ROOT-KNOT NEMATODE DEVELOPMENT AND ROOT TISSUE RESPONSES OF THE ROSE

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ROOT-KNOT M M A T G D E DEVELOPMENT AMD ROOT TISSUE RESPONSES OF THE ROSE A Thesis Submitted to the Faculty of Purdue University by Harold William Reynolds In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy June, 1950

PURDUE UNIVERSITY

THIS IS TO CERTIFY THAT THE THESIS PREPARED U N D E R M Y SUPERVISION

by

Harold William Reynolds

entitled

ROOT-KNOT imiATOna DBTELOPliBNT A N D ROOT

T ISSUE aSSPONSSS O F the r o s e _____________________

COMPLIES WITH THE UNIVERSITY REGULATIONS O N GRADUATION THESES

A N D IS APPROVED BY M E AS FULFILLING THIS PART O F THE REQUIREMENTS

F O R THE D E G R E E OF

Doctor of Philosophy

P r o f e s s o r in C h a r g e o f T h e s is

cl? ;

f

H ead o f S ch o o l o r D epa r tm en t

K a y 29________ 19 50

TO THE LIBRARIAN:--

-ty—. THIS THESIS IS N O T TO B E R E G A R D E D AS CONFIDENTIAL.

PE0PES80H TBT OHAEGB

GI?AD. SCHOOL FORM O—3.40-1M

VITA Harold William Heynolds was born near Salem, Indiana on September 10, 1907.

His elementary and high school training was

received at the Reid Grade School and Salem High School.

He entered

Purdue University in September 1927 and was graduated with the degree of B.S.A., with a major in horticulture, in June 1931.

He

spent the following year at the University of California at Davis, with an assistantship in plant pathology.

In June 1933 he accepted

an appointment with the U. S. Department of Agriculture in emergency conservation work, and in 1936 was transferred to the Soil Conserva­ tion Service as nursery manager of the Soil Conservation Service nursery at Washington, Indiana.

Starting in February 1939, he

attended Purdue University and received the degree of M.S. in Feb­ ruary 1940.

In May 1940 he was elected to associate membership in

the society of Sigma 23.•

During the two succeeding years he con­

tinued as nursery manager at Washington, Indiana.

In February 1942

he was transferred to the Special Guayule Research Project at Salinas, California, where he worked until March 1946.

He then resumed graduate

study toward the Ph.D. degree at Purdue University until November 1947. During December of 1947 he resumed work with the U. S. Department of Agriculture in the Division of Hematology of the Bureau of Plant Industry, Soils and Agricultural Engineering, and is presently employed in this work at Sacaton, Arizona.

AGKMOOTJEDCMENTS

The writer wishes to acknowledge his indebtedness to Dr. C. L. Porter, Purdue University, for suggestions and super­ vision in the prosecution of these studies; to Dr. J, A. McClintock, Purdue University, for suggesting this problem and making available plant materials; and to Mr. Milton M* Evans of Saeaton, Arizona for assistance in the photography.

TABLE OF CONTENTS Page ABSTRACT INTRODUCTION

..........................................

REVTEW OF LITERATURE MATERIALS AND METHODS

1 3

. . . . . . . . . . . . . . .

. . . . . .

3

Sources and Preparation of the Inoculum . . . . . . . . . . . Making the Inoculations . . . . . . . . . . . . a . . . . . . . Preparation of Roots for Observation ..........

6 7 8

EXPERIMENTAL RESULTS

...............................

12

Observations on Rose Roots ............ . • 4, 8, 12, 16, 20 and 24 Days After Inoculation..........

12 12-16

Observations on Tomato R o o t s ................ 4, 8, 12 and 16 Days After Inoculation . • • . . . . • • •

17 17-19

DISCUSSION SUMMARY AND CONCLUSIONS BIBLIOGRAPHY AND CITED REFERENCES ABBREVIATIONS USED IN THE ILLUSTRATIONS APPENDIX A.

. . . . . . .

20

........................

24

.......... .................... *

PLATES AND EXPLANATIONS . . . . . . . . . . . . . .

28 30 31-56

LIST OF ELATES

Plate

II.

Rose -Egg masses and preparasitic larva

Rose -Normal stained and sectioned root t i p s

III.

Rose -4 days after inoculation

IV.

Rose -8 days after inoculation

V.

Rose - 12 days after inoculation

VT.

Rose -16 days after inoculation

VII.

Rose -20 days after inoculation

VIII. IX.

. . . . . . . .

.......... ............

38

...

40 42

..........

Tomato - Normal stained and sectioned root tips Tomato - 4 days after inoculation

XI.

Tomato - 8 days after inoculation

36

.

Rose —24 days after inoculation.........

X*

32

34

.

I.

-?-a-ge.

44

....

46

....

48

.... ..

50 52

XII.

Tomato - 12 days after inoculation

. . . . . . . . . . .

54

XIII.

Tomato - 16 days after inoculation

. . . . . . . . . . .

56

ABSTRACT

ROOT-KNOT NEMATODE DEVELOPMENT AND ROOT TISSUE RESPONSES OF TBS ROSE A study is presented dealing with the parasitism of the root-knot nematode, Meloidogyne incognito Goeldi, on the roots of Rosa multiflora Thunb.

Particular attention is given to host tissue responses and

development of the parasite from the time it entered the root until fully developed.

An identical and simultaneous experiment was conducted

on ah herbaceous plant, the tomato5 Lyoopersicum esculentum Mill., primarily as a comparative study*

The length of the life cycle was

determined for the parasite in each host, for the environmental condi­ tions under which these studies were conducted. Experimental data were secured by inoculating simultaneously a number of rose and tomato seedlings with a water suspension of viable larvae.

Twenty-five to thirty roots were excised for study at four-day

intervals until egg masses appeared on the surface of the roots for each host.

One third of the excised roots were used as nematode stained

and cleared root specimens; one third for demonstration of host cell and tissue responsesj and the balance used for studying growth and development of the parasite.

These three lots of excised roots were

prepared for observations, photomicrographs, and camera lucida drawings in accordance with the standard laboratory techniques accepted for studies of this nature. These studies show that the parasite entered the tomato root and established a nutritive relationship more quickly than in the rose. However, once the parasite became established, host tissue responses

and development of the parasite progressed at about the same rate in each host. The length of the life cycle of the parasite was 30 days in the rose and 22 days with the tomato.

This is based on the time which

elapsed between inoculation and the presence of larvae in egg masses resulting from the inoculations.

Lateral roots were formed frequently

from near the point of infestation on the rose*

However, lateral root

formation did not occur as often in the tomato.

Gall formation was

evident in the tomato soon after inoculation and this condition became more pronounced as the parasite developed.

With the rose, gall forma­

tion did not occur at any stage of development of the parasite; however, egg masses were produced in abundance on the surface of the roots. Egg mass production on roots of plants without gall formation should be of interest to the growers and plant inspectors.

It is pos­

sible to overlook egg masses, in a casual macroscopic examination, and thus to mistakenly judge a plant free of the root-knot nematode. The staining and sectioning technique used in this study demon­ strated the presence of the parasites and the response of the host tissues and cells to good advantage.

It is believed that this same

technique may be used as an aid in determining the nature of resistance of certain plants to the root-knot nematode.

ROOT-KNOT NEMATODE DEVELOPMENT AND ROOT TISSUE RESPONSES OF THE ROSE

INTRODUCTION The root-knot nematode, which includes species of the genus Meloidogyne Goeldi, is a serious and destructive pest.

It attacks roots

and subterranean stems, and is almost world wide in distribution.

Neal (15)

states that this pest has been known to occur in the United States since 1805.

In England it was discovered by Berkeley (2) in 1855, on the roots

on greenhouse cucumbers.

It attracted the attention of greenhouse men in

the United States as early as 1876, when damage was reported on roses and violets.

In areas which have a mild winter climate, it is a serious pest

on many of the economic crops, and under greenhouse conditions it is troublesome everywhere.

In the hot arid Southwest, where irrigation is

practiced extensively, heavy losses likewise occur similar to those ex­ perienced in southern and southeastern states. The root-knot nematode has long been a problem in the culture of roses to the nurseryman, gardener, and state and federal inspector whose duty it is to prevent inter- and intra-state shipment of infested plants. In Arizona alone approximately 40,000 rose bushes were intercepted by state inspectors and condemned in one season.

However, within the past

few years this condition has been somewhat alleviated by the use of standard nematocides in nursery soils, to the extent that much cleaner stock is now offered for sale. Rosa multiflora Thunb. is now used extensively in many parts of the country as a hedge or fence plant against livestock, and it also has its value as an erosion control and wildlife plant.

Heavy losses from

the root-knot nematode have recently been encountered in a Soil Conser­ vation Service nursery where a great deal of this stock is propagated. Considerable work has been published on the root-knot nematode relative to control measures, life history and morphology, and environ­ mental factors affecting the parasite.

Also studies have been made on

the length of the life cycle and response of the host tissues on a few herbaceous plants.

However, very little work has been conducted on

host-parasite relationships of perennial plants like the rose. The present work, therefore, deals with the response of the root tissues of Rosa multiflora Thunb. and the growth, development, and length of life cycle of the parasite.

A simultaneous experiment was

conducted on an herbaceous plant, the tomato, Lycopersicum esculentum Mill., primarily for comparison. KEVIEN OF LITERATURE Streets (19) regards the root-knot nematode as a frequent and often unrecognized cause of poor growth of roses in the southern states. Lyle (12) indicates that root-knot infested rose bushes have a much shorter life v/ith fewer and smaller sized flowers. Massey (13) states that this parasite causes a serious disease of roses, both under glass and in the home garden. Alstatt (1), in a study of the susceptibility of rose understocks to attack by the root-knot nematode, reports that of thirteen different species and strains of roses tested, only one was highly resistant.

The

others showed infestation ranging from slight to severe. In studies dealing with the length of the life cycle of the parasite, Bessey (3) states that the time required for the development from the egg to the mature, egg laying individual depends to a great extent upon the temperature and upon the plant affected.

Godfrey and Oliveira (10), working with pineapple and cowpea, found that the length of time elapsing from the initial inoculation to the first eggs observed was 35 days for pineapple and 19 days for cowpea. They report an additional five days required for hatching the eggs at a temperature around the optimum, Tyler (22) found that the minimum time required for the life cycle from larva to larva in experiments on tomato roots was 25 days at 27.0° C., increasing to 87 days at 16.5° G.

She further states that

the minimum time from the appearance of a gall to the beginning of egg laying was 15 days at 27.0° C. and 79 days at 14.5° C. Christie (5) deals with development of root-knot nematode galls and host tissue responses of the roots of the tomato.

He notes that

the larvae entering the root tend to pass between the cells, and that injury to the root through cell destruction is slight.

When the parasite

is permanently located in the root, the head usually is in the plerome near the beginning of the region of elongation. the immediate effects on the root cells are:

He further notes that

hypertrophy of cells in

the cortical region; slight hypertrophy of cells of the pericycle and endodermis when lying near the path of the parasite; a stimulation of cell division in the pericycle; and sometimes a suppression of cell division in the apical meristem.

This author points out that the divi­

sion of the cells of the pericycle, as stimulated by the presence of the parasite, results in a layer of small-cell parenchyma, outgrowths of which form the lateral roots so common when this parasite invades the root.

This tendency for lateral roots to form at the point of infesta­

tion was also reported by Treub (21), Molliard(14), and Saran (17). With regard to giant cell formation, Christie (5) further reports

that during the first 40 to 60 hours after the parasite invades the root, cells of the central cylinder lying in the region of the para­ site’s head remain undifferentiated.

After about three days, these

undifferentiated cells enlarge slightly, their nuclei swell, and their walls disintegrate.

The protoplasmic content of adjacent cells then

coalesce to form a giant cell.

Frequently these cells are members of

rows that normally would have contributed to the formation of a vessel. The giant cell invades adjacent areas and other cells are absorbed after dissolution of cell walls.

Eventually nuclear membranes break down and

giant-cell nuclei coalesce and finally disintegrate. Tischler (20) devotes his attention primarily to the cytologieal aspects of giant cell formation, especially the method of nuclear divi­ sion.

He reports that during early stages of development the division

of giant-cell nuclei is by normal mitosis.

However, he concludes that

after a certain period division occurs by amitosis. N§mec (16) and Kostoff and Kendall (11) all agree that nuclear division in early stages of giant cell forroation is by mitosis.

They

believe that the later amitotic divisions discussed by Tischler were either mitotic divisions of an abnormal character or nuclei in early stages of fusion. With regard to the cause of gall formation, Kostoff and Kendall (11), working with roots of Nicotiana hybrids, believe that a secretion pro­ duced by the oesophageal glands of the parasite increase permeability in the plant tissues, and an exosmosis results, bringing about an accumulation of food in the region of invasion.

They further state, ,fAs a consequence

of the presence of this accumulation of nutrition, the growth of the plant tissues is accelerated in these regions and is morphologically expressed by the swelling, or gall, on the root.

At the same time, however, the

other organs of the plant are deprived, of nutrition and their growth is inhibited.

The increased permeability is instrumental in bringing about

a localization of the foreign substances, but the plant suffers a loss of nutrition in the substance so carried from surrounding tissues to the area of invasion, while the parasite receives a great store of nutritive material in a very small region about its head, as is shown in the rich granular cytoplasmic content in these regions.” Concerning the development and metamorphosis of the parasite, Christie and Cobb (6) point out that the first molt takes place within the egg.

They note that the molts of the female at the time the caudal

spike is lost, correspond to the molts of the male at the time of meta­ morphosis.

Two cuticles are simultaneously detached and loosen about

the body of the female; then, very shortly thereafter, another cuticle is detached.

They observe that the male molts a fourth time immediately

on completing its metamorphosis and while it is still within the sac formed by the second and third cuticles.

There is, therefore, a dis­

tinct fourth stage, during which metamorphosis takes place in the male. ”In the female,” they state, ”there appears to be a very brief interval between the detachment of the second and third cuticles and the detach­ ment of the fourth cuticle, hence, strictly speaking, there is a fourth larval stage.

However, these last three molts are so nearly simultaneous

that the fourth stage is largely theoretical.” MATERIALS AND METHODS Seed of Rosa multiflora was furnished by the Department of Horti­ culture of Purdue University.

The seeds were germinated in a flat of

sterile sphagnum moss under laboratory conditions starting August 30, 1948, and after six weeks the seedlings were transferred to 4-inch pots of sterile soil and removed to the greenhouse for further growth.

Tomato seedlings were grown by seeding directly in pots of sterilized soil.

These, however, were not seeded until about ten days

before the time planned for making inoculations, because of their more rapid rate of growth. Source and Preparation of the Inoculum The source of the nematode population used in these inoculations was a plot at the U. S. Field Station, Sacaton, Arizona.

This nematode

has been identified by Chitwood (4) as Meloidogyne incognito Goeldi. He further states that this species appears to be general (probably native) in the southern United States and various subtropical American islands.

This species readily infests tomato, cotton, okra, grape,

mulberry, fig and other commercial crops and several weed hosts at this station. Several rose seedlings were transferred to pots containing soil in which heavily infested okra was growing, and placed in the lathhouse for about 75 days. infested.

During this time, the rose seedlings became heavily

However, there were very few galls formed and these occurred

only at the ends of long roots, where apparently a great number of larvae had entered at the same point. abundant on the rose roots.

Egg masses, however, were very

The egg masses were smaller than root galls,

and might easily have been overlooked unless one used low power magnifi­ cation while examining the roots.

One rose plant, for example, showed

approximately 500 egg masses and the inoculum for the experiment was selected from this plant (Plate I, A). By aid of the stereoscopic mici*oscope and a laboratory dissecting needle and scalpel, the egg masses were removed from the surface of the rose roots and placed on small pieces of cloth, supported by similar sized

pieces of screen wire, and then placed in Petri dishes.

Usually about

200 egg masses were placed on each cloth and wire in each Petri dish. Enough water was added to each dish to keep the cloth moist by capil­ larity but not submerge the egg masses.

These dishes were then placed

in an electric incubator at 26° 0. for hatching the eggs.

The daily

hatch was removed and held in the refrigerator at 7° 0. for later use. This method for securing large quantities of root-knot nematode larvae is similar to that described by Godfrey (9) except that he placed the roots which showed numerous, well developed egg masses directly on the woven wire screens. Making the Inoculations Just prior to July 25, 1949, on which date inoculations were made, both the rose and tomato seedlings were transferred from the lathhouse to the nematology laboratory.

This minimized the exposure of the plants

to excessively high soil and air temperatures such as prevail in southern Arizona during the summer months.

The laboratory, equipped with an

evaporative cooler, provided a more optimum temperature for development of the parasite.

The pots containing the roses and tomatoes were placed

in a large metal tray on the west side of the laboratory under artificial light.

Moist sand was firmed about the pots to a depth of four inches,

and this moisture was maintained for the duration of the experiment.

A

weekly recording soil thermograph was used to determine soil temperature ranges.

Very little fluctuation in soil temperatures was recorded

throughout the pex*iod of the entire experiment.

This record shows a

range from 26.1° to 27.8° with a mean temperature of 27*1° C.

This

temperature is considered optimum for development of the root-knot nema­ tode by Tyler (22), who determined that the most rapid development occurs around 81° E. (27.2° C.).

The inoculum, which consisted of viable larvae in water (Plate 2, B), was made by introducing 400 to 500 larvae at several different places in each pot one to two inches below the soil surface. Thirty roses and thirty tomato seedlings were inoculated simultaneously on July 25, 1949.

This procedure subjected all seedlings to the same

temperature range for the duration of the experiment.

No definite

amount of water was applied to the seedlings, but an attempt was made to provide optimum moisture conditions throughout the period of obser­ vation.

Godfrey (8) demonstrated that through the range of soil

moistures favorable to plant growth, little or no difference in sus­ ceptibility is obtained. Preparation of Roots for Observation With the rose seedlings, 25 to 30 roots were excised at the following intervals after inoculation: 24 days.

4, 8, 12, 16, 20, 22, 23 and

On the 24th day, egg masses were present on the surface of

some of the roots. With the tomato seedlings, 25 to 30 excised roots were taken just as in the case of the rose.

However, these were taken at 4, 8,

12, 15 and 16 day intervals, as egg masses were present on the 16th day with the tomato.

All adhering soil particles were removed, and the

roots at each collection interval and for each host were divided into three equal lots. One third of the roots for both hosts at each collection interval were treated as stained nematode and cleared root specimens.

Killing

was in warm (55° C.), weak Flemming’s solution, as described by Godfrey and Oliveira (10). the nematodes black.

The osmic acid of the Flemming’s solution stained The root specimens containing the nematodes were

placed in the Flemming’s solution for one hour, and then washed for a

period of at least four hours in running water. in the following series of alcohol: percent.

They were dehydrated

15, 35, 50, 70, 85, 95 and 100

The roots were then cleared in clove oil and mounted in thin

balsam for later observation.

Photomicrographs were made with a 5x7”

camera in which the lens had been removed and the lens board fitted with the microscope tube and a IX ocular.

Objectives of 16 and 38 mm.

were used, depending upon the magnification desired, for all photo­ micrographs shown in this paper. Another one third of the excised root specimens of both the rose and the tomato were sectioned preparatory to host tissue studies. These roots were first killed and fixed in formol-acetic-alcohol, after which they were dehydrated in a series of alcohol of 15, 35, 50, 70, 85, 95 and 100 percent.

From the 100 percent alcohol, they were placed in

a solution of half alcohol and half chloroform for a period of four hours and then transferred to 100 percent chloroform for four to five hours.

This chloroform was discarded and a fresh supply poured on, to

which vi&s added paraffin (60-62° C. melting point), a small piece at a time until saturation occurred at room temperature.

The vials were

then placed on top of the 62° C. oven and a few additional pieces of paraffin were added.

The contents of the vials were poured into

evaporative dishes, placed again on top of the oven, protected from dust and left there until the surface had solidified.

The evaporative

dishes were then placed in the oven until all traces of chloroform had escaped.

A new supply of paraffin was added and the specimens were

arranged and imbedded. All sections were cut 14-16 microns in thickness with the sliding microtome.

They were stained with safranin in 70 percent alcohol and

cleared in xyol, and mounted as permanent preparations in thin balsam.

The remaining one third of the excised roots were used in con­ nection with studies of development and growth of the parasite in the two hosts.

The nematodes were meticuously removed at the regular

four-day intervals from the excised roots with a slender eye knife and a laboratory dissecting needle.

These were transferred by means

of a cactus spine pick to a well slide containing water and sealed with a cover slip.

The well slide was placed in an electric oven at

50° C. for a period of 20—30 minutes for killing through gradual heat. With the aid of a medicine dropper containing a very fine orifice, the water was drawn off and replaced with formol-acetic-aleohol, which also contained about 1 percent glycerine. solution for 48 hours.

The specimens were left in this

They were then transferred to a small nematode

preparation dish (1 ml. capacity), whieh was filled with a solution of 30 percent alcohol and 1-1/4 percent glycerine.

This small preparation

dish, together with 2 grams of calcium carbonate contained in a small screw cap vial with an orifice 1 mm. in diameter, were placed in a 50 ml. preparation dish and sealed with vaseline.

The tiny orifice of the vial

permitted only a gradual withdrawal of water and therefore did not cause shrinkage of the specimens.

After two weeks of dehydration and glycerine

infiltration, the small 1 ml. preparation dish containing the specimens was placed in a large calcium chloride desiccator for a period of ap­ proximately one week for final desiccation. Aluminum slides were used in mounting all specimens.

These slides

were prepared by inserting a 25 mm. square cover slip in the groove of the aluminum slide and moving the cover glass toward the middle, where it \vas centered over an opening 20 mm. in diameter.

The cover glass was

held in place by two cardboards inserted from the ends through the slide

grooves.

The specimens were mounted as permanent preparations in a

drop of pure glycerine placed at the center of the cover slip.

The

size of the drop of glycerine varied with the size and thickness of the specimen or specimens mounted on the particular slide, the larger ones requiring a little more glycerine. Next, three short glass rods were selected with a thickness the same as the specimens, and placed in the drop of glycerine as a cover glass support.

These prevented crushing or distortion of the specimens

after the 18 mm. round cover slip was ringed in place.

The glass rods

from which the cover glass support was cut were always kept in a small amount of glycerine.

These rods, as well as the pure mounting glycerine,

were stored in a desiccator in order to eliminate any traces of mois­ ture. The thickness of both the 25 mm. square mounting cover slip and the 18 mm. round one was selected on the basis of the thickness of the specimens for any particular slide.

Very thin cover slips of 0.11 to

0.13 mm. were used with larger specimens, and this thickness did not interfere when using the 1-1/2 mm. oil immersion, microscope objective. However, on the smaller specimens a little greater eover glass thick­ ness did not interfere when the 1-1/2 ram. microscope objective was used. The aluminum slide proved to. be of great value in this study because specimens could be observed through either the 18 mm. cover slip or the 25 mm. one, by inverting the slide.

Microscopic observations have been

made through 4 mm. dry objectives, and 2 mm. and 1-1/2 mm. oil immersion objectives, with 5X and 10X oculars.

Drawings have been prepared by

aid of the camera lucida and transferred to Ross scratch board for final tracing with india ink.

EXPERIMENTAL RESULTS As stated previously, the rose and tomato seedlings were inocu­ lated simultaneously with viable larvae of the same root-knot nematode population (Plate I, B) and therefore subjected to the same temperature and moisture relationships throughout the entire experiment.

Also in

all instances a large number of roots were excised and studied, and observations recorded only on the most advanced stages of root tissue responses and parasite development for each collection interval.

It

is assumed, therefore, that any differences presented here in root tissue responses or rate of parasite development were inherent in the two hosts studied. Observations on Rose Roots All observations made on rose roots at the different collection intervals indicated will be presented first.

This includes observa­

tions on stained nematodes in cleared root tissues, studies of root tissue and cell responses, and development and growth of the pax*asite. Next, these same observations and studies will be presented for the toroato, giving particular attention to any differences which were noted between the two hosts. 4 Days After Inoculation. The 4-day observation (Plate III, A) on stained nematodes and cleared root tissues shows that several nematodes have entered the roots and are migrating in the cortex parallel with the stele.

In a

few instances, migration occurs within the stele and this is demon­ strated in the longitudinal section in Plate III, C.

Also in this case

the parasite has not yet ceased migration and has therefore not started to feed.

Immediately after feeding starts, one of the first things

observed in the immediate vicinity of the parasite’s head is the forma­ tion of several multinueleate giant cells, sometimes called nourishing cells.

A comparison of Plate III, 0 with a normal longitudinal section

of a root (Plate II, B) shows no evidence of any changes taking place in cells and tissues adjacent to the parasite’s head.

Plate III, D

shows one of several migrating larvae removed from the root four days after inoculation.

When this larva is compared with the one shown in

Plate I, B, which was drawn immediately after emerging from the egg, there is no evidence of growth or development in the former. Generally the larvae penetrate the roots just behind the root cap in the meristematic region.

Plate III, B shows a larva about half

way in the root near the meristematic region.

Occasionally penetration

takes place directly through the root cap parallel with the main axis of the root, and in other instances penetration occurs in the region of maturation. 8 Pays After Inoculation. After eight days there is evidence that the larvae have ceased migration and are starting to feed.

Of the figures on Plate 17, the

longitudinal section (Figure B) is about the only one which demonstrates that the parasite has established a nutritive relationship with the host. Here may be seen a few cells which are somewhat larger than the surround­ ing or adjacent cells. giant cell formation.

These appear to be the very earliest stages of Note that the parasite may be seen in the lower

portion of this group of enlarged cells.

It appears as a small black

spot because it was cut transversly when the root was sectioned.

An

examination of the larva which was taken from the root eight days after inoculation (Plate IIT, C) shows very little, if any, change in growth or development as compared with the four-day larva in Plate III, D .

12 Days After Inoculation. An examination of the 12-day-old stained nematodes and cleared root specimens (Plate V, A and B) shows several nematodes, some of which are within the stele and lie parallel with the main axis of the root. The bodies of some of the parasites extend obliquely in the cortex with their heads in direct contact with the periphery of the stele.

The

longitudinal section twelve days after inoculation shows a few multinucleate giant cells in close proximity with the parasite’s head.

An

examination of the nematode at the 12-day interval shows that some development has taken place (Plate V , D) .

The parasite has become

somewhat thicker and the genital primordium near the center of the body occupies about three fourths the width of the body cavity. 16 Days After Inoculation. Changes in host tissues progressed at a rapid rate between the 12th and 16th day after inoculation.

The stained nematode and cleared

root specimen shows that the parasites have become somewhat thicker (Plate VI, A).

Note that the posterior end of the nematode extends out

in the cortex, with the head in the central cylinder.

The longitudinal

section shows the relative size and thickness of the parasite to better advantage (Plate VI, B ) . Also the giant cells, which have developed considerably, parallel with the main axis of the root, may be seen. There is no evidence of gall formation in these 16-day-old studies. An examination of Plate VI, C indicates that growth and develop­ ment of the parasite likewise was very rapid between the 12th and 16th day.

There is considerable increase in thickness and the developing

ovaries, which contain several developing eggs, extend anteriorly to almost half the length of the body.

There is also present a distinct

spike tail, and according to Christie and Gobb (6), this specimen is

still a second stage larva; that is, the second molt has not yet occurred.

When this molt occurs, the spike tail is cast with the

larval skin.

There is yet no evidence of uteri, vagina or vulva

formation. 20 Days After Inoculation. In the stained nematode and cleared root preparations, it has been difficult to demonstrate the host-parasite relationship because these older root tissues stain about as deeply as does the parasite. However, Plate VII, A and B show that the parasite in this instance lies entirely within the stele.

Also there is little if any evidence

of gall formation even 20 days after inoculation, when the parasite is almost mature.

Lateral root development has taken place in the

immediate vicinity of the parasite.

This condition has been observed

by the writer repeatedly, and other workers (as mentioned in the literature review) have reported the same observation. The longitudinal section at the 20-day interval (Plate VII, 0) shows portions of the darker multinucleate giant cells near the head of the parasite, while the parasite’s body lies in the cortex almost parallel with the longitudinal axis of the root.

In this section there

is a slight tendency toward gall formation on the side of the root occupied by the parasite. In Plate VII, D several changes have taken place in the nema­ tode between the 16th and 20th day after inoculation.

The ovaries

have become considerably longer and coiled, and extend well up in the anterior part of the body.

At the posterior end of the ovaries may

be observed the uteri which are just starting to coil and elongate. These converge to a common tube in which the vagina is just forming, and finally the vulva may be seen at the surface and slightly on the

ventral side of the body.

Through molting, the spike tail which was

characteristic of the 16-day-old larva has been lost. 24 Days After Inoculation. Nematode stained and cleared root specimens again show heavily stained root tissues to the extent that differentiation between the host and parasite is difficult.

Nevertheless, Plate VIII, A and B

show an egg mass on the posterior end of the parasite which has broken through the surface of the root. Plate I, A.

Compare Plate VTII, A and B with

There is no evidence of gall formation even at maturity

of the nematode.

Again a lateral root has developed near the place

occupied by the parasite. The longitudinal section (Plate VIII, C) shows a portion of the multinucleate giant cells near the anterior end of the parasite.

In

this instance, an egg mass has not yet broken through the root surface but the gelatinous matrix, in which the eggs are extruded and imbedded, has started to appear.

There is no evidence of gall formation or lateral

root development in this section. Plate VIII, D shows a mature female with segmented and unsegmented eggs in the uteri.

The ovaries and uteri extend over almost all of the

body cavity and contain eggs in different stages of development.

This

specimen represents an early stage of a mature, egg laying female. As mentioned previously, roots were excised 22 and 23 days after inoculation, but these are not shown here.

Some of the roots showed

the presence of gelatinous matrix on the 23rd day, but no eggs were observed until the roots were excised and examined on the 24th day. On the 30th day after inoculation, an occasional larva was observed in a few of the egg masses examined, thus showing that the eggs were just starting to hatch.

Therefore, the minimum length of

the life cycle on the rose under the conditions of this experiment may be regarded as 30 days.

This is based on the time which elapsed from

inoculation to. the hatching of the very first eggs. Observations on Tomato Roots As previously stated, the procedure with tomato roots throughout the entire experiment was identical to that of the rose.

However,

because of the more rapid development of the parasite in the tomato, the excised root specimens were taken a fewer number of times as there was a shorter life cycle of the parasite. 4 Days After Inoculation. The parasite penetrated the tomato roots readily, and after a short migration period of probably less than two days, it began to feed. The nematode stained and cleared root specimen (Plate X, A) shows the head of the parasite well within the stele, with the posterior end extending obliquely and downward in the cortex.

A slight root swelling

may already be observed in the area occupied by the parasite.

Compare

this with Plate IX, A, B and C, which show a stained and cleared root and longitudinal and cross sections respectively of normal tomato roots. The four-day longitudinal section (Plate X, B) shows that the multi­ nucleate giant cells have already formed and that the head of the parasite lies in the immediate vicinity of these cells.

Slight swell­

ing or early stages of gall formation is also evident in this section. Plate X, C (a cross section made through the region of the giant cells) shows the width of the giant cells in relation to other root cells. Compare this with Plate IX, C.

Plate X, D, which represents the larva

four days after inoculation, shows a thicker and more developed specimen than the preparasitic stage shown in Plate I, B.

Host tissue responses and development of the parasite thus proceed at a rapid rate in the early stages of infestation as compared with the rose.

The 4-day stage in the tomato is comparable to the

12-day stage in the rose* 8 Days After Inoculation. The stained nematode and cleared root specimen (Plate XI, A) indicates that changes in the tissues of the tomato as well as growth of the parasite were rapid during this four-day interval.

Note that

gall formation and giant cells are very evident at this stage.

The

longitudinal section (Plate XI, B) shows the multinucleate giant cells to good advantage.

Also gall formation is quite evident in this section.

An examination of the parasite taken from a root eight days after inoculation shows that the nematode has become much thicker is developing rapidly (Plate XI, C). half the length of the body.

The ovaries extend anteriorly to

The spike tail, which is cast at the time

of the second molt, is also present.

The development of this parasite

eight days after inoculation in the tomato is comparable to 16 days* development in the rose (Plats VI, C).

However, gall formation is much

more evident in the tomato root. 12 Pays After Inoculation. The stained nematode and cleared root specimen shows that de­ velopment is still progressing rapidly, with gall formation and the giant cell region quite evident (Plate XII, A).

The longitudinal

section (Plate XII, B) shows the multinucleate giant cells and the parasite* s body somewhat below this region.

This section was appar­

ently cut obliquely, as the parasite* s head does not appear to be adjacent to the giant cells. formation.

This section also shows evidence of gall

An examination of the parasite twelve days after inoculation (Plate XII, 0) shows that the body is thicker and that very rapid development has taken place in the reproductive organs between the 8th and 12th day.

The coiled ovaries extend over most of the body

cavity, and the uteri, vagina and vulva have formed.

The 12-day stage

in the tomato appears to be comparable with the 20-day stage in the rose. 16 Pays After Inoculation. Stained nematode and cleared root specimens show the typical mature, flask-shaped parasites with egg masses extruding from the posterior end of their bodies.

With this number of parasites in the

root tip, all forward growth was stopped soon after invasion and the root tip ends abruptly, forming a point just in advance of the posi­ tion of the parasites.

This condition also exists in Plate XII, A,

twelve days after inoculation.

In this case only three parasites,

however, entered the root and cessation of terminal root growth was not as abrupt as in the root shown in Plate XIII, A, where at least six parasites were feeding from the same general area. In the longitudinal section (Plate XIII, B)are shown portions of the bodies of two parasites.

With one, an egg mass may be observed

which has already broken through the tissues at the surface of the root.

Giant cells and gall formation also show to good advantage

in this section. Plate XIII, C shows a young, full grown, egg laying female. The parasite has become typically flask shaped and the ovaries and uteri lie coiled and extend over most of the body cavity, and contain eggs in various stages of development. An examination of tomato roots xvas made 15 days after inocula-

tion and gelatinous matrix was present, but no eggs were observed until the roots were excised and examined on the 16th day.

Thus the

16th day after inoculation in the tomato is comparable with the 24th day in the rose.

As xvith the rose, within six days after egg masses

were present a few larvae were observed, thus showing that egg hatching was just beginning.

Therefore, the minimum length of the life cycle

on the tomato, based on the time elapsed from inoculation to the hatch­ ing of the first eggs, is 22 days. DISCUSSION The root—knot nematode is an obligate parasite, and it therefore does not develop to any extent or complete its life cycle outside of the root or subterranean stem.

It is also a sedentary parasite; that

is, once it enters the root and after a migration period of possibly one to several days, depending upon the host invaded, it becomes stationary and does not move through the tissues in search of its food.

Instead, a highly developed interrelationship exists between

the plant tissues and the parasite.

The parasite causes large multi­

nucleate giant cells to form from undifferentiated cells in the immediate vicinity of the vascular tissues, and it is through these giant cells that the parasite establishes a nutritive relationship with the host. From this study, it is evident that the period of time required for root penetration of the larvae and the migration period within the root differs with the rose and the tomato.

With the rose, there was

no indication that the parasite had started to feed four days after inoculation.

This conclusion is based on the absence of cellular

changes and giant cell formation in root specimens examined at the four-day interval.

Eight days after inoculation there was some evidence that giant cells were iust starting to form, but there was no apparent change in thickness of the parasite or other indication of growth at this time. At the 12-day interval, giant multinucleate cells were quite prominent and the parasite had become thicker, with development starting in the reproductive organs. The length of time required for entrance of larvae and migration in the tomato was very short when compared with, the rose.

After a

period of only four days, multinucleate giant cells had formed and were rapidly developing, and growth and development of the parasite were already evident. With regard to the rate of growth of the parasite in the two hosts, there was little difference observed once the parasite estab­ lished a nutritive relationship with its host.

While it is true that

egg masses were present on the rose roots 24 days after inoculation and this required only 16 days in the tomato, it should be recalled that most of this eight day interval was used in larval entrance and migra­ tion in the case of the rose.

It therefore appears that once giant

cells have formed, there is supplied, in the case of both hosts, an adequate amount of food for continued and rapid growth of the parasite. Lateral roots formed readily in the rose from near the point of infestation.

This phenomenon has been reported by other workers,

particularly while dealing with herbaceous plants.

The cause of these

numerous lateral roots has generally been ascribed to a stimulation of mitotic activity in the pericycle by the parasite.

When numerous

lateral roots are formed, this often gives the root system a ’’bearded appearance as referred to in the literature.

This tendency for lateial

root formation was not observed as frequently in the tomato. Only one male nematode was observed throughout the entire ex­ periment.

This specimen was found emerging from a tomato gall in

one of the stained root preparations, and was therefore not a suitable specimen for a camera lucida drawing because of heavy staining. Root-knot n e m t o d e infestation without gall formation as demonstrated in this investigation with the rose is a matter of con­ siderable significance.

Growers and plant inspectors may mistakenly

judge plants to be free of the root-knot nematode in a casual macro­ scopic examination, basing their decision on the absence of galls. It is possible with the staining technique employed in this study to demonstrate the presence of the parasites regardless of their stage of development or whether or not galls have formed.

Also it has been

demonstrated voith this staining technique in studies on both the rose and tomato, as well as recent investigations by the writer on young cotton seedlings, that there are usually many more root-knot nematode larvae in the roots than would be suspected even though the root examinations were made with the aid of a stereoscopic microscope. The sectioning and staining technique has shown to good advantage the reaction of the root tissues and cells progressively as the parasite develops.

It appears to the writer that the use of these different

techniques may aid in explaining the nature of root-knot nematode resistance which several species and varieties of plants possess. That the root-knot nematode may be present without causing the formation of galls has been reported by Steiner (18), who states that this condition is often observed on cotton and corn, where the nematode breaks through the surface of a root without forming galls.

The writer

has likewise observed this condition on cotton seedlings in the South­

west.

In one instance 15 mature, female root-knot nematodes were taken

from the surface of one primary root of a young cotton seedling, and there was no evidence of galling. It is not to be implied here that gall formation never occurs when rose roots are attacked by the root-knot nematode.

Massey (13)

shows a root system of Rosa multiflora, which he secured through the courtesy of the Texas Agricultural Experiment Station, that was severely galled.

The writer has observed rose roots from various other places

(including eastern, midwestern and western United States) which showed conspicuous galling. The fact that different species of the root-knot nematode (formerly regarded as different races or strains) cause different types of galling as to size and number must not be overlooked.

Chitwood (4)

reports that Meloidogyne incognito var. acrita did not infest Jumbo peanuts but developed readily on cotton with rather small galls, the egg masses subglobular, outside the roots.

He further reports that on

tomatoes the galls were massive, confluent, with egg masses chiefly inside the roots. Steiner (IS) in comparing the difference in appearance of galls on tobacco and the peanut states that such variations in character of root galling and disfiguration may be related to different root-knot nematode strains or races, but may also be the result of different reactions by the host. Christie and Albin (7), working with various races of the rootknot nematode, demonstrated that a plant may be susceptible to each of two races, but the type of root galling produced by one race may differ from that produced by the other.

It is possible that had other species and populations of* the root-knot nematode been used in parallel experiments, different root tissue responses would have resulted in jRosa multiflora roots.

This,

however, remains to be demonstrated. SUMMARY AND CONCLUSIONS Rose and tomato seedlings were simultaneously inoculated with root-knot nematode larvae and placed in the laboratory under practically constant soil temperature and moisture conditions for development of the parasite and further growth of the plants.

Twenty-five to thirty excised

roots were taken for examination at each four day interval until egg masses were formed on the roots of each host. divided into three lots.

These excised roots were

One third were used as stained nematode and

cleared root specimens for demonstration of the presence and size of the parasites in root tissues.

They were immediately killed in Flemming’s

weak solution, which stained the nematodes black.

After this, they were

washed, dehydrated in a series of alcohol, cleared in clove oil, and finally mounted in thin balsam for microscopic examinations and photo­ micrographs. Another one third of the excised roots were used as sectioned material in the demonstration of progressive root tissue and cellular responses of the two hosts.

These were killed in formol-acetic-alcohol

and dehydrated in the usual series of alcohol, and finally embedded in paraffin preparatory to sectioning on the sliding microtome.

The sec­

tions were stained with safranin in 70 percent alcohol and mounted in thin balsam as permanent preparations for observations and photomicro­ graphs . The remaining one third of the excised roots were used to study

the progressive development of the parasites in the two hosts.

These

were removed from the root, killed by gradual heat, and mo tinted in glycerine as permanent preparations for camera lucida drawings.

The

observations made from these different preparations for the two hosts are recorded as follows: 1.

Generally the larvae penetrated the roots just behind the

root cap in the meristematic region in both hosts.

Occasionally pene­

tration was observed taking place directly through the root cap parallel with the main axis of the root, and in a few cases they entered the root in the region of maturation.

The length of time required for penetration

and migration of the larvae in the root tissues of the rose was six to seven days as compared with approximately two days for the tomato.

There

was no evidence of changes taking place in the rose root tissues or growth of the parasite four days after inoculation.

With the tomato,

however, the four day examination showed that the parasite had caused the formation of multinucleate giant cells through which it establishes a nutritive relationship with the host.

Growth had also taken place in

the parasite. 2«

Multinucleate giant cells were just forming eight days after

inoculation in the rose, but there was no evidence of growth of the parasite.

At this same interval in the tomato, changes in root tissues

and growth of the parasite vneve progressing rapidly, including pronounced gall formation. 3.

Twelve days after inoculation rose roots showed definite

orientation of the parasite with the vascular tissues and further development of multinucleate giant cells. the parasite was also evident.

Growth and development of

With the tomato, further changes in

host tissues and cells occurred between the 8th and 12th day.

The

parasite became thicker and there were significant changes and develop­ ment of the reproductive organs. 4.

Between the 12th and

16th day, certain changes in host

tissues and development and growth of the

parasite in the rose pro­

ceeded at about the same rate as occurred in the tomato between the 4th and 8th day. in the rose.

However, there was yet no evidence of gall formation

Sixteen days after inoculation, the tomato roots showed

the presence of egg masses and the parasite had become typically flask shaped with mature eggs in the uteri. 5.

The 20-day stage in the rose compared favorably with the

12-day stage in the tomato with site.

regard to the development of the para­

Gall formation w a s stillnot evident in the rose. 6.

Twenty-four days after inoculation the rose roots showed egg

masses on the surface of the roots,

but there was no evidence of

formation.

had developed to the flask shaped

The 24-day-old parasite

stage with mature eggs in the uteri.

gall

The 24—day stage in the rose was

comparable to the 16-day stage in the tomato. 7.

A few larvae were observed in egg masses of both the rose

and the tomato six days after eggs first appeared*

Therefore, under

the conditions of this experiment, the length of the life cycle of the nematode on the rose was 30 days, while with the tomato it was 22 days. This is based on the length of time

elapsing from inoculation to

the

hatching of the first eggs from the

egg masses that developed

asa

result of these inoculations. 8.

Lateral roots formed frequently in the rose from near the

point of infestation of the parasite. as frequently on the tomato.

This condition was not observed

9.

Root-knot nematode infestation without gall formation, as

demonstrated in this investigation, may lead growers and plant inspectors (through a casual macroscopic examination) to mistakenly judge plants to be free of the root-knot nematode, basing their decision on the absence of galls.

The staining technique employed in this study can be used to

demonstrate the presence of the parasite regardless of its stage of development or whether or not galls have formed.

The sectioning and

staining technique followed showed to good advantage the progressive changes in the root tissues and cells as the parasite develops.

Also

the use of these different techniques may aid in determining the nature of root-knot nematode resistance which several species and varieties of plants possess.

28

BIBLIOGRAPHY AMD CITED REFERENCES

1

.

ALSTATT, G. E. Susceptibility of some common rose understocks to nematode root-knot. Pit. Dis. Rpt. 26: 371. 1942.

2

.

BERKELEY, M. J. Vibrio forming cysts on the roots of cucumbers. Gdnrs*. Chron. April 7th: 220. 1855.

3.

BESSEY, ERNST A. Root-knot and its control. Bur. Plant Ind. Bui. 217. 1911.

4.

CHITWOOD, BEN. G. A revision of the genus Meloidogyne Goeldi. Proc. Helminth. Soc. Wash. 16: 90-104. 1949.

5.

CHRISTIE, J. R. The development of root-knot nematode galls. Phytopath. 26: 1-22. 1936.

6.

_______________ , and GRACE SHERMAN COBB. Notes on the life history of the root-knot nematode, Heterodera marioni. Proc. Helminth. Soc. Wash. 8: 23-26. 1941.

7.

_______________ , and FLORENCE E. ALBIN. Host-parasite relation­ ships of the root-knot nematode, Heterodera marioni. I. The question of races. Proc. Helminth. Soc. Wash. 11: 31-37. 1944.

8

.

9

. _____________ „

U. S. Dept. Agr.

GODFREY, G. H. Effect of temperature and moisture on nematode root knot. Jour. Agr. Res* 33: 223-254. 1926. Some technique used in the study of the root-knot nematode, Heterodera radicicola. Phytopath. 21: 323-329. 1931.

The development of the 10 . ______ _______, and JULIETTE OLIVEIRA. root-knot nematode in relation to root tissues of pineapple and coTAipea. Phytopath. 22: 325—348. 1932. 11

. K0ST0FF,

DONTCIiO and JAMES KENDALL. Cytology of nematode galls on Nicotiana roots. Centralbl. Bakt., II Abt. 81: 86-91. 1930.

12 .

LYLE, E. W. Nematode resistance in rose understocks. Annual 28: 157-158. 1943.

13.

MASSEY, L. M. Soil fumigants for root-knot control. Annual 32: 119-124. 1947.

14.

M0LLIAED, MARIN. Sur quelques caraeteres histologiciues des cecidies produites par 1*Heterodera radicicola Greef* Rev. Gen. Bot. 12: 156-165. 1900.

Am. Rose Am. Rose

15.

MEAL, J. C. The root-knot disease of the peach, orange, and other plants in Florida, due to the work of the Anguillula. U. S. Dept. Agr. Div. Entom* Bui. 20. 1889.

16.

NEMEC, B. Das Problem der Befruchtungsvorgange und andere zytologische Fragen. Gebruder Borntraeger, Berlin. 1910. Part VI. Vielkernige Riesenzellen in Heterodera-Gallen: 151-173. 1910.

17.

SARAH, A. B. On some anatomical deformities in the root-tissue of Hibiscus esculentus Linn, brought about by the infection of eelworms and their bearing on the growth of the plant. Jour. Indian Bot. Soc. 13: 197-199. 1934.

18.

STEINER, G. Plant nematodes the grower should know. Sci. Soc. of Fla. Proceed. 4-B: 72-117. 1942.

19.

STREETS, R. B. Control of Arizona rose diseases. Exp. Sta. Bui. 213. 1948®

.

20

21

.

22 .

The Soil

Arizona Agr.

TISCHLER, G. Ueber Heterodera-Gallen an den Wurzeln von Circaea lutetiana L. Ber® Deut. Bot* Gesell. (1901) 19, Gen.Versammi.: 95— 107. 1902. TREUB, M. Quelques mots sur les effets du parasitisme^ de l fHeterodera .javanica dans les racines de la eanne a sucre. Ann. Jard. Bot. Buitenzorg. 6: 93-96. 1887. TYLER, JOCELYN. Development of the root-knot nematode as affected by temperature. Hilgardia 7: 391-415. 1933*

2 3 . ___

_. Circ. 330.

The root-knot nematode. 1944.

Cal. Agr. Exp. Sta.

ABBREVIATIONS USED IN THE ILDJSTRATCONS

a n ....................... .. anus ap dsl gl ........ .. dor-sal gland aperture ........... .. lateral field ar lat bib ined . . . . . . . . . . . . . . . . . median bulb cor . . . . . . . . . . . . . . . . . . . corpus cut . . . . . . . . . . . . . . . . . . . cuticle gl ........................... .. glands gl dsl ............ .. dorsal gland gl subm ............... . . . . . . . . . submedial glands grn int . . . . . . . . . intestinal granule l b . lips lum oe. . . * ............ .............. oesophageal lumen ncl dslg l . . . . . . . . . . . . . . . . dorsal gland nucleus nrv r .................................... nerve ring on . . . . . . . . . . . . . . . . . . . . spear on b i b .................................... spear bulb ovr dev ........................... developing ovary ov dev ........................... developing egg o v u t .................................... uterine egg por ex .................................. excretory pore prm gen . . . . . . . . . . . . . . . . . genital primordium r e t ...................................... rectum st cut . . . . . . . . . cuticle striation trm ................. .. terminus ut . . .................................... uteri vag ................... . . . . . . . . . vagina vlv ................................valve vu.1 .......... .. vulva

EXEExAHATION OF PLATS I »

(Over)

ELATE I (HOSE)

Egg masses formed on the surface of roots. Drawing of preparasitie larva.

3B30.

.

210

3

t rtn...

o n bib a p dsl Ql

lu m

b lb

oe

w ed

nrv

r

£/ d s l

ncl d s l $ I g/

subm

Qrn ini

B

PLABE I

4

PLATE II (ROSE)

A.

Young normal root tip excised just before tbe inoculations were made* X25.

B.

Longitudinal section of young normal root tip.

C.

Gross section of a young normal root tip.

X?0.

X70.

34

i i

B

A

C

PLATE II

PLATE III (ROSE)

4 Days A fter Inoculation. A*

Portion of young root showing migration of parasites.

X25„

B.

Root tip showing a larva entering root at about the growing p o i n t . X25.

G.

Longitudinal section showing migration of larva in stele. X70.

D.

A drawing of the parasite which shows that no growth or development has yet taken place. X660.

.bfb rued Wv

r,? ?

^>ore*

A

ELATE IV (ROSE)

8 Lays After Inoculation. A.

Portion of* root showing position of* parasite*

X25,

B*

Longitudinal section of* root tip showing very early stage of giant cell formation* X70.

G.

A drawing of the parasite which shows that little, if any growth or development has taken place, X660,

f /'

IM l

A

B

bib med

ELATE V (HOSE)

12 Days A fter Inoculation* A.

Portion of a stained root showing orientation of* parasites with regard to vascular tissues of* root. XSO.

B.

Same as A.

C.

Longitudinal section showing multinucleate giant cells in the immediate vicinity of the parasite’s head. The posterior end of the parasite was cut away when the section w as prepared. XL25.

D.

A drawing of the parasite which shows that some growth and development have occurred. X660.

X25.

ELATE VI (ROSE)

16 Days A fter Inoculation. A.

Portion of stained root showing size and position of parasites. X25.

B.

Longitudinal section of a root ■showing position of parasite with. rela.tion to stele, cortex and the giant cells. X70.

C.

A drawing of the parasite which, shows development of ovaries and presence of spike tail. X235.

PLATE 7 H

(ROSE)

20 Says After xnoculation. A*

Part of stained root the stele* There Note lateral root of the parasite.

showing parasite entirely within is no evidence of gall formation* formation in the immediate vicinity X6Q.

B.

Same as A.

0.

Longitudinal section of root which shows the parasite in the eortez almost parallel with the longitudinal a;-:is of the root. A portion of the mnltinucleate giant cells m a y be seen. Also a lateral root has formed but at sone distance from the head of the parasite. X70.

D.

A drawing of the parasite which shows developing ovaries, eggs, uteri and presence of vagina and vulva. Note that the spike tail has been lost with the second molt. X235,

X25.

.ovr dev _ov d e v

PLATE VTII (ROSE)

24 Days After Inoculation. A.

A portion of stained root which shows an egg mass on the posterior end of the parasite. Differentiation between the parasite and host is difficult because of the deep staining character of the older root tissues. Note lateral root development from near the place occupied by the parasite. XSO.

B.

Same as A.

C.

A longitudinal section of root showing position of para­ site and giant cells. The gelatinous matrix is just starting to form at the posterior end of the parasite. Note that there is no evidence of gall formation. X70.

D.

A drawing of a mature parasite. Note coiled ovaries and uteri containing eggs in various stages of development. X235.

X25.

ELATE VIII

PLATE IX (TOMATO)

A.

A very young, normal root tip excised just before the inoculations were made. X25.

B«

A longitudinal section of a very young, normal root. X70.

C.

Gross section of a very young, normal root.

X125.

PLATE IX (TOMATO)

A.

A very young, normal root tip excised just before the inoculations were made. X25.

B.

A longitudinal section of a v e r y young, normal root. X70 .

C.

Gross section of a very young, normal root.

XL25.

W.* " ' p' ^ aX v* C

.ELATE IX

ELATE X (TOMATO )

4 Days After Inoculation. A.

A stained root tip showing the head of the parasite in the vascular tissues with the posterior end lying obliquely and downward in the cortex. Forward growth of root has continued after the parasite entered the root, as the parasite lies some distance from the root tip. X25.

B.

A longitudinal section showing that rnultinucleate giant cells have already formed. The parasite* s head may be observed in the immediate vicinity of these cells. The posterior end of the parasite was removed in sectioning. X70.

C.

Cross section showing several rnultinucleate giant cells. X70.

D.

A draxving of the parasite which shows that growth and develop­ ment have already begun. X660.

port?x

prm pen.

PLATE XI (TOMATO)

8 Days After Inoculation, A.

A stained root tip which, shows the darkened parasite and the region occupied by the giant cells. Note that gall formation is quite evident. X25.

B.

A longitudinal section which shows the position of the parasite in relation to the rnultinucleate giant cells. X70.

C.

A drawing of the parasite which shows that considerable growth and development have already taken place. X235.

on ..bib wed ...vlv ..por

ex

&vr d e v

.... o v c/ev

...TCt

..a n

ELATE XIX (TOMATO)

IS Days After Inoculation. A stained root showing the presence of three parasites. Note that forward growth of root has been stopped, probably during the early stages of infestation. XS5.

B.

A longitudinal section through a gall, showing multinucleate giant cells and a part of the parasite. Apparently this section was cut obliquely and, as a result, the head of the parasite was removed. X70.

C.

A drawing which shows the growth and development of the parasite. Note that the coiled ovaries extend to anterior end of the body. Uteri as well as the va and vulva are already formed. XS35. GQ d-

A.

ELATE XIII (TOMATO)

16 Days After Inoculation. A.

A stained root tip which, shows several mature parasites with the egg masses present on the root surface, Bote that forward growth of the root was stopped, apparently soon after the large number of parasites started feeding, as the root tip extends only a short distance from the region of infestation. X25„

B.

A longitudinal section through a gall which shows multinucleate giant cells and two parasites. With one pnrasit the egg mass has broken through the surface of the root. X70.

C.

A drawing of a mature parasite showing coiled uteri and ovaries with eggs in various stages of development. X235

56

m

B A

ut

Ov Ut

\V

ret

vul

~C PLATE XIII