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you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. AN I11VESTIGATION OF THE SUSCEPTIBILITY OF VARIETIES
OF LUCER]\;']; r-IEiJICAGO .;;;;.;...;...;;..;:;;.;..;... L. TO THE JlIEltATODE
DITYLENCHUS
(KUHN)
A thesis
submitted in partial fulfilment
of the requirements for the Degree
of
Naster of Agricultural Science
in the
University of Canterbury
by
P.A.
Burnett
Lincoln College
F'HCNTISP1!!:C
. i el
of lu c r ne i n Mid CEnt 0r hury
U ght er are rs i .d i c F e nr>r.a tor.e
d a;-l~ge
CONTENTS
PAGE
CRAPrER
I.
II.
III.
IV.
INTRODUCTION •
1
REVIEW OF THE LITERATURE •
3
1.
Taxonany
3
2.
Host Range
4
3.
Biological Races
6
4.
Life History
8
5.
Resistance Testing Techniques
11
19
1.
Nematode Collection, Preservation and Extraction
19
2.
Plant Preparation
25
3.
,Plant Inoculation
29
RESULTS
,2.
'VI.
•
MATERIALS AND METHODS •
1.
V.
•
Results fran soil flats trials
Resul ts fran filter paper rolls •
·
·
35
·
3$
64
DISCUSSION AND CONCLUSION
SUMMARY
35
69
•
ACKNO',lLEDGF.MENTS
•
70
REFERENCES
·
71
APPENDICES
·
75
•
75
.,
Appendix 1.
Appendix 2.
•
64
LIST OF FIGURES
FIGURE
PAGE
l.
An outline of the life history of Ditylenchus dipsaci
2.
Appa.ratus for the extraction of nematodes from plant
materia.l
3.
10
21
Apparatus for separating nematodes from soil and plant
debris
4.
.
23
Appara.tus for separating nematodes from soil and plant
debris
.
24
LIST OF PLATES
1.
Covered trays used to hold the filter paper rolls in the
growth ca.binet .
27
2.
Syringe used for inoculation of lucerne seedlings
30
3.
Lucerne pla.nts in filter pa.per rolls three weeks after
inoculation .
4.
~:ematode
infected and uninfected plants after three weeks
FRONTISPIECE
Field of lucerne in Mid Cpnterbury.
32
33
LIST OF TABLES
TABLE
1.
PAGE
Condition of young lucerne plants (Wairau) ~wo weeks after
infection with nematodes in 1% solution of methyl
cellulose •
2.
36
Condition of young lucerne plants (Wairau) three weeks
after infection with nematodes in 1% solution of
methyl cellulose
3.
37
Condition of young lucerne pla.nts (vlairau) four weeks
after infection with nematodes in 1% solution of
methyl cellulose
37
4.
Results from filter paper rolls in Experiment 1
5.
Resul ts from rolls inoculated with nematodes in 1%
solution of methyl cellulose
6.
Mortalities in rolls inoculated with
39
41
1%
solution of methyl cellulose
42
7.
Mortalities in control rolls.
43
8.
Results fran three lucerne varieties tested for nematode
susceptibility
9.
Hesult::: from three lucerne varieties tested for nematode
susceptibility
10.
Results from the macerated seedlings from Experiment 11
11.
Results from three lucerne varieties tested for nematode
susceptibility
12. ,
44
Results from the ma,cerated seedlings from Experiment 12
47
49
51
53
LIST OF Tt;BLES
(Continued)
PAGE
TABLE
13.
Results from three lucerne varieties tested for
nematode susceptibility .
14.
55
Results from the Iill3cer",ted lucerne seedlings from
ExperJrnent 13
15.
}{e~n
stem diEJmeters in mm. of 45 seedlings of three
v~rieties
16.
57
of lucerne in three experiments .
58
Transformed data from Table 15 - (loglO of mean stem
diameter)
59
17.
Analysis of variance of results in Table 16
Hl.
Analysis of variance of plants containing nematodes
and eggs •
60
61
19.
Percentage of plants containing reproducing nematodes
20.
Comparison of stem diameter
61
of seedlings with ratings
of nema.tode numbers and reprodllction within the plants
for 30 plants from Experiment 13 .
63
1
CHAPI'ER I
INTRODUCTION
The stem nematode, Ditylenchus dipsHci (Kiihn), is an important pest of
lucerne (Medicago sativa L.), in Canterbury.
Because of the way in which
lucerne is ero"m, the nature of the crop and its monetary value, the
introduction or development of varieties resistant to nematode attack seems
to be the most promising method of combating this pest at present.
In other P!?TtS of the world nemGtodes simihr to Di tylenchus dipsaci
hllve been successfully controlled on some crops by the use of systemic
orgtno-phosphpte insecticides.
However, the cost of these chemicals usually
exceeds the velue of the lucerne crop, a factor which also excludes the use
of soil fumigRnts.
Crop rotB.tion with non host plants also affords an economical method
of controlling Ditylenchus dipsaci.
However, frequent crop rotation is not
favoured in Canterbury as the cost of lucerne establishment is high and, on
the lighter lands, profitable non host plants are difficult to find.
Control of nematodes by the introduction or maintenance of their
parrsites, predators or diseases ha-s been tried in other countries several
times.
When it has been possible to alter the soil environment to favour
parasites or predators, control has been occasionally achievec}.
However,
just what cOD6it:ions are necessary for the successful propagation of the
enemies of gitylenchus dipsaci are unknown at present,
Several nematode resistant strains of lucerne have been developed in
North $nd South America.
Unfortunately, none of these are agronomically
2
suited to New Zealand conditions.
New
Zealand
Further, just how susceptible the major
variety of lucerne, Wairau, is to nematode attack in comparison
to the resista.nt varieties developed overseas, has not been determined.
It
w::s decided, therefore, to develop a technique to test the ability of various
varieties of lucerne to withstand the attacks of the CAnterbury race of
Ditylenchus dipsp.ci.
Because of the large host range and the many different
r1!ces of Di tylenchus dipSfl.ci reported in literature, it wa.s deemed important
to obtBin
nem~todes
grown lucerne.
belonging to the
particul~r
race attacking Canterbury
3
CHAPTER II
REVIEW OF THE LI1'ERt.TVRE
1.
TAXONCMY
The ma.jor stem nematode disea.se of lucerne, Medicago sativa L. is
cllused by the obligate endoparasitic nematode, Ditylenchus
dipsac~
(Kuhn).
Its classific~tion after Thorne (1949) and Goodey (1963) is as follows:
Phylwn
Aschelminthes
Class
Nematonen
Order
Tylenchida
Superfamily
Tylenchoidea
Family
Tylenchidae
Subfamily
Tylenchinae
Genus
Dit;y:lenchus Filipjev, 1936
Type species Ditylenchus dipsaci (Kuhn, 1857) Filipjev, 1936.
Thorne (1949) states that this nematode ranges in length fran 1.0 rrun.
to 1.3 mm. and gives a detailed description of the species.
The nematode
weB first described by Kuhn as Anguilla dipsaci from specimens taken in
Fuller1s teasel, Dipsacus fullonum L. (Bingefors, 1957, and Barker and
Sa.sser, 1959).
Due to the large host range of this species many synonyms
were described in early literature.
include synonym lists.
Thorne (19~.5) and Goodey (1963)
4
2.
HOST RANGE
This stem infecting nematode is believed to have been present in
Germ/my at least as early as 11319, when Schweiz (Edwards, 1932) described
8
disease of red clover very similar to the condition now known to be
cAused by Ditllenchus dipsaci.
The first record of the occurrence of
Ditylenchus dipsaci on lucerne was made by Kuhn in 11381 (Edwards, 1932),
who described it as a new species Tllenchus navensteinii.
Later work
by Ritzema Bos (Edwards, 1932) showed this form was in fact what is now
known as Ditl1enchus dipsaci.
It was first identified from diseased
clover in England by Ritzema Bos in 18137 (Edwards, 1932) and was first
recorded in lucerne in Britain in 19413 (Brown, 1953).
Edwards (1932)
states that Lounsbury reported it from South Africa in 1909 and also says
that it may have been present for some years before this.
McKay (1922)
isolated Ditl1enchus dipsaci from lucerne in Oregon and stated that it had
not previously been recorded fram this host in America.
Godfrey (1922)
reported tha.t during the previous season it occurred in the United States
of America in red clover, lucerne, strawberries and daffodils.
Cobb
(19213-29) recorded that Ditllenchus dipsaci was known to be widespread in
the United States of P,merica, occurring in twenty-six states all told.
Noble (1925) called attention to the presence of the disease in lucerne in
the Hunter River district of New South VJales, Australit, and stated that
growers were of the opinion that Ditllenchus dipsaci had been present in
their fields for mtJny years.
Edwards (1932) states thf!t it was widespread
throughout New South Wales.
Kirk (1903 and 1907) recorded this nematode in New Zealand from
potatoes.
Edwards (1932) stated that this record was not Ditllenchu~
dipsaci and Clark (1963) also questions it.
Kirk (1908) published an
5
account of nemBtodes in New Zealand and in this the confusion over the
potato nematode still persists, but Ditylenchus dipsaci was recorded from
oats ~nd wheat.
Morrison (1957) was the first to record it in lucerne
in New Zealand, but Smith and Pa.lmer (pers. comm.) say that it was
recognised before this.
The distribution of this nematode in New
Zea.land is not well documented, but from the author's experience it is
widespread throughout the Marlborough and Canterbury regions.
Ritzema. Bos (Edwards, 1932) stated in 1888 that Ditylenchus dipsa~
was known as a parasite of 34 species representing 14 natural orders of
pla,nts.
Goodey (1929) lists it as occurring in 126 plant species
representing 30 natural orders.
the host range was 135 plants.
In 1931 the same worker reported that
Christie (1959) stated that the host range
of Ditylenchus dipsaci was approaching 375 species.
Altogether some 400
plants, both monocotyledons and dicotyledons, have been listed as hosts
(Goodey, 1956 and Goodey et al., 1959).
Dingley (1969) made a thorough review of the host range in New
Zealand and her list is as follows:
Allium ceEa
Onion
Avena sativa.
Oats
Endymoin sp
English Bluebell
Gladiolus sE
Gladiolus
Hyacinthus orientalis
Hyacinth
Medicago sativa
Lucerne
Narcissus sE
Daffodils
Phlox paniculata
Phlox
Plantago
Narrow leaved Plantain
lanceol~ta
Plantago major
Broad leaved Plantain
continued/ ...
6
3.
Secale cereale
Ryecorn
Solanum tuberosum
Potato
Trifolium pratense
Red Clover
Trifolium repens
White Clover
Triticum aestium
Wheat
Tulipa sp cult
Tulip
BIOLCGICAL RACES
In 1888 Ritzema Bos grouped a number of nominal species together
under the name of Tylenchus devastatris (the present Ditylenchus dipsaci)
and at the same time noted that stem eelworms from different host plants
showed different host preferences (Seinhorst, 1957).
This was the first
indication of the existence of biological races and since then many workers
have investigated this phenomenon.
Cross inoculation experiments by
Goodey (1922), Hodson (1926) and others, have shown that these host
preferences are constant and that the biological races are distinguishable
biological units.
Steiner (1925) in his discussion pointed out that
different populations of a nematode species may vary in their relations to
the SRme host species.
Be believed that a nematode population would
1:ilways prefer that particuler host species and the variety or strain in
which its parents lived.
He considered that this preference for one host
species increased with the number of generations which had lived in it.
Hhen
!3.
popUlation hl'd grown long enough in a host plant he considered tha,t
it becrune so speciAlised that i t could not easily attack other plant species.
Quanjer (1928) (Bingefors, 1957) thought that there were IIbridging
species" which made possible the transition from one plant species to
7
another.
Goodey (1931) supposed a type of "food memory" as causing the
preference of a nematode population for one host species.
Steiner (1956) (Eriksson, 1965) changed his ide~s and said that he
w~s
of the opinion
th~t
most of the races are morphologically different
t1nd should be given specific rank.
Eriksson (1965) noted that the
Russian workers also held this view and had given specific names to sane
of the races.
Seinhorst (1957) said that this idea of Steiners was not
substantiated and he distinguished 11 races of Ditylenchus dipsaci each
with its more or less restricted host range.
Sturhan (1964) using suitable host plants succeeded in interbreeding
at lep',st six biological races, in ten different combinations, sUbstantiating
that they were only races of the same nematode species.
frequent occurrences
He obtained
of abnormalities in the shape of body and the tail
in two hybrid populations, demonstrating the existence of remarkable
genetical differences between the biological races.
He suggested that the
possibility of interbreeding is probably an
reason for the frequent
L~portant
occurrences of different rf:'\.ces and of strains with different physiological
behaviour within the biological races of one host plant species.
It seems
likely thrt new biological races could originate in this wPy.
Webster (1964) find Eriksson (1965) both managed to produce race
hybrids using callous tissue cultures.
Webster (1967) carried out crossing
experiments with six different races of Ditylenchus dipsaci Bnd produced
10 fertile hybrids.
He tested the host range of five of these hybrids and
found no relation between the host range of the hybrids and that of the
pa.rents, which contrasted with Eriksson IS (1965) findings.
He also found
th1Jt on the B.verage, the hybrids multiplied more slowly than their parents.
Webster noted further that mixed popUlations must occur in nature, and his
B
results suggested that such mixtures would contain parental races, their
hybrids Bnd backcrosses.
He says that back crossing, together with
infertility or poor reproductive rate, may explain the slight variation in
host range of the known races, and the absence of records of new races.
Webster (1967) sums up by saying,
ttAt one time Ditylenchus dipsaci
populations were probably separated geographically and ecologically on
different Mtura.l hosts and so tend to form biological races that are
Fanning brought together these hosts and their nematode
host specific.
popula.tions and created a dynamic si tua tion with mixed populations of
races, their hybrids and backcrosses, leading to populations with a host
nnge spectrum differing slightly according to their genetical make Up,lI
Smith (1951) recorded the presence of two biological races of
Ditylenchus
that rttacked lucerne.
In spite of nll the investigations thr t have been carried out, it
cannot be
s~id
th!'t the problems of race differentiation and of the
stl'bility of the biological races of Ditylenchus dipsf'ci have been solved.
4.
LIFE HISTORY
Very little work has been carried out on the life history of
Ditylenchus dipsaci in its lucerne host,
Yuskel (1960) however, gives an
excellent description of its life history in onions at 15°C. and a brief
resume of this follows.
The young Ditylenchus dipsaci females usually begin laying between
three and seven dtl.Ys after the final moult, at a rate of eight to ten eggs
per day, giving a range of 200 to 500 eggs per adult female.
substantiated this egg range.
Webster (1967)
Immediately after la.ying, the eggs are
9
greyish in colour, but this colour pales with age to a very light grey hue.
The first larval
~fter
egg.
st~ge
egg deposition.
within the egg is thought to occur 5 - 5.5 days
The larva.e is able to move slightly wi thin the
Before this stage is rea.ched no movement h(ls been observed.
This
full grown first shge larvae moults almost immediately pnd the hB.tching
of the second stl'.ge larvl'e fran the egg takes place seven days after the
b eginnin g of the cycle.
The second moult occurs after another 2 - 2.5 days, the third moult
~fter
3 - 3.5 dBYs, and the fourth after another 4 - 5 days.
Adult
feml'lles and males were present 9 - 11 days from the time of egg hatch.
o
One cycle in onions took between 19 &nd 23 days to complete at 15 C. and
the longevity of both females and males ranged between
L~5
to 73 days.
The sex of the second stage larvae could not be determined in
Yuskel's experiments but the sex of the third and fourth larvae stages
could be ascertained by exa.mina.tion of the genital primordium.
Females
do not oviposit without being mated, nor do they continue to lay eggs
without feeding (Palo 1962 and Webster 1967).
"~.
male may copulate with,
{'nd successfully fertili7e, more than one female.
Fig. 1. shows an outline of the life history of Ditylenchus dipsaci.
The length of life of the stem nematode seems to depend very largely
on environmental conditions.
of moist,
time.
dec~.ying
Hodson (1926) has shown that in the presence
tissue the organism dies within a very short period of
It is possible, however, to keep I'l,dul ts and hrvae alive for
between four a.nd five weeks at a temperature between 2 o C. and 40 C.
Nematodes which leave the host tissue and migrate into the soil are unable
to become quiescent while they remain in a moist environment and, moreover,
they are unable to feed until they locate a suitable host.
The
10
ig. 1
Mature, Male
x
Mature F~male ~
4 days after final
moult
days
\
oviposition
4th larval
stage
3-
3~
5 days
- 5· 5 days
3rd larval
stage
lst larval stage
still in egg
I
2nd larval stage
st ill in egg
hatch about 7 days
after oviposition
An
outli
of
of the
Life
History
11
length of time during which this enforced starvation can be withstood is
not known, but Goodey (1951) considers one year the maximum hostless
period for Di tylenchus dipsaci.
However, in the absence of a moist environment, Ditylenchus dipsaci
in common with mlmy other nematodes, is able to become quiescent and
rempin in this dormrnt condition for long periods.
Goodey (1923) showed
thr!t the pre-f1dult or fourth larval st:,\ge of Ditylenchus dipsaci was the
form which
appe~red
to resist desiccation for the longest periods.
He
found thpt quiescent nematodes from diseased onions and narcissus bulbs
were readily revived in water after periods of desiccation ranging from
24 to 27 months.
The same worker in 1930 showed that the period of
desiccation for the larvae of Ditylenchus dipsaci was six years.
Fielding
(1951) (Van Grundy, 1965) sets the survival of Ditylenchus dipsaci in dried
teasel at 20 - 23 years.
5.
RESISTANCE TESTING TECHNIQUES
Determinations of resistance to Ditylenchus dipsaci may be carried
out in different ways.
The determination may be made by growing the
plants to be investigated for resistance in field trials in naturally
infected fields.
Bingefors (1957) reviews this work and says that some
workers compa.red the area infected with the total aren covered by the
v~riety,
whi1e others looked at the yield differences between infected and
non-infected plots,
(Bingefors in his work with red clover both charted
the arees of infection ~nd also measured yields.)
Efforts to produce more even infestations in field plots by spreading
infected soil or plB.nts over them were made by Frandsen (1951) on red clover
12
fields, and by Burkart
(1937) (Bingefors, 1957) and Smith (1955) with
lucerne.
The first triclls in which plants were grown under relatively
constant glasshouse conditions were those of Rostrup
(1913) (Bingefors,
1957) in which soil from infested fields was used to provide the source
of nematodes.
Goodey (1922) used pots with infected soil and also added
. pieces of infected plants to ensure an overall uniform infection.
He
also used an inoculation technique in which nematode eggs were placed with
a fine capillary glass pipette onto red clover plants that were at the
first true leaf stage.
Seinhorst
(1945) (Bingefors, 1957) described a
method whereby rye seedlings were inoculated with a number of nematodes in
an incision in the coleoptile.
Bingefors
(1957) stntes that his original laboratory technique for
testing red clover for resistance to Ditylenchus dipsaci was to sow the
red clover in boxes of soil infected with Ditylenchus dipsaci, or soil
into which infected plants had been mixed.
However, fungal attack on the
plants Wt:s found to be difficult to overcome, so a modified method was used.
The pltmts to be infected were grown in pots and infection w/"',s brought
I'lbout by pll'cing a folded piece of filter paper, which had been dipped in a
suspension of nematodes in water, between the young shoots.
fl.fter being
inoculated the pots were placed in moist peatmould in a forcing pit covered
by gla.ss windows to produce a high humidity around the plant.
This was
found to be necessary during the first period of inoculation if even results
were to be obtained.
This technique was found to be very wasteful of space,
to take a. long time from inoculation to determination and not to give
reliable results.
13
Bingefors (1957) in reviewing his e~rlier work discusses a seedling
technique in which clover seeds were germinated and then as soon as possible
transferred to filter paper strips.
The seedlings were placed along the
edge of one strip and covered with another.
were then rolled into a compact cylinder.
The two strips and plants
Infection was originally
carried out by dipping these rolls, seedlings downwards, into a suspension
of nemtltodes containing 2000 - 3000 nematodes per ml.
immersed in the suspension for about an hour.
motion by the passage of a weak air current.
The plants remained
The suspension was kept in
Frandsen (1951) modified
this method and by means of a syringe dripped the nematode suspension onto
the embryo plant before the filter papers were rolled together.
adv~ntrge
The
of this method was the direct placement of the nematodes onto the
phnt rnd filter paper, thus providing favourable conditions for nematode
movement ...,nd entry into the plant.
Frandsen carried out tests with
different numbers of nematodes in the inoculum and found that about 30
nem~todes
per drop gave the best results.
Bingefors (1957) states that in his earlier work he found that it was
an .!ldvantage to apply the nematode suspension as a drop between the
cotyledons with a pipette or hypodermic syringe.
The best concentration
here appeJ"red to be about 2,500 - 3,500 nematodes per ml, or about 50
nematodes per drop, which was a suitable number to ensure infection.
Di,jkstra (1956) used this method of inoculation with only 15 - 20 nematodes
per drop.
He added carboxymethyl cellulose to the water to help prevent
aggregation of the nematodes.
After inocula.tion, the plants were placed under cover to keep the
humidity high and inoculation
later.
Wi;S
repeated in all cases one or two days
The inoculated rolls of seedlings were usually kept in pots or
14
o
beakers with wtter in a greenhouse with a temperature in the range of 12 C.
o
(Frandsen, 1951;
- 20 C.
Dijkstra, 1956;
Bingefors, 1957).
The nematodes to be used in the inoculum by these workers were
obtained by collecting infested plant material from the field and extracting
the worms either from the fresh or dried material.
Separation of the
nematodes from the plant matter in these experiments (Frandsen, 1951;
Dijkstra, 1956;
appar~tus,
Bingefors, 1957) was carried out with a modified Seinhorst
Seinhorst, (1950) (Bingefors, 1957) in which diseased plants are
placed in
l~rge
plants.
\~en
funnels and.a fine mist of water is sprayed onto these
the plants are wetted the nematodes leave the tissue and
collect in receptacles at the base of the funnels.
Frandsen scored his
plflnts 10 - 15 days tofter the inocula.tion, on the swellings of the embryo
stem.
The plants were divided into responding (susceptible) and non-
responding (resistant or presumably resistant).
develop norma.lly
w~s
Any seedling that did not
disregarded.
Dijkstra (1956) cautions that in an assessment of resistant and
susceptible plants all sorts of transitional stages can be found, making it
difficult to distinguish susceptible and resistant plants.
For further
breeding work Dijkstra selected plants that were resistant (had no swelling)
from his initial tests.
Bingefors (1957) carried out his initial assessment after two weeks
and the plants were divided into resistant and susceptible categories
according to their symptoms.
Plants without swelling were considered
resistant and plants with swelling were considered susceptible.
He
cautioned tha.t some of the assessments did not give an entirely satisfactory
result, but for direct selection he pointed out that one has to depend on
visual symptoms because tpe plants must be retained alive.
Bingefors (1957)
15
also stressed the importance of reproduction in the susceptible plants,
that is the number of eggs that were produced by the nematodes in the plant.
He recommended standardisation of the type of infected material that was
used to provide the nematodes for inoculation and he used only dried
material.
Any plant that died was thought to be very susceptible because
depths were much hifher in susceptible varieties than resistant ones.
In 10 week old plants Bingefors used the following scale to rate
resist,·"'nce.
a
=
no nematodes
1
very small number nematodes
and no eggs
2
a few nematodes and some eggs
3
many nematodes and eggs or the
plant was killed and possessed
typical symptoms.
Bingefors then calculated an index of resistance from the formula
Index of resista.nce
="
(1 - ~~)
x
100
3n
where
a
is the classification of each individual plant
n
is the number of observed plants
Host of this work was with red clover, but Bingefors did carry out some work
with lucerne (Bingefors, 1961).
Frandsen (1951), Dijkstra (1956) and Bingefors (1957) all used
a.rtificial light in their greenhouses.
Grundbacher (1962) in his work with lucerne slightly rnodified the
filter paper roll technique that had been used by the earlier workers.
He
germinated his seeds in moist vermiculite and then transferred them as
seedlings to the filter
0
p~per
rolls which were placed in r,rowth cabinets at
11.1 C. - 15.6 °C., and at these temperatures he found thrt he could keep
16
them a.li ve "for several months".
The rolls were supplied with either tap
or a dilute HOBglands solution and assessed for resistpnce after a
w~ter
month at 11.1 0 0. or three weeks at 15.6°C.
used
w~s
5,400
The
nem~todes
resist~nt
The strength of the inoculum
per ml and all the seedlings were inoculated twice.
seedlings usually exhibited little swelling, but due
to the presence of plants with intermediate swellings, Grundba.cher maintains
it
w~s
impossible to have an accurate classification on swellings alone.
He found thl1t the most critical evaluation of resistance was the amount of
nematode reproduction taking place in the shoot apex region, even though
reproduction could take place in the hypocotyl, the cotyledons and the
Susceptible plants usua.lly contained adult nematodes, eggs and
petioles.
sorne la.rvae in the shoot apex, while resistant plants had very few eggs
present and the nematodes were smaller.
Grundb~cher
(1962) placed the seedlings in continuous light for an
initial period after inoculation.
He mainta.ined that this prevented the
cotyledori3?losing and facilitated the penetration of the nematodes into the
plant.
Eanna and Hawn (1965) who also used a fil ter paper roll technique,
found that there was no necessity for a. period of continuous light after
inoculation.
Grundbacher (1962) stated that a visual assessment for resistance
could be made by elimintting all plants that had any swelling.
(pers. comm. 1969) believed that a swelling score for
within a
v!'lriety.
v~riety
8
Stanford
series of plants
would give a good evaluation of the resistance of that
He mlso srid that in selection work, the swelling of the plants
wes 8 very satisfRctory basis for division, provided an effective inoculation
had been obtained.
Because of this type of selection the level of
resistance in the laboratory Was found to be lower than the level of
17
resisttmce obtained in thEt field (Bingefors, 1957;
Frandsen, 1951;
Grundbacher, 1962).
Hann~
and Hawn (1965) tested to determine the most suitable level of
inoculum and found that a.dequate infestation was assured with 50 to 70
nematodes per drop.
They finally used 40 to 50 nematodes per drop,
applied on two successive days.
They also found that using carboxymethyl
cellulose as a carrier for nematodes had no significant effect upon the
entry of nematodes into shoot apices.
tJ.
Griffin (1967) found that there was
greater percentage of plants galled when the cotyledons were inoculated
with
8
methyl cellulose solution of nematodes, when the seedlings were
maint:eined at
no% -
100% relative humidity, than when they were maintained
fit 30% - 6f$ relative humidity, and so established the necessity for high
humidity thl'lt was used by earlier workers.
He also found that an inoculum
level of 50 nematodes per drop at the cotyledon stage was sufficient to
give maximum galling.
Wynne and Busbice (1968), who grew the lucerne seedlings for
inoculation in moist vermiculite, used a one to nine scale to rate the
nematode drunage to the plants, two weeks after inoculation.
They then
checked the infections by macerating the apical meristem and counting the
nematodes and eggs present.
They found that they could correlate swelling
with susceptibility, but maintained that reproduction within the plant was a
more direct criterion for susceptibility.
Temperature was found to influence the resistance of one lucerne
va.riety. (Grundbacher and Stanford, 1962;
Griffin, 1968)
Lahontan became
more susceptible as temperature rose, but the resistl',nce of other varieties
remained stable.
Wynne and Busbice (1968) found that 19°C. appeared to be
the most suitable temperature at which to carry out their tests.
l~
It appears that nematodes enter all plants.
In susceptible plants
nerrlatode multiplication is rapid, while in resistant plants there may be
no reproduction at all (Bingefors, 1957;
and Stanford, 1962;
Grundbacher, 1962;
Wynne and Busbice~ 196~).
Grundbacher
19
CHAPTER III
MATERULS AND
1.
~1ETHODS
NEM:ATODE COLLECTION, PRESERVATION AND EXTRACTION
The
s~nptoms
recognis~ble
of Ditylenchus dipsaci attack on lucerne are most
in Canterbury during the spring (September and
l1utumn (Mr rch and April).
circuIt r or
ov~l p~tches
The typical field symptoms
~t
Oct~ber)
and
these times are
where plants ha.ve been killed or badly stunted.
These I'lffected !'IreBS make very little growth, a.re usually very slow in
recovering after cutting, and invariably fail to show any appreciable new
growth in the following spring,
The affeqted plants present a typical
dwarfed "nd distorted appearance.
There is a marked reduction in the
number of stems a,nd these are swollen and generally show a distinct brownish
colouration especially at the
b~se,
the stem for some distance.
Generally, affected stems are very brittle
and easily broken off.
although in many cases it extends up
The new buds arising from the crowns of affected
plnnts are usually swollen, spongy in texture and pale greenish brown in
colour.
In cases of severe infestation, the crowns rot away and the plants
eventua,lly die out, leaving a stand of unprofitable thinness which rapidly
becomes overgrown with weeds and gra,sses.
Infected stands of lucerne on properties at North Loburn and Rakaia
were used ns the sources of
nem~tode
infected plants.
The stunted plants
were hl'rvested either by hrnd or with a small rotary mower.
w~s
then
pl~ced
This material
in cotton bngs and carried back to Lincoln College where it
w~s dried in ovens set at
30 0 C. for 24 hours.
This temperature killed all
20
nematodes, apart from the fourth larval stage.
It was found that this was
more satisfactory than air drying the material, due to a tendency of the
ml'lterial to heat and rot.
bags in
~
This material, once dry, was stored in cotton
dry place until inoculum was required.
The apparptus used to separate the nematodes from the dried lucerne
is illustrated in Figure 2.
proof container.
one and
~
It consisted of a two foot cube metal water-
This container was mounted on legs to produce a fall of
half inches from the rear to the forward edge, and three outlets
were incorporated at six, twelve and eighteen inches along its lower
forward edge.
Brackets were provided in the container to hold a nylon
gauze covered wooden frame.
A spray nozzle TN3V manufacturer's number
(Spraying Systems Co.), with a spray rate of 3.75 gallons per hour at a
wa,ter pressure of 60 lbs. per square inch, was positioned on an adjustable
bracket above the container.
The spray nozzle was at the end of a water
supply line in which the wB,ter flow was regulated by a solonoid controlled
valve activl'l.ted by a time switch which opened the solenoid control valve for
one minute in every four minutes.
to under one gallon per hour.
This diminished the nozzle 1 s rated flow
Three plastic funnels of six inch diffineter
with cll'1mp s8{1led ends were positioned below the outlets, so thpt the run
off wnter could be retained.
As water was allowed to overflow these
funnels the whole ;.1.ppl),rlltus had to be positioned on a freely draining floor.
The infected dried lucerne was spread on the gauze covered frame.
The r,ction of dempening the infected material revives the C"Juiescent fourth
lr:,rval sts.ge nemlltodes, which move with the water through the outlets of
the container into the funnels in which they settle under the influence of
gravity.
The low rate of water flow used was to prevent the possibility
21
Fig.
~valve
water
pply
aid
spay
no
...... tim
swi
s
n to
ho1dplant
material
1---
ppa atu
n
atodes f
colle ti
funnel
n
extroctio
plant m
ri L
22
of the nematodes being carried away with the overflowing water from the
funnels.
After 24 hours, nematodes could be drawn off from the funnels.
WilS
It
found that the best draw off of nematodes was after 36 hours, and that
even after seven days it was still possible to obtain nematodes from the one
sample of lucerne.
Nematodes extracted after 36 hours were usually
sluggish due to the lack of oxygen in the water.
These were revived by
treating with an aerator, which simply bubbled air through the suspension
of nematodes, giving them additional oxygen.
Soil and plant material were also carried into the funnels and
were separ,"ted from the nematodes by filtering through a coarse mesh sieve
1
covered with toilet paper, as shown in Figure 3.
containing the nematodes
sieve, which
WI'tS
extraneous material was poured into the
positioned in a dish in such a manner thnt the water level
reached the toilet
le~ving
~nd
The suspension
This enabled the nematodes to swim through,
p~per.
the debris behind on the upper surface of the pnper.
A second method was to pour the suspension of nemetodes, soil and
plBnt materiAl onto R filter paper in a filter funnel.
the debris were retAined on the filter paper.
The
p~per
The nematodes and
was then laid
over an inverted watch glass within a petri dish, and water was added until
it just lapped the filter paper.
The nematodes would then move down the
filter paper to the water, while the extraneous matter remained held on the
filter paper (Fig.
g~ve s~tisf~ctory
4).
Neither method was found to be superior and both
results.
The nematodes were then allowed to sink to the
bottom of the vessel and excess water was decanted.
1
Jeyes Baby Soft Toilet Paper.
·I .
u p
n
and
f
tOilet
paper
coJlection
dih
wate
-
level
sieve
Apparatu for
po ating
nematode from soil and pi nt
debris.
Fi .
. ilter paper
with nematode
nd rubbish
~-----'watch glass
-petri di h
a
I
v
pparatus for
pa,rati
nematode from 501' and plant
debris
25
The nematodes were stored in water at 2°C. - 4°C. until needed.
The storage time
W8S
normally for one or two days only, but viable nematodes
have been kept as long as four weeks at this temperature.
2.
PLANT PREPARATION
The lucerne seedlings used during this project were grown in rolls
of filter paper or in soil flats.
The seeds of all varieties (lines) used during this work were
surface sterilized before germination.
The seed was soaked in
30%
hydrogen peroxide for one hour and then, without rinsing, were placed on
filter pAper in petri dishes to germinate.
was
th~t
there
w~s
The advantage of this method
no necessity to rinse the seed with water, Bnd so there
wr.s very little chance of contamination after the sterilization.
It was
found thrt this method did not give a complete kill of seed coat carried
p~.thogens,
llnd consequently a second method, which consisted of treating
the seed with a solution composed of equal parts of lN HCL and conunercial
1
.
This was found to be satisfactory, even
bleach for 12 minutes, WI".S used.
though rinsing with distilled water had to be carried out, thus increasing
the
poss~bility
of recontamination.
A test germination to estimate the proportion of diseased seed was
undertaken.
Due to the difficulties experienced in controlling disease
of the seedlings, a.ny seed line with a high level of contamination was
rejected.
The germination of the seed for use in the filter paper rolls was
carried out in covered petri dishes on a double layer of filter paper
1
Commercial bleach used was Janole..
26
soaked with water containing the fungicide captan.
Many more seeds than necessary were germinated so that selection
could be made for seedlings that were at the same stage of development.
This selection was made ffter two to three days and the seedlings chosen
were transferred to filter paper rolls.
Filter paper rolls were made from strips 11.5 cm. x 2g.5 em. of
Whptml?ns chromAtography pp.per No.1 (soft) and No. 50 (hard).
seedlings were plta.ced on
:':>
The lucerne
strip of hard paper, moistened with a captan
solution, so tha.t their cotyledons extended flbove the mf.'rgins of the paper.
The strips were rolled using a gll'l.ss tube for guidance, and the roll was
secured with
8.
rubber band.
Fifteen seedlings were grown per roll.
A
method employing 50 seedlings on strips 11.5 cm. X 57 cm. was found to be
unsa.tisf~.ctory
due to the large number of seedlings requiring attention at
inoculation.
The completed rolls were then placed in plastic pots of 1.5 in. in
diameter and 2 in. in height.
Water was added to these pots to provide
moisture for the first seven days, and then a Hoaglands solution (Hoagland
and Arnon, 1950) was added to provide nutrients for continued growth.
The completed pots were placed in trays in a growth cabinet which
o
provided a 16 hour day at a temperature of 16 C.
The trays were covered
by pla.stic sheets which were supported by a frame manufactured from eight
gruge wire (Plate 1.).
room for the
h~ndling
These frames had to be high enough to allow ample
of the pots.
The trays contained water so that the
seedlings in the rolls were held at a relative humidity approaching 100.
This wrs to f[. cili b.te the entry of the nematodes into the plant after
inocul.t,Jtion.
PLAT E 1
Co ered t r ays use d to hol d t he f i l e r
pa
r rolls in t he gr owth c8bi e t
The seedlings were then left until the development of the first
The inoculations were then carried out.
true
norm~lly
about two dnys after the seedlings had been placed in the rolls.
and the rolls were sprayed with
The
c~ptan
This was
~
small amount of a
solution every day throughout the experiments, to help prevent
fungal attack.
grown in soil were raised in flats 15 in. x 18 in.,
The
which had been surface sterili;-,ed by the
each sown with 100 lucerne
same method
described.
r:s
The seed had then been air dried and
rubbed with captan as an added precaution against fungal attack.
The seeds were planted in 10 rows of 10 each.
obts.ined by
100 lead head
The spacing was
a board the same area as the surface of the flat, with
driven into it.
This could be pressed into the soil
in the flett and the 100 indentations obtained were evenly spaced.
In each of these 10 rows, five of the indentations were sown with
single seeds, and the remaining five were sown with three seeds each, to
r..llow for seedling mortality.
Transplanting took place where necessa.ry as
soon P.s the seedlings had fully developed cotyledons.
Extra seedlings that
were not required for transplanting were retained, because at this stage
there would be no competition between plants.
The fh ts were pIt ced
trays in a glasshouse, the temperature of
which v~ried d~ily between 16°C.
29
o
c.
Artificial lighting in this
gh.sshouse wts controlled by a time switch to operate between seven a.m.
and seven p.m.
Overhead watering of the seedlings was avoided by standing
the flats in metal trays.
requir~d.
This allowed the seedlings to draw water as
29
The seedlings were inoculated
two
d~.ys
lp.ter.
~t
The relative humidity
the first true
~bout
le~f
stage and again
the plants at inoculation was
increased towards the 100% level by covering the flnts and the trays with
trF'.nsparent plastic sheeting supported by frames of eight gauge wire.
The seedlings in the flats were sprayed with a small amount of captan
dll.ily to reduce fungal attack.
3.
PLANT INOCULATION
Clean nematodes that had been stored under refrigeration (pp.25 )
were revived by aerating the water in which they were stored.
cellulose was added to increase the viscosity of the solution.
Methyl
Tbis helped
to retain the nemC'.todes on the seedlings, thus enhancing their infective
~bility.
It also tended to slow the aggregation of the nematodes, a.nd so
the nmnber of neml"todes in the inoculum was more constant from the beginning
to the end of en inoculation series.
Both three and one percent solutions of methyl cellulose were used;
however, it
w~s
found that the one percent solution
w~s
the better, due to the
tendency of the three percent solution to form a hard skin of methyl
cellulose which inhibited the growth of the seedling.
The inoculum was applied to the seedling by the use of a syringe
with an 1$ gauge needle (Plate 2).
One drop of inoculum was added to each
plant, the drop being positioned between the cotyledons at the apical
region.
The number of nematodes in a sample of ten drops of inoculum was
deter'mined by examination under a steroscopic microscope, and the number of
nematodes in the solution was adjusted until each drop contained approximately
50 nemlltodes.
PLATE 2
~yrin~e
used for inoculetion of lucerne seedlings.
31
The inoculation of the seedlings on the rolls was carried out in a
sterile cabinet,
The rolls were then replaced in the humidity chamber in
the growth ca.binet, left for two days, and then reinoculated.
Each
seedling received, therefore, a total inoculation of approximately lob
nematodes over a time period of two days.
The seedlings in soil flats were inoculated in the glasshouse.
The flats were covered with ple..stic sheets as des cribed,
Elnc]
then left
for two drys before reinoculation,
It
W::1S
found that during inoculations it Wp,s necessary to keep the
inocullIffi slightly chilled and also to stir it frequently to stop any
aggregation of the nematodes.
The aerator could not be used for stopping
aggregation because it caused too mAny bubbles in the inoculum solution.
/' fter inocul.:,tion the plants were left for three weeks (Plete 3)
f'.nd their symptoms (swelling in the apicr.l region) were assessed either
visup..lly, or by mensuring the width of the stem between the cotyledons
(Plate 4).
This measurement was carried out with an eyepiece graticule
in a steroscopic microscope.
A sample of seedlings was then macerated
individually and a check was made for the presence of nematodes and eggs.
All the preliminary work in this resear.ch project was carried out
using the lucerne variety Wairau.
This is the most cammon variety of
lucerne grown in New Zealand, but no information is available on its
resistnnce to stem nematode.
In the experiments where the relative resistance of lucerne varieties
to stem nematodes was determined, three lucerne varieties were used.
were Wairau, Grimm and ltlashoe.
These
Grimm is an old variety of lucerne which is
known to be highly susceptible to attack by stem nematode (Bingefors, 1961).
1,vtlshoe, which
WI'S
released by the United States Department of Agriculture in
32
c
c
c
PLAT E 3
Ll.;cerne pl1:>nts in f i t e l' paper rolls thr 'e wf.' eks after inoculation
Ke y:
c
i
control
infected
33
PUITE 4
Nema tode infected and uninfected lu ce r ne plants after three weeks
Top
lin~
~
i-\ot tom line -
Infected
Swollen stem
Uninfected
34
1966 (Hunt, et al, 1966), is an eight clone synthetic variety derived
mainly of Nemstan and known to have a high degree of resistance to stem
nematode.
35
CHAPTER IV
HESULTS
1.
R.ESULTS FR(l\1 SOIL FLt:T TRIALS
Although eventually it wps possible to successfully grow lucerne
seedlings in filter paper rolls, ini tia.lly the extensive losses due to
fungal attack caused some doubt as to whether this method could be used
It was decided, therefore, to set up a trial in
in this study at alL
soil filled flats to find out something about the efficiency of inoculation
techniques, the effect, if any, of using a methyl cellulose solution on the
seedlings, and the accuracy of judging whether or not infection of the
seedlings had taken place.
Twelve flats were sown with T1JEdrau lucerne by the method described,
and grouped
M
follows.
(A replication consisted of 200 plants).
(i)
Flats 1 and 2 (Control) were not inoculated.
(ii)
Flats 3 and 4 (Methyl cellulose) were inoculated with one
percent solution of methyl cellulose alone.
Flf1ts
( iii)
nem~todes
5 to 12 (.rteplications 1 to 4) were inoculated with
in a one percent solution of methyl cellulose.
Three estimates of infection of the seedlings by the nematodes were
m~de;
the first
iElt four weeks.
~fter
two weeks, the second at three weeks, and the third
These
estim~tes
r.pical region of the plant;
infected.
were carried out on the condition of the
plants with swollen stems being regarded as
The results of the test are tabulated in Tables 1, 2 and 3.
About 65 percent of the seedlings became infected, 35 percent remained
uninfected.
Losses from causes other than nematodes approximated those of
seedlings in control flats.
The plants were grown for a further month in the flats after these
tests were finished, and the ability of some of the stunted plants to recover
wp.s noted.
At times
B.
lateral shoot became the ma.in growing stem, leaving
This meant that although the plants harboured a
the apical stem stunted.
popul!!1tion of nematodes, they did not have overa.ll stunting.
TABLE 1
CONDITION OF YOUNG LUCERNE PLANTS (WAIRA U) TWO v..'EEKS AFTER
INFECTION WITH NEMATODES IN 1% SOLUTION OF METHYL CELLULOSE
Infected
PIa n t
Nu mb e r s
Normal
Dled
Total
'-"-,,-~
.....
-
Control
0
255
2
257
Methyl Cellulose
0
255
5
260
Rep. 1
109
184
4
297
Rep. 2
142
125
5
272
Rep. 3
122
165
0
289
Rep. 4
134
127
4
265
37
TABLE 2
CONDITION OF YOUNG LUCERNE PLANTS (WAIRAU) THREE WEEKS AFTER
INFECTION WITH NEMATODES ~l% SOLUTION OF METHYL CELLULOSE.
Infected
Pla.nt
Numbers
Normal
Died
Total
----,
Control
0
253
4
257
Methyl Cellulose
0
252
8
260
Rep. 1
175
113
9
297
Rep. 2
144
112
6
262
• 3
157
131
1
289
Rep. 4
154
105
6
265
TABLE 3
CONDITION OF YOUNG LUCERNE PLANTS (WAIRAU) FOUR WEEKS AFTER
INFECTION WITH NEMATODES IN 1% SOLUTION OF METHYL CELLULOSE
Infected
PIa n t
Nu mb e r 5
Normal
Died
Total
Control
0
249
9
260
Methyl Cellulose
a
251
8
257
• 1
183
103
11
297
Rep. 2
153
110
9
272
• 3
Rep. 4
209
78
2
289
167
92
6
265
I
2.
RESULTS FRCN FILTER PAPER ROLL TRIALS
In the filter paper roll trials a series of experiments, Nos. 1 - 13,
were carried out and these are dealt with in sequence.
Experiment 1
The first experiment using filter paper rolls to hold the seedlings
employed strips of Vlhatma,ns No. 1 and No. 50 chromatography paper, 11. 5 cm.
x 57 em..
Each roll contained 50 seedlings.
of these being kept as a control.
Nine rolls were made, one
The seedlings in the remaining eight
rolls were inoculated twice, each inoculation drop containing approximately
50 nematodes suspended in a 3% solution of methyl cellulose.
These rolls
were then left for three weeks in the growth cabinet At 16°c. and then
e.ssessed on the
b~sis
plrnts were normE'll.
wrter in
t>.
that swollen plants were susceptible and non swollen
To check these results the plents were mAcerated in
wl'tch glass e,nd examined under e steroscopic microscope for
nemttodes and eggs.
The results are given in Table
4.
39
TABLE 4
RESULTS FR(l.I _F~LT]!Jl. PAP]:R ROLLS IN EXPERIMENT 1
No.Plants
showing
nematode
symptoms
No.Plants
containing
nematodes
Normal
Plants
11
a
a
39
16
34
14
14
2
50
16
34
16
16
4
50
5
45
5
50
34
16
31
30
6
50
15
35
15
15
7
50
8
42
8
8
8
50
10
40
10
10
9.
50
31
19
26
26
Initial
tTo. of
Phnts
Survival
after 3
\-reeks
1 ( Control)
50
39
2
50
3
Roll No.
--...............
-.'-,~..,..
No.Plants
killed by
fungal
attack
3
5
-"--....
Of the total of 400 nematode treated plants, 135 survived and 120 of
these plants had symptoms of nematode attack.
(Roll 4 was eliminated).
Of these 120, when macerated in water, 119 had nemAtodes and eggs present.
One plant had nemr-todes but no eggs.
The 10 plants that had no swelling had
no nem1'.todes present when macera,ted.
Experiments 2 - 8
Seven experiments (Nos. 2 - 8) were attempted but all were unsuccessful
because of seedling mortality.
de~ths.
Fungal attack caused the majority of the
Surface sterilization of the seed was carried out (pp.25 ).
The
tempera.ture in the grm-rth cabinet was lowered to 12 0 C to try and help prevent
40
the fungAl
Att~ck,
but without success.
The three msin pa.thogens that
attacked the seedlings were Aspergillus niger, Phoma medicaginis and mildew
(Peronosporl'l trifoliorium).
Wl1S
The seed line of Wairau used in these trials
tested by the Plant DiseElse Division, D.S.LR., Lincoln, and found to
carry
~
very heavy infection of the seed born pa.thogen Phoma medicaginis.
This line was discarded and a clean seed line was obtained for the rest of
the research work.
In experiments 2 - 8 it was realised that 50 plants per roll were too
many, bec8.use the paper si ze used in the rolls (ll. 5 em. x 57 cm.) was too
large to work with efficiently.
In addition to this the 3% solution of
methyl cellulose caused the plants to stick together, and also caused the
binding of the apica.l region, thus inhibiting growth.
The number of
seedlings was subsequently reduced to 15 per roll and a 1% solution of
methyl cellulose
WI',S
used instE?ad of the 3% solution.
Experiment 9·
Forty filter paper rolls were made from papers 11 .. 5 cm. x 22.5 cm.
with 15 seedlings of 1;.!Birau lucerne in each.
(i)
These were grouped as follows:
Rolls 1 - 14 were inoculated with nematodes in a 1% solution
of methyl cellulose.
(ii)
Rolls 15 - 27 were inoculated with 1% methyl cellulose alone.
(iii)
Rolls 28 - 40 were not inoculated.
Rolls 1 - 27 were inoculated by the method described.
All the
seedlings were supplied with Hoaglands nutrient solution after seven days of
being in the rolls, and sprayed with a solution of captan daily.
The
seedlings were then left for three weeks in a growth c~binet at l6°c. and
~ssessment
T~bles
of infection was cBrried out visually.
5, 6 and 7.
The results are given in
41
TABLE 5
RESULTS FROM ROLLS INOCULATED WITH NEMATODES IN 1% METHYL CELLULOSE
_
Roll No.
Initial
Plant
Numbers
Number
Plants
Surviving
,_'~~
~-
____
~
___ ..... _, __ _____
~
Mortality
,~,'._.~
__
~
_ _...
No.Plants
showing
nematode
symptoms
~'
_
_
........
~d_.n.,,'·
Normal
Plants
-~------~,~-,~
1
15
14
1
14
2
15
12
3
12
3
15
15
0
15
4
15
14
1
14
5
15
12
3
12
6
15
14
1
14
7
15
15
0
15
B
15
15
0
15
9
15
13
2
13
10
15
14
1
14
11
15
14
1
14
12
15
15
0
15
13
15
12
3
11
14
15
15
0
15
210
194
16
193
Totals:
___
1
1
42
There was no obvious swelling in any seedlings in the reme-ining
rolls so
only were recorded:
TJ'BLE 6
MORTALITIES IN ROLLS
INOCULA TED 1tJITH
Roll No.
1% METHYL CELLULOSE
Initial
Plant Nos.
Mortality
15
15
0
16
15
1
17
15
3
18
15
4
19
15
2
20
15
1
21
15
0
22
15
3
23
15
1
24
15
1
25
15
2
26
15
2
27
15
0
19.,5
21
Totals:
---~.'"
43
TABLE 7
MORTALITIES IN THE CONTROL ROLLS
Roll No.
Initial
Plant Nos.
JlIortality
--~~~-.
28
15
0
29
15
1
30
15
3
31
15
4
32
15
2
33
15
1
34
15
0
35
15
3
36
15
1
37
15
1
38
15
2
39
15
2
40
15
-.....
0
Totals:
... .......
""~~-,....,-
~
21
195
'------"--.-~-
Experiment 10
Fifteen filter paper rolls with 15 lucerne seedlings in each were
set up
~s
(i)
follows:
Rolls 1 ~ 5 contained seedlings of the lucerne variety Wairau.
The lucerne seedlings in !loll 1 were not
inocul~"ted.
in rolls 2 - 5 were inocuhted with nemA.todes in
I"
The lucerne seedlings
1% solution of methyl
44
(ii) Rolls 6 - 10 conta.ined seedlings of the lucerne variety Washoe.
The lucerne seedlings in Roll 6 were not inoculated.
The lucerne seedlings
in Rolls 7 - 10 were inoculated with nematodes in a 1% solution of methyl
cellulose.
(iii) Rolls 11 - 15 contained seedlings of the lucerne va.riety tahonta.,n.
The lucerne seedlings in Roll 11 were not inoculated.
The lucerne seedlings
in qolls 12 - 15 were inoculated with nematodes in a. 1% solution of methyl
cellulose.
All seedlings were grown for a period of three weeks in a growth
c{l,binet, cmd rlgain Msessment of infection
tiB.S
The results a.re
visual.
given in Table 8.
RESULTS FROM THREE LUCERNE
VJ~,RIETIES
TESTED FOR NEHATODE SUSCEPTIBILITY
v.!oira.u
Roll No.
Initial
Pla,nt' .
Numbers
Number
Plants
Surviving
Mortality
No.Plants
showing
nematode
attack
1 ( Control
15
10
5
0
2
1'5
12
3
11
3
15
13
2
13
4
15
15
0
15
5
15
11
4
11
Totf.11s:
75
61
14
50
continued/ ...
Normal
Plants
45
TABLE g (Continued)
1tJr.shoe
Initiel
Plant
Numbers
Number
Plants
Surviving
Mortality
No.Plants
showing
nematode
a.tta.ck
6 (Control)
15
12
3
0
7
15
13
2
13
g
15
15
0
15
9
15
g
7
g
10
15
9
6
7
2
18
43
2
Roll No.
Norma.l
Plants
-->-,-~----.,
Totals:
57
75
..
---.------,--.---.~~,
Lt1hontAn
11 (Control)
15
10
5
0
12
15
11
4
11
13
15
14
1
13
14
15
9
6
9
15
15
7
8
7
Totl'1ls:
75
51
24
40
-----'''''"',-.---.---~.-...--~-.--~--.
1
1
Experiment 11
Fifty-four filter paper rolls with 15 lucerne seedlings in each were
set up ss follows:
(A
unit was taken as 45 plants and thus made up of
three rolls.)
(i)
Rolls 1 - 18 contained seedlings of the lucerne variety Washoe.
The seedlings in Rolls 1 - 6 (controls) were not inoculated.
in Rolls
The seedlings
7 - 18 (treatments) were inoculated with nematodes in a 1% solution
of methyl cellulose.
(ii)
Rolls 19 - 36 contained seedlings of the lucerne variety Hairau.
The seedlings in Rolls 19 - 24 (controls) were not inoculated.
i~
The seedlings
Rolls 25 - 36 (treatments) were inoculated with nematodes in a 1% solution
of methyl cellulose.
(iii) Rolls 37 - 54 contained seedlings of the lucer~e variety Grimm.
The seedlings in Rolls 37 - 42 (controls) were not inoculated.
The seedlings
in Rolls 43 - 54 (treatments) were inoculated with nematodes in a 1% solution
of methyl cellulose.
All seedlings were then left for three weeks in a grmvth cabinet at
The results of this experiment are given in Table 9.
Assessment was
by mel"!surement of the stem diAmeter and the measurements for the whole sample
pre given in Appendix 1.
47
9
RESULTS FROM THREE LUCERNE VARIETIES
TESTED FOR tJEMATODE SUSCEPrIBILITY
Initial No.
of Plants
Plants
Surviving
Mortality
Mean Stem
Diameter in mm.
Controls
Rolls
1- 3
45
40
5
2.72
Rolls
6
45
35
10
2.83
.9
45
35
10
3998
Rolls 10-12
45
37
8
3.76
Rolls 13-15
45
36
9
3.88
Rolls 16-18
45
29
16
3.79
270
212
58
Treatments
Rolls
Totds:
48
TABLE 9 (Continued)
Initial No.
of Plants
Plants
Surviving
Mortali ty
Mean Stem
Diameter in mm.
A sample of tne measured plants from each of the three varieties was
macerated with water a.nd examined to see "rhether there 1'1-ere nematodes (larvae
08nd 8.clul ts bulked) and eggs present.
in Table 10.
The means of this sample are given
(The plants used are categorized in Appendix
2).
49
TABLE 10
RESULTS FRCM THE MACERATED LUCERNE SEEDLINGS FRON EXPERIHENT 11
Washoe
Variety
Number of plants tested
Wairau
Grimm
73
131
[57
7
70
46
4.96
5.43
6.43
Number of plants with nematodes
only
51
32
12
Averp,ge stem diameter ff plants
with nematodes only in mm.)
3.92
4.03
5.00
15
29
24
2.84
3.22
3.36
Number of plants with
nematodes and eggs
Avera~e
wit
stem diameter of ptants )
nematodes and eggs in mm.
Plants with no Attack at all
Aver(j~e stem diflm{ter of llants
wit no /:l,ttack in mm.
If the ability of the nematodes to reproduce within a plant is a
sign that the plant is susceptible to attack, then the percentage of plants
conta.ining reproducing nematodes should give an indication of the
susceptibility of the variety.
These percentages are as follows:
Washoe
9.6%
Wairau
53.4%
Grimm
52.9%
50
Experiment 12
Thirty-six filter paper rolls with 15 lucerne seedlings in each were
set up as follows:
(A unit was taken as 45 plants and thus made up of
three rolls.)
(i)
Holls 1 - 12 contained seedlings of the lucerne variety l.vashoe.
The seedlings in Rolls 1 - 6 (controls) were not inoculated.
The seedlings
in Holls 7 - 12 (trea,tments) were inoculated with nemAtodes in a 1% solution
of methyl cellulose.
(ii)
Rolls 13 - 24 contained seedlings of the lucerne variety Wairau.
The seedlings in Rolls 13 - 19 (controls) were not inoculated.
The seedlings
in Rolls 19 - 24 (trea.tments) were inoculCi.ted with nematodes in a 1% solution
of methyl cellulose.
(iii) Rolls 25 - 36 contained seedlings of the lucerne variety Grimm.
The seedlines in Rolls 25 - 30 (controls) were not inoculr:ted.
The seedlings
in ~olls 31 - 36 (tre8tments) were inocuhted with nematodes in a 1% solution
of methyl cellulose.
All seedlings in this experiJnent were grown for a period of three
\<Teeks in a growth cabinet at 16°C.
given in
the
T~ble
11.
me~surements
The results of this experiment are
Assessment was by measurement of the stem diameters and
for the Whole sample are given in Appendix 1.
51
TABLE 11
RESULTS FROM THREE LUCERNE VARIETIES
TESTED FOR NEMATODE SUSCEPTIBILITY
Initial No.
of Plants
Plants
Surviving
Mortality
Mean Stem
Diameter in mm.
Washoe
Controls
Rolls
1- 3
45
42
3
2.75
Rolls
4- 6
45
41
4
2.88
7- 9
45
40
5
Rolls 10-12
45
39
6
Treatments
Rolls
"--"-~-'---'~'-~-
180
162
18
Rolls 13-15
45
40
5
2.83
Rolls 16-18
45
43
2
2.89
Rolls 19-21
45
40
5
5.74
Rolls 22-24
45
41
4
180
164
16
Totals:
iiairau
Controls
Treatments
Totals:
continued/ ...
52
'l'ABLE
11 (Continued)
Initial No.
of Plants
Plants
Surviving
Mortality
Mean Stem
Diameter in mm.
,---_-.--'----""
Grimm
Controls
Rolls
25-27
45
40
5
2.90
Rolls
28-30
45
38
7
2.99
Rolls
31-33
45
41
4
5.96
Rolls
34-36
45
39
6
5.89
180
158
22
Treatments
Totals:
A sample of the measured plants from each of the three varieties
wes macerated lrith water and examined to see whether there were nematodes
(le.Tvae and adults bulked) and eggs present.
given in Table
12.
The means of this sample are
53
TABLE 12
RESULTS FRCM THE MACERATED LUCERNE SEEDLINGS FRCM EXPERIMENT 12
Washoe
Variety
Wairau
Grimm
Nwnber of plants tested
77
80
81
NUmber of plants with nematodes
Il. nd eggs
32
47
52
5.81
6.52
6.79
Number of plants with nematodes
only
29
24
21
f+Inplanys
nun.
4.25
4.82
4.74
16
9
8
3.19
3.09
3.38
Aver~~e
wit
Avera~e
wit
stem diameter of Prants )
and eggs in mm.
nem~todes
stem diameter
nematodes only
Plants with no attack at all
Average stem diameter of plants
with no attack (in mm.)
,
--..
---,~----."----
If the ability of the nematodes to reproduce within a plant is a
sign that the plant is susceptible to attack, then the percentage of plants
containing reproducing nematodes should give an indication of the
susceptibili ty of the variety.
These percentages are as follows:
Washoe
41.6%
Wairau
58.8%
Grirrrrn
64.2%
54
Experiment lJ.
Fifty-four filter paper rolls with 15 lucerne seedlings in each were
set up
(ft, unit
follows:
~s
Wl'lS
taken as L,5 pla.nts lind thus made up of
three rolls.)
(i)
Rolls 1 - 18 contained seedlings of the lucerne variety Washoe.
The seedlings in Rolls 1 - 6 (controls) were not inoculated.
in Rolls
7-
The seedlings
18 (treatments) were inoculated with nematodes in a 1% solution
of methyl cellulose.
(ii)
Rolls 19 - 36 contained seedlings of the lucerne variety Hairau.
The seedlings in Rolls 19 - 24 (control~ were not inoculated.
The seedlings
in Rolls 25 - 36 (treatments) were inoculated with nematodes in a 1% solution
of methyl cellulose.
(iii) Rolls 37 - 54 contained seedlings of the lucerne variety Grinm.
The seedlings in Rolls 37 - 42 (controls) were not inocula.ted.
The seedlings
in Rolls 43 - 54 (treatments) were inoculated with nematodes in a 1% solution
of methyl cellulose.
'.11 seedlings in this experiment were grown for three weeks in a
growth cAbinet at 16
13.
t"ssessment
W/1S
o
c.
The results of this experiment Are given in Table
by measurement of the stem dil'lmeters, pnd the
mepsurements for the whole sample f1.re given in
.~ppendix
1.
55
TABLE 13
RESULTS FROM THREE LUCERNE VARIETIES
TESTED FOR NEMATODE SUSCEPTIBILITY
Initial No.
of Plants
Plants
Surviving
Mortality
Mea.n Stem
Diameter in nun.
~veshoe
Controls
Rolls
1- 3
45
39
6
2.75
Rolls
4- 6
45
39
6
2.78
7- 9
45
38
7
4.34
Rolls 10-12
45
36
9
4.38
Rolls 13-15
45
37
8
4.25
37
8
4.32
226
44
Treatments
Rolls
Rolls 16-18
~5
-",,-
Totals:
"'"-
-
-
~~
-' -"'-
~,~
,
270
,-",,,,,,,,,,-.~.----"-'-----'"""
'.
Wairau
Controls
Rolls 19-21
45
41
4
2.78
Rolls 22-24
45
43
2
2.69
Rolls 25-27
45
35
10
4.92
Rolls 28-30
45
44
1
4.82
Rolls 31-33
45
40
5
Rolls 34-36
45
38
7
Treatments
rn_.L_'
~,
5.05
4.95
TABLE 13 (Continued)
Initial No.
of Plants
Plants
surviving
Jl.iortali ty
Mean Stem
Diameter in rum.
Rolls 37-39
45
40
5
2.91
Rolls 40-42
45
40
5
2.94
39
6
5.41
7
5.27
Grimm
Controls
TreAtments
Rolls 43-45
Rolls 46-48
45
Rolls 49-51
45
38
7
5.37
Rolls 52-54
45
35
10
5.45
270
230
40
Tota.ls:
A sample of the measured plants from each of the three varieties wa.s
macerated with water aNti examined to see whether there were nematodes
(larvae and adults bulked) and eggs present.
nre given in Table 14.
The means of this sample
57
TABLE 14
RESULTS FRCM THE MACERATED LUCERl\lE SEEDLINGS FRCM EXPERIMENT 13
\lJ"ashoe
"
~
_
Wairau
~
Variety
Grimm
_'-0."_-",,", '--" ___ , ___ , _ _ ._.
Number of plants tested
72
78
76
Number of pla.nts with nematodes
and eggs
28
45
47
5.23
5.84
5.94
Number of plants with nematodes
only
26
19
20
Average stem di3meter 9[ plants
witfi nem~todes only ~ln mm.)
4.19
3.95
4.70
17
15
9
3.18
2.97
3.36
Average stem di~eter ofcPlants)
,dth nemntodes and eggs In mrn.
Plants with no attack at a.ll
Averr,ge stem diameter of pl~nts
with no atta.ck (in mm.)
-------------_._--_._---------"_. --_.
If the ability of the nematodes to reproduce within a plant is a
sign that the plant is susceptible to attack, then the percentage of
plants containing reproducing nematodes should give an indication of the
susceptibility of the variety.
These percentages are as follows:
Washoe
38.9%
Wairau
57.6%
Grirrun
61.8%
The mea.ns of the stem diameters for all seedlings in Experiments 11,
12 and 13 are given in Table 15.
TABLE 15
HEAN STEM DIAHETERS IN rom. OF 45 SEEDLINGS
OF THREE VARIETIES OF LUCEfnJE IN THI1.EE EXPERIHENTS
Treatment
\iashoe
1vairau
Controls
2.72
2.96
2.98
Inoe.
3.98
4.53
5.21.
3.76
4.58
5.40
3.88
4.57
4.97
& Variety
Experiment 11
3 .79__________,~~rr_
5.38
2.75
2.83
2.90
2.88
2.82
2.99
Inoe.
4.67
5.74
5 ·96
Controls
2.75
2.78
2.91
2.78
2.69
2.94
4.34
4.92
5·41
4.38
4.82
5.27
4.25
5.05
5.37
4.32
4.95
5.45
"
ExEeriment 12
Experiment 13
Grimm
...
.-., ,.
.. '-
Controls
Inoe.
._--".00-- -
~-.-.----
.
59
The da,ta. in Table
Was
transformed to log 10 of the mean
diAmeter as shm.ffi in Table 16.
TABLE
TRANSFOnMED DATA FRCl1 TABLE 15 - (LOG 10
Treatment
& Variety
\'Jashoe
DIA~fErER)
OF MEAN
Weir-au
Controls
Grimm
.474
e481
Inoculations
Total:
l£f.eeriment 12
Controls
.600
.656
.717
.575
.661
.737
.589
.660
.696
.579
.678
.731
3.230
3.607
10.673
.439
.459
Inoculations
Total:
continued/. , •
.669
.759
.770
.661
.742
.768
2.228
7.119
60
TABLE 16 (Continued)
Treatment
Washoe
Hairau
Controls
.439
.444
.464
.444
.430
.46fJ
. fJ74
.932
& Variety
Experiment 13
Grimm
Subtotal
• fJfJ,2
Inoculations
.638
.692
.733
.642
.683
.722
.626
.703
.730
.636
.695
.736
Subtotal
2,244
____ 2·773
2.921
__§_~.?38
Total:
3.427
3.647
3.853
10.927
Grand Total:
fJ.fJ85
9.669
10.165
_~
_2.6 89
28.719
-~~-,-
,.,..,
.---
An analysis of variance follbwing Sokal and Eohlf (1969) was then
cll.rried out and this is shown in Table 17.
TABLE 17
ANALYSIS OF VARIANCE OF RESULTS IN TABLE 16
df
Source
.-~~
Among experiments
Among varieties
Between treatments
Error
Total
..
---~
..... -"
ss
ms
.002202
,,052064
.57.952
.040054
.666272
.001101
.026032
.571952
.000954
F
2
2
1
42
47
1.75 N.S.
27.3'" * *
599.3***
,
.......
,-..-----".....
---~.
61
A further analysis of variance (Sakal and Rohlf, 1969) was carried
out on the mean stem diameters of the plants in Experiments 11, 12 and 13
that contained nema.todes and eggs.
listed in Tables 10, 12 and 14.
included in Appendix 2.
The numbers of plants infected are
The stem diameters of these plants are
The analysis of variance of these is shown in
Ta.ble 18.
TABLE 18
ANALYSIS OF VARIANCE OF PLANTS CONTAINING NEMATODES AND EGGS
...- - - - -
------~--------------------
ss
df
Source
Among varieties
Error
Total
ms
22.25
1.01
44.49
375.92
420.41
2
371
373
F
22.03***
The percentage of plants that had reproducing nema.todes in them when
they were macerated in Experiments 11, 12 and 13 is shown in Table 19.
TABLE 19
PERCENTAGE OF PLANTS CONTAINING REPRODUCING NEMATODES
--------------------
--------~------,-.-------
Experiment
Experiment
Experiment
11
12
13
111)"~shoe
9.6
41.6
3EL9
lv~ira.u
53.4
5f3.8
57.6
Grimm
52.9
64.2
61.8
Lucerne Variety
-.. ..,.."'....
~----.-~.-~"--
62
In Expel"iment 13 a subsample
b~
was
taken and a score on the follol-ling
s wrs given to the number of nem~todes.
v~rieties,
the
(1957) ,
of
This was. with 30 plants of
Hr-shoe, i-lrirau and Grimm.
The
syst~m
used was that
(pp.
1
= No nematodes or
= Nematodes but no
2
=
A few nematodes and
3
~
Many nematodes and
o
eggs
An index of resistance can be calculated fram the following
Index of resistance
where
a
the classification of each
tl.ud
n
the number of observed plants,
=
X 100
indi\~dual
Using the results tabulated in Table 20, the following indexes were
obhined:
ivpshoe
48
Hdrau
28
Grimm
26
63
TABLE 20
CQ~PARISON
OF STEM DIAMETER OF SEEDLINGS WITH RATINGS OF NEMATODE NUMBERS
AND REPRODUCTION WITHIN THE PLANTS FOR 30 PLANTS FRGf EXPERIMENT 13
, ~---,----.----
Ylashoe
Diameter
of plant
in mm.
Rating
--">--~".-"
,
Grinun
Diameter Rating
of plant
in mm.
Diameter
of plant
in mm.
--.----.-~-"-~---
1
2
3
4
5
6
7
8
9
10
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
2.8
29
30
4.2
5.0
3.3
5.2
4.2
3.6
5.2
5.2
3.8
2.3
3.6
4.2
7.3
5.8
5.2
3.6
6.2
5.8
5.0
5.2
5.6
2.7
3.1
4.0
3.2
6.4
5.1
3.8
3.6
3.6
2
3
2
2
1
1
3
2
1
0
0
2
3
2
2
2
3
3
1
3
1
0
0
1
0
2
I
1
0
0
~.8
.4
5.4
5.3
6.0
4.5
4.8
5.8
3.8
5.0
4.0
4.0
4.8
3.0
5.8
3.3
6.4
7.0
6.0
7.0
6.3
5.5
7.8
2.8
7.8
7.2
6.8
3.5
3.8
3.1
0
3
3
3
3
3
2
3
2
3
3
0
1
1
3
1
3
3
3
3
3
2
3
1
3
3
3
0
1
0
.......,----~,-~-"-"'
7.0
6.8
6.3
3.5
3.3
6.8
6.5
4.6
4.3
6·3
7.0
6.7
7.2
6.8
5.5
7.0
4.5
4.0
4.2
4.0
6.3
4.0
4.0
5.4
5.8
5.2
5.0
6.8
6.3
4.6
3
3
3
2
0
3
3
0
1
3,.,
;)
3
2
2
1
3
3
2
1
1
3
3
1
3
2
3
1
3
3
3
CHAPTER
V
DISCUSSION MJD CONCLUSION
Initially in this research project, time was devoted to perfecting a
technirue for growing lucerne seedlings under conditions in which they could
be tested for resistence to the stem nematode Ditylenchus dipsaci.
ways of growing seedlings were tried.
These were, growing the seedlings
in filter paper rolls, a technique similar to that used by
(Bingefors, 1957;
Dijkstra, 1956;
Two
Frandsen, 1951;
e~rlier
workers
Grundbacher, 1962;
Hannl'l I".nd Hawn, 1965), or, growing the seedlings in soil fll'lts.
lvork was
origintllly concentrr>ted on the soil flats beccuse of diser>se problems that
occurred in initif)l l'1ttempts to grow the seedlinr;s in filter pflper rolls.
Eventul'llly these disease problems were eliminated and the filter paper roll
techninu8 was used in preference to the soil flat technique for the following
reasons.
The filter paper rolls were more convenient to handle and work with
tha.n the soil flats;
many more plants could be dealt with;
the environment
could be controlled more accurately by the use of growth cabinets instead of
having the plants in
9.
gl,sshouse as was necessary with the soil flats;
the time period for ea.ch experiment was shortened.
Assessment of the plants
in soil flats was ca,rried out on a visual basis, swollen plants being
classified es susceptible.
The results of these assessments, which were
c1:'l.rried out hTO, three and four weeks after inoculation, are given in Tables
1, 2
~nd
3. There W;,1s no uniformity of infection obtained between replications
1, 2, 3 tlnd L, in this experiment, but i t is possible to say that about 65%
of the BOO seedlings inoculated became infected.
The fact that uniformity
65
of infection between replications in this experiment could not be
obt1.1ined WI'lS ..mother important reason for the rejection of the soil flat
technirme by the author.
The plants that were inoculated with a 1%
solution of methyl cellulose only in the soil flat trials showed no swelling,
so that it is possible to say that it was definitely the nematodes that caused
the swelling in the plants inoculated with nematodes in a 1% solution of
methyl cellulose.
The results for the work using the filter paper roll technique are
listed in Chapter IV in a sequence of experiments from 1 - 13.
findings for Experiment 1 are given in Table
The
4 and it can be seen that the
mortality of seedlings due to fungal attack was so high that no worthwhile
results on resistance of the lucerne could be obtained.
Experiments 2 - 8
were 13.11 failures due to fungal attack, which was found to have originated
from a heavily infected seed line.
9,
This line was rejected before Experiment
However, during these experiments many useful points of technique
evolved.
It wllS found thpt 50 plants per roll were too many because the
pr>per si""e used in the rolls (11.5 cm. x 57 cm.) w~s too l.<irge to work with
efficiently.
Ps well IlS this the
3%
solution of methyl cellulose caused
the plr.nts to stick together and also caused the binding of the apical
regions, thus inhibiting growth.
The paper size for the rolls was
subsequently reduced to 11.5 cm. x 22.5 cm., the number of seedlings per
roll reduced to 15 and a 1% solution of methyl cellulose was used instead
of the
3%
solution.
Throughout all these experiments an average of 50 nematodes per drop
of inoculum w[-ls used and each plant was inoculated twice.
In Experiment 9, 15 seedlings were used in rolls made from filter
p~)peY's
of the new dimensions.
The plants were inoculated and grown
66
successfully for three weeks.
The symptoms were visua.lly assessed and
the results are given in Tables 5, 6 and 7.
Fungal disease was not a
problem in this experiment because the seed line had been changed and an
efficient method for the surface sterilization of the seed was being used.
In this experiment there was no swelling in the plants that were inoculated
with a methyl cellulose solution only, so again it can be said that swelling
in the plants in the filter paper rolls was due to the nematodes.
This
experiment brought to an end the phase of work on the technique of growing
seedlings in filter paper rolls.
c~rried
Up to this stage all the work had been
out with V·TF.'irau lucerne,
In Experiment 10 three varieties of lucerne were grown in filter
ptper rolls.
These were Hairau, the relative resistance of which is
unknown, Lahontr::n fwd \',T:l.shoe, which I'tre varieties that have been bred in
North lImericA for resistnnce to stem nematode attack.
ViSUAl Rssessment are given in Table 8.
The results of a
This experiment showed that it
w8s quite possible to grow lucerne varieties other than Wairau in filter
pAper rolls.
Experiments 11, 12 and 13 were designed to compare three different
varieties of lucerne, H8shoe, Vlairau and Grimm, for their resistance to
stem nematode.
vlashoe has been bred for resistance to nematode attack,
\'[airau he s an unknown relative resistance, while Grimm is known to be
susceptible.
In these three experiments the system of rating the plants
on visual symptoms was improved upon by using an eyepiece graticule in a
steroscopic microscope to measure the stem diameters.
The plant
mortalities and the average stem diameters are given in Tables 9, 11 and
13 for Experiments 11, 12 and 13 respectively.
A sample of plants of each
VAriety from every experiment was macerated with w.?ter, eXI3.mined under a
67
steroscopic microscope Bnd split into three categories as follows:
those
,rl th nematodes and eggs present, those with nematodes only present and those
with neither nematodes nor eggs present.
Tables 10, 12 and 14 express the
proportions of the samples in each category and also give the means of the
sample.
Table 15 gives the mean stem diameters in mm. of 45 seedlings of
three varieties of lucerne in three experiments, and Table 16 shows these
figures transformed to log
10
An ana.lysis of variance of the transformed means of the stem
diameters (Table 17) showed that there were highly significant differences
between the varieties in the degree of swelling due to nematode attack.
Grimm showed the greatest swelling, Washoe the least, while Wairau was
intermediate.
There were also highly significant differences between
control and treated plants which was expected.
This analysis showed no
significant differences between the experiments.
An analysis of variance was carried out on the diameters of those
plants in Experiments 11, 12 and 13 that did have nematode reproduction
occurring.
varieties,
Again, highly significant differences were obtained between
1'l.8
shown in Table 18.
Table 19 is a summary table of the percentages of plants in which
nematode reproduction occurred in the three Experiments 11, 12 and 13.
From these figures it can be seen that the percentages of the plants tested
thl'lt
h~\d
reproducing nematodes were very similar for Wairau and Grimm, but
were much lower for Hashoe.
The fact that the percentages of plants
infected in Experiment 11 were generally lower, with Washoe being much
lower, can be explained by the fact that this was the first experiment
where testing for reproduction was carried out and the technique of scanning
68
for eggs and nematodes had not been perfected.
r~tings
Table 20 shows that the
given to V.Jashoe seedlings are, in general, lower than the ratings
gi ven to 1rJa i ra.u ;}nd Grimm seedlings.
This also helps explain why the
percentflges of Nnshoe seedlings with nematode reproduction are so much
lower in Experiment 11.
The method of obtaining a resistance rating for a variety of red
clover, as used by Bingefors (1957), was used on a sample of 30 plants of
each
v~riety
in Experiment 130
The ratings obtained placed Wairau and
Grimm very close in their susceptibility, while Washoe was much more
resistant.
This rating agrees with the figures for the percentage of
plants in each variety in which nematode reproduction occurred in
Experiments 12 and 13, where Washoe was found to be more resistant than
Grimm and lHairau, which were close together.
From these figures it ca.n be seen that swelling alone cannot be used
to give? the relative resistance of different lucerne varieties, but from
the results shown in Tables 10, 12 and lh it can be seen that selections
could be made within a variety on the basis of swelling.
The differences
hebleen the a.verl'lge stem diameters of the control seedlings and those that
were recorded ns having no nematodes or eggs when they were tested, could
be due to the fact that nematodes may have entered the seedlings and though
they rlid not survive, nevertheless caused some slight swelling,
Compl'lring resistance of lucerne vaTieties to Di tylenchus dipsaci
attnck would be best
in eDch
v~Tiety
c~rried
out on the basis of the percentage of plants
that contain reproducing nematodes.
must be classified as susceptible.
On this basis liIairau
CHAPTER VI
SUMMARY
The results of testing lucerne for resistance to the stem nematode
Ditylenchus
dipsaci are recorded.
~
perfecting
nematodes.
B
A major part of the study was given to
technique for growing lucerne seedlings for inoculation with
Two techniques were tried and filter paper rolls were found
to be superior to soil flats.
The technique of groldng seedlings in filter paper rolls and
inoculating them with nematodes was used to produce a rating of the
resistance for the New Zealand variety of lucerne, t'Tairau, when compared
with a known resistant variety, 1iTashoe and a susceptible variety, Grimm,
From the results obtained \':airB.u must be classified as a susceptible
va.riety.
It A.pper:rs that a selection wi thin a variety of lucerne for
resistance to Ditylenchus dipsB.ci could be made by elimina.ting the swollen
plBnts.
However, to obtain the relative resistances of several varieties,
it would be necessary to obtain a measure of the reproductive rates of
the nematodes in the plants.
69
70
ACKNOWLEDGEMENTS
I wish to thank Dr. C.P. Hoyt for his supervision
and his advice on the preparation of this manuscript.
Professor
this project
I
o wish to thank
R.A. Harrison for his valuable assistance during
The co-opera.tion of
~Tr.
work.
A. 1<vallB ce during the sta.tistical
is gr:=tefully acknmvledged and appreciation is expressed to Hr. J.
for his help \vi th the photography,
ThAnks are due to r.1r. Rex Copland a.nd Mr. A. T. Hetherell for
I>.llowing me to collect nemAtode infected lucerne from their
Finp.nciPI e ssist~:;nce from the N.?:. Wool Board "md the D. S, 1. R,
grrtefully
~.cknowledged.
FinAlly I wish to thlmk Mr, D, rves for proof rel'lding and Mrs. V.
Judd for her very
cnp~ble
of the manuscript.
71
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Phytopathology 49: 664-670.
BARKER, K.R. and
nematode Ditylenchus
BINGEFORS, S.
1957.
to stem nemBtodes.
Studies on breeding red clover for resistance
Vaxtodling 8: 123p.
BINGEFORS, S.
1961.
Stem nematode in lucerne in Sweden.
in lucerne aga.inst stem nematode.
f(. LantbrHogsk. Annll'.
BR Ci'lfN , E .B.
1953.
Stem eelworm in lucerne.
Pl. Path.
II
27:
2: 54.
CHRISTIE, J.R.
1959.
Plant nematodes: their bionomics and control.
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CU.HK, H.C.
Zeahnd.
1963.
A review of ph.nt oerasitic nemA.todes in :;ew
Proc. 11th. N.Z. Weed and Pest Control Conf. p91-95.
COBB, N.A.
1928-29.
The stem nema ~enchus d~psp.ci its movement
in commerce ~nd its reaction to hydrocy~nlc BCl g~87
J. Parasit.
15:
DIJK,STRA, J.
1956.
Experiences with the breeding of red clover
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DINGLEYL J .~L. 1969.
N.?.. u.S.LR. Bull.
Records of plant diseases in New Zealand.
192: 297p.
review
EDVURDS t E.T.
1932.
Stem
diseBse of lucerne,
of lit.erature concerning the
organism Tylenchus
(Kuhn)
Bast.
Agric. Gaz. N.S.W. 43: 305-314, 3/+5-356.
~~~
ERIKSSON J K.B.
1965.
Crossing experiments with races of Di~lenchus
dipsacl on callus tissue cultures.
Nematologies 11: 244-24 .
FRANDSEN~
K.J,) 1951.
Studies on the clover stem nematode (Tylenchus
dipsacl Kuhn.
Acta Agric. Scand. 1: 203-270.
GODFREY, G.H.
1922.
The stem and bulb infesting nematode in America.
Phytopathology
12: 52-53.
GOCDEY 1 T.
to stem-d
Fuhn.
J.
GOODEY, T.
reference
1: 47-52.
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c.':.used by the eelworm T,;y:1enchus
syn. devastatrix
. ScL Carob.
12: 20-30.
~===;;:;.
scence Elnd revivisence in nematodes with special
tri ~ and T;y:lenchus ~~".
J. Helminth.
72
GOODEY, T.
1929.
The stem eelworm Tylenchus dips~ci; observations
on its attack on pota.toes and marigolds, with a host list of plants
parasitized by it.
J. Helminth.
7: 183-200.
(lOODEY, T.
Biological races in nematodes and their significance
1931.
in evolution.
Ann. Appl. Diol.
18: 414-419
GOODEY, T.
1951.
Soil and freshwater nematodes.
London, Methuen.
390p.
GOODEY, T.
1956.
The nematode parasites of plants catalogued under their
hosts; revised by J.B. Goodey and M.T. Franklin.
Farnham Royal Commonw.
Agric. Bur.
14Op.
GOODEY, T.
1963.
by J.B. Goodey.
Soil and Freshwater nematodes.
London, Methuen. 544p.
2d ed. rewritten
GOODEY, J.B; FRANKLIN, M.T. and HOOPER, D.J.
1959.
Supplement to the
nematode parasites of plants catalogued under their hosts.
1955-1958.
Fnrnham Royal Commonw. Agric. Bur.
66p.
GRIFFIN, G.D.
1967.
Evaluation of several techniques for screening
alfalfa for resistance to Ditylenchus dipsaci.
Pl. Dis. Reptr.
51: 651-654.
GRIFFIN, G~D.
1968.
The pathogenicity of Ditylenchus dipsa<?i. to alfalfa
and the rellltionship of temperature to plant infection and susceptibility.
Phytop~.thology
58: 929-932.
GRUNDBACHER, F.J.
1962.
Testing alfalfa seedlings for resistance to
the stem nem~tode Ditylenchus dipsaci (Kuhn) Filipjev.
Proc. Helminth.
Soc. Wrsh.
29: 152-158.
GRUNDBA.CHER, F. J., and STANFORD, E.H.
1962.
Effects of temperature on
resistance of Alfalfa to the stem nematode Ditylenchus dipsaci.
Phytopathology
52: 791-794·
HANNP, H.R., and HA V'TN , E.J.
the alfalfa stem nematode.
1965.
Seedling inoculation studies with
Can. J. Pl. Sci.
45: 357-363.
HODSON, W.E.H.
1926.
Observations on the biology of Tylenchus dipsaci
(Kuhn) Bastian and on the occurrence of biologic strains of the nematode.
Ann. Appl. Biol.
13: 219-228.
HOAGLAND, D.R., and ARNON, D.l.
growing plants without soil.
1938.
The water culture method for
Circ. Calif. Agric. Exp. Stn.
347: 39p.
HUNT, C.J; PEA DONS, R.M; CARNAHAN, H.L; and LIEBEID1AN, F.V.
Registration of Washoe alfalfa.
Crop Science
6: 610.
KIRK, T.W.
1903.
Potato gall-worm.
1966.
Rep. Dep. Agric. N.Z.
KIRK, T.lv.
1907.
Eelworms - (Tylenchus devastatrix).
N,Z.
15: 175-176.
11: 436-437.
Rep. Dep. Agric.
73
KIRK, T .l~! .
MCKAY, M.B.
Oregon.
Eelwonns.
1909.
Rep. Dep. Agric. N.Z.
16: 123-126.
1922.
Occurrence
Phytopathology
12:
MORRISON, L.
1957.
Agropyron sCAbrum.
NOBLE, R.J.
36: g27·
1925·
New records of eelwonn infestations of lucerne and
A Rev. Canterbury Agric. ColI. N.Z.
p62-63.
A
diseRse affecting lucerne.
PALO, A•V • 1962.
Translocation and devalopment of stem eelworm
Ditylenchus dipS8Ci (Kuhn) in lucerne Medic~ sativa.
Nematologica
7: 122-132.
SEINHORST, J .lV.
1957.
Some aspects of the biology and ecology of stem
nematode.
Nema.tologica (Suppl.)
2: 355-361 S.
SMITH, C.F.
1951.
Biological races of Ditylenchus dipsaci on alfalfa.
Phytopathology
41: 199-190.
~UTH,
C.F.
1955.
And stem nematode.
Breeding alfalfa for resistance to bacterial wilt
Nevada
. Expt. St. Bull.
198: 15p.
SOKAL, R.R. and ROHLF, F.J.
1969.
Biometry:
practice of statistics in biological
The principles Bnd
Freeman 776p.
STEINER, G.
1925.
The problem of host selection and host specialization
of certain plant infesting nemas and its application in the study of
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Ph)~opathology
15: 499-534.
STURHAN, D.
1964.
Kreuzungsversuche mit biologischen RAB
Stengelalchens (Ditylenchus dipsaci).
Nematologica
10:
THORtm, G.
1945.
Di
nchus destructor n. sp.
The potato rot nematode
pnd Ditllenchu5 dipsAcl ,u n, 1857) Fi1ipjev 1936; the teazel nematode
(Nemr-toda: Tylenchidae).
Proc. Helminth. Soc. \~rash.
12: 27-34.
THQRNE,. G.
(NemFltod~,
1949.
On the classification of the T*lenchida new order
Phasmidia).
Proc. Helminth. Soc. Was.
16: 37-73.
VAN GRUNDY, S.D.
Phytopeth.
l\iEBSTER, J.M.
nem~todes.
~JEBSTER,
3:
1965.
43-bg.
Factors in survival of nematodes.
Ann. Rev.
1964.
Biological races in species of plant parasitic
PArasitology
54: gpo
J.M.
1967.
The significance of biological races of Dity1enchus
dipsaci and their hybrids.
Ann. Appl. BioI.
59: 77-g3.
74
t'TYNNE, J.C. and BUSBICE, T.H.
1968,
Effects of temperature and
incubetion period on the expression of resistance to stem nematode.
Crop Science
8: 179-183.
1960.
Observations on the life cycle of Ditylenchus
dipsaci on onion seedlings,
Nematologica
5: 289-296.
YUSKEL, H.S.
75
APPENDIX
STEM
P1a.nt
No.
2
3
4
5
6
7
8
9
10
11
12
14
15
16
17
19
20
2,1
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
)-1-1
42
~1EASUREMENTS
1,
FOR INDIVIDUAL LUCERNE SEEDLINGS IN mm.
Controls
Rolls
Rolls
4-6
2.
2.6
3.0
3.0
2.5
2.2
3.0
3.5
2.5
3.0
3.4
2.6
2.6
2.1
2.5
2.0
2.5
2.4
3.0
2.0
3.3
3.0
3.5
2.6
3.6
3.5
2.0
2.5
3.1
2.5
2.6
3.5
2.0
2.5
2.8
3.0
2.5
2.3
3.0
4.
3.3
3.3
3.5
2.4
2.5
2.6
2.6
2.8
3.2
2.9
3.2
3.0
3·1
2.5
2.5
2.8
2.0
3.0
2.6
2.4
2.5
2.9
3.1
2.6
3.0
3.2
2.6
3.0
2.2
2.8
3.0
4.2
2.7
3.5
4.0
7.5
3.5
3.5
3.8
3.0
5·0
4.5
3.5
5.5
3.5
4.5
5·4
4.3
4.8
3.2
2.5
2.1
3.5
6.0
5.5
6.0
2.6
3.2
4.3
2.7
2·3
4.0
3.0
2.2
2.3
3.5
4.0
3.8
4.5
3.3
2.1
5.0
2.2
6.0
4.0
2.1
5.0
5.0
3.8
3.2
3.1
2.8
3.3
3.5
4.5
4.0
5.0
3.8
5.1
2.5
' ,4.3
2.5
3.5
7.0
3.2
3.0
3.8
4.3
.3
3.4
4.0
2.9
3.1
4.2
5.0
4.2
5.3
4.1
5.1
3.6
4.5
5.6
4.3
5.4
3.5
3.9
1..2
4.6
4.2
2.6
2.8
3.6
3.6
3.7
3.5
2.6
3.4
3.2
3.2
4.5
4.1
2.5
4.9
•
5.
3.4
4.0
2.6
6.2
5.2
3.4
2.1
5.8
3.0
4.0
3.5
2.9
3.8
5.3
3.2
3.3
5.4
3.8
3.2
4.6
3.4
3.5
3.1
4.5
2.8
3.4
1.9
L;3
~4
45
M", .. n·
-----~-
')
'7 ')
_
..
_ .--"-----,----~-.-.-~~--,-----"~---,-
') O?
') 06
'"} "1<-
"
";'6
..,
",f)
76
APPENDIX 1
(Continued)
l'Jairau
Plant
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Mean:
Experiment 11
Controls
Rolls
Rolls
19-21
22-24
2•.4
3·1
3.5
3·0
4.0
3.8
3.5
3.5
3.0
3.3
3.2
3.2
3.4
3.3
3.1
3.5
2.8
2.5
2.7
4.0
2.5
3·0
2.5
3·1
2.8
2.5
2.5
3.7
2.5
3.5
2.6
3.0
2.5
3.0
2.2
3.5
2.6
3.2
3.2
3.0
2.6
3.2
2.9
2.9
3.0
3.1
2.1
3.7
2.8
2.0
2.6
3.1
2.5
3.0
2.6
2.5
3.0
2.3
3.0
3.5
3.0
3.5
2.7
3.2
2.6
3.5
2.4
3.6
2.8
4.0
3.0
3.3
3.6
3.0
3.1
2.5
2.4
2.7
3.2
3.0
2.6
3.2
2.5
3.0
3.2
3.0
2.5
3.2
.2
2.96
3.02
Rolls
25-27
5.0
3.7
3·3
5.5
8.0
3.6
4.2
2.4
5.3
5.5
4.8
5.5
5.0
3.3
7.6
3.8
3.2
5.0
4.3
5.0
6.5
5.5
5·0
2.0
4.5
3.0
5.5
4.6
5·5
7.0
5.9
2.2
3.0
6.0
2.4
3·2
6.0
7.0
3.5
2.6
2.2
3.0
Treatments
Rolls
Rolls
28-30
31-3]
3.7
4.5
3.2
3.5
2.8
3.0
6.0
6.5
4.8
6.0
2.7
3.5
4.5
5·3
4.0
4.3
5.0
4.1
3.0
5.0
5.5
4.5
6.0
7.0
3.4
4.5
6.2
3.9
3.8
4.5
7.0
4·0
4.5
3·3
6.5
4.5
5.0
3·1
6.5
4.3
2.8
6.0
5.5
4.5
4.8
6.5
4.3
5·5
2.8
5.6
5.0
2.4
2.5
4.6
4.0
3.4
6.5
6.5
4.8
5·5
2.9
4.8
4.0
5.5
2.6
8.0
6.5
4.5
3.2
3.6
2.1
3.7
5.0
3.8
2.1
6.5
6.3
2.9
6.0
4.5
2.7
5.2
6.5
---"._-' .---. - -" -." _.-' --. 4.53
4.58
~~-
Rolls
34-36
7.5
2.5
4·0
5·0
5.2
5.2
4.5
4.8
5.0
4.2
6.2
4.2
7.0
3.0
4·9
6.3
4.6
4.0
4.7
5.3
3.2
5.6
5.8
3.7
5.2
4.9
5·1
2.0
4.3
4.9
5.8
4.1
7.8
4.6
2.0
5.3
3.9
4.8
.. -.""".--~
4.57
4.77
77
APPENDIX 1
(Continued)
Experime~
Gvimm
Plant
No.
1
2
"
.J
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Mean:
Controls
Rolls
Rolls
40-42
37-39
2.9
3:8
3.6
3·2
3.0
3.0
2.8
2.2
2.8
2.B
2.8
3.0
2.6
3.4
3.1
3.5
2.6
3.0
2.6
3.3
3.0
3.0
2.8
2.6
2.1
3.2
3.2
3.2
; 2.7
3.0
2.6
3.1
3.8
3.3
2.7
2.5
3.2
3.0
3.6
3.0
2.9
3.1
2.9
3.0
3.2
3.0
3.8
3.4
3.0
3.2
3.1
3.9
2.6
3.5
2.8
3.0
3.8
3.3
2.2
2.9
2.7
2.9
2.S
2.6
2.8
2.7
2.2
3.3
3.1
2.7
3.2
2·5
3.5
3·3
2.4
3.1
3.1
2.913
3·0,3
Rolls
43-45
5.5
7.3
4.8
10.0
5.8
5.5
4.8
3·0
5.0
6.0
4.5
4.0
4.0
2.8
7.0
8.0
4.7
5.0
6.0
5.3
3.0
5.7
4.3
6.5
4.3
2.3
5.5
5.21
Trea.tments
Rolls
Rolls
46-4B
49-51
3.B
6.5
6.0
7.2
7.0
4.4
5.2
5.0
6.2
2.3
3.2
3.4
3.4
4·5
2.5
5.1 '
6.5
4.6
6.2
6.5
4.0
6.3
5.6
7.5
2.6
5.0
6.2
7.0
5.0
3·2
5.8
4.0
6.0
3.3
7.0
4·5
4.0
3.6
6.2
5.5
5.0
3.6
5.8
3.5
6.5
6.4
6.5
5.8
8.0
4.2
4.5
4.5
5.8
7·5
rp.6
7.0
2.0
3.4
7.1
4.0
2.6
6.5
7.0
3.7
4.6
5·2
6.0
5·7
6.0
4.2
7.0
8.5
7.0
3.8
3.5
6.0
5.40
4.97
Rolls
52-24
3.5
3.9
7.3
3.7
7.2
6.3
4.0
4.2
5.5
7.0
7.6
8.2
3.8
7.6
6.7
6.7
4.8
3.5
4.0
5.8
2.9
8.0
5.1
3.1
6.8
3.3
7.5
6.0
6.9
7.5
4.6
6.0
2.2
2.8
6.8
3.4
5.0
5,38
APPENDIX 1
78
(Continued)
Experiment 12
Trea.tments
Rolls
Rolls
Plant
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
}1efln:
.
3.1
3.5
2.6
2.2
3.4
2.7
2.9
3.0
3.0
2.9
3.0
2.4
2.6
3·2
2.2
2.8
3.1
2.7
2.4
2.6
2.7
3·1
2.3
2.6
2.8
2.9
3.2
2.7
2.6
3.4
3.2
2.1
2.7
2.4
3.0
2.3
2.8
2.1
2.3
2.9
2.5
4.0
3.3
3.3
3.5
2.4
2.5
2.6
3.6
2.8
3.2
3.4
2.9
3,6
3·0
2.8
2.7
2.8
3·5
3·0
2.5
2.6
2.9
2.8
2.9
3
2.4
2.5
2
2
2.5
3.0
2.6
2.8
2.5
2.5
3.0
2.6
2.3
3.3
3.3
7.8
3.5
3.1
5.6
5.3
4.5
4.1
6.0
3·5
4.0
3.0
3.0
3.0
6.8
3.0
4.6
6.5
5.0
4.0
5.0
5·0
5·1
7.0
6.0
4.5
3.6
2.8
4.5
7.0
6.1
3.9
5.6
6.5
4.5
3.8
4.5
3·2
4.8
3·4
7.3
3.1
2.5
4.6
4.5
4.2
6.0
2.7
4.0
3.0
2.8
6.5
2.7
5.8
4.6
4.5
7.2
2.8
4.6
4.3
4.0
2.1
6.0
5.4
6.3
2.6
7.8
6.1
6.7
5.3
2.8
3.6
6.5
5.0
4.5
3.1
3'.2
5·4
2.75
2.88
4.6':(
4.58
79
APPENDIX 1
(Continued)
Experiment· 12
Wairau
PIHnt
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
29
30
31
32
33
34
35
36
37
38
39
40
Controls
Rolls
Rolls
.
3.2
2.9
2.6
2.6
2.6
2.3
2.5
2.6
2.0
3.3
2.6
3.0
3
2.6
2.9
2.2
2.6
2.2
3.6
3.4
2.8
2.8
2.1
2.2
2.4
3.5
2·5
2.5
3.8
3.5
2.5
2.5
3.0
3.2
3.3
3.5
2.5
4.0
2.5
3.0
4.0
3.5
3.3
3.2
3.4
3.1
2.8
2.7
3.0
3,1
3.7
3.5
2.5
3·5
2.7
3·2
2.6
3·0
2.6
3·0
2.7
2.5
3·0
2.S
2.4
2.6
3.0
3.0
2.2
2.6
3.0
2.6
2.9
3·1
2·3
2.0
2.6
2.5
2.5
2.3
3.2
8.0
5.0
7.6
5.0
7.4
4·5
4.5
5.5
4·5
6.5
4.5
6.0
5.8
6.8
7.0
7.2
7.5
5.0
4.0
5.0
6.3
6.8
6.5
4.0
3.5
4.0
6.0
4.7
6.3
5.5
8.0
4.0
5.2
2.4
6.3
6.5
5.8
6.5
6.0
6.0
5.S
6.7
S.o
7.5
5.6
3.6
7.2
4.0
5.2
4.6
4.5
5.6
7.2
2.8
6.9
7.2
4.8
3.7
5.2
5.6
4.0
6.5
6
7.2
2.4
3.5
4.7
7.8
6.8
5.3
6.6
6.7
8.0
3.2
6.4
5.9
6.0
4.0
4.8
2.83
2.89
5.74
5·52
41
42
43
44
45
~1e8n:
Treatments
Rolls
Rolls
~,PPENDIX
80
1
(Continued)
Plant
No.
Controls
Rolls
Rolls
0
2
3
4
5
6
7
8
9
10
12
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Mean:
Experiment 12
Treatments
Rolls
Rolls
•
2.2
2.9
3.0
2.4
2.9
2.8
3.5
3.0
3.0
3.2
3·0
2.9
4.0
2.8
3.4
2.8
2.8
2.6
2.5
3.6
3.4
2.2
3.6
2.2
3.4
2.2
2.2
3.2
2.8
2.9
2.6
3.0
3-4
2.8
3.6
2.7
3.1
2.1
2.9
2.8
3.4
2.2
3.8
2.6
3.2
2.9
2.9
3.3
3.2
3.6
3.1
2.9
2.9
3·1
3.4
2.7
2.4
3·1
2.7
2.6
3.1
3.8
2.3
3.1
2.8
3.6
3.2
3.0
3.8
2.6
2.1
3.4
3.1
2.9
2.8
2.9
6.0
8.0
7.5
3.0
9.0
6.0
6.0
6.6
8.6
8.3
5.7
2.9
6.3
6.5
6.8
7.0
6.0
4·0
6.0
3.5
6.0
7.0
8.3
3.0
5.6
5.5
6.0
6.5
7.0
7.0
3.6
6.8
5.5
5.5
6.0
8.5
6.3
2.5
6.0
4.0
·5
5.6
2.5
8.3
4.5
5.0
6.5
7.2
3.6
5·8
6.4
7.2
5.0
7.5
4.5
6.0
4.0
7.0
4·0
8.0
6.5
5.5
5,0
5.8
6.5
2.3
3.6
8.6
7.5
7.3
6.5
5.7
6.2
4.6
5.8
6.8
7.1
6.8
5·2
2.90
2.99
5.96
5.89
81
APPENDIX 1
(Continued)
Washoe
Experiment 13
Rolls
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
I
T,.featments
R.olls
Rolls
2.4
3.6
3.0
3.0
2.7
2.8
3.1
2.3
2.8
2.8
3.0
2,4
3.0
2.9
2.6
2.6
2.2
2.1
2.9
2.6
2.8
2.9
3.0
2.6
2.4
3.4
2.6
2.6
2.7
2.7
3.2
2.6
2.8
2.8
2.3
2.9
2.8
2.1
2.5
3.0
3·2
2.3
2.6
3.0
2.5
2.5
2.8
2.6
2.3
2.5
2.9
2.5
2.8
2.6
2.7
2.7
2.3
2.8
3.3
2.9
3.3
3.2
3.5
3.6
2.8
2.5
3.0
2.5
2.5
2.9
2.8
2.7
3.3
2.3
2.8
2.4
5.2
4.3
3.6
4.2
3.2
5.2
5.6
4.2
5.0
5.2
3·3
3.6
3.6
3.8
2.7
2.3
3.9
4.0
3.1
3.6
4.2
3.6
6.2
3.6
3·2
5.8
3·2
3.4
5.2
7.3
6.4
5.2
4.5
5.1
3.8
3.8
5.0
5.2
5·0
5.6
4.2
6.3
5.0
4.8
5.2
4.8
2.6
3.3
2.6
5.8
2.6
4.6
4.2
4.7
3·1
5.0
4.2
3.8
5.5
5.4
4.0
4.5
6.3
2.8
3.4
5.8
3.5
3.0
4.2
5.4
3.8
3.5
2.75
2.78
4.34
4.38
Rolls
3.5
3.8
3.5
2.8
4.5
3.8
5.6
4·1
3.0
2.8
5.4
2.7
3.2
7.0
4.3
4.2
8.0
6.5
5·0
3.2
6.5
3.0
6.5
5.2
4.5
3·3
4.0
4.5
.3
3.6
3.6
3.8
2.3
6.3
2.8
3.8
7.8
3.2
4.8
5.0
2.3
6.5
4.3
5.1
3.8
3.1
2.8
5.2
6.8
3.1
4.4
5.2
2.7
5.4
6.2
3·1
2.8
3.9
5.1
2,7
3.1
4.3
6.3
5.1
4.4
4.
4·32
2·3
6.4
2·5
4·5
3·3
3.5
3.2
4~3
41
42
43
44
45
r1ean:
APPENDIX 1
(Continued)
Plant
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3)
32
33
34
35
36
37
38
39
40
41
h.2
Rolls
19-21
2.7
2
2.5
3.2
2.8
2.6
2.6
3.0
3.0
2.7
2.7
2.2
3.1
2.9
3.1
3.3
2.6
2.3
3·1
3.1
3.4
2.3
3.0
3.2
2.8
3.0
2.8
2.5
2.5
2.8
2.9
2.6
2.5
2.6
2.7
2.7
2.6
3.0
3.2
2.5
2·5
22-24
3.1
2.9
2.3
2.5
2.$
2.9
2.7
3.4
2.9
2.6
2.6
2.5
2.2
2.6
2.3
2·5
2.7
3.1
3.0
3.3 .
2.5
2.3
2.6
2·5
3.1
2·5
2.2
2.7
3.2
2.2
3.0
2.9
2.8
3.0
3.0
2.8
2.8
2.6
2.3
2.4
2.7
2.4
2.2
25-27
6.8
3.1
3.8
7.2
3.5
2.8
6.4
7.8
5.0
7.0
5.4
6.0
5.3
4.0
4.0
2.78
2.69
4·92
43
2,15
4.5
7.0
4.0
4.0
6.0
6.5
5.5
4.8
5·4
5.5
3·8
3.3
4.8
6.3
3.8
5·8
3.0
3.5
3.8
h4
Treatments
Rolls
Rolls
28-30_
5.0
4.8
5.7
4.8
6.$
4· 5
5.4
7.7
5.8
4.8
5.5
6.3
3.2
3.8
2.4
5.3
2.6
6.3
4.5
2.6
2.5
2.4
2.9
4.8
3.0
4.8
5.0
5.0
7.0
4.6
7.6
6.8
4.0
6.8
6.4
5.6
2.9
6.4
2.4
3.0
4.6
5.1
4.0
6.8
Rolls
31-33
6.8
2.8
3.8
3.3
4.5
7.0
4.8
6.3
7.2
4·0
3.5
4.0
6.0
5.8
4.0
4.0
5.3
6.0
5.4
5.5
5.4
7.0
;.0
A·$
3.$
3·5
5.5
7.8
6.4
6.5
3.0
5·8
6.0
2.8
3.5
4.0
3·8
6.3
4.0
7.2
34-36
2.6
5.0
5.3
7.6
4.8
6.8
5.7
6.3
4.0
6.8
4.5
5.05
4.95
4.15
6.8
2.6
6.4
4.5
2.5
5.6
5.4
2.4
2.9
6.4
7.7
2.9
. 5.$
4.8
2·4
4.8
5·5
4.4
4.6
6·3
5.0
5.1
3.2
5.• 0
4.0
6.8
47
Menn:
4.
APPENDIX 1
(Continued)
2.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
113'
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
2..9
2.5
2.7
3.0
2.B
3.0
2·5
3.5
3.1
2.7
2·5
2.5
3.0
3.2
2.5
3·0
3·0
2.6
3.0
2.5
3.6
2·5
2.4
2.8
3·5
2.7
3.3
2.8
3.6
3·3
2.9
2.6
2.7
2.9
3·3
3.8
2·3
2.9
2.8
2.9
2.8
3.6
3.2
3.0
3.8
2.6
2.1
4.0
3.4
3.1
7
2.8
3.2
3.0
3.3
2.9
2.5
2.8
3.6
2.5
3.0
2.7
2.6
2.9
3.3
2.5
2.7
2.8
2.6
3·5
2.9
2.4
2.8
2.7
3.1
3.3
2.2
2.8
5.5
3.5
6.3
4.5
4.0
7.0
3.0
4.0
4.6
3.0
4.3
6.3
7.0
4.2
5.8
6.8
4.0
5.5
5.8
5.8
3.3
4.2
5.4
6.5
6·7
5.2
5·0
6.2
6.0
7.2
6.8
6.8
4.2
6.3
5·5
7.0
4.6
6.3
6.3
6.7
5.2
7.0
5.3
3.5
4.2
5·0
6.3
4.5
4.0
4.8
6.3
4.6
6.5
4.3
5.4
7.0
5·5
4.2
3.0
4.6
3.3
6.3
5.8
4.6
4.0
5.8
4.2
6.8
5.8
3.0
7.0
5·5
6.8
4.0
6.3
6.2
3.4
5.8
6
5.7
4.8
4.1
4.3
4.9
5. a
6.3
6.8
7.1
5·7
6.4
3.8
3.2
5.7
6.3
4.8
4.2
5.3
5.8
6.7
2.9
4.2
7·5
6.1
5.9
4.7
5.1
6.3
4.2
4.0
5.1
6.0
6.3
6.7
6.3
5.8
6.0
5.1
5.3
4.0
4.2
4.9
6.3
4.2
7.2
6.8
2.9
4.2
7.1
6.2
3·4
5.7
7.5
5.9
3.8
6.3
6.3
4.8
5.9
4.7
5·7
3·2
5.1
4.8
6.3
6.8
5.3
2.91
2.94
5.
5.27
5·37
5.45
h2
43
44
45
Mean:
-.---
84
APPENDIX 2
STEM DIAMETERS OF INOCULATED LUCEHNE SEEDLINGS
TESTED FOR NEM,~TODE REPRODUCTION IN rom"
Reproduction
Occurring
Normal
6.0
2.7
3.8
3.2
2.1
2.6
2.7
2.3
2.2
2.3
3.5
3.3
2.1
2.9
3.4
3·5
7·5
3.5
4.5
5.2
5.4
2.6
number
of plants 7
Hean stem
diameter 4.96
Total number
of plants 15
Mes,n stem
diameter 2.84
Nematodes
Only
3.4
4·2
4.0
3.5
3.5
3.0
2.3
5. a
4.5
3.5
5.5
3.5
4.5
5.4
4.3
4.8
3.2
2.5
3.5
6.0
5.5
6.0
3.2
4.3
4.0
3·0
3.5
4·0
3.8
4.5
3.5
5.0
3.4
4.0
6.2
3.4
2.1
3.0
4.0
3.5
3.8
5.3
3.2
3.3
3.8
4.6
3.1
4.5
2.8
3.5
1.9
Total number
of plants
51
Mean stem
diameter 3.92
APPENDIX 2
(Continued)
Experiment 11
Wairau
Reproduction
Occurring
5.0
6.5
5.5
5.0
4.5
5.5
5.5
7.0
5.9
3.0
6.0
6.0
7.0
6.0
5.3
5·5
6.0
7.0
4.5
6.5
5.5
5.0
6.5
5.5
4.5
6.5
6.3
6.0
6.5
4.5
6.5
6.0
4.5
4.3
5.0
4~ 5
7.0
4.5
6.2
5.0
4.3
6.0
6.5
5.6
4.6
7·5
4.0
5·0
5.2
4.5
5.0
4.2
6.2
4.2
7.0
4.9
6.3
4.7
3.2
5.6
5.g
5.2
4.9
5.1
4.9
5.S
4.1
7.S
4.6
5.3
Normal
Nematodes
Only
2.0
3.0
4.6
3.2
2.6
2.2
3.7
3.0
3.5
4.0
3.0
3.4
3.1
2.g
4.8
2·5
4.0
2.9
2.6
3.2
3.7
2.S
2.7
4.0
3.3
4.5
2.4
4.0
2.0
3.g
3.2
5.0
4.3
2.2
2.4
3.5
3.0
3.5
4.S
5.0
3.9
3.S
6.5
5.5
2.S
5.5
5.0
2.7
3.2
4.1
4·5
4.5
4.3
2·5
4.8
3.0
4.6
5.3
3.7
4.3
3.9
Total number
of plants
70
Total number
of plants
29
Total number
of plants
32
Mell.n stem
dinmeter 5.4)
Mean stem
diameter 3.22
Mean stem
diameter 4.03
86
APPENDIX 2
(Continued)
Grimm
Reproduction
Occurring
7.3
10.0
5.S
5.5
6.0
4.5
4.0
7·0
8.0
6.0
5.3
5.7
6.5
6.0
7.0
6.5
6.5
5.0
6.0
7·0
5.0
5.8
6.5
6.5
8.0
4.5
7·5
7.0
7.0
6.0
7.0
8.5
7.0
7.6
6.7
6.7
4.8
5.8
8.0
5.1
6.8
6.9
7.5
6.0
6.8
5.0
Total numher
of plants
46
Mean stem
diameter 6.43
Normal
Nematodes
Only
5.5
3.0
2.8
5.0
3.0
4.3
4.3
2·3
2.3
3.2
2.5
4.0
4.0
4.0
2.0
2.6
3.8
3.5
3.8
3.5
2.9
2.2
2.8
3.4
4.8
4.8
5.0
4.0
4.7
6.5
5.0
4.5
7.5
5.0
5.5
4.0
6.0
6.0
4.0
3.1
4.6
Total number
of plants
24
Mean stem
diameter 3.36
Total number
of plants
17
Hea.n stem
diameter 5.00
APPENDIX 2
(Continued)
Experiment 12
1<Jashoe
Reproduction
Occurring
Normal
4.0
7.g
5.6
5.3
4.1
6.0
3.5
6.g
6.5
5,0
4.0
5.0
5.0
5.1
7.0
7.0
6.1
5.6
6.5
4.5
7.3
6.0
6.5
5.8
7.2
6.0
5.4
3·1
3.0
3.6
2.g
3.2
3.4
2.5
4.5
2.7
2.8
2.7
2.8
2.1
2.6
2.8
3.7
6~.3
7.8
6.1
6.7
hl
Total number
of pla.nts
32
Hel'ln stem
diameter 5.81
Nema.todes
Only
Total number
of plants
16
Mean stem
diameter 3.19
3.5
4.5
4.0
3·0
3.0
3.0
4.6
6.5
4.5
3.9
4.5
3.g
4.g
3.1
4.6
4.2
4·0
3.0
4.6
4.5
4.6
4.3
4.0
5.3
3.6
6.5
5.0
3·1
5.4
Total number
of plant:;;
29
Mean stem
dill1lleter 4.25
SS
APPENDIX 2
(Continued)
Experiment 12
Reproduction
Occurring
S.. O
S.o
7.6
5.0
7.4
5.5
6.5
6.0
6.B
7.2
6.9
7.2
5.2
5.6
6.5
6.3
7.2
4.7
Normal
Only
3.5
5.0
2 .. 4
4.5
4.5
4.5
4.5
4.0
2.4
3.6
2.B
2.4
3.5
3.2
7.0
7.2
7.B
7.5
6.6
6.3
6.S
6.7
S.O
Total number
of plants
9
6.0
6.3
5.5
S.O
6.3
6.5
6.5
6.0
6.7
S.O
7.5
5.6
7.2
4.6
6.4
6.0
4.S
Mean stem
diameter 3.09
6.B
4.5
5.6
Total number
of plants
47
Me09.n stem
diruneter 6.52
Nematodes
5.0
4.0
5.0
6.5
4.0
4.7
4.0
5.2
5.S
6.0
5.B
4.0
5.2
4.S
3.7
4.0
5.3
5.9
'4.0
Total number
of plants
24
Mean stem
diameter· 4.S2
APPENDIX 2
( Continued)
Experiment 12
Reproduction
Occurring
6.0
B.O
7.5
9.0
6.0
6.0
6.6
9.6
8.3
6.3
6.5
6.S
7.0
6.0
4.0
6.0
6.0
7.0
8.3
5.6
5.5
6.5
7.0
7.0
6.S
5·5
8.5
6.3
6.0
7.0
6.5
S.3
6.5
7.2
6.4
7.2
7.5
6.0
7.0
8.0
6.5
5.5
6.5
S.6
7.5
7.3
6.5
5.7
6.2
6.S
7.1
Norma.l
Nema.todes
Only
3.5
4.0
3.0
5.7
3.0
6.0
2 .. 5
2 .. 9
2.5
3.6
2.3
3.6
3.6
5.5
Total number
of pla.nts
8
Mean stem
diameter 3.38
6.0
4.0
5.6
4.5
5.0
5.S
5·0
4.5
4.0
4.0
5.0
5.S
4.6
5.S
5.2
6.8
Total number
of plants
52
'fo.lean stem
diameter 6.79
Total number
of plants 21
Mean stem
diameter 4.74
90
APPENDIX 2
(Continued)
Washoe
Experiment 13
Reproduction
Occurring
Normal
Nematodes
Only
5.B
5.2
4.2
5.2
5.6
5.0
5.2
3.3
3.6
6.2
5.8
5.2
7.3
6.4
5.2
4.5
5.6
4.2
6.3
5.0
5.8
4.6
5.0
4.2
4.5
6.3
5.8
3.6
3.2
3.6
4.3
4.2
3.9
4.0
3.1
3.6
4.2
3.6
3.4
5.1
3.8
3.8
5.0
5.2
5.0
4.8
3.3
4.2
4.7
3.8
5.5
5.4
4.0
3.0
4.2
3.B
Total number
of plants 28
Mea.n stem
diameter 5.23
3.B
2.7
2.3
3.6
3.2
3.2
4.0
2.6
2.6
2.6
3.1
2.8
3.4
3.5
3.5
Total number
of plants 18
Mean stem
diameter 3.18
Total number
of plants 26
Mean stem
diameter 4.19
91
APPENDIX 2
(Continued)
Wairau
Normal
Nematodes
only
6.3
5.3
6w3
3.1
3.5
4wB
2.8
3.8
4.0
4.0
3.8
5.0
3.5
3.2
3.8
Reproduction
Occurring
6.B
7.2
6.4
7.B
5.0
7.0
5.4
6.0
5.3
4.0
4.0
4.5
7.0
6.0
6.5
5.5
5.0
5.0
7.6
6.8
6.8
6.4
5.6
6.4
5.1
6.B
2.$
2.4
2.6
2.5
4.8
2~6
2.4
4.0
2.9
2.4
4.B
5.4
5.5
4.8
6.3
5.8
3.• 3
3.8
3.0
3.8
Total number
of plants 15
Mean stem
diameter 2.97
4.8
4.5
4.5
2.9
4.8
3.0
4.6
3.0
4.6
4.0
5.0
Total number
of plants
19
5.7
6.8
Mean stem
diameter 3.95
4.B
5.4
7.7
5.$
5.5
Total number
of plants
45
Mean stem
diameter 5.B4