Pafhogenicify of the Nematodes Rotylenchulus reniformis,
Helicotylenchus dihystera, and Tylenchorhynchus claytoni
to Shade Tobacco
By
SAW ART RATANAWORABHAN
A DISSERTATION PRESENTED TO THE GRADUATE COUNOL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1969
ACKNOVTLEDGEMEHTS
The author expresses his sincere appreciation to members of his super-
visory committee: Dr. Grover C. Smart, Jr., Chairman; Dr. V. G. Perry; and
Dr. Howard N. Miller. A special debt of gratitude is due Dr. Smart for his
guidance, consultation, and criticism of manuscript.
He is indebted to Dr. William G. Eden, Chairman of the Department of
Entomology and Hematology^ and Dr. Perry, Prof-.:3sor of Kematology, who made a
teaching assistantship available making it possible for the author to con-
tinue his education.
Appreciation is extended to ^Ir. William B. Tappan, Associate Entomologist,
North Florida Experiment Station, for providing soil, original nematode popu-
lations, tobacco seeds and valuable information regarding shade-grown
tobacco .
He expresses his gratitude to Mr. R. W. Patrick for assistance on several
occasions during the investigation.
A recommendation of Mr. A. L. Taylor, Nematologist , formerly with FAO
mission in Bangkok, to AID officials in Bangkok granting the extension of
training to the author in order to complete this investigation is greatly
appreciated.
Finally, he thanks his wife for her understanding, encouragement, and
assistance.
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CONTENTS
Page
ACKKOWLEDGEffiNTS
LIST OF TABLES
LIST OF FIGLTRES
ABSTRACT
INTRODUCTION
LITERATURE BEVim
MATERIALS AND METHODS
Pathogenicity
General Methods
Experiment I •
Experiment II » III, and IV ■^^
Histopathology of Rotylenchulus rentformis
on Tobacco Roots ^'
RESULTS ^'
Pathogenicity ^'
Experiment I ^
Experiment II ^
Experiment III ^'
Experiment IV 1'
Histopathology ^
DISCUSSION
3
Pathogenicity Experiments 3
ExDeriment I 3
Experiment II 3
Exneriment III 3
Experiment IV 3
Histopathology
SUT^MARY ^
LITERATURE CITED ?
BIOGRAPHICAL SKETCH 5f
LIST OF TABLES
Table Pag
1. Means of heights (cm) of tobacco plants measured on four
different dates after inoculation on October ih^ I966 15
2. Means .of tobacco leaf and root veights (g) and the popu-
lations of nematodes 128 days after inoculation on
October l^t, I966 . .* I6
3. Means of plant heights (cn) and green leaf and root weights
(g) of tobacco, and the teminal populations of nematodes,
80 days after inoculation on J\xn.e. I6, I967 I8
h. Means of plant heights (cm), green leaf and root weights (g)
of tobacco and terminal populations of nematodes, TO days
after inoculation on June 3, 196? 20
5. Means of plant heights (cm), green leaf and fresh root
weights (g) of tobacco, and terminal populations of
nematodes, k2 days after inoculation on July 28, I968 22
o. Comparison of terminal populations of Helicotylenchus
dihystera inoculated alone and in combinations as indicated. . kO
7. Comparison of terminal populations of Rotylenchulus
reniformis inoculated alone and in combinations as indicated , 1+1
8. Comparison of the terminal populations of Tylenchorhynchus
claytoni inociaated alone and in combinations as indicated . . 2+3
9. Comparison of the terminal populations of Helicotylenchus "
dihystera inoculated alone and in combination with •
Rotylenchulus reniformis and T^'lenchorhynchus claytoni .... hk
10. Comparison of terminal populations of Rotylenchulus , , ■ ^ '." ^
reniformis inoculated alone and in combination with ' '
Helicotylenchus dihystera and Tylenchorhynchus claytoni. . . . 1»5
11. Comparison of terminal populations of Tylenchorhynchus
claytoni inoculated alone and in combination with
Helicotylenchus dihystera and Rotylenchulus reniformis .... 1+6
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I •
LIST OF FIGURES
Page
Figure ' t
1. Photomicrographs of adult females of Rotylenchulus i
reniformis. A, eelvorm shape; B, C, D, developing
stages; E, mature kidney shape. F, an enlargement
of t shoving vulva (v). Didelphic reflexed ovarxes
are not discernible in this photograph
2. A, Cross section view of a tobacco root from an
uninoculated plant. B, An enlarged viev of A
3. A-D, Photomicrographs of cross sections of tobacco
roots infected vith Rotylenchulus reniformis shoving _
various shapes of hypertrophied^ cells in the pericyclic
region surrounding vascular cylinders
U. Photomicrograph of two females of Rotylenchulus.
X^r^^ formis feeding in the same area of a tobacco root
5. Cross section view of a tobacco root infected with
Rotylenchulus reniformis showing the nematode head
positioned near the hypertrophied pericycle
28
6. An enlarged view of a portion of Fig. 5
T. A, Cross section view of a tobacco root infected
with Rotylenchulus reniformis
8. A, View of a longitudinal section of tobacco root
infected by Rotylenchulus reniformis showing
hypertrophied cells and enlarged nuclei
9. A-F, Photomicrographs of serial sections of a tobacco
root infected by Rotylenchulus reniformis showing the
development of hypertrophied cells (arrows) which
apparently originated in the pericycle
10. Photomicrograph of a tobacco root showing two nuclei
(a) of a hypertrophied cell which had been fed upon
by Rotylenchulus reniformis ^
11. Comparison of terminal population levels of three
nematode species at different levels of inoculum
1*2 days after inoculation
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Abstract of Dissertation Presented to the Graduate Council
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
PATHOGENICITY OF THE IMEMATODFS P0TYLENCHULU5 RENIFORMIS,
HELICOTYLEITCHUS DIHYvSTERA. MID TYLENCHORHYiNCHUS CLAYTONI
TO SHADE TOBACCO
Savart Ratanavorabhan
March, I969
Chairman: Dr. Grover C. Smart, Jr.
Major Department: Entomology and Nematology
Helicotylenchus dihystera, Rotylenchulus reniformis , and
Tylenchorhynchus claytoni are plant parasitic nematodes frequently
encountered in shade-grown tobacco fields in North Florida. Pathogeni-
city experiments vere conducted in the greenhouse using each species
alone and in all combinations with the other tvo species at different
inoculum levels. T. claytoni significantly retarded the root system
of shade-grown tobacco variety 'Dixie Shade' with an exposure period of
128 days. However, height and yield of inoculated plants were not
affected by root retardation. Smaller root systems of plants were
always accompanied by some discoloration and shriveled appearance of
roots. At exposure periods of 80, 70, or k?. days at inoculum levels
comparable to those used for 1?8 days' exposure, root retardation by T.
.^^-^-y^^"^ ^i^- "O't °<^cur. H. dihystera and R. reniformis inoculated
alone or in all combinations at any inoculum level did not affect T>lant
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growth. At any given time of exposure T. claytoni inoculated to tobacco
alone or in combination vith the other species reproduced at a greater
rate than did the other nematodes. Terminal populations increased
several times over the inoculum. Terminal populations of H. dihystera
and R_. reniformis inoculated alone or in combination vere in most cases
greater than the inoculum, but nvmbers were not as great as were terminal
populations of T. claytoni. Inoculation with 1,500 specimens each of
T, claytoni , _H. dihystera, and reniformis resulted in significantly
fewer numbers of R. reniformis U2 days after inoculation than when 1,500
specimens of R^. reniformis were inoculated alone. Similarly, inocxila-
tion with 1,500 specimens each of If. dihystera, R^, reniformis , and T..
claytoni resulted in fewer numbers of T. claytoni h2 days after inocvila-
tion than when 1,500 specimens of T. claytoni were inoculated alone.
Histopathological studies of tobacco roots infected by R. reniformis
revealed that eelworm-shaped females penetrated tobacco roots near the
root tips to initiate infection. The parasites penetrated the epidermis,
cortical parenchyma, and endodermis and fed in the pericyclic region.
Only the stylet penetrated into peri cycle cells. Hypertrophy and en-
largement of nuclei in the pericyclic region extended from the cell
where a nematode stylet was inserted to adjacent pericyclic cells. Cyto-
plasmic content of hypertrophied cells stained darker than uninfected
surrounding cells. The above-ground portions of parasitized plants
exhibited no symptoms.
- vii -
imODUCTION
Records maintained at the Department of Entomology and Nematology,
University of Florida, Gainesville and the North Florida Experiment Station,
Quincy, shov that plant parasitic nematodes of the genera Helicotylenchus ,
Rotylenchulus and Tylenchorhynchus are frequently encountered in shade-grown
tobacco areas in North Florida. Experiments at Quincy indicated that yields
were increased and a better quality of tobacco was produced when nematicides
were used (Tappan and Kincaid, 1962 and 196^+). Records also revealed that
Helicotylenchus dihystera (Cobb) Sher (syn. H. nannus Steiner ) , definitely
affected crop growth. This parasite damaged shade-grown tobacco roots re-
ducing plant height, yield, and quality of the tobacco (Tappan and Kincaid,
1963). The reniform nematode, Rotylenchulus reniformis Linford and Oliveira,
has been suspected of reducing yield and quality of shade-grown tobacco.
Tylenchorhynchus claytoni Steiner , though not reported in the Gainesville
and Quincy records as a pathogen of tobacco, hsis been reported as a pest of
tobacco in other areas.
The purposes of these investigations were to determine the effects of
Helicotylenchus dihystera, Rotylenchulus reniformis and Tylenchorhynchus
claytoni on shade-grown tobacco variety 'Dixie Shade' by using each species
alone and in all combinations with the other species, and to investigate the
histopathology of R. reniformis on tobacco.
1
LITERATURE REVIEW
Members of the nematode genus Helicotylenchus are encoxmtered frequently
in the roots and surroxmding soil of many host plants. The parasites have
•been found repeatedly in soil samples from turf grasses, ornamental plants
and field crops by various workers (Perry et al . , 1959; Taylor, I960;
McGlohon et al . , 196I; Springer, 196^*; Roman, 1965; Sher, 1966). K. dihystera
is the predominant species found in shade-grown tobacco areas of North Florida
(Tappan and Kincaid, 1963), yet little is known about its pathogenicity to
tobacco.
Sledge (1956) studied the pathogenicity of H. dihystera to certain
varieties of com and found that the nematode multiplied readily but caused
little damage. The symptoms included those of stunting and some root necrosis
at high inocxilum levels. Minton and Cairns (1957) showed that H. dihystera
decreased root production by 'Ogden' soybean. However, seed yields of the
infected and control plants were not significantly different. H. dihystera
was reported to be associated with gardenia decline by Konicek and Jensen
(1961). The decline plants showed stunting, yellowing, premature dropping
of leaves, failure to form flower buds, premature dropping of buds, and
eventual death of plants. Ruehle (I966) reported that H. dihystera para-
sitized and reproduced on six species of pine seedlings native to the south-
eastern United States. However, effects of this parasite on the pine seedlings
were not determined.
Pathogenicity of a few other species of the genus have been studied by
Perry et_ al . (1959). They demonstrated that H. digonicus Perry and, to a
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- 3 -
lesser extent^ K. nicrolo"bus Perry [ = H. pseudorobustus (Steiner) Golden]
and H. pimilus Perry [ = Rotylenchus punilus (Perry) Sher] parasitized roots
of common Kentucky blue grass, Poa pratensis L., and produced a condition
called "summer dormancy".
The reniform nematode, Rotylenchulus reniformis vas described from roots
of cowpea, Vigna sinensis Endl. , in Hawaii (Linford and Oliveira, 19^0).
Since then the species has been reported to occur in various tropical and
semi tropical areas of the world and in the southern United States (Smith,
19liO; Smith and Taylor, 19^1; Steiner, 19^9; Martin, 1955; Peacock, 1956;
Timm, 1956; Minton and Kopper, 1959; Thorne, 196l; Sasser et al . , 1962;
Lambe and Home, 1963; Swarup et_ al , , 196?; Fassuliotis and Rau, 1967; and
Dasgupta et al. , 1968). This species attacks various host plants (Linford
and Yap, 19^0; Peacock, 1956; Luc and de Guiran, I96O; Birchfield and
Brister, 1962; Ayala and Ramirez, 196i;). Among the economic crops are cotton,
sweet potato, pineapple, tomato and tobacco.
The importance of R_. reniformis was recognized when Neal (195^) reported
that the presence of the reniform nematode increased the incidence of
Fusarium wilt of cotton in the wilt susceptible varieties. Birchfield and
Jones (1961) stated that R. reniformis was found associated with cotton
failures distributed over 2,000 to 2,500 acres in Louisiana. This parasite
was associated with severe disease symptoms of dwarfing, premature decay with
loss of secondary roots, and death of young cotton plants. These conditions
resulted in poor stands, "grassy" areas in the fields, and ultimate yield
reductions of Uo to 60 per cent. Jones et_ al . (1959) reported that the
infestation of R. reniformis at the rate of 320 specimens per 1+73 ml (one
pint) of soil caused a reduction in the green weights of cotton plants by
12.6 per cent, a 21.3 per cent reduction in total boll weights and 20.7 per
cent reduction in number of bolls. Cotton seedling growth was reduced when
_ It «
roots were parasitized by R_. reniformis according to Brodie and Cooper (196^4).
Minton et al. (I96U) reported that R_. reniformis reduced emergence, plant
height, and yield of cotton, sind hastened crop maturity. They also noted
that the incidence of Fusarium wilt of cotton was significantly increased by
the presence of R_. reniformis. Kost-parasite relationships of R_. reniformis
on cotton were studied by Birchfield (19^2) . He found that young females
initiated infection by extending the anterior portion of their bodies
through the epidermis and cortical parenchyma to feed in phloem tissues of
young roots. Phloem cells near the feeding site of the nematode stained
darker thsui normal tissues; damage to the phloem extended several cells
along the eixis of the root. Birchfield concluded that necrosis in the phloem
and parenchyma apparently resulted in severe root "pruning" of seedlings and
subseq^uent dwarfing of cotton.
In Florida, R_. reniformis was first reported by Steiner (I9U9) on
tomato and coffee weed (Cassia tora L^. ) from Quincy. Later, the parasite
was found parasitizing roots of mango tree (Manr^ifera indica L. ) on the
groxinds of the University of Florida Subtropical Experiment Station at
Homestead according to Van Weerdt et_ al . (1959). However, the damage by
R. reniformis to mango in Florida has not been investigated.
Tylenchorhynchus claytoni , the tobacco stunt nematode, was described by
Steiner (1937) from specimens collected around and in the roots of stunted
tobacco plants growing near Florence, South Carolina. Graham (195U) fovmd
that species in 67 per cent of a series of soil samples taken from tobacco
fields in Eastern South Carolina. He also stated that the species was en-
countered frequently in soil samples from cotton and com fields. As an
ectoparasite of tobacco, Graham reported that T. claytoni caused stunting
of the tobacco plant and a much retarded root system as compared with normal
growth in nematode- free soil. Graham further stated that the roots did not
show definite symptoms "but became shriveled and sparsely developed. Drolsom
and Moore (1955) reported that T. claytoni was one of the principal plant
parasitic nematodes foxind in tobacco fields in North Carolina where stunting
of plants occurred. Holdeman (1956) stated that the presence of T. claytoni
attacking the roots of wilt susceptible tobacco variety 'Oxford I-I8I,'
greatly increased the incidence of wilt caused by Fusarium oxysporim.
Krusberg (1959) reported that T. claytoni completed its life cycle in about
one month on alfalfa roots growing on nutrient agar. He also found that males
are necessary for reproduction. Root systems of tobacco growing in pots were
considerably retarded by large populations of T_. claytoni. No nematode injury
to the roots of tobacco variety 'Dixie Bright 101' was observed. Host range
studies, including 1*1 plant varieties, indicated that the tobacco variety
'Bottom Special' is the most suitable host. Reproduction of the nematode
was best at 29.1+° - 35.0° C on tobacco and at 21.1° - 26.6° C on wheat.
Nelson (1956) tested several corn hybrid lines in the greenhouse against
T. claytoni and found that at high population levels root and shoot weights
of plants were reduced,
Brodie and Cooper (1961*) reported that T. claytoni reproduced in the
presence of cotton seedling roots but did not cause excessive root injury or
measurable retardation in gro'^h of seedlings.
Pathogenicity of T. claytoni on turf grasses was investigated by Troll
and Rhode (I966). They observed the parasite feeding on the roots of annual
rye grass, creeping red fescue, and Kentucky blue grass but root injury was
not discernible. Shoots of the three grasses inoculated with T. claytoni
were not affected by the nematode. The lower dry weights of roots and shoots
of inoculated plants compared with non-inoculated controls indicated that
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T. claytonj was pathogenic to creeping red fescue (Festuca rubra L. )
Sumner (196T) reported that T^. clnytoni was one of the predominant stylet-
bearing nematodes found in Kentucky blue grass lawns in Lincoln, Nebraska.
However, attempts to demonstrate its pathogenicity on blue grass in green-
house and laboratory experiments were unsuccessful.
■ dtiytoni is also found frequently in forest tree nuseries. Hopper
(1958) reported that population levels of 1,500 to 2,000 specimens per
^73 ml of soil caused severe injury to seedlings of slash pine, Pinus
elliottii Engelm. He described the symptoms as ranging from green-, yellow-,
and brown-colored seedlings to dead seedlings. However, low infestation of
T. claytonj did not appear to affect pine seedlings. Sutherland and Adams
(196^*) reported that T. claytoni was parasitic on but not pathogenic to red
pine seedlings (Pinus resinosa Ait.). Ruehle (1966) established that T_,
claytoni is parasitic on loblolly, longleaf, pond, sand slash, shortleaf,
and Virginia pines.
Sher (1958), and Barker and Worf (1966) reported that parasitism by
T. cla:/-boni resulted in retarded growth of azalea plants. Barker and Worf
also found that T_. claytoni accentuates the appearance of foliage chlorosis
of azalea plants.
MATERIALS AND METHODS
Pathogenicity
General Methods
In December, I965, soil infested with moderate numbers of Rotylenehulus
reniformis , Helicotylenchus dihy stera , and lov numbers of Tylenchorhynchus
claytoni vas collected from the North Florida Experiment Station at Quincy,
Florida. The soil was processed using the elutriation technique (Seinhorst,
1956) and a modified Baermann funnel technique (Christie and Perry, 1951).
Larvae , eelworm-shaped females and males of R_. reniformis were isolated
individually and randomly and each of several pots were inoculated with a
total of 100 specimens. The pots were divided into five groups and each group
was planted to bean (Phaseolus vulgaris Savi . ) , cotton (Gossypivim hirsutum
L. ) , sweet potato (impomoea batatus Lam.), tobacco (Nicotiana tebacvur. L. )
or tomato (Lycopersicon esculentm Mill.). Large numbers were recovered from
tomato and thus this host was selected for the increase and maintenance of
populations of R. reniformis .
One hundred gravid females (males are not known to occur) of H. dihystera
were placed in 15-cm plastic pots and either tobacco or tomato seedlings
were planted. Reproduction was greater on tomato and thus this host was used
to maintain populations of H. dihystera also. This soil did not contain suf-
ficient numbers of T. claytoni to establish colonies.
In Jiine, I966, soil containing large numbers of T. claytoni was collected
near Quincy, Florida. The samples were processed by the elutriation technique
- T -
- 8 -
(Seinhorst, 1956) and large numbers vere recovered. Kales and females vere
individually isolated and each of several pots vaB inoculated vith a total
of 200 specimens. A tobacco seedling then was planted in each pot and after
three months populations of the parasites increased several times the original
inoculm.
All nematode colonies vcrc maintained in an air-conditioned greenhouse
at 26+ h°C, About 28-1*2 days prior to initiating the experiments, tobacco
seeds were sown in a flat containing autoclaved soil. The flats of seedlings
were maintained in the greenhouse and received fertilizer and water as needed.
The soil used to colonize the nematodes and used in Experiment I was
collected from the North Florida Experiment Station, autoclaved at 18 psi for
30 minutes, and allowed to aerate a few days before use. Then 2,000 ml was
placed in each of several 15-cm plastic pots.
Experiment I. - '
This experiment was initiated on October 14, 1966, and terminated on
February 20, 196?. Tobacco seedlings were 36 days old at the time of
inoculation. The following nine treatments for this experiment were repli-
cated fovr times:
1. Inoculated with 500H^
2. " " 500T
3. tt t, 1,000H
^. " " 1,000T
5. " " 500H +
500T
6. " " 2,000H
7. " " 2,000T
8. Inoculated \ ./ h ! ;
1,000H +
1,000T
9.
Uninocxilated
a,
'H = Helicotyle; -.us dihystera, T = Tylr..\ohorhynchus claytoni
Pot arrangement vas a completely randomized block design. Specimens
for inoculations were obtained from the greenhouse colonies described above.
Nematodes were extracted from the soil by using an elutriation technique
(Seinhorst, 1956). The specimens were used within U8 hours after isolation
from the soil. Nematodes in all treatments were hand-picked with the aid
of a dissecting microscope.
Inoculation was performed by adding the requisite numbers of nematodes
as a water suspension into a hole made in the soil in a pot, then a tobacco
seedling was planted in the same hole and water was added. Pots then were
randomly arranged on a greenhouse bench. A liquid fertilizer solution (Orthci^
12-6-6) vas applied every two weeks, and plants were watered as required.
Plants heights were recorded at monthly intervals, and "ripe" leaves
were primed on three different dates and the green weights recorded.
Upon termination of the experiment the remaining leaves were primed and
weighed. Weights of leaves at the three different primings and at the termin-
ation of the experiment were combined. The stalks then were cut off at the
soil line and plant heights were measured. After the stalks were cut the
roots were removed from the soil, washed gently in r\mning water, blotted
with paper towels, and weighed. The roots then were examined for nematode
injury with the aid of a dissecting microscope. The soil was removed from
the pots, thoroughly mixed, and two 100 ml samples were processed by using
a rapid centrifugal- flotation technique (Jenkins, 196h). The nematodes re-
covered were rinsed into a graduate cylinder and enough water was added to
- 10 -
make 250 ml. Specimens were counted in five aliquot s of 10 ml each and
populations for each sample determined. An average vas taken of the tvo
samples and the numbers of nematodes per pot calculated.
Experiments II, III, and IV
Soil used in these experiments vas collected from a field near the
North Florida Experiment Station. This soil was fumigated with methyl bromide
at the rate of two pounds per cubic yard for hB hours and allowed to aerate
in an open shade area for two weeks. Then 2,000 ml were placed in each of
several 15-cm plastic pots. At this time sor.e of the soil was processed for
nematodes and none was found.
Experiment II, a repetition of Experiment I, was initiated on Jvme l6,
1967, and terminated on September U, I967. The tobacco seedlings were hi days
old when transplanted and inoculated.
Experiment III vas initiated on June 3, 19^7, and terminated on August
12, 1967. The seedlings used were 28 days old. The 15 treatments each
replicated four times were as follow:
1. Inoculated with 500H
2. " " 5OOR
3. " " 5OOT
I*. " " 1,000H
5. " " 1,000R
6. " " 1,000T
7. " " 5OOH +
5OOR
8. " " 50OR +
5OOT
9. " " 5OOH +
5OOT
10. Inoculated with
500H +
500R +
50OT
11.
II
n
1,500H
12.
n
M
1,500R
13.
n
ft
1,500T
n
n
1,500H +
1,500R +
1,500T
15. Uninoculated
s-H = Helicotylenchus dihystera, R = Rotylenchulus rerxifornis,
T = Tylenchorhynchus claytoni
Experiment IV, a repetition of Experiment III, was initiated on
July 28, 1968, and terminated on September 8, I968. Tobacco seedlings were
1+2 days old at the time of inoculation. .
The pot arrangements in these three experiments were completely
randomised block designs. Nematode specimens for inoculations were obtained
from the greenhouse colonies described above. Specimens were isolated from
the soil by using either an elutriation technique (Seinhorst, 1956) or a
modified Baermann funnel technique (Christie amd Perry, 1951) or both. The
specimens were used within 36 hours after isolated from the soil. To obtain
the ntmbers of specimens needed for inoculation, the number of specimens in
one ml of water was determined and the correct number of milliliters of water
was used for each treatment. Inoculations and maintenances were performed
as previously described for Experiment I. On termination of the experiments
leaves were primed and weighed. The stalks were cut off at the soil line and
plant heights were measured. The soil and roots were removed from the individ-
ual pots and placed in a 10-liter plastic pail to which 5,000 ml of water were
- 12 -
added. The roots vere moved about in the water to separate them from the
soil after vhich they were removed from the pail, vashed in running vater,
blotted with paper towels, weighed and excmined. The water and soil in each
pail was roiled, an aliquot of 1,000 ml taken immediately and then processed
using a modified Baermann funnel technique (Christie and Perry, 1951) for
Experiments II and III, and a rapid centrifugal-flotation techniq.ue (Jenkins,
196k) for Experiment IV. The specimens recovered were counted by the aid of
a dissecting microscope and the numbers of specimens in each pot were ccnputed.
Females of R. reniformis which were embedded in the roots were not included
in the computations.
Histopathology of Rotylenchulus reniformis on Tobacco Roots
On July U, 1968, thirty day-old seedlings of the shade-grown tobacco
variety 'Dixie Shade' were placed in ih-cm plastic petri dishes painted
black. The plants were placed through slits made in the edge of the petri
dishes with stems and leaves outside. At the point of exit, the stems were
wrapped in cotton. Nematode inoculum composed of a mixture of larvae, males
and eelwona-shaped females of R_. reniformis was pipetted onto the roots which
then were covered with autoclaved builder's white sand and watered with rain
water which had been collected for this purpose. Uninoculated plants served
as controls . Plants were watered alternately with rain water and Hoagland
solution as required by using polyethylene squeeze bottles. The experimental
plants were kept in a growth room at approximately 2T°C. A l6-hour photo
period was used.
One month after inoculation the plants were removed from the petri dishes
and the roots were examined for nematode infection and injury. Some of the
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inoculated roots were stained in acid fuchsin-lactophenol and cleared in
lactophenol (McBeth et al . , 19^1)- Sections of roots 2-3 cm in length in
vhich saccate females were embedded and comparalDle sections of iininfected
roots were placed for at least 2h hours in Craf III fixative (Sass, 1958)
containing chromic acid (30 ml of one per cent), acetic acid (20 ml of
10 per cent), formaldehyde (lO ml of 37. 5 per cent) and vater {hO ml). They
were dehydrated "by a normal butyl alcohol series, embedded in 'Tissuemat'
(60°C. m.p.) and sliced in cross- and logitudinal-sections , 10 micron thick,
with a rotary microtome. Sections were affixed to glass slides with Haupts
adhesive and stained using Johansen's quadruple stain method (Johansen, 19^0).
The stained sections were mounted in 'Permount' and examined with the aid of
a compound microscope.
RESULTS
Pathogenicity
Experiment I
Plant growth was almost uniform throughout the experiment and no
symptoms on the above-ground plant portions were observed at any time.
Data on plant heights taken at 30, 73, 90 and 128 (terminal) days after
inoculation showed no significant differences between inoculated and un-
inoculated plants (Table l). Green weights of leaves taken at three dif-
ferent primings and at the termination of the experiment were combined. An
analysis of variance of the data on leaf weights disclosed no significant
differences between the inoculated and uninoculated plants (Table 2). An
analysis of variance of the data on root weights disclosed highly significant
differences between treatments (Table 2). Duncan's multiple range test re-
vealed that the root weights of the uninoculated plants were significantly
higher at the one per cent level than those of plants inoculated with each
of the three population levels of T. claytoni . Root weights of the
uninoculated plants were also significantly higher at the five per cent
level than those of plants inoculated with 500 specimens of K. dihystera +
500 specimens of T. claytoni . Differences were not significant between the
roots of uninoculated plants and those of plants inoculated with 1,000 and
2,000 specimens of H. dihystera or 1,000 specimens of H. dihystera + 1,000
specimens of T. claytoni »
- Ih -
Table 1. — Means of heights (cm) of tobacco plants measured on four dif'
ferent dates after inoculation on October lU, 1966.
Treatments
Days after
inoculation
30*
, Y3*
90*
1.
500H^
26.0
56.0
77.2
113.5
2.
50 OT
27.5
57.2
77.5
113.7
3.
1,000H
25.2
55.2
76.2
112.7
k.
l.OOOT
29.2
53.7
71.7
103.7
5.
5OOH +
118.5
5OOT
27.0
58.7
80.7
6.
2,000H
31.2
60.2
81.0
- 120.2
7.
2, COOT
26.7
52.7
72.5
101.2
8.
l.OOOH +
1,OOOT
29.2
55.7
- 73.2
102.2
9.
Uninoculated
29.5
61.0
81+. 0
122.2
* = Differences not significant at the 5 per cent level of probability.
^ = Heli cotylenchus dihystera, T = Tylenchorhynchus claytoni
- 16 -
Table 2. — Means of tobacco leaf and root veights (g) and the populations
of nematodes 128 days after inoculation on October ll+, I966.
Treatments
b
Nematodes
Leaves^
Roots
Populations
Increas(
1.
500H*^
80.2
28.9^
3,3^5
6.5X
2.
5OOT
87.2
28.1,
29.9^"
32.9^^
58,000
ll6x
3.
k.
1,000H
l.OOOT
85.3
83.6
1^,250
117,875
^X
118X
5.
5OOK +
86.9
1,995
kx
5OOT
93,625
187X
6.
2,000H
83.6
10,830
5.5X
7.
2,000T
87.9
211,500 .
105X
8.
1,000H +
86.2
!+,337
hx
l.OOOT
115,000
115X
9.
Uninoculated
Qk.k
h2.f
0
OX
Differences not significant at the 5 per cent level of probability-
Differences isgnificant at the 1 per cent level of probability.
Treatment means having the same letter are not significantly
different.
H = Heli cotylenchus dil?ystera, T = Tylenchorhynchus claytoni
- IT -
Root systems of pleints inoculated with T. claytoni exhibited some dis-
coloration and shriveled appearances. Well defined lesions or extensive
root necrosis vere not observed.
In every case the number of nematodes increased over the initial
inoculum. The numbers of H. dihystera increased as follows: on plants
inoculated with 500 specimens about six and one-half times, with 1,000
specimens about four times, and with 2,000 specimens about five and one-half
times. The nmbers of T. claytoni increased as follows: on those plants
inoculated with 500 specimens about ll6 times, with 1,000 specimens about
ll8 times and with 2,000 specimens about 105 times. Nematodes on those plants
inoculated with H_. dihystera + T. claytoni increased as follows : with 500
specimens of each, H_. dihystera increased about four times whereas T_. claytoni
increased about 18? times; and with 1,000 specimens of each, H. dihystera
increased about four times and T. claytoni about 115 times (Table 2).
. . I ' . , . iv, .
Experiment II 'r ■ .• -■
^ Symptoms of injury were not detected during the experiment. Inoculated
and uninoculated plants were uniform in general appearances in all treatments
and plant heights were not significantly different (Table 3). Green weights
of the leaves and weights of the roots at termination of the experiment were
not significantly greater for uninoculated plants than for inoci;dated plants
(Table 3). Examination of the root systems of inoculated plants revealed no
discoloration or other symptoms of root injury.
Populations of T. claytoni increased during the course of the experiment
at all three levels of inoculum. The nematodes increased about 30 times
when 500 specimens were used as inoculum, about 17 times when 1,000 specimens
were used and about 10 times when 2,000 specimens were used. Numbers of
Table 3. — Means of plant heights (cm), and green leaf and root weights (g)
of tobacco, and the teminal populations of nematodes, 80 days
after inoculation on June l6, I96T.
Treatments
Heights^
3»
Weights
Leaves
Roots
Nematode
Populations
1.
500H^ .
160.0
197.6
75.7
257
2.
5OOT
162.0
200.2
78.8
15,063
3.
1,000H
158.5
190. U
75.lt '
725
k.
1,000T
171.2
138.1
86.9
17,6!;8
5.
5OOH +
163.7
197.9
72.5
182
5OOT
. 9,572
6.
2,000H
161.7
188.9
81.8
1,617
7.
2,000T
157.7
192.8
69.6
20,552
8.
1,000H +
169.5
196.8
81. U
393
1,000T
89.1*
17,71*1
9.
Uninoculated
I6J+.7
190.0
0
^ Differences not significant at the 5 per cent level of probability.
H = Helicotylenchus dihystera, T = Tylenchorhynchus claytoni
Each treatment replicated four times.
H. dihystera at termination of the experiment averaged fever than the initial
num'bers in all cases . In combination with K_. dihystera > T_. claytoni in-
creased about 19 times vhen 500 specimens of each species were used as
inoculum, and about I8 times vhen 1,000 specimens of each species vere used
(Table 3).
Experiment III
Differences in grovth between the inocxilated and tininoculated plants
could not be detected during the covirse of the experiment. Symptoms of
injury to above-groimd portions of the inoculated plants vere not observed.
The heights, green veights of leaves and fresh weights of roots of inoc\ilated
and iininoculated plants were not significantly different (Table h) . No dis-
coloration or other symptoms of root injury were evident.
Saccate females , some with egg masses , of R. reniformis were found
embedded in roots of plants inociilated with specimens of this parasite.
Malformation or swelling of the infected root, at or near the feeding sites
were not observed. Neither necrosis nor lesions were evident.
Final populations of T^. claytoni in every case increased over the
inoculum but the numbers of H. dihystera and R. reniformis decreased whether
inoc^llated alone or in combination (Table h) .
Experiment IV
The results of this experiment were very similar to those of Experiment
III, differing only in the final populations of the parasites. Symptoms of
damage to the above-ground plant parts due to nematodes were not observed.
Data on plant heights, green weights of leaves and fresh veights of roots
revealed no significant differences betveen inoculated and uninoculated plants
(Table 5).
-to-
llable h. — Means of plant heights (cm) green leaf and root veights (g) of
tobacco and terminal populations of nematodes, 70 days after
inoculation on June 3, 19^1-
Treatments
Heights^
Weip;hts^
Leaves
Hoots
Kematode
Populations
pUUii
1 lift 9
1+8 Q
175
2 .
xdi, (
±?0. ?
k"^ ?
86
3.
500T
±d.ti .d
14 ( . p
P J.
4.
IjOOOH
13'+.T
±>4. f
5.
1 jUUUr!
■l? 0
108
D .
X ,UUUi
XcLO, ?
n ^li n
XP'+ . u
k'^, 8
8 025
T.
C AnTJ -1.
pOOH +
Hp .
X
Ann
U5.6
125
o
u.
CAAR 4.
1 Sli 7
X^H . 1
70
500T
2,1+75
0
CAAU +
ISl* 2
52.1
120
500T
1,971
10.
500H +
.130.7
11^7.7
I49.5
160
500R +
96
500T
2,752
11.
1,500H
122.5
11*2.5
1+6.8
388
12.
1,500R
126.5
li*5.5
1*5.1
130
13.
1,500T
131.5
161.7
52.3
13,137
l!+.
1,500H +
133.2
151.7
50.7
560
1,500R +
2I+7
1,500T
38.1
12,271
15.
Uninoculated
123.5
153.0
0
* Differences not significant at the 5 per cent level of probability.
H = Helicotylenchus dihystera, R = Rotylenchulus reniformis ,
T = Tylenchorhynchus claytoni
Each treatment replicated four times.
- 21 -
In. each case the average numher of nematodes on termination of the
experiment was greater than the nimber used as inoculum (Table 5).
Histopathology
Adult females of Rotylenchulus reniformis ranging from eelvorm to
kidney-shaped (Fig. 1 A-E) were found in the stained tobacco roots. No life
cycle stage was found with the body totally within a root. The majority of
specimens were found in the secondary roots with developing females near the
root tips and more mature kidney-shaped females (Fig. 1 D, E) some distance
away from the root tips. Some of these females had deposited eggs into the
gelatinous matrices, others had secreted the matrices but had not deposited
eggs, and still others had not yet secreted the matrix. In all cases the
posterior paxt of the female and the matrix and eggs were outside the roots
(Fig. 3). On several occasions two or more specimens were found feeding in
the same area (Fig. 1;), but this was not the usual case.
This parasite fed in the stele of the roots, apparently on pericyclic
cells (Fig. 5, 6, 7 A). The head was never found in the pericycle but
appeared to remain in the endodermal region with the stylet penetrating the
pericyclic cells. Cells fed upon enlarged in size to crush cells of the
stele and endodermis. The hypertrophied pericycle cells in many cases en-
circled the vascular cylinder (Fig. 3) and extended longitudinally 15-19 cells
along the root (Fig. 8). This condition was most often associated with
females which had produced eggs. A lesser number of cells was affected
where the females were immature (Fig. 9 A-E).
The enlarged cells stained darker than other root cells and contained
enlarged nuclei (Fig. T A, 8, A, B). Affected cells near the head of a
Table 5. — Means of plant heights (cm) green leaf and fresh root weights (g)
of tobacco, and terminal populations of nematodes, k2 days after
inoculation on July 28, I968.
Treatments Heights^ Weights^ Nematode
Leaves
Roots
Populations
1.
5OOH
70.7
71.6
27.5
l,Ji00
2.
5OOR
66.7
72.1
31.0
1,356
3.
5OOT
70.5
76.0
31.1
19,293
h.
1,000H
69.7
72. u
29.5
2,8U3
5.
l.OOOR
69.7
73.2
31.7
3,517
6.
1,000T
67.0
73.1
32,558
T.
5OOH +
70.2
76.1
3h.2
866
5OOR
1,566
8.
5OOR +
67.0
72.0
30.9
1,251
5OOT
9,878
9.
5OOH +
67.2
73.3
35.0
761
5OOT
10.
5OOH +
69.0
70.1*
31.5
866
5OOR +
1,1488
5OOT
7,761
11.
1,500H
69.5
70.3
31.6
3,576
12.
1,500R
69.5
80.5
' 36.0
5,136
13.
1,500T
6h.o
Ih.l
35.3
li8,92l
Ik.
1,500H +
65.7
73.3
36.5
1,767
1,500R +
2,563
1,500T
16,021
15.
Uninoctilated
67.7
Ih.l
35.7
0
Differences vere not significant at the 5 per cent level of probability.
H = Heli cotylenchus dihystera.R = Rotylenchulus renif ormis ,
T = Tylenchorhynchus claytoni
Figxire 1. — Photomicrographs of adiilt females of Rotylenchulus
reniformis. A, Eelvorm shape; B, C, D, developing stages; E, mature
kidney- shape. F, An enlargement of A, showing vulva (v). Didelphic
reflexed ovaries are not discernible in this photograph.
Figure 2.
culated plant.
— A, Cross section viev of a tobacco root from an unino-
B, An enlarged view of A.
Figure h. — Photomicrograph of two females of Rotylenchulus
reniformis feeding in the same area of a tobacco root. Note the
dissolution of cell walls in the hypertrophied cells.
- 28 -
Figure 5. — Cross section view of a tobacco root infected with
Kotylenchulus reniforais showing the nematode head positioned near the
hjrpertrophied peri cycle. Note the dark stained structure (a) in the cell
near the nematode head (b).
.tMnlJ'^T of a portion of Fig. 5. Ifote the dark
^M^r./ '"i^^* ^ f ^ ^^^^^-^^ connecting from the
point of stylet insertion (b) into the hyoertrophied cell
- 29 -
Figure 7.— A, Cross section view of a tobacco root infected vith
Rotylenchulus reniformis. Note that some hypertrophied cells are in
double layers. B, A comparable uninfected root.
nematode vere larger than those more distant, giving a spindle-shaped appear-
ance in longitudinal section (Fig. 8 A). Occasionally cell vails near the
feeding site were dissolved (Fig. 10), a condition vhich was more prevalent
and usually more extensive where more than one specimen fed at the same site
(Fig. U).
The vails of two or more cells near the head of a parasite were thickened
(Fig. 10). Pink-stained tubular strands were sometimes seen in a cell to
which the lips of a nematode were appressed (Fig. 6). These strands vere
curved and appeared to be connected to or near the stylet of the nematode.
Figure 8.— A, Viev of a longitudinal section of tobacco root
infec.ed by Rotylenchulus reniformis shovin/r hyrertrophied cells and
enlarged nuclei. Note the decreasing size of the hypertrophied cells
avay from the site of feeding (a). B, An enlarged viev of a portion
1
Figure 9. — A-E, Photomicrographs of serial sections of a tohacco
root infected by Rotylenchulus reniformis showing the developnent of
hypertrophied cells (arrows) which apparently originated in the pericycle
A is more distant and E is nearest the feeding site. Note the increasing
numbers of hypertrophied cells from A to E. Anterior parts of the nema-
tode are not seen but posterior portions can be seen at the top of D and
E. F, A comparable cross section view of an uninfected root.
I- '
- 3h -
Figure 10. — Photomicrof^raph of a tobacco root shoving two nuclei
(a) of a hypertrophied cell vhich had been fed upon "by Rot;ylenchulus
reniformis. Note the thick cell vails near the stylet insertion (b).
The nematode head (c) was moved away from the point of stylet insertion
during the preparation process.
DISCUSSION
Pathogenicity Experiments
Experiment I
Specimens of T. claytcni fed as ectoparasites on toTsacco and reproduced
in large numbers under the conditions of the experiments. The spiral
nema.tode, H_. dihystera, also parasitized tobacco roots but did not reproduce
in ntimbers as large as did T. claytoni. Single aJid multiple inoculations of
these tvo species caused no differences in top growth of plants when com-
pared to the uninoculated plants. Single or multiple inoculations with
different inoculum levels of T^. claytoni caused no stunting of plants, in
contrast to the results reported by Graham (195^). Quantitative yields of
the plants as indicated by green weights of leaves were not affected by this
parasite. It is possible that quality of the tobacco might have been af-
fected due to parasitism by T_. claytoni.
J ' All three inoculum levels of T_. claytoni with an exposure period of 128
days resulted in reduced root systems of parasitized plants when compared to
those of the control plants. Growth of the above-ground portions of the
plants was not affected by the root reduction.
J i No explanation can be offered for the root symptoms expressed in the
experiment when the plants were each inoculated with 500 specimens of H.
dihystera. The fact that no symptoms were visible at the higher population
levels indicates, however, that the root retardation was due to factors
- 36 -
other than the presence of this parasite. Root retardation in the T.
claytoni treatments was always accompanied by some discoloration and
shriveled appearance.
Inoculations with 500 specimens of T. claytoni + 500 specimens of H.
dihystera also resulted in retarded root systems of the plants. It is
probable that T. claytoni was primarily responsible for the retardation.
\\'here T. claytoni and H. dihystera were inoculated in combinations (500 or
1,000 specimens each), terminal populations of each species were about the
same as when each was inoculated alone at comparable levels. It was con-
cluded that neither T. claytoni nor H_. dihystera suppressed or stim\ilated
reproduction of the other species.
Experiment II '
Treatments in this experiment were similar to those in Experiment I
but differed in time of year, a shorter period of exposure and the use of
ftmigated soil for Experiment II. Nevertheless, plant growth was better
than that in Experiment I. The better growth was probably due to a longer
day length and the use of fumigated soil.
Terminal populations of T^. claytoni in every case were several times
greater than the inoculum level (Table 3). However, the terminal numbers
were not so great as those of Experiment I, possibly due to the shorter
duration of the experiment, the different method of extracting nematodes
from soil and the use of fumigated soil.
The numbers of H_. dihystera recovered on termination of the experiment
were less than the inoculum used, whether inoculated alone or in combination
with T. claytoni (Table 3). Nonetheless, all the recovered specimens had
dark intestines indicating continuous feedinR. It is probable that the small
number recovered was due to inefficiency of the extraction technique. V/eber
and Williams (1968) reported recovery of only 53.3 per cent of H_, erythrinae
vith the centrifu^!;al- flotation technique. Reproductive potential of H.
dihystera might also be low. The life cycle of this species is not known,
but Taylor (19d1) reported that the life cycle of H, microlobus , a similar
species, was completed on tomato in 35 days at 22.80C and at least 30 days
at 26.T°C. If the life cycle of H. dihystera is of similar duration, the ex-
posure period of 80 days would have been sufficient for two or at most three
generations in this experiment. Furthermore, tobacco may not be a favorable
host for H. dihystera.
Experiment III
All three nematode species were used in this experiment in single and
multiple inoculations. No significant differences occurred between inoculated
and uninoculated plants (Table h) but means of plant heights, leaf weights
and root weights were smaller than those of plants in Experiment II, pre-
sunably due to the shorter period of the experiment. Less space was allowed
between the pots and this may have hampered plant growth. Terminal popula-
tions of nematodes were determined using the modified Baermann funnel
technique (Christie and Perry, 1951). Treatments that were inoculated with
T. claytoni alone or in combination with one or both of the other species
resulted in terminal populations of T. claytoni greater than the inoculvtm.
However, terminal populations of H. dihystera and R. reniformis recovered
were less than the inoculum whether inoculated alone or in combination with
one or both of the other species. The Baermann funnel as used was not an
efficient method of extracting nematodes from soil and this was undoubtedly
- 38 -
the primary reason for the smaller numters recovered. The relatively-
short period of exposure (JO days) might have heen a contributing factor
also. Other possible reasons were discussed under Experiment II.
Experiment IV
This experiment was a repetition of Experiment III except that the ex-
posure period was shortened to k2 days. While plant heights, leaf weights
and root weights were less than those of plants in previous experiment s, no
symptoms of injury were observed. In this experiment the rapid centrifugsil-
flotation technique (Jenkins, 196k) was used to isolate nematodes from the
soil. In spite of the relatively short duration of the experiment all three
nematode species were recovered in greater numbers than used as inocxilum in-
dicating that this method of removal is more efficient than the Baermann
funnel. Also no determination was made of the number of females of R.
reniformis embedded in the roots . The fact that T. claytoni was always re-
covered in larger numbers than were the other species demonstrates that the
reproductive potential of T. claytoni obviously is greater than that of H.
dihystera and R_. reniformis under the conditions of these experiments.
When all three nematode species were inocxxlated alone at 500, 1,000
and 1,500 specimens terminal populations of each k2 days after inoculation
increased in proportion to the inoculum level (Fig. 11). Helicotylenchus
dihystera increased an average of about 2.6 times, Rotylenchulus reniformis
about 3 times and Tylen chorhyn chus claytoni about 3^ . 5 times .
Populations of H. dihystera k2 days after inoctaation were not signifi-
cantly different when 500 specimens were inoculated alone than when 500
specimens were inoculated in combination with 500 T. claytoni or 500 R.
reniformis or both (Table 6). The same relationship held when 1,500 specimens
- 39 -
50,000
1*0,000
30 ,000
20,000
10,000
I I Number recovered
Original numbers
m
H_. dihystera
R. reniformis
2ES
:v"'"'t
T. claytoni
Figure 11. —Comparison of terminal population levels of three nematode
species at different levels of inoculum k2 days after inoculation.
WW
- ho -
Table 6. — Comparison of terminal popiilations of Helicotylenchus
dihystera inoculated alone and in combinations as indicated.
Replications
Treatments I II III IV Means
500H
9^45
1,295
1,260
2,100
8U0
1,1+00
5OOH +
525
665
1,1*35
866
5OOR
T61
5OOH +
805
560
1,155
525
5OOT
866
5OOH +
595
735
92+5
1,190
500R +
500T
Differences were not significant at the 5 per cent level of
probability.
H = Heli cotylenchus dihystera, R = Rotylenchulus reniformis
T = Tylenchorhynchus claytoni
- l4l -
Table 7. — Coaparison of terminal popiilations of Hotylenchulus renifonais
inoculated alone and in combinations as indicated.
Treatments
I
Replications
II III
IV
1
Means
500R^
1,1*70
910
665
1,365
500R +
980
1,995
1,925
1,365
1,556
500H
500R +
' 630
770
2,625
980
■ 1,251
500T
5OOR +
1,715
1,020
1,785
1,1+35
1,1*88
5OOH +
5OOT
Larvae, males and eelworm-shaped females vere included.
Differences vere not significant at the 5 per cent level of
probability .
R = Rotylenchulus reniformis , H = Heli coty lenchus dihystera
T = Tylenchorhynchus claytoni .
of each species vere used (Table 9). In both cases, however, the means
were hiRher when H_. dihystcra was used alone.
/ Populations of n_. reniformis h2 days after inoculation were not sipp:iifi-
cantly greater when 500 specimens were inoculated alone than when 500
specimens were inoculated in combination with 500 specimens of T. claytoni ,
500 specimens of H. dihystera or both (Table 7). However, when 1,500
specimens of R. reniformis were inoculated alone and when 1,500 were inocu-
lated with 1,500 T. claytoni and 1,500 H. dihystera differences were signifi-
cant at the 5 per cent level (Table 10). While the presence of the other
nematodes did not effect reproduction of R. reniformis at the lower inoculum
levels, they did have an apparent effect at the higher level.
Populations of T. claytoni k2 days after inoculation were not signifi-
cantly different when 500 specimens were inoculated alone than when 500
specimens were inoculated with 500 H. dihystera. 500 R. reniformis or both.
The means, however, were higher when inoculated alone (Table 8). When 1,500
T. claytoni were inoculated alone and when 1,500 were inoculated with 1,500
ii* ^i^-ystera and 1,500 R_. reniformis differences were significant at the 1
per cent level (Table 11). The presence of the other two nematodes definitely
influenced reproduction by T. claytoni. The influence probably was competi-
tion for feeding sites and/or food.
Histopathology
Penetration of roots by the parasite was not observed, but since
eelworm-shaped females were observed near the root tip and more mature
females farther away, it seems apparent that the eelworm-shaped females
penetrated near the root tips. The anterior portion of their bodies
- 1*3 -
Table 8. — Comparison of the terminal populations of Tylenchorhynchus
claytoni inoculated alone and in combinations as indicated.
Replications
Treatments
I
11
III
IV
Means^
500T^
5,635
18,31+0
10,850
1+2,350
19,293
500T +
l+,itlO
It ,690
8,365
l+,305
5,1+1+2
500H
500T +
7,105
11,970
10,780
9,660
9,878
500R
500T +
1,9^3
8,925
8,015
6,160
7,761
5OOH +
5OOR
Difference were not significant at the 5 per cent level of probability.
T = Tylenchorhynchus claytoni , H = Heli cotylenchus dihystera,
R = Rotylenchulus reniformis.
Table 9. — Comparison of the terminal populations of Heli cotylenchus
dihystera inoculated alone and in combination with Rotylenchulus
reniformis and Tylenchorhynchus claytoni .
Replications
Means ^
Treatments
I
II
III
IV
1,500H^
1,500H +
1,500R +
1,500T
1,995
1^,165
1,330
2,730
2,205
1,995
1,5^0
3,576
1,767
Differences were not significant at the 5 per cent level of
probability.
H = Helicotylenchus dihystera, R = Rotylenchulus reniformis
T = Tylenchorhynchus claytoni .
Table 10.— Comparison of terminal populations of Rotylenchulus renlformis
inoculated alone and in combination with Helicotylenchus
dihystera and Tylenchorhynchus claytoni.
Replications
Treatments
I
II III
IV
Means
1,500R° !
1,500R + {
1,500H +
1,500T
3,7^5
2,555
3,955 6,U05
1,715 3,500
6,Ul40
2,U85
5,136
2,563
a Only larvae.
males
and eelworm-shaped females
vera included.
^ Differences significant at the 5 per cent level of probability.
° R = Rotylenchulus reniformis, H = Helicotylenchus dihystera
T = Tylenchorhynchus claytoni.
- 1+6 -
Table 11. — Comparison of terminal populations of Tylenchorhynchus claytoni
inoculated alone and in combination vith Helicotylenchus
dihystera and Rotylenchulus reniformis.
Replications
Treatments
I
II
III
IV
Keans^
1,500T^
1,500T +
1,500H +
1,500R
28,560
11,795
62,1*75
5,390
27,650
20,125
77,000
26,775
1*8,921
16 ,021
^ Differences significant at the one per cent level of probability.
^ T = Tylenchorhynchus claytoni , H = Helicotylenchus dihystera
R = Rotylenchulus reniformis .
pentrated the epidermis, cortical parenchyma and endodermis and only the
stylet penetrated the stele. As the nematode "began to feed on a pericyclic
cell its esophsigeal gland secretions entered the cell to alter the proto-
plasmic contents. The cell began to enlarge and then stained darker than
surrounding cells. As feeding continued other pericyclic cells were affected
hoth in a circular and in a longitudinal direction. The cells neeir the head
of the nematode were larger than those away from the head, due apparently to .
the fact that these cells received the stimulatory esophageal gland secretions
in more concentrated form than did the more distant cells. In a few cases
cells other than pericycle became involved, probably protophloem or protoxylem
cells .
As the females matured the root continued to grow so that mature females
were always found some distance from the root tip.
Males were never observed feeding on tobacco roots^ which agrees with the
statement of Linford and Oliveira (19^0). Occasionally, males were found in
the gelatinous matrix secreted by the female, but copulation was not observed.
Birchfield (I962) reported that R. reniformis fed on phloem in the
cotton plant, and Linford and Oliveira (19^0) reported that it fed on the
cortex in cowpea. Apparently different tissue is used as the feeding site,
depending on the host plant, the population of the nematode or both.
Cytoplasmic response of pericyclic cells to the feeding of R_. reniformis
on tobacco roots was similar to the response of cortical cells of susceptible
citrus roots fed upon by Tylenchulus semipenetrans Cobb (Van Gundy and
Kirkpatrick, 1961*). However, no cell enlargement occurred in citrus nor was
the area involved so large as in tobacco.
Mankau and Linford (1960) reported tubular strands in the syncytium of
•Ladino' clover fed on by Heterodera trifolii Goffart, but they reported
- 1*8 -
these strands as "being hyaline. The strands in tobacco stained pink. The
strands are apparently related in some way to feeding as suggested by Mankau
and Linford (196O) but their exact nattire remains unknown.
i
SUMMARY
Tylenchorhynchus clnytoni , Helicotylenchus dihystera, and
Rotylenchulus reniformis are parasites of shade-grown tobacco, and are
found frequently in shade-Rrown tobacco areas of North Florida.
Pathogenicity experiments using shade-groxm tobacco variety 'Dixie
Shade' indicated that single inoculation of T. claytoni at levels of
500, 1,000, or 2,000 specimens per 2,000 ml of soil significantly re-
tarded root production of tobacco plants 128 days after inoculation.
Some discoloration and shriveled appearance accompanied root retardation,
but lesions or necrosis were not evident. Exposure periods of U2, TO
or 80 days did not result in root retardation.
Multiple inoculations of specimens of T. claytoni with either
^* dihystera or R_. reniformis or both at any inoculum level usually did
not affect root production of plants compared to the uninoctilated plants.
Inoculations with H. dihystera or H. reniformis alone or in combina-
tion did not affect top growth when compared to xminoculated plants.
Terminal populations of T« claytoni when inoculated alone or in
combination with ff. dlhyatera, rem 1 feral s or both increased several
times over the inoculvim levels. Terminal populations of II. dihystera and
R_. reniformis were in most cases greater than the inoculum levels but num-
bers were not as great as with T^. claytoni . Populations of li. dihystera
h2 days after inoculation were not significantly different when 500
specimens were inoculated alone than when 500 specimens were inocvilated in
_ 1»9 -
combination vith 500 T. claytoni, 500 H. reniformis or both. The sa^-ne re-
lationships held vhen 1,500 specimens of each species vere used. Populations
of R. reniformis ^2 days after inoculation vere significantly greater vhen
1,500 specimens were inoculated alone than when 1,500 specimens were in-
oculated in combination with the equal numbers of T. claytoni and H.
dihystera. The same relationships did not hold true when 500 specimens of
each species vere used. Populations of T. claytoni U2 days after inocula-
tion were significantly greater when 1,500 specimens were inoculated alone
than when 1,500 specimens were inoculated with 1,500 H. dihystera and 1,500
R. reniformis. However, when 500 specimens of each species were used there
were no significant differences in terminal populations of T. cla>-toni.
Histopathological studies of tobacco roots infected by Rotylench^xLus
reniformis disclosed:
1. Eelworm-shaped females penetrated tobacco roots near the root tips
to initiate infection.
S. Papasltea penetrated the epiderrais, cortical parenchyma, and
endodermis and fed in the pericyclic region. Only the stylet penetrated
into pericyclic cells.
3. Hypertrophy and enlargement of nuclei occurred in the pericyclic
region due to the feeding of the nematodes.
h, Hypertrophied cells extended from a cell in which the nematode sty-
let was inserted to cells in both a longitudinal and a circular direction.
5. The cytoplasm of hypertrophied cells stained darker than did unin-
fected surrounding cells.
Considering the data on pathogenicity and the results of histological
studies, H. dihystera, T. claytoni , and R_. reniformis all appear to be weak
pathogens of tobacco but none a severe pathogen.
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BIOGRAPHICAL SKETCH
Sawart Ratanaworabhan was born on March lU, 1936 in Prae, northern
Thailand. After attending public schools at Prae and Chiengmai, he en-
rolled at Kasetsart University, Bangkok, where he received the degree of
Bachelor of Science in Agriculture in I96O. He worked as plant quaran-
tine inspector until 1961, when he joined the staff of the Rice Protection
Research and Training Center, Department of Rice, Bangkhen, Bangkok. He
came to the United States in March, I963 and enrolled in the College of
Agriculture, University of Florida in the summer trimester seeking a
second bachelor's degree. He was awarded a B. S. A. degree in April,
I96U. He entered the Graduate School in the spring of I96U and was
awarded the degree of Master of Science in December, I965. He continued
his graduate work toward the degree of Doctor of Philosophy while holding
a teaching assistantship in the Department of Entomology and Hematology
until he completed the requirements for the degree in March, I969.
He married the former Niphan Chanthawanich on August 9, I967 in
Gainesville, Florida. He is a member of the Newell Entomological Society,
the Helminthological Society of Washington and the Society of Nematolo-
gists.
- 56 -
This dissertation vas prepared under the direction of the chairman
of the candidate's supervisory committee and has been approved by all
members of that committee. It was submitted to the Dean of the College
of Agriculture and to the Graduate Council, and was approved as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
March , I969
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