis of variance of the effect of different pot sizes and nematode densities on the growth of PC and PD at 35 and 80 days after planting are presented in Table 72. This table shows that pot size and nematode density had effects (significant mostly at the 1% level) on the development of potato plants. (see also Fig. 41). Pot size had a positive affect: the larger the pot 21:1 Table 72,, Effect of not size and nematode density on observations made on two potato varieties at 35 and 80 days after planting and on yield (Figures are levels of signif4nce). Observations Single effects Interactions At 35 days Pot size Density Variety PxD PxV DxV PxDxV (P) (D) (V) Height (cm) 1% 1% 1% 1% N. S 5% N.S. No.leaves/plant 1% 1% N.S 1% N.S N.S N.S No.leaflets/plant 1% 5% 1% 1% N.S 5% 5% Leaf area (sq.cm) 1% 1% N.S 1% N.S N.S N.S At 80 days Height (cm) 1% 1% 1% 1% N.S 5% N.S No.tubers 1% N.S N.S N.S N.S N.S N.S Weight tubers (g) 1% 1% N.S 1% N.S N.S N.S Fig. 41. Effects of different .size pots on development of potato plants grown in soil infested with different G. rostochi- ensis densities. Effect of different pot sizes on top development. Effect of different nematode densities on top development of plants grown in small pots (350 g. soil). Effect of different nematode densities on top development of plants grown in mediam pots (1000 g. soil). Compensatory top development of P. Dell and P. Crown potato plants grown in a low infested soil (10 eggs/g soil) in large pots (1900 g. soil). ) PENTLAND DELL PENTLAND CROWN 1111- -40 1000 PENTLAND CROWN 1111 PENTLAND CROWN 243 the larger the values of height, numbers of leaves and leaflets and leaf area. Nematode density in general had a negative effect: as nematode density increased the values were lower. However, leaf area at 10 eggs/g soil was higher than that of uninfested plants, indicating reparative growth. (Fig. 41) Variety did not affect the number of leaves or leaf area, suggesting that both varieties responded almost equally. Although PD was taller and had more leaflets than PC, it seems that this was due to inherent varietal differences. Results for two-factor interactions show that pot size x nematode density had an affect significant at the 1% level on the development of both varieties. Although some of the other interactions were also significant (at the 5% level) they only showed differences between controls and the highest infestation density. The interaction of pot size x density showed that in small pots (9cm diameter) the effect of nematode density on height, numbers of leaves and leaflets and leaf area is not as clear as in medium or large size pots (12 and 16 cm diameter respectively). Although no significant effects were found with the multiple interaction of pot size x density x variety, the treatment means are represented in graphs a,b,c and d of Fig. 42. It can be seen that becr.use two PC plants (at 100 eggs/g soil in small pots) were badly damaged during the growing period the mean values were decreased. However, the general pattern was not much affected. Although the varieties have different mean values for the growth indices, the trends for medium and large pot sizes are similar, but different from those for small pots. Pot size had an effect significant at the 1% level on number and weight of tubers (Table 72): the larger the pot the larger the number and weight of tubers. Nematode density significantly 241+ affected (at the 1% level) only weiht and not numbers of tubers, suggesting thA the initiation of tubers was not affected by nematode attack. There were no significant differences between PC and PD in number and weight of tubers. The only two factor interaction which significantly affected tuber weight was pot size x density. Plants in medium and large sized pots at low densities gave higher yields than those in small pots, obviously because of the larger volume of soil which allowed better development. Although the reduction in yield at the highest nematode density was much greater in large pots than- in medium size pots, the same significant differences were found for both these sizes, but not for the small pots. The effects of multiple-factor interactions on number and weight of tubers can be seen in Fig. 42 (g and h) the number of tubers for each variety in each pot size is not much affected by different nematode densities. However, weight of tubers in plants of both varieties grown in medium and large pots declines sharply at the highest density (1000 egirs/g soil). This fall in yield is not observed in plants grown in small pots, which show an erratic response. PD yielded slightly better than PC at lower densities but the situation is reversed at the highest nematode density. Figs. 42e and 42i show the effects of different size containers on nematode multiplication. Multiplication was generally greatest for both varieties when they were grown in medium sized pots (1000g) These results as a whole indicate that although potato varieties grow better in large pots (1900 g) and the effects of different nematode densities on growth indices are larger, these effects are proportionally similar to those obtained from plants grown in medium sized pots (1000g). Also, potato cyst-nematodes reproduce - 245 ) !0 -. m 25 25 0 E (c O 0- (a) s (1) 0- _ 0 20 20 al0 M 80 day 15 IS r 0 o I 0 • ht 10 cr ig He 5 5 52 a. 15- - - - -0 0 , (b) v e' CL 6 (9) te, 0 10- 5 4T5 4 z 5- 2 0 -6 — ▪ ‘J'a •- -- -4 (c) Let ► 80 (h) 13 45- 1 60 ,7;; 25 _c -S-72 40 20 I— 0 0 10 100 1000 0 10 100 1000 INITIAL INFESTATION DENSITY (eqqs I9 soil ) Fig. 42. Effect of different pot sizes (+=small;°=medium and •=large) on growth and yield of Pentland Dell ( ) and Pentland Crown (--___) inoculated with different nematode densities and on nematode multiplication. 246 best in medium sized pots over the range of population densities tested. Therefore, under the conditions of this study pots of 1000 gram capacity seem to be the minimum size which may be used to assess the degree of tolerance of potato plants at different potato cyst- nematode densities. Furthermore, as pots of this size are easy to handle large scale tests may be carried out without problems of space. B Field experiment Few field trials to estimate the degree of tolerance of potato varieties to cyst-nematode attack have been carried out. Reasons for this may be a lack of interest or perhaps the requirements for this type of study. Amongst the factors which must be avoided are: an uneven distribution of potato cyst-nematodes on the experimental site, aifferences in soil type or climatological conditions between the infested and uninfested sites which may modify results, presence of other pest or diseases etc. The only records of tolerant varieties so far are from Holland (Huijsman, et al, 1969: Seinhorst, et al, 1971) Canada (Morris, 1971), England (Evans, 1976) and Peru (CIP, 1975). (i) Materials and methods Ten potato varieties were choosen and grown in a potato cyst- nematode infested and an uninfested field at Woburn Experimental Farm. Both fields were sampled to check the degree of infestation. The G. rostochiensis infested field gave an average of 101 eggs/g of soil, more or less evenly distributed. The experimental design was a randomized block with three-fold 2 replication and plots of 52 m , with 4 rows of plants per plot. During the growing season the percentage of leaf cover ("ground cover index") was estimated using an apparatus consisting of two 247 bars 15 cm apart mounted one above the other. Each bar had twenty 3 mm holes drilled at 7 cm intervals, and the observer looked down through the aligned holes when the apparatus was held above two crop rows. The number of pairs of holes through which green leaf could be seen was recorded at 5 positions in the central rows of each plot on both sites every two weeks to maturation, beginning &weeks from planting. The final tuber yields were measured at the end of the season. During the growing season, at ten weeks, leaf samples were taken to analize the amounts of N,P,K,Ca and Mg present by the methods already described in IV.1 (ii) Results and discussion Results of the measurements of ground cover for each variety on both fields at two week intervals (from the 6th to the 20th) are presented in Fig.43. The varieties Desiree (DE), Majestic(MJ), Naris Peer (MR), Pentland Dell (PD) and Ulster Lancer (UL) showed greatly reduced top development and earlier senescence of foliage on the infested site compared to the uninfested site, indicating poor tolerance of the damage caused by G. rostochiensis The haulms of Maris Piper (MP) and Pentland Crown (PC), although somewhat reduced in size by nematodes, persisted longer than the rest of the varieties, so favouring tuber bulking and therefore showing the best tolerance of G. rostochiene,is attack. Results of analysis of variance of the effects of single factors and their interaction on percentages of N,P,K,Ca and Mg in haulm dry matter and yield are presented in Table 73 and the treatment means are in Table 74. Nematodes had an effect significant at the 1% level on N,P,K and Ca and at the 5% level on Mg. Nutrient content also varied significantly between varieties (1% level). The interaction of varieties and nematodes was significant at the 1% level on the yield of tubers and of K content of the leaves; on N,P and Mg was significant at the 5% level. Ca was not affected. Nematodes had 2113 Desiree Mans Pi per 80 x - LONG MEAD (Infested) 0 - FAR FIELD (Un-infested) 60 40 20 X -X X x -. 0 Majestic Mans Peer 80 60 40 ,x 20 x .\i-x-x 0 Pentland Crown Pentland Ivory 80 60 GROUND 40 ,x COVER )( \x 20 0 Pentland Dell Record 80 60 C 40 20 6 0 x-x X----X Stormont Enterprise Ulster Lancer 80 60 40 20 x x x I I 1-X 0 8 10 12 14 lb 1 8 20 6 8 10 12 14 16 1 8 20 WEEKS AFTER PLANTING, Fig. 43. Yeasurement of top development of ten potato varieties grown in infested (101 ec- gs/g soil) and uninvested fields by a "ground cover index" (p). 24-9 Table 73. Level of significance of effect of nematodes, potato variety and their interaction on % of nutrient content in haulm dry matter and yield of 10 potato varieties. FACTORS N P K Ca Mg YIELD Nematodes 1% 1% 1% 1% 5% 1% Vriety 1% 1% 1% 1% 1% 1% N x V 5% 5% 1% NS 5% 1% Table 75. Correlation factors between nutrient concentration in haulm dry matter (%) and yield (tons•/ ha) of 10 potato varieties. P K Ca Mg YIELD N 0.530 0.257 -0.346 -0.124 0.470 P * 0.572 -0.577 -0.268 0.895' K * -0.336 -0.278 0.544 Ca * 0.573 -0.6664 Mg * -0.313 Yield * Significant 0.05 250 Table 74. Percentages of nutrients in hatam dry matter and yield (ton/ha) of potato varieties grown in uninfested and infested fields. INF.LEVEL VAR. N P K Ca Mg Yield 0 eggqg MP 4.94 0.29 4.07 1.88 0.39 28.4 Soil DE 5.68 0.32 4.27 1.67 0.43 27.3 MJ 5.28 0.29 3.83 1.62 0.42 27.6 MR 5.49 0.28 4.65 2.05 0.45 20.0 PC 5.72 0.35 3.86 1.33 0.39 35.6 PI 5.21 0.34 4.29 1.26 0.31 32.2 PD 5.20 0.28 3.80 1.54 0.35 33.5 RE .47 0.30 4.41 1.46 0.31 30.9 SE 5.33 0.34 4.16 1.54 0.36 28.8 UL 5.68 0.30 4.00 1.72 0.34 28.8 Means 5.40 0.31 4.13 1.61 0.37 29.3 101 eggs/g soil MP 5.22 0.24 4.59 1.87 0.39 17.1 DE 5.02 0.18 3.71 2.23 0.42 3.9 MJ 4.97 0.16 3.05 2.01 0.50 4.4 MR 4.56 0.14 3.75 2.46 0.42 0.9 PC 5.47 0.25 3.48 1.91 0.38 11.9 PI 4.82 0.19 3.19 1.81 0.35 7.8 PD 4.77 0.17 3.03 2.33 o.46 6.0 RE 5.37 0.18 3.45 1.57 0.29 7.6 SE 5.24 0.19 3.43 2.43 0.45 6.1 UL 5.23 0.20 3.79 2.05 0.36 3.7 Means 5.07 0.19 3.55 2.07 0.40 6.9 Level significance 5% 5% 1;„ NS 5% 1% S.E.D. 0.22 0.02 0.25 0.03 2.0 L.S.D. 0.45 0.04 0.68 0.06 5.4 251 a negative effect on N, P and K in haulm dry matter (significant at 1% level), but not so on Ca and Mg, which were increased by nematodes. Yield of all varieties was significantly reduced (at the 1% level) in the infested field. Figure 44, shows the effect of nematode attack on the concentration of each element in the potato varieties grown in the infested field as a percentage of those grown in the uninfested field. Nitrogen (N) was reduced in all varieties with the exception of the resistant variety MP; phosphorus (P) was affected in all varieties and more than other nutrients, but least in the varieties MP and PC. Potassium (K) was affected in much the same way as N, perhaps suggesting that the potato cyst-nematode induced syncytia in susceptible varieties may be involved in the uptake or translocation of these two elements because infested plants of MP (in which no syncytia are developed) showed slightly increased contents of N and K. Calcium (Ca) was the only nutrient whose concentration increased in all susceptible varieties but Mg increased in MJ, PI, PD, SE and UL, although it decreased in MP, PC and RE. The concentrations of Ca and Mg were not affected in MP. Figure 45, shows how yield was affected by potato cyst-nematodes and how the nematodes multiplied on each variety. Yield reduction between fie)ds is shown as a percentage of that obtained for each variety in the uninfested field. Fig. 45a shows that although most varieties were badly affected due to their low tolerance limits,MP and to a lesser extent PC were able to tolerate the damage and produce tubers. Although the percentage yield reduction of PC was greater it showed a better ability to produce tubers than MP in uninfested soil (Fig.45c) Potato cyst-nematode populations (Fig.45b) were reduced only with 252 I0 0 n N L.] 1 1 LJ (70) !Or 201- 0 i0- P 20 30- 40- 50- 20- 10- K 0 e/o) 10- 20- 30- 60- Co 45- (0/0) 30- IS- O 40- 30_ Mg 20- (70) 10- 0 10 MP DE MJ MR PC PI PD RE SE UL POTATO VARIETIES Fig. LIf . Nutrient concentration in haulm dry Tratter of plants grown on the infested site as percentages of those in plants grown on the uninfested site. 253 oc ° 467 20- 40- b0- -° 80r 100L .1111wID (b) 40 • Uninfested field O Infested field 30 Ilm••■•■ c 20 (c) O -0 10 w. - 0 171 MP DE M.! MR PC PI PD RE SE UL POTATO VARIETIES Fig. 45. Percentage yield reduction (a),neriatode multiplication (b), and actual yield (c) of ton potato varieties grown on infes- ted and uninfested fields. 254 ' MP as was expected. The early variety MR did not allow good multiplication because of its great intolerance, which was reflected in early senescence compared with the other varieties (Fig. 43 and 46), The greatest multiplication occurred on DE and MJ, indicating that very although they were nctAtolerant, they were able to support larger numbers of nematodes in their root systems than the other susceptible varieties. Finally, resultsofa correlation analysis (Table 75) show that yield was strongly positively correlated with % P content (0.895) and negatively with Ca content (-0.666). Fig. 46. Top growth of some potato varieties showing different level of tolerance to G. rostochiensis. General view of potato varieties 12 weeks old grown in a clean soil (left) ana an iniested soil (right). Top development of G. rostochiensis-free Maris Piper (left) and infested (right). Top development of G. rostochiensis-free Maris Peer (left) and infested (right). Top development of G. rostochiensis-free Pentland Crown (left) and infested (right). 256 SUi•?NAflY' OF CO21CLUSIONS the Variations of„characters of second-stage larvae and cyst-terminal areas used for identification of potato cyst-nematode species are due both to intrinsic genetic variation and to the influence of external factors such as: density of nematodes in the roots, temperature, daylength and the techniques used to prepare the specimen to be measured. The characters which are least altered are stylet length and distance from head tip to excretory pore in second-stage larvae and fenestral length and number of ridges between the anal pore and vulval fenestra in cyst-terminal areas. Results show that no more than If observations per population of these characters would permit identifi- cation of the populations but it is recommended that this number is raised to 15. When areas and perimeters of second stage larvae and males of previously identified populations were compared using an Image Analyzing computer, it was found that all values overlapped and no identification was possible. Canonical variate analysis of measurements of second stage larvae and cyst-terminal areas showed that populations of the same species coming from the same geographical area were usually very similar, but that sometimes they differed greatly. This occurred mainly with samples collected in southern areas of Peru (Lake Titicaca) and in Bolivia. The use of colour changes in developing females of potato cyst- nematodes, although a good method for differentiating the species of limited value when "cream" types of G. pallida or mixtures of species are present. However, it can be used as a preliminary identification, at the same time as populations are multiplied for further studies. Amd--ngst the South American populations some "cream" types of G. Pallida were found, indicating that their 257 distribution is not so restricted as was thought. When some populations were tested by using cuttings of Maris Piper (Higene, ex-andigena), and other plants containing different resistance genes (H2 and H3), the presence of G. rostochiensis pathotypes which overcame the H, resistance gene was confirmed in populations from Peru, Bolivia and Italy. Populations of G. pallida tested on plants with the H2 and H3 resistance genes behaved variably; or them; m any reproduced freely. The use of cuttings for these tests is a convenient vmy of obtaining test plants in large numbers. Studies using protein gel electrophoresis of immature females, scanning electron microscopy and controlled mating of selected British and Peruvian populations of G. rostochiensis showed no differences between them, in spite of the Peruvian population being an aggresive pathotype. The Peruvian "white" G. pallida was slightly differentfroma white British G. pallida when bands of protein pattern were compared. Intraspecific mating between populations from different places of origin were successful. The comparative study of the biology of the same selected ' populations showed no fundamental differences between British and Peruvian populations of the same species. However, the Peruvian populations of G. pallida seems to develop successfully at a wider range of temperatures than the "white" British G. pallida. In general, G. pallida populations showed better adaptation to low temperatures than those of G. rostochiensis, which in turn seems to develop better than G. pallida at temperatures above 20°C. Daylength had an indirect effect through the host on the rate of invasion and development of both species as daylength increased (16 hrs.) the development of plant roots also increased and consequently less competition for root space might have occurred. However, when 258 root diffusates from potato plants (S. tuberosum sub sp.tuberosum) grown under short day conditions (12 hrs) were added to cysts formed on plants grown under long day conditions, hatching of the British G. rostochiensis was greater than that of the Peruvian. When the suitability as plant host of some British commercial potato varieties was tested by using British populations of G. rostochiensis and G. pallida, the cultivar Record showed an unusual effectjlehatching and multiplication rate, of the G. pallida on Record were less than on other varieties, suggesting some degree of resistance. Based on . the geographical distribution of the two species of potato cyst-nematodes found in Andean countries,a 'hypothesis- of their evolution is suggested. Certainly G. pallida and probably G. rostochiensis too, occupies a distinct geographical area. The probable border between species almost coincides with the political border of Peru and Bolivia, where the Altiplano and Lake Titicaca are located (15.6°S). North of this line most populations are exclusively G. pallida; in southern areas along the Andes and especially in those surrounding Lake Titicaca, G. rostochiensis predominates, although G. pallida occurs too. This speciation might have occurred originally on the Eastern slopes of the Central Andes during the early Pleistocene, when several glaciation and inter- glaciation events modified the distribution of flora and fauna in the Andes of South America. These events together with geographical barriers which still remain (e.g. the Altiplano and Lake Titicaca) may have isolated populations from one another. After speciation a co-evolution with potato species may have occurred on the eastern side of the Andes (probably between Bolivia and Argentina). G. pallida, a species already better adapted by this time to low temperatures, may have moved through the Altiplano to northern areas with frost- 259. resistant potato species (S. stenotomum). Later on when the tetraploid potato S. tuberosum ssp. andigena evolved from S. stenotonium, potato and • nematode moved together further north, both of them becoming widely distributed in the Andes. The distribution pattern of races and species in European countries seems to have been little- changed by climate, potato varieties or cultivation; the introduction of current races and soecies seems to have been from the Andes where both species occur. This introduction, it has been suggested, happened in the late 1840's, when wild and cultivated potatoes were brought to Europe in an effort to breed late blight resistant varieties. Although the date of this suggested introduction fits more or less with the time required for the pest to establish and become noticed (1831), there is another possible route of introduction. Potato cyst-nematodes have been detected in organic fertilizer ("Guano") collected from shores and offshore islands of the Peruvian coast, so they might have been introduced to England and Germany in this way, as large amounts of guano were imported for ap,lication to farmlands from about 1040. Further dissemination through the potato-growing areas of the Old World probably occur:•ed with newly bred potato varieties, as has happened in other countries which have imported potato tubers recently. Dpvement of agricultural produce and goods with adhering soil doubtless also played a role. Results of experiments on the effects of potato cyst-.nematodes (G. rostochiensis Rol) on the physiology of susceptible and resistant potato cultivars indicate that both types are affected adversely, although they responded differently. Root systems of susceptible potato plants grown in heavily infested soil were shortened, matted and debilitated. Root systems of resistant plants were also damaged, mainly bY. invading larvae with weight hardly affected. Resistant plants were able to grow better and produce more tubers than susceptible ones. Earls Piper plants grown in infested soil used water more efficiently than Pentland Dell, 260 due to their greater stomatal resistances and leaf water potentials, factors which favour growth. Rate of photosynthesis of resistant and susceptible plants was decreased by nematodes. The rate in Naris Piper declined steadily as nematode infestation increased but in Earis Peer add Pentland Dell it increased at low nematode densities, perhaps to compensate for the damage and so divert photosynthates to parts of the plant other than the tubers. The total photosynthesis per plant was reduced in all potato varieties as nematode density increased, but this effect is mainly a reflection of leaf area, which was reduced in all heavily infested plants. The percentages of nutrients in haulm dry matter of susceptible and resistant potato plants was variably affected. Nitrogen and potassium were decreased by nematodes in susceptible plants but not in liaris Piper perhaps suggesting that the potato cyst-nematode induced syncytia in the susceptible plants may be involved in the diversion of these two elements to the exterior. Phosphorus was reduced in both types of Plants by nematode infestation. Although magnesium was also affected, the effect upon it was not as clear as that of calcium, which increased as nematode density increased. This chane in uptake of Ca is probably due to the increased transpiration rate observed in the susceptible potato plants because Ca is known to move through the plants with the transpiration water stream. Results on the study of tolerance to potato cyst-nematodes (G. rostochiensis Rol) suggested that one Kilogram pots might be used in large scale tests, since the growth pattern of the varieties studied and the biology of the nematodes were not much affected ( but they were in smaller pots). 2inaily, the potato cult_,.vars Loris Piper and to a les_;er c,xtent Pentland Crown have been shown to tolerate the damage caused by G. rostochiensis better than other commercial varieties, which were unable to produce 261 tubers due to their poor root growth and top development and their early senescence. 4 - f 262 APPENDIX TABLE 1. Populations under study. he capital letters in the code represent the country of orisin, e=eyt that the second letter of the Peruvian populations corresponds to the department from which they were collected. The number in the code represents the population number within a country and populations are further identified below by a locality or race name. CCUNT2Y LOCALITY or CODE LATITUDE RACE NILE ICELAND Pathotype A IC-1 63.0°N Pathotype I - Chreinsot IC=2 65.0°N Eyrar - Eyr I IC-3 65.0°N Pathotype II - Chreinsot IC-4 65.0°N Pathotype I - Fimex IC-5 65.00N 1-athotype I • idranda IC-6 65.0°N Population II - laranda IC-7 65.0°N Population II - Crobro IC-3 65.0°N DEI LARK Farpd'er 89 DE-1 56.0°N UNITED KIN3DON 2eltwell UK-3 52.3°N Cadishead UK-2 53.4°N Dunminning UK-1 54.0°N GERil.i.NY Rasse B GE-1 50.0°N CA:?ADA Vancouver CA-1 49.4°N .:E“ZOLNDLAND Newfoundland NF-1 48.0°N JAPAN Hokkaido JA-1 43.0°N Hokkaido JA-2 43.0°N Hokkaido JA-3 43.0°N 263 COUNTRY LOCALITY or CODE LATITUDE RACE NAME ITALY Brusciano IT-1 41.0°N Mariglianello IT-2 41.0°N SPAIN Valencia SP-1 39.4°N Granada SP-2 37.0°N GREECE Douneika GR-1 37.1°N Naxos GR-2 37.1°N Mantinia GR-3 37.1°N MALTA Malta MA-1 35.8°N CYPRUS Achillidis CY-1 35.0°N VENEZUELA Merida VE-1 8.4°N Tachira VE-Z 7.6°N INDIA Thalayaltmond IN-1 11.5°N COLOMBIA Narino CO-1 1.5°N Pasto CO-2 1.2°N o Pasto CO-3 1.2 N o Pasto co-4 1.2 N o Tuquerres CO-5 1.0 N o Tuquerres co-6 1.0 N o Tuquerres CO-7 1.0 N Cumbal CO-8 l.0°N o Cumbal CO-9 1.0 N o Gualmatan CO-10 1.0 N Gualmatan CO-11 1.0°N ECUADOR Mejia EC-1 3.0°S Mejia EC-2 3.0°S PERU Huambos PC-1 6.5°S Hualgayoc PC-2 6.7°S Huamachuco PL-1 7.8°S Huamachuco PL-2 7.8°S 264 COUNTRY LOCALITY or CODE LATITUDE RACE NAME PERU Huamachuco PL-3 7.8°S Otuzco PL-4 7.9°S Chaclla 1 PH-1 9.8°S Chaclla 2 PH-2 9.8°S Huanuco PH-3 9.8°S Huasahuasi PJ-1 11.2°S Huasahuasi PJ-2 11.2°S Huasahuasi PJ-3 11.2°S Huasahuasi PJ-4 11.2°S Huasahuasi PJ-5 11.2°S Huasahuasi PJ-6 11.2°S Huasahuasi PJ-7 11.2°S Huasahuasi PJ-8 11.2°S o Huasahuasi PJ-9 11.2 S Huasahuasi PJ-10 11.2°S Huasahuasi PJ-11 11.2°S Huasahuasi PJ-12 11.2°S Huasahuasi PJ-13 11.2°S Huasahuasi PJ-14 11.2°S Huasahuasi PJ-15 11.2°S Tarma PJ-16 11.5°S Jauja PJ-17 11.7°S Jauja PJ-18 11.7°S Jauja PJ-19 11.7°S Jauja PJ-20 11.7°S Jauja PJ-21 11.7°S Jauja PJ-22 11.7°S Jauja PJ-23 11.7°S Comas PJ-24 11.75°S Comas PJ-25 11.75°S Comas PJ-26 11.75°S 265 COUNTRY LOCALITY or CODE LATITUDE RACE NAME PERU Comas PJ-27 11.75°S Comas PJ-28 11.75°S Comas PJ-29 11.75°S Acolla PJ-30 11.75°S Chocon PJ-31 11.8°S Choeon PJ-32 11.8°S Comas PJ-33 11.8°S Comas PJ-34 11.8°S Comas PJ-35 11.8°S Comas PJ-36 11.8°S Comas PJ-37 11.8°S Comas PJ-38 11.8°S Comas PJ-39 11.8°S Concepcion PJ-40 11.9°S Concepcion PJ-41 11.9°S Concepcion PJ-42 11.9°S Orcotuna PJ-43 11.95°S Chupaca PJ-44 12.1°S Chupaca PJ-45 12.1°S Chupaca PJ-46 12.1°S Chupaca PJ-47 12.1°S Chupaca PJ-48 12.1°S Sta.Ana PJ-49 12.1°S Umusbamba PK-1 13.2°S Ccotatoclla PK-2 13.3°S Andenes 3 PK-3 13.5°S Andenes 2 PK-4 13.5°S 266 COUNTRY LOCALITY or CODE LATITUDE RACE NAME PERU Sandia PP-1 14.30°S Sandia PP-2 14.30°S Sandia PP-3 14.30°S Sandia PP-4 14.30°S Sandia PP-5 14.30°S Sandia PP-6 14.30°S Capa Chica PP-7 15.600S Ichu PP-8 15.85°S Ichu PP-9 15.85°S Ichu PP-10 15.85°S Ichu PP-11 15.85°S Juli PP-12 15.90°S Juli PP-13 15.90°S Salcedo PP-14 15.90°S Juli PP-15 15.95°S Juli PP-16 15.95°S Ilave PP-17 16.10°S Ilave PP-18 16.10°S Ilave PP-19 16.10°S Yunguyo PP-20 16.20°S Yunguyo PP-21 16.20°S Yunguyo PP-22 16.20°S Yunguyo PP-23 16.20°S Pomata PP-24 16.30°S Tiabaya PA-1 16.40°S Copo I, Pocsi PA-2 16.50°S Copo II, Pocsi PA-3 16.50°S Piaca PA-4 16.50°S 267 COUNTRY LOCALITY or CODE LATITUDE RACE NAME BOLIVIA Puerto Acosta BO-1 15.60°S Puerto Acosta B0-2 15.60°S Puerto Acosta BO-3 15.60°S Achacachi B0-4 15.70°S Achacachi BO-5 15.70°S Achacachi BO-6 15.70°S Belen B0-7 16.00°S Belen BO-8 16.00°S Belen BO-9 16.20°S Achacachi B0-10 16.20°S Copacabana B0-11 16.25°S Copacabana BO-12 16.30°S Copacabana BO-13 16.30°S Batallas B0-14 16.30°S Batallas BO-15 16.35°S Chinole BO-16 17.20°S Sayari BO-17 17.20°S Colomi BO-18 17.25°S Morachata BO-19 17.25°S Cochabamba BO-20 17.30°S Prusila BO-21 17.30°S Quillacollo BO-22 17.35°S Coari BO-23 17.45°S Potosi B0-24 19.60°S Moraya B0-25 21.80°S SOUTH AFRICA Pretoria SA-1 25.80°S CHILE La Serena CH-1 30.00°S NEW ZEALAND Pukekohe NZ-1 37.10°S 268 TABLE 2 - Distribution of potato cyst-nematode species in some Andean countries. (Determined by female colour.). CODE FEMALE COLOUR SPECIES LATITUDE 4 VE-1 Yellow G.rostochiensis 8.4°N VE-2 Yellow G.rostochiensis 7.6°N CO-1 Cream G.pallida 1.5°N CO-2 White-Cream G.pallida 1.2°N CO-3 White G.pallida 1.2°N CO-4 Cream-Yellow G.pallida/G.rostochiensis 1.2°N CO-5 White G.pallida 1.0°N CO-6 White G.pallida 1.0°N CO-7 White G.pallida 1.0°N co-8 White G.pallida 1.0°N CO-9 White G.pallida 1.0°N CO-10 White G.pallida 1.0°N CO-11 White G.pallida 1.0°N EC-1 Cream G.pallida 3.0°S EC-2 Cream G.pallida 3.0°S PC-1 Cream-White G.pallida 6.5°S PC-2 White G.pallida 6.7°S PL-1 Cream G.pallida 7.8°S PL-2 Cream G.pallida 7.8°S PL-3 White-Cream G.pallida 7.8°S PL-4 White G.pallida 7.9°S PH-1 White-Cream G.pallida 9.8°S PH-2 White G.pallida 9.8°S PH-3 White G.pallida 9.8°S PJ-1 White-Cream G.pallida 11.2°S * Nay have been introduced from Europe. 269 TABLE ') (Contined). CODE FEMALE COLOUR SPECIES LATITUDE PJ-2 Cream G.pallida 11.2°S PJ-3 Cream G.pallida 11.2°S PJ-4 White-Cream G.pallida 11.2°S PJ-5 White-Cream G.pallida 11.2°S PJ-6 Cream G.pallida 11.2°S PJ-7 White G.pallida 11.2°S PJ-8 White-Cream-Yellow G.pallida/G.rostochiensis _11.2°S PJ-9 Cream G.pallida 11.2°S PJ-10 White G.pallida 11.2°S o PJ-11 White G.pallida 11.2 S PJ-12 White G.pallida li.es PJ-13 White G.pallida 11.2°S PJ-14 White-Cream G.pallida 11.2°S o PJ-15 White G.pallida 11.2 S PJ-16 White G.pallida 11.5°S PJ-17 White G.pallida 11.7°S PJ-18 White G.pallida 11.7°S PJ-19 Cream G.pallida 11.7°S PJ-20 White G.pallida 11.7°S PJ-21 White G.pallida 11.7°S 0 PJ-22 White G.pallida 11.7 S PJ-23 Cream G.pallida 11.7°S PJ-24 Cream G.pallida 11.7°S PJ-25 White-Cream G.pallida 11.75°S PJ-26 Cream G.pallida 11.75°S PJ-27 White G.pallida 11.75°S PJ-28 , White G.pallida 11.75°S 270 TABLE 2 (Continued) CODE FEMALE COLOUR SPECIES LATITUDE PJ-29 Yellow-White G.rostochiensis/G.pallida 11.75°S PJ-30 White-Cream G.pallida 11.75°S PJ-31 Cream G.pallida 11.8°S PJ-32 White G.pallida 11.8°S PJ-33 Cream G.pallida 11.8°S PJ-34 White G.pallida 11.8°S PJ-35 White-Cream G.pallida 11.8°S PJ-36 White-Cream-Yellow G.pallida/G.rostochiensis 11.8°S PJ-37 Cream G.pallida 11.8°S PJ-38 Cream G.pallida 11.8°S PJ-39 Yellow-White G.rostochiensis/G.pallida 11.8°S PJ-40 White G.pallida 11.9°S PJ-41 White G.pallida 11.9°S PJ-42 White G.pallida 11.9°S PJ-43 White G.pallida 11.95°S o PJ-44 White-Cream G.pallida 12.1 S o PJ-45 White G.pallida 12.1 S PJ-46 White G.pallida 12.1°S o PJ-47 White G.pallida 12.1 S PJ-48 White G.pallida 12.1°S o PJ-49 White G.pallida 12.1 S PK-1 White G.pallida 13.2°S PK-2 White-Cream G.pallida 13.3°S PK-3 Cream G.pallida 13.5°S PK-4 White G.pallida 13.5°S PP-1 White G.pallida 14.3°S PP-2 White G.pallida 14.3°S 27.1 TABLE 2 (Continued) CODE FEMALE COLOUR SPECIES LATITUDE PP-3 White G.pallida 14.3°S PP-4 White G.pallida 14.3°S PP-5 White G.pallida 14.3°S PP-6 White G.pallida 14.3°S PP-7 White-Cream G.pallida 15.6°S B0-1 Yellow G.rostochiensis 15.6°S B0-2 White-Cream-Yellow G.pallida/G.rostochiensis 15.6°S B0-3 White G.pallida 15.6°S B0-4 White G.pallida 15.7°S B0-5 Yellow G.rostochiensis 15.7°S B0-6 White G.pallida 15.7°S PP-8 Yellow G.rostochiensis 15.85°S PP-9 Yellow G.rostochiensis 15.85°S PP-10 Yellow G.rostochiensis 15.85°S PP-11 Yellow G.rostochiensis 15.85°S PP-12 White-Cream G.pallida 15.9°S PP-13 White G.pallida 15.9°S PP-14 White G.pallida 15.9°S PP-15 Yellow G.rostochiensis 15.95°S PP-16 Yellow G.rostochiensis 15.95°S B0-7 White G.pallida 16.0°S B0-8 White G.pallida 16.0°S PP-17 White G.pallida 16.1°S PP-18 White G.pallida 16.1°S PP-19 Yellow G.rostochiensis 16.1°S PP-20 Yellow G.rostochiensis 16.2°S PP-21 Cream-Yellow G.pallida/G.rostochiensis 16.2°S PP-22 White-Cream-Yellow G.pallida/G.rostochiensis 16.2°S 272 TABLE 2 (Continued) CODE FEMALE COLOUR SPECIES LATITUDE PP-23 Yellow G.rostochiensis 16.2°S B0-9 Yellow G.rostochiensis 16.2°S B0-10 White G.pallida 16.2°S B0-11 White-Cream-Yellow G.pallida/G.rostochiensis 16.25°S PP-24 Cream G.pallida 16.3°S B0-12 White G.pallida _16.3°S B0-13 White-Cream-Yellow G.pallida/G.rostochiensis 16.3°S B0-14 White G.pallida 16.3°s B0-15 White-Cream G.pallida 16.35°S PA-1 Yellow G.rostochiensis 16.4°S PA-2 White-Yellow G.pallida/G.rostochiensis 16.5°S PA-3 Yellow G.rostochiensis 16.5°S PA-4 Yellow G.rostochiensis 16.5°S B0-16 Yellow G.rostochiensis 17.2°S B0-17 White G.pallida 17.2°S B0-18 White-Yellow G.pallida/G.rostochiensis 17.25°S B0-19 Yellow-White G.rostochiensis/G.pallida 17.25°S B0-20 Cream-Yellow G.rostochiensis/G.pallida 17.3°S B0-21 Cream-Yellow G.pallida/G.rostochiensis 17.3°S B0-22 Yellow G.rostochiensis 17.35°S B0-23 Yellow G.rostochiensis 17.45°S B0-24 Yellow G.rostochiensis 19.60°S B0-25 Yellow-Cream G..rostochiensis/G.pallida 21.80°S CH-1 Yellow G.rostochiensis 30.0°S 273 TABLE 3 - Distribution of potato cyst-nematode species in some sothery countries. CODE FEMALE COLOURg. SPECIES LATITUDE IC-1 Yellow G.rostochiensis 65.0°N IC-2 Cream G.pallida 65.0°N IC-3 Cream G.pallida 65.0°N IC-4 Yellow G.rostochiensis 65.0°N IC-5 Cream G.pallida 65.0°N IC-6 White G.pallida -65.0°N IC-7 Yellow-Cream G.rostochiensis/G.pallida 65.0°N IC-8 Yellow G.rostochiensis 65.0°N DE-1 White G.pallida 56.0°N UK-1 Cream G.pallida 54.0°N UK-2 White G.pallida 53.4°N UK-3 Yellow G.rostochiensis 52.3°N GE-1 Yellow-Cream G.rostochiensis/G.pallida 50.0°N CA-1 Yellow G.rostochiensis 49.4°N NF-1 Yellow G.rostochiensis 48.0°N -JA-1 Yellow G.rostochiensis 43:0°N JA-2 Yellow G.rostochiensis 43.0°N JA-3 Yellow G.rostochiensis 43.0°N IT-1 Yellow G.rostochiensis 41.0°N IT-2 Yellow G.rostochiensis 41.0°N SP-1 White G.pallida 39.4°N SP-2 Yellow G.rostochiensis 37.0°N GR-1 Yellow G.rostochiensis 37.1°N GR-2 Yellow G.rostochiensis 37.1°N GR-3 Yellow G.rostochiensis 37.1°N MA-1 Yellow G.rostochiensis 35.8°N 274 TA3LE 3 (Continued) CODE ' 7ZEAT:77, COLOUR * SPECIES LATITUDE CY-1 Yellow G.rostochiensis 35.0°N IN-1 White G.pallida 11.5°N SA-1 Yellow G.rostochiensis 25.8°S NZ-1 .1hite1 G.pallida 37.1°S * Describes the predominant colour during female development. However, when two colours were preseJt the first one describes the predominant colour and the second indicates the presence of some females of different colour and these were considered as mixed populations. 275 Table 4 Means and standard deviations of larval measurements of some Andean populations (n 15) Popul- Body length Stylet length Head tip Tail length Specie ations (Am) BL (pm) SL to Exc.Pore (pm)TL (pm) EP C0-1 471.8 ± 14.0 24.5 1 0.5 104.9 A 3.3 52.4 t 2.5 G.pallida EC-1 464.2 ± 20.7 23.5 t 0.4 101.7 t 4.6 51.6 t 2.7 G.pallida EC-2 458.8 ± 13.5 23.8 .1- 0.2 102.4 t 2.7 50.6 ± 2.4 G. pallida PC-1 446.5 ± 14.4 23.3 1. 0.2 99.9 ± 2.7 47.9 t 2.7 G.pallida PL-1 436.0 ± 15.8 22.8 ± 0.7 102.5 t 4.5 51.3 t 4.5 -G.pallida PL-2 457.1 ± 32.0 23.4 ± 0.4 107.6 I 6.0 54.5 ± 4.5 G.pallida PL-3 473.9 ± 15.0 24.1 ± 0.4 102.0 I 2.7 52.1 t 3.0 G.pallida PL-4 461.8 ± 16.4 23.7 t 0.3 104.7 t 6.8 53.6 t 1.7 G.pallida PH-1 466.3 ± 20.4 23.4 ± 0.5 122.1 t 5.8 53.5 t 3.7 G.pallida PH-2 478.5 ± 19.6 23.3 ± 0.5 109.7 t 3.7 55.2 t 2.8 G.pallida PH-3 456.4 ± 26.5 23.3 ± 0.7 106.6 t 4.5 53.4 t 3.9 G.pallida PJ-1 454.1 ± 27.7 23.4 t 0.6 109.6 I 4.8 57.1 t 3.4 G.pallida PJ-2 441.1 ± 14.6 23.8 t 0.5 109.3 t 4.1 54.7 t 3.4 G.pallida 4 PJ-3 476.1 ± 21.2 23.9 ± 0.6 110.1 - 6.0 56.1 t 3.8 G.pallida PJ-4 510.1 ± 28.0 24.0 ± 0.7 113.1 t 4.9 56.7 t 2.3 G.pallida ■ PJ-5 538.3 ± 21.5 24.5 - 0.7 115.2 t 4.6 62.0 t 2.9 G.pallida PJ-6 497 3 ± 29.2 23.9 t 0.7 111.3 I 5.9 58.8 t 4.1 G.pallida PJ-7 485.8 ± 24.8 24.0 t 0.6 109.8 t 6.6 59.9 t 3.8 G.pallida PJ-8 481.9 ± 40.8 23.2 t 1.8 108.4 t 9.7 54.6 t 5.1 G.pail/ G.rost. PJ-9 481.2 t 37.2 24.3 t 1.0 110.1 t 8.1 55.4 t 4.6 G.pallida PJ-14 495.6 ± 14.0 23.8 t 0.6 111.1 t 3.8 60.3 t 4.3 G.pallida PJ-16 445.4 t 22.0 23.9 t 0.5 108.5 t 4.3 54.0 t 3.3 G.pallida PJ-17 547.3 t 28.9 24.9 t 0.6 120.0 t 5.6 63.3 t 3.4 G.pallida PJ-18 487.1 t 22.2 24.3 t 0.3 113.1 t 4.3 55.4 t 3.3 G.pallida PJ-19 480.7 f 28.7 24.3 f 0.7 108.4 t 5.1 56.4 t 3.0 G.pallida 276 Table 4 (Contd) Popul- Body length Stylet length Head tip Tail length Species ations (pm) BL (pm) SL to Exc.Pore (Alm)TL (Am) EP PJ-20 486.7 ± 29.2 24.4 ± 0.6 112.6 t 6.6 54.9 ± 4.3 G.pallida PJ-23 484.9 ± 18.6 24.1 ± 0.7 108.8 ± 4.7 57.0 ± 4.5 G.pallida PJ-24 472.8 ± 226 23.6 ± 1.0 106.9 f 3.4 53.4 ± 2.7 G.pall/ G.rost. P3-25 480.4 ± 35.6 24.2 ± 0.6 114.3 t 5.6 54.2 a. 3.0 G.pallida PJ-26 500.9 t 42.7 23.9 ± 0.8 117.1 ± 7.2 56.6 ± 2.9 G.pallida PJ-29 459.8 ± 26.0 22.1 f 2.0 102.5 ± 4.5 51.4 ± 4.3 G.rost/ G.pall. - PJ-31 508.6 ± 21.4 23.6 - 0.6 108.3 ± 3.4 55.5 f 3.0 G.pallida PJ-32 481.9 - 13.6 24.7 ± 0.3 103.5 ± 4.0 56.4 .1- 2.7 G.pallida PJ-33 463.6 ± 20.0 23.7 ± 0.4 106.0 ± 4.0 55.0 t 3.4 G.pallida PJ-35 469.8 ± 13.5 24.1 ± 0.6 109.3 ± 5.0 54.2 ± 3.0 G.pallida PJ-36 468.2 t 37.1 23.3 ± 1.3 105.8 ± 5.5 54.5 ± 4.6 G.pall/ G.rost. PJ-37 485.7 ± 21.3 24.1 1 0.6 110.9 ± 4.4 56.8 ± 2.3 G.pallida PJ-38 500.9 ± 17.0 23.6 00.5 101.1 ± 2.2 54.4 ± 3.4 G.pallida PJ-39 44o.8 ± 26.1 21.4 ± 1.2 99.6 ± 6.2 50.4 ± 4.3 G.rost/ G.pall. PJ-40 444.3 ± 22.8 23.2 ± 0.7 105.0 ± 5.3 54.8 ± 3.6 G.pallida PJ-44 490.3 ± 23.8 23.5 ± 0.7 108.0 ± 4.1 57.7 ± 2.2 G.pallida PJ-46 518.4 ± 24.3 23.6 ± 0.6 114.4 ± 6.5 56.1 = 3.3 G.pa]lida PK-2 481.1 t 21.2 24.8 ± 0.4 103.9 ± 4.6 53.2 ± 5.5 G.pallida PK-3 520.3 ± 22.4 23.8 ± 0.6 115.4 I 4.8 55.6 ± 2.6 G.pallida PP-1 473.5 ± 17.7 23.6 ± 1.3 108.0 ± 4.8 55.0 ± 3.9 G.pall/ G.rost. PP-2 477.8 t 23.6 23.8 ± 0.8 109.5 ± 4.1 54.0 ± 4.6 G.pallida. PP-7 473.5 ± 21.6 23.0 ± 0.5 103.7 t 3.0 52.9 ± 1.9 G.pallida B0-1 462.9 f 26.5 23.1 ::I 0.7 106.8 ± 6.8 52.3 ± 3.4 G.pall/ G.rost. 277 Table4- (Contd.) Popul- Body length Stylet length Head tip Tail length Specie ations (pm) BL (pm) SL to Exc.Pore (Am)TL (pm) EP B0-2 485.3 ± 33.7 23.1 0.6 108.4 ± 4.4 53.4 = 3.8 G.pallida B0-5 458.3 ± 18.4 22.3 ± 1.6 102.7 t 3.3 51.4 = 4.3 G.rost/ G.pall. PP-8 448.4 ± 19.3 21.6 ± 0.5 98.7 f 3.5 49.7 = 2.6 G.rost. PP-9 462.1 ± 15.1 21.9 ± 0.4 99.4 ± 2.3 51.3 ± 3.6 G.rost. PP-12 477.0 ± 11.3 23.3 ± 0.8 111.5 ± 3.7 49.6 = 2.1 G.pallida PP-21 484.5 ± 30.5 23.8 ± 1.0 107.9 ± 6.6 54.3 = 2.6 -G.pallida PP-22 453.7 ± 20.7= 22.8 ± 1.5 104.3 ± 5.3 51.8 t 3.2 G.pall/ G.rost. PP-23 470.9 ± 27.9 21.8 ± 0.8 105.3 ± 3.8 51.9 2.8 G.rost. B0-9 453.7 ± 20.2 20.8 ± 0.5 104.5 3.0 50.8 f 4.5 G.rost. PP-24 486.8 ± 19.0 23.9 ± 0.5 109.1 f 5.1 52.9 = 2.7 G.pallida B0-12 529.7 ± 30.7 23.4 ± 0.6 121.2 ± 7.9 56.4 = 3.7 G.pallida B0-13 487.2 ± 24.9 22.9 t 1.2 106.8 ± 3.5 55.2 ± 4.0 G.pall/ G.rost. Bo-15 465.9 ± 22.4 22.8 - 0.6 110.5 ± 2.9 53.9 ± 1.5 G.pallida PA-1 419.8 ± 22.1 21.5 - 0.8 87.7 t 4.3 46.7 ± 3.1 G.rost. PA-2 482.7 ± 19.2 22.3 - 1.8 109.5 ± 5.9 52.6 ± 5.2 G.pall/ G.rost. PA-3 454.1 ± 24.5 21.8 - 1.0 103.7 ± 5.4 50.5 ± 2.6 G.rost. G.pall. PA-4 463.8 ±10.5 23.4 t 0.3 112.4 ± 2.3 56.3 ± 1.6 G.pallida B0-18 490.8 ± 19.7 21.8 t 0.5 100.3 ± 4.3 52.2 -1 2.8 G.rost. B0-19 467.3 ± 24.0 21.8 t 1.1 105.2 ± 5.4 53.8 = 3.9 G.rost/ G.pall. B0-20 470.1 ± 20.6 21.3 t 0.9 105.9 ± 5.6 52.7 3.4 G.rost/ G.pall. B0-21 470.5 ± 22.8 22.0 t 0.4 104.2 ± 4.6 54.7 = 2.6 G.pall/ G.rost. B0-22 500.3 ±24.7 22.5 t 1.1 101.3 2" 5.7 53.4 ± 2.1 G.pall/ G.rost. B0-23 445.4 ± 11.2 21.3 t 0.6 99.5 ± 2.7 49.9 = 2.9 G.rost. 273 Table 4 (Contd.) Popul- Body length Stylet length Heat tip Tail length Specie ations (gm) BL (um) SL to Exc.Pore (um)TL (gm) EP B0-24 467.2 t 30.0 22.3 t 1.2 105.6 t 6.8 55.1 3.4 G.rost/ G.pall B0-25 461.2 ± 16.6 21.2 t 0.8 102.5 t 3.7 50.2 t 2.5 G.rost/ G.pall CH-1 450.7 I 19.7 21.1 t 0.5 102.5 t 3.0 48.2 t 2.1 G.rost. 279 Table 5 Means and standard deviations of larval measurements of some "Others" populations (n.15) Popul- Body length Stylet length Head tip Tail length Specie ations (pm)BL (pm)SL to Exc.Pore (pm)TL (pm)EP IC-1 467.1 ± 12.2 21.6 ± 0.4 100.5 I 2.9 48.9 t 2.9 G. rost. IC-2 481.1 ± 20.7 23.3 f 0.3 104.6 ± 3.2 47.0 1- 3.3 G. pallida IC-3 496.6 I 19.4 23.5 t 0.4 106.3 I 5.5 51.2 t 2.7 G. pallida IC-4 444.3 t 17.0 20.6 t 0.5 91.8 I 3.3 42.9 .1 3.8 G. rost. IC-5 517.6 ± 18.4 24.1 t 0.2 109.2 f 3.5 56.5 t 3.2 G. pallida UK-1 499.1 ± 16.9 24.3 -f- 0.4 101.8 1. 4.5 56.0 t 2.1 G. pallida UK- 2 451.1 ± 21.3 23.6 ±`0.5 102.7 t 3.5 49.8 I 3.3 G. pallida UK-3 433.5 f 16.5 20.6 t 0.5 96.2 f 3.2 46.8 t 2.5 G. rost. GE-1 433.9 t- 16.7 21.3 I 0.5 103.4 t 3.1 50.3 t 1.7 G. rost. NF-1 567.6 ± 24.6 25.0 f 0.4 - - G. pallida + + JA-1 437.0 I 12.0 21.5 f 0.4 100.1 - 3.1 49.0 - 1.4 G. rost. JA-2 455.2 1. 16.2 21.8 I 0-5 103.2 I 3.1 50.6 ± 2.4 G. rost. IT-1 447.6 t 20.9 20.9 t 1.0 100.7 t 4.4 50.2 t 3.8 G.rost./ G.pall. IT-2 453.7 I 15.2 21.1 t 0.4 96.0 t 3.0 47.0 t 2.1 G. rost. SP-1 473.9 t 18.4 23.4 f o.7 112.3 t 3.9 54.4 t 3.0 G. pallida SP-2 472.1 I 15.2 21.2 I 0.4 99.8 ± 3.4 47.7 I 2.4 G. rost. GR-1 440.8 ± 19.2 20.5 t 0.6 101.5 t 5.4 48.6 1.- 3.0 G. rost. GR-3 449.9 t 16.9 20.4 I 0.7 102.4 ± 4.8 49.4 t 2.4 G. rost. CY-1 475.3 t 8.4 21.2 f 0.5 93.2 ± 2.6 47.8 I 2.0 G. rost. IN-1 437.7 f 18.3 21.9 t 0.8 106.1 I 3.2 49.6 I 3.1 G. rost/ G. pall SA-1 441.9 t 21.7 21.0 1 0.5 101.0 t 3.9 50.6 I 3.6 G. rost. NZ-1 494.4 ± 10.2 23.6 ± 0.3 105.0 - 2.1 50.9 - 3.3 G. pallida Table 6 Means and standard deviations of cyst measurement of some Andean and other populations (n=15) Andean Populations Fenestral Fenestral Number of Dis.Betw Granek's Fenestral Specie length width ridges Fenestral ratio area 3.14 (um) L (um) W (n+ni/2) to Anus(B) (B/L) (L x W/4) CO - 1 24.8 ± 2.9 t 22.4 ± 1.9 10.3 ± 2.0 50.8 ± 2.9 2.1 ± 0.2 439.2 2.- 12.9 G.p. EC - 1 25.3 ± 4.6 22.0 ± 2.9 8.3 ± 2.2 51.6 ± 4.6 2.1 2: 0.3 440.8 ± 9.0 G.p. EC - 2 25.4 ± 3.0 22.5 ± 2.8 8.4 ± 2.1 54.9 ± 3.0 2.2 ± 0.3 453.8 ±11.5 G.p. PC - 1 22.9 ± 1.6 21.7 ± 1.6 9.8 ± 2.0 47.1 ± 1.6 2.1 ± 0.2 389.6 ± 8.6 G.p. PL - 3 21.8 ± 2.3 21.1 ± 1.9 11.5 ± 2.8 55.5 ± 2.3 2.6 ± 0.2 363.7 ±14.6 G.p. PL - 4 24.1 ± 2.4 21.2 ± 2.7 9.0 ± 2.3 51.8 ± 2.4 2.1 ± 0.3 404.3 ±14.5 G.p. PJ - 32 25.7 ± 2.8 21.2 ± 2.5 8.9 ± 2.0 50.5 ± 2.8 2.0 ± 0.2 429.1 ±10.7 G.p. 24.2 22.6 G PJ - 38 ± 3.0 ± 1.9 8.3 ± 2.0 39.4 ± 3.0 1.6 ± 0.2 433.1 ±13.8 .p. IN) Co 0 PK - 2 25.2 ± 3.2 22.4 ± 1.5 8.9 ± 2.4 55.4 ± 3.2 2.2 ± 0.2 445.9 1.18.1 G.p. PP - 8 19.4 ± 2.4 19.0 ± 2.3 14.8 ± 1.4 70.1 ± 2.4 3.6 ± 0.2 293.6 ±13.9 G.r. PP -9 18.4 ±-4-3.2 17.0 ± 2.4 11.6 ± 3.3 44.8 ± 3.2 2.5 ± 0.2 250.9 ±13.3 G.r/G.p. BO - 18 18.9 ± 2.9 17.8 ± 2.3 12.7 ± 2.5 49.2 ± 2.9 2.7 ± 0.2 268.5 ±13.2 G.r/G.p. BO - 22 22.5 ± 2.6 20.7 ± 2.7 9.6 ± 2.6 46.0 ± 2.6 2,1 ± 0.3 370.0 ± 9.9 G.p. Others IC - 1 20.6 ± 2.5 20.0 ± 2.4 15.9 ± 2.7 71.6 ± 2.5 3.5 ± 0.2 326.5 ±15.3 G.r. IC - 2 24.8 ± 3.0 24.0 ± 2.1 14.0 ± 5.6 57.6 ± 3.0 2.3 ± 0.2 472.6 ±19.5 G.p/G.r Table 6 (Contd) Others Fenestral Fenestral Number of Dis.Betw Granek's Fenestral Specie length width ridges Fenestral ratio area 3.14 (um)L (um)W (n+n'/2) to Anus(B) (B/L) (L x W/4) IC - 3 23.4 ± 3.2 22.0 ± 2.3 12.4 ± 4.1 46.8 ± 3.2 2.1 ± 0.2 407.2 ± 15.5 G.p/G.r IC - 4 20.4 ± 1.9 20.4 ± 1.4 21.0 ± 3.6 66.9 ± 1.9 3.3 ± 0.1 329.1 ± 11.7 G.r. IC - 5 22.3 ± 1.9 21.2 ± 1.3 10.2 ± 3.1 39.0 ± 1.9 1.7 ± 0.1 372.8 ± 13.7 G.p. UK - 1 26.4 ± 3.0 22.2 ± 2.1 8.6 ± 2.1 43.6 ± 3.0 1.7 ± 0.2 463.2 ± 11.8 G.p. UK - 2 26.3 2 2.0 22.7 ± 1.7 11.3 ± 2.6 52.9 ± 2.0 2.0 ± 0.2 470.5 ± 12.3 G.p. UK - 3 20.6 ± 1.6 20.8 ± 1.4 17.4 ± 2.9 64.6 ± 1.6 3.1 ± 0.1 339.4 ± 21.4 G.r. IT - 2 21.6 ± 1.8 21.2 ± 1.6 16.9 ± 3.0 80.7 ± 1.8 3.8 ± 0.2 361.4 ± 16.6 G.r. SP - 2 20.8 ± 2.6 20.6 ± 2.9 15.2 ± 3.4 65.6 ± 2.6 3.2 ± 0.3 341.9 ± 15.3 G.r. CY - 1 21.0 I 1.1 20.7 ± 1.2 18.4 ± 5.7 76.2 ± 1.1 3.6 ± 0.1 341.9 ± 24.0 G.r. NZ - 1 23.7 ± 2.3 22.2 ± 2.0 12.1 ± 2.3 58.5 ± 2.3 2.5 ± 0.2 415.3 ± 13.8 G.r. Table? Identification of species of potato cyst-nematodes and pathotypes in Andean populations from results on female colour, measurements on cyst terminal areas and second stage larvae, and differential clones. Popu- Female colour Cyst-Terminal 2nd std;. Differential Actual lations area larvae clones status VE-1 G. rost. G. rost. VE-2 G. rost G. rost. CO-1 G.pallida G.pallida G.pallida G.pallida G.pallida C0-2 G.pallida G.pallida G.pallida C0-3 G.pallida G.pallida G.pallida EC-1 G.pallida G.pallida G.pallida G.pallida G.pallida EC-2 G.pallida G.pallida G.pallida G.pallida G.pallida PC-1 G.pallida G.pallida G.pallida G.pallida G.pallida PL-1 G.pallida G.pallida G.pallida G.pallida PL-2 G.pallida G.pallida G.pallida G.pallida PL-3 G.pallida G.pallida G.pallida G.pallida G.pallida PL-4 G.pallida G.pallida G.pallida G.pallida G.pallida PH-1 G.pallida G.pallida G.pall/ G.pallida G.rost. PH-2 G.pallida G.pallida G.pallida PH-3 G.pallida G.pallida G.pallida PJ-1 G.pallida G.pallida G.pallida G.pallida PJ-2 G.pallida G.pallida G.pallida G.pallida PJ-3 G.pallida G.pallida G.pallida G.pallida PJ-4 G.pallida G.pallida G.pallida G.pallida PJ -5 G.pallida G.pallida G.pallida G.pallida PJ-6 G.pallida G.pallida G.pallida G.pallida PJ-7 G.pallida G.pallida G.pallida PJ-8 G.pall/ G.pall/ G.pallida G.pallida G.rost. G.rost. 283 Table? • (contd) Popu- Female colour Cyst-Terminal 2nd stg. Differential Actual lations area larvae clones status PJ-9 G.pallida G.pallida G.pallida G.pallida PJ-14 G.pallida G.pallida G.pallida G.pallida PJ-16 G.pallida G.pallida G.pallida G.pallida PJ-17 G.pallida G.pallida G.pallida PJ-18 G.pallida G.pallida G.pallida G.pallida PJ-19 G.pallida G.pallida G.pallida -G.pallida PJ-20 G.pallida G.pallida G.pallida PJ-23 G.pallida G.pallida G.pallida G.pallida PJ=24 G.pallida G.pall./ G.pallida G.pall./ G.rost. G.rost. PJ-25 G.pallida G.pallida G.pallida G.pallida PJ-26 G.pallida G.pallida G.pallida G.pallida PJ-29 G.rost./ G.rost./ G.rost.1 G.pall. G.pall. G.pall. PJ-31 G.pallida G.pallida G.pallida G.pallida PJ-32 G.pallida G.pallida G.pallida G.pallida PJ-33 G.pallida G.pallida G.pallida G.pallida PJ-35 G.pallida G.pallida G.pallida PJ-36 G.pall./ G.pall./ G.pall./ G.rost. G.rost. G.rost. PJ-37 G.pallida G.pallida G.pallida G.pallida PJ-38 G.pallida G.pallida G.pallida G.pallida G.pallida. PJ-39 G.rost./ G.rost./ G.rost./ G.pall. G.pall. G.pall. PJ-40 G.pallida G.pallida G.pallida G.pallida PJ-44 G.pallida G.pallida G.pallida G.pallida PJ-46 G.pallida G.pallida G.pallida PK-2 G.pallida G.pallida G.pallida G.pallida PK-3 G.pallida G.pallida G.pallida G.pallida PP-1 G.pallida G.pall./ . G.pallida G.rost. 234 Table (contd) Popu- Female colour Cyst-Terminal 2nd stg. Differential Actual lations area larvae clones status PP-2 G.pallida G.pallida G.pallida PP-7 G.pallida G.pallida G.pallida G.pallida B0-1 G.rost. G.pall./ G.rost. G.pall./ G.rost. G.rost. B0-2 G.pall./ G.pallida G.pallida G.rost. B0-5 G.rost. G.rost./ G.rost./ G.pall. G.pall. PP-8 G.rost. G.rost. G.rost. G.pallida G.rost. PP-9 G.rost. G.rost. G.rost. G.rost. PP-12 G.pallida G.pallida G.pallida PP-21 G.pall./ G.pallida G.pallida G.pallida G.rost. PP-22 G.pall./ G.pall./ G.pallida G.rost. G.rost. G.rost. PP-23 G.rost. G.rost. G.pallida G.rost. B0-9 G.rost. G.rost. G.rost. PP-24 G.pallida G.pallida G.pallida G.pallida B0-12 G.pallida G.pallida G.pallida B0-13 G.pall./ G.pall./ G.pall./ G.rost. G.rost. G.rost. B0-15 G.pallida G.pallida G.pallida PA-1 G.rost. G.rost. G.rost. G.rost. PA-2 G.pall./ G.pall./ G.pall./ G.rost. G.rost. G.rost. PA-3 G.rost. G.rost./ G.rost. G.pall. PA-4 G.rost. G.pallida G.pallida B0-18 G.pall./ G.rost./ G.rost. G.pallida G.rost./ G.rost. G.pall. G.pall. B0-19 G.rost./ G.rost./ G.rost./ G.pall. G.pall. G.pall. B0-20 G.rost./ G.rost./ G.rost./ G.pall. G.pall. G.pall. 285 Table? (contd) Popu- Female colour Cyst-Terminal 2nd stc. Differential Actual lations area larvae clones status B0-21 G.palR./ G.pall./ G.pall./ G.rost. G.rost. G.rost. B0-22 G.rost. G.pallida G.pall./ G.pallida G.pall./ G.rost. G.rost. B0-23 G.rost. G.rost. G.pallida G.rost. B0-24 G.rost. G.rost./ G.rost./ G.pall. G,pall. B0-25 G.rost./ G.rost./ G.rost./ G.pall. G.pall. G.pall. CH-1 G.rost. G.rost. G.rost. 236 Table 8 Identification of species and pathotypes of potato cyst-nematodes in "Other" populations from results on female colour, terminal cyst area and second stage larvae measurements and differential clones. Popu- Female colour Cyst-Terminal 2nd stg. Differential Actual lations area larvae clones status IC-1 G.rost. G.rost. G.rost. G.rost. IC-2 G.pallida G.pall./ G.pallida G.pallida G.pallida G.rost. IC-3 G.pallida G.pall./ G.pallida G.pallida G.pallida G.rost. IC-4 G.rost. G.rost. G.rost. - -G.rost. IC-5 G.pallida G.pallida G.pallida G.pallida G.pallida IC-6 G.pallida - - G.pallida IC-7 G.rost./ - - - G.rost./ G.pall. G.pall. IC-8 G.rost. - - G.rost. DE-1 G.pallida - - G.pallida UK-1 G.pallida G.pallida G.pallida G.pallida G.pallida UK-2 G.pallida G.pallida G.pallida G.pallida G.pallida UK-3 G.rost. G.rost. G.rost. G.rost. G.rost. GE-1 G.rost./ G.rost. G.rost. G.pall. CA-1 G.rost. G.rost. NF-1 G.rost. - G.pallida - G.pallida JA-1 G.rost. - G.rost. G.rost. JA-2 G.rost. - G.rost. - G.rost. JA-3 G.rost. - - - G.rost. IT-1 G.rost. - G.rost./ G.rost. G.rost./ G.pall. G.pall. IT-2 G.rost. G.rost. G.rost. G.pallida G.rost.* SP-1 G.pallida - G.pallida - G.pallida SP-2 G.rost. G.rost. G.rost. G.rost. G.rost. GR-1 G.rost. - G.rost. - G.rost. GR-2 G.rost. - - - G.rost. 287 Table 8 (contd) Popu- Female colour Cyst-Terminal 2nd sto- Differential Actual lations area larvae clones status GR-3 G.rost. G.rost. G.rost. MA-1 G.rost. G.rost. CY-1 G.rost. 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