STUDIES ON EESISTANCE IN CERTAIN POTATO HYBRIDS TO BACTERIAL RINGROT CAUSED BY

CORYNEBACTEF.IUM SEPEDONICUM (SPIECKERMANN AND KOTTHOEE) SNAPTASON AND BURKHOLDER

Frank Vernon Stevenson

A THESIS

Submitted to the Graduate School of Michigan State College of Agriculture and Applied

Science in partial fulfilment of the requirements for the degree of

MASTER OP SCIENCE

Department of Botany and Plant Pathology

19UH

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Acknowl edgment

The writer is indebted, to Dr. J. H. Muncie for his help

and suggestions in handling the material and to him and Dr.

E. A. Bessey for their valuable suggestions incorporated in

the preparation of the manuscript.

Table of Contents Page

Introduction....................... ........................... 1Name of the Disease.............................. 1Economic Importance, ......... . 2Distribution.................................................. 2Transmission and Spread*....... .................... ........ 2Causal Organism..................... ......................... 3

Nutritional Requirements.............. UBurkholder1 s Medium............. U

Colony Characteristics,...... ............. ............... 5Synonymy............. ....................... . 5Host Range, ......................... .......... 3

Symptoms.............................. ..................... . 6Foliar, ........... 6Tuber.............. 6Diagnosis. ........ ................................ 7Ultra Violet Light Diagnosis...................... 10Oram Stain. ................................. 10

Gram's Modif ic ations of Lugol' s I odine.............. . 11Burke’s Modification of the Gram Stain..................... 11

Varietal Resistance. .... .................. 13Inoculation Technic ...... lHMethod of Reading. .............. l6Results of 19^2 Experiments.................... 17

Emergence,................................................. 17Michigan State Seedling Varieties.......................... 1JU.S.D.A. Seedling Varieties............. 18

Varieties Used in 19^3** .............. 20Results.... * ................ 21Discussion and C o n c l u s i o n s ......... 22Summary. .... ..................................... , 2hLiterature Cited................ 26Table 1. Emergence and Presence or Absence of Ringrot in "

Seedling Varieties of Two U.S.D.A. Crosses Pedigree Nos.185and B186 in Tests at East Lansing, Michigan, in 19^3...... 29

Table 2. Presence or Absence of Ringrot Infection in 32......Selections Prom 18 Hybrid Varieties and Selections Carried, at Michigan State College Inoculated and Tested at East....Lansing, Michigan, in 19̂ -2............ 30

Table 3* Emergence and Type of Response to Ringrot Inoculatim of Seventeen Crosses or Varietal Selections, Mostly ObtainedFrom the United States Department of Agriculture, as......Determined in Field Trials at East Lansing, Michigan, in...19U3............................................ 31

Figure 1. Prevalence of Ringrot in the United States of.....America in 19^0....... 33

Figure 2. Ringrot in Michigan Including I9UU Reports — by... Counties. .............. 3^

INTRODUCTION

The potato occupies sixth place among the crops grown in the

United States as determined by acreage and value of the crop pro­

duced, As a table food product, it undoubtedly is only second to

that of wheat (28),

The potato is a very large factor in the economic set-up of

Michigan as it is the number one cash crop of the state (17). A

considerable part of this acreage is in certified seed production

both for use in the state and in adjacent areas.

In 1931* Conners reported a serious new disease of potatoes

in Canada (8). The first published report regarding this disease

was by Spieckermann (2̂ -) in an article in a German illustrated

farming paper in 1913* Donde stated in 1937 that he had seen it in this country as early as 1932 (l). It was first suspected to

be present in Michigan by Muncie in 1939 and the suspicion was confirmed in I9H0 (18). Considerable attention has been given

this disease because of its rapid spread and its heavy toll in

those fields wherever it has taken hold.

Name of the Disease

In first publishing on this disease, Savile and Racicot (22)

called it "Bacterial Wilt and Rot," Bonde used the name "Bac­

terial Wilt and Soft Rot" (l). Since "bacterial wilt" was already

in use for a disease of potatoes caused by Phytomonas solanacearum

and "soft rot" for one caused by Erwinia carotovora. it was de­

cided by the Committee to Coordinate Research on New and Unusual

Potato Diseases to translate the descriptive German "Ringfaule"

and call the new disease "Ringrot" or "Bacterial Ringrot" (lh) and

thus avoid the confusion which would result from using names which

were already in use for diseases caused by other organisms.

Economic Importance Losses due to the disease have been extensive and severe in

localities where it has become established. The above-named com­

mittee states that: "It is evident from the letters received by

the Committee that ringrot is rapidly becoming one of the most de­

structive diseases of the potato in this country. — — The impera­

tive necessity for both extensive and intensive research has been

emphasized by all collaborators" (10),

Distribution

The known distribution of ringrot in the United States is

shown in Figure 1. This shows it to be present in greater or

lesser degree in 37 of the US states; nine of them for the first

time in 19^0 (29), Further search will probably show it to be in

more of the remaining 11 states since many of those now reported

as free are bounded by states already reporting the disease.

In Michigan, the disease has been reported from 2U widely

scattered counties. These are shown in Figure 2. These findings

are based on stain tests of material received in the Botanical

Section of the Experiment Station from county agents and potato

inspectors.Transmission and Spread

ProbabLy the principle method of spread is in diseased tubers

although it may be carried along for considerable time on machin­

ery such as the grader, planter, and digger, in sacks, bins,

crates, warehouses and other articles coming in contact with

diseased potatoes,

Dykstra, in discussing the means by which the pathogen sur­

vives from season to season, states that the California station

found little if any over-wintering of the pathogen in the soil,

Bonde showed, however, that in Maine diseased tubers may survive

and give rise to volunteer plants (2) which may harbor the disease.

Brentzel and Munro working in North Dakota reported that in­

sect transmission experiments, with grasshoppers as vectors, gave

uniformly negative results (U), list and Kreutzer, of Colorado,

indicate that grasshoppers, Colorado potato beetle, and black

blister beetle are capable of transmitting the disease, but it is

doubtful if insect transmission in the field is a problem (15).Experiments at various experiment stations wherein a cutting

knife was contaminated and then used to cut a series of healthy

tubers which were planted in the order cut have shown ringrot

promiscuously throughout the lots and as high as the 2hth hill,

Dykstra reported that California workers showed the cutting knife

to be the most effective agent in the spread of bacterial ringrot

(10), Kreutzer et al of Colorado (12) and Starr and Riedl (26) of Wyoming report good results in holding down spread by the

cutting knife through the use of a power-driven revolving knife

which is partially immersed in a disinfecting bath.

Causal Organism

The organism causing ringrot is a small (,~{2p by 1,25jj), non-encapsulated, non-motile, non-sporulating, rod-shaped bac-

-U~

terium occurring singly but rarely. Most often, the rods are

paired and occasionally chains of four or more will be seen.

The staining reaction is Gram-positive although variations are

quite often present. Pleomorohism is frequent and enlarged, club-

shaped cells result. According to Skaptason and Burkholder, divi­

sion is by "snapping" and V- or L-shaped pairs are fairly common

(23).Nutritional requirements

The organism is quite fastidious and this factor makes it

difficult to grow on ordinary nutrient media as the rate of growth

is very slow and colonies are quite small. The use of an enriched

medium devised by Burkholder gives quicker growth and more certain

results (5).Burkholder's Medium:

300 grams of sliced potatoes boiled, filtered and broth made up to one liter with distilled water.

Peptone 5 grsN«gBP0 2 11

NaCl 2 H

Sodium Citrate 1 II

Asparagine 6 If

Dextrose 6 II

Agar 12 II

Marten et al of West Virginia showed that the addition of a

solution of C. P. potassium dichromate at the rates of 1:9000,

1:12,000 or l:15*000 to the above agar is very helpful in keeping

-5-down Gram-negative contaminants when making isolations (l6).

Colony Characteristics

Colonies average about 2mm. in diameter. They are cream

colored, wet, glistening, and rather sticky in consistency.

Synonymy

The organism was first called Bacterium sepedonicum Spieck.

and Kot., in 191^. E. F. Smith then named it Aplanobacter sepe­

donicum (Spieck. and Kot.) E. F, S., in 1920; Magrou put it in

the genus Pbytomonas as Phytoroonas sepedonica (Spieck. and Kot.)

Magrou in 1937* ond. later in 1937* Savile and Racicot again con­

sidered it to be a member of the Phytomonas group. The name

Corynebacterlum sepedonicum (Spieck. and Kot.) Skap. and Burk,

was given by Skaptason and Burkholder in 19^2 because the presence

of numerous pleomorphic forms and the "snapping" type of division

taken together with its other characteristics seemed to indicate

the organism to be a member of the diphtheria group (23). Dowson

places all Gram-positive plant pathogens in the genus Corynebac-

terium (9)»

Host Range

All commercial varieties of the potato are susceptible (10).

Rupert (20) tried inoculations on eight other related hosts and

of these he found the tomato (Lycopersicum esculentum) to be the

only one showing definite infection. Stapp, working in Germany,

reported infection of tomato, Pjsuffl arvense. and Phaseolus vulgaris

by artificial inoculation (27).

- 6-

Symptoms

Foliar

Symptoms on the potato plant in the field are a slight graying

with a later yellowing and wilting of the leaves. Frequently, not

all branches are infected, but the infected branches droop or curl

downward on the ends and the leaves roll upward. Often all that

is affected is a part of a shoot or as little as one side of a

single leaflet. Ag the condition advances, the parts turn brown

and dry. There is no apparent browning of the stem although the

organism may be found in abundance up to a height of three inches.

Kreutzer and McLean found that a rapid decrease in numbers oc­

curred as the height above that point increased (13).Tuber

In the tubers, the symptoms of severe infection are a shrink­

ing and cracking of the skin. These cracks may extend below the

skin as far in as the vascular ring. If such a tuber is cut

across, especially near the stem end, it will frequently show a

yellowing of the vascular ring, although the disease may be present

without showing this coloring. In advanced stages, a soft cheese­

like exudate may be squeezed out of the ring in a manner suggest­

ing toothpaste out of a tube. It is often possible to "peel" the

cortex from the vascular ring. If there are no secondary rots,

such as that caused by Erwlnla carotovora. the tuber will become

a hard leathery mummy.

Often the decay is more general than that described above and

the crumbly condition extends far into and often entirely through

the tuher# In such cases, a honey-combed mummy is all that is left#

This mummy is also firm and dry.

One striking difference between ringrot and other potato rots

is that it is entirely devoid of a disagreeable odor. Many tubers

having ringrot are a stinking, slimy mass, but this is due to the

presence of secondary invaders.

The disease is not readily identified by the field symptoms

as they are not distinct and may be confused in certain stages by

the presence of blackleg or the browning caused by Fusarium wilt.

Because symptoms appear so late in the growing season, early and

late blights, insect injuries and especially maturation of the

plant tissue, serve to confuse the picture further. Many tubers

do not break down, if at all, until they have been in storage for

many weeks, but even though they may fail to show any symptoms,

the presence of typical Gram-positive staining bacteria in the

vascular ring proves them to be affected with the disease.

Diagnosis

Because of the uncertainty of identification of ringrot from

symptoms visible on the tops and tubers,, due to its highly variable

character, and through the presence of other conditions which serve

to confuse the observer, it becomes necessaiy to rely on some other

technic for certainty in diagnosing this disease. The slow growth

and inactive biochemical nature of the organism make the use of

fermentation indicator technics practically an impossibility. The

Gram-stain seems to be the method of choice. This is unfortunate

because the Gram-stain is not intended to be a final diagnostic

test. The Gram-stain is considered to he reliable only for young

actively growing cultures as it has been shown, according to Salle

that older cultures become quite Gram-variable (2l). The age of

the organism in a stored tuber can only be guessed at. Since the

identification of the ringrot organism is usually made from smears

of diseased tubers and because these bacteria are so slow-growing,

much variation is bound to occur*

According to Salle, "The change of Gram character with age is

especially true of those organisms which are only weakly Gram-

positive and are cultivated in media containing fermentable sub­

stances that become acid in reaction as growth proceeds." He also

shows that little of either stain is taken up at the iso-electric

point and that the Gram character of many organisms can be shifted

by the addition of suitable acid or basic substances to the medium

One or more of these conditions may well explain such stained

material as is found where every other condition except the Gram-

stain reaction indicates the presence of ringrot. The author has

had stained material wherein all but a few pairs of bacteria were

red, or one half of the pair stained red and one blue. In others

which were all doubtfully reddish-blue or negative, all other con­

siderations such as size, shape, numbers, and condition- of the

tuber were strongly indicative of ringrot infection.

In this laboratory, most of these would be marked + indicat­

ing them to be, at most, suspicious. This author believes that

seed lots containing a number of tubers unrecognized as infected,

thus may be allowed to continue in growers 1 hands for a consider­

able period until conditions become such that the bacteria recover

their Gram-positive expression and are then recognized. A definite

indication of this being possible was to be seen in a case involv­

ing one grower's certified seed. The author stained some smears

which gave a + reading because of their reddish-blue color, as

above, and was told this same rating had been given this grower's

potatoes in previous readings, later in the year, material was

returned from a car lot of the same grower's seed that had been

shipped out of state and refused because of ringrot. On staining,

the organism was found in abundance and was clearly recognizable

so that this lot which had merely been under suspicion was now

proved to have the disease* The return to an active growing state

of the organisms present or the influence of other more obscure

balances of factors such as oxidation of products, or change in

the metabolic rate, or some other conditions, had produced a condi­

tion more nearly optimum for the recovery of expression of the Gram

character at some time subsequent to the first examination. It is

also possible that in selecting rotted tubers to be sent in for

diagnosis, the inspector did not originally select the tubers badly

infected with the ringrot organism. The presence, however, of

organisms, which were morphologically those of ringrot but doubt­

ful as to staining reaction strongly indicate some change in dye

absorption, and such behavior, coupled with the uncertainties of

the Gram^-stain technic mentioned before, may thus be accountable

for failure of recognition in the stain test. It is possible,

therefore, for an incorrect reading to be made if too much stress

-10-is laid on Grsm reaction color,

"Ultra Violet Light Diagnosis

A technic for use in the H0°F. seed storage at the time of

cutting seed is based on the fact that ringrot tubers fluoresce a

characteristic greenish color, in the vascular ring, under ultra

violet black light (ll). It should be emphasized that thorough

experience with the technic combined with a tendency toward a

heavy discard of all suspicious looking tubers seems to give good

results* It cannot, however, take the place of the stain technic

for laboratory diagnosis. Burkholder reports that Skaptason found

the fluorescing substance to be riboflavin, and he says that this

is by no means a specific test as other bacteria synthesize this

same vitamin. It is further pointed out that riboflavin is rapidly

destroyed at high temperatures and especially under ultra violet

light (6).

Gram Stain

In working on a new tecbnic for staining bacteria in tissues,

Christian Gram (188H) discovered the differential principle of

the stain which now bears his name (2l),

"The usual procedure calls for anilin gentian violet for 1 to 2 minutes; blotting without washing; Gram's iodine solution for 1 to 2 minutes; blotting without washing decolorization in 95 per cent ethyl alcohol for 30 to SO seconds; blotting, and counterstaining briefly ip eosin, safranin, fuchsin, or Bismark brown( 7)•"

Gram’s solutions:Anilin Gentian Violet

Sat, Ale, Sol, gentian violet..........520 c.c.Anilin water (2 c.c, anilin shaken with

9S c.c. water and filtered).........100 c.c.

— 11-Gram*s Modifications of Lugol*s Iodine

Iodine... 1 g.Potassium Iodide..................... 2 g.Distilled Water.......................3^0 c.c.

Counterstain

Safranin (2.5 per cent solution in 95 Per centethyl alcohol).................. 10 c.c.

Distilled Water 100 c.c.

In this study, a modification of the Gram stain by Burke was

used as a basis for further modification (25).Burke*s Modification of the Gram Stain

•'Hood a thin film with a 1 per cent aqueous solution of methyl violet. (Add to and) Mis’ with the dye 3 to 8 drops of 5 Pe** cent solution of HaHCO,* Allow to stand 2 to 3 minutes. Plush off excess^stain with Lugol's iodine solution. Cover with fresh solution and let stand one minute or longer. Wash in water briefly and remove practically all of the water by blotting, but do not allow smear to become dry. De­colorize with acetone-ether (3 to l) until decolorizer flows from the slide almost uncolored (usually less than 10 seconds). Wash with water, blot or air-dry. Counter-stain, using 2 per cent solution of Safranin 0. Wash briefly with water, blot and dry quickly."

This was somewhat changed in that the recommendations

of Professor G. B. Reed as given by Racicot, Savile and Conners

(19) were followed in making up the following solutions:Solution 1. Crystal violet............... .. 2.5 gms.

Water................................1000 c.c." 2. Sodium bicarbonate................... 12.5 gms.

Water. .1000 c.c." 3* Iodine 20 gms.

Sodium hydroxide (molar solution)... 100 c.c.Water............. ..... ........... 900 c.c.

" U. Ethyl alcohol, 95 per cent.......... 750 c.c.Acetone........................... 25O c.c.

" 5* Basic fuchsin, saturated solutionin 95 Per cent alcohol...... .. 100 c.c.Water........................ ....... 900 c.c.

- 12-

Tha last five solutions mentioned above were originally in­

tended to be used in a very rapid staining technic, but this author

returned to more conventional periods of time for allowing the

solutions to remain on the smears.

T^e slide, with the smear affixed, was first flooded with the

sodium bicarbonate solution because less trouble from crystallizing

out of the crystal violet seemed to result than when the usual

order of crystal violet followed by sodium bicarbonate was used.

The crystal violet solution was then added drop by drop until the

smears were well covered and this mixture was allowed to stand

exactly two minutes. The slide was then drained and the iodine

mordant flooded on and left for one minute. The slide was flushed

using a washing bottle and then decolorized using a medicine

dropper to apply the acetone-alcohol mixture. Decolorizing was

continued for some time until the solution ran clear from the

slide. The slide was washed thoroughly, using the wash bottle and

then flooded with the counterstain. This was washed off almost

immediately and the slide blotted and dried by holding it above the

flame of a Bunsen burner. The stained smear was then examined

under oil immersion using oil of cedar because of its excellent

light conducting qualities.The smears were prepared by scraping out the whole of the

vascular ring in the area exposed by cutting off the stem end of

the tuber, as near the end as possible, with a sharp-pointed scalpel,

and then pressing this material on the slide and squeezing out some

of the juice until it appeared milky. Each slide was marked off

- 13-into ten squares with a wax pencil, and a small area on one end was

left to allow for identitying the slide and for gripping it during

staining operations. Tor ease of examination, the order on the

slide was from one through five across the "bottom from left to

right, then up to the right-hand square above for six, and thence

to the left through number ten, thus finishing above number one.

Since so much stress in this method is placed on color, the

microscope light is an important factor. In this study a Spencer

370 lamp, using a 100 watt projection type bulb, was used. It has

an iris arrangement whereby the light can be focused. The dis­

tance from filament to the flat side of the mirror was ten inches.

A blue filter was used. The beam of light was focused with the

iris until it just filled the field using the low power objective.

The substage condenser of the microscope was kept at its highest

level. All readings of stained material were made with the light

as above and using the oil immersion lens.Varietal Resistance

Attempts so far made to control ringrot by treating the seed

pieces have been too uncertain in results and have in many cases

materially reduced the stand, and, as a consequence, emphasis is

being placed on the problem of developing or finding varieties re­

sistant to ringrot.

Results from Wyoming, based on a. reading of field symptoms,

as reported by Dykstra (10), show only United States Department of

Agriculture seedling as having no symptoms by the time of

frost which occurred shortly after the September lgth reading.

-lH~

Bonde et al also have reported, in preliminary observations

of hybrid potato material, a possibility of inherited differences

with regard to resistance to bacterial ringrot infection (3). In

order to see if such differences do exist and to determine if

some of these strains might be suitable for parent material, which

could be used in developing higher resistance, it seemed desirable

to test some of them more fully.

In the author^ test, stress was placed on the actual pre­

sence of the bacteria in the tuber rather than on the presence

or absence of field symptoms.

The potatoes used in the 19U2 study were grown from tubers of United States Department of Agriculture seedlings from the crosses

President x Earlaine (Pedigree No, B185) and President x 96 - 56

(Pedigree No, Blg6). There were 797 tubers of the first and 776

of the second cross. In addition, 32 selections of 18 hybrids

carried at Michigan State College for scab resistance were in­

cluded. Chippewa was used as the check variety.

Inoculation Technic

In the Wyoming study cited above the inoculation technic was

as follows: "The cut surface of the seed piece was smeared with

bacterial ooze from a ringrot diseased tuber, and the whole seed

piece was then submerged in a water suspension (of) bacterial ooze

taken from diseased tubers." Bonde et al omitted the submersion

in ooze and inoculation was affected by rubbing slices of infected

Green Mountain tubers on the freshly cut surfaces of the healthy

seed pieces.

The needle method of inoculation was used in the present in­

vestigation because it had given 'quite a high incidence of in­

fection in previous tests at this station. All of the tubers

tested in 19^2 were inoculated into each of two eyes per tuber. A

"spear" point needle was used because it gives greater access to

vascular tissue than does the stab of a straight needle. The

needle was dipped into the exudate from a known ringrot tuber,

just prior to inoculating each potato, and it was then thrust into

the "eyebrow" and well into the flesh of the tuber in order to make certain that the vascular tissue had been penetrated and that an

adequate amount of inoculum had. been placed sufficiently deep to

secure infection.

After inoculation, an incubation period of a week at room tem­

perature was given in order to allow the slow-growing bacteria to

become established. The tubers were planted in the experimental

plots at East Lansing on May 20, I9U2. The txibers were spaced

1^—15 inches apart and covered with a hoe. The rows were three

feet apart and were approximately 135 feet long. The plants were

dusted as needed through the season using a fixed copper-calcium

arsenate "Cuke and Melon" dust or "Talcla" with calcium arsenate

added at the rate of one pound to twenty of dust to keep down the

injury from early blight, hopperburn, and the Colorado potato

beetle.

The potatoes were dug the first week in October in separate

hill lots. The tubers from each hill were put in a two-pound

paper bag and these were collected in potato crates and stored in

— 16—a fruit cellar. They were allowed to stand until Februaiy because

it was thought that this would allow the disease to progress to a

recognizable degree in those tubers having the organisms present,

but not showing any symptoms. They were then removed to the labora­

tory and the readings made.

Method of Reading

To reduce the number of microscopic examinations necessary,

each lot was examined macroscopically and the hill eliminated, if

possible, by that means. The macroscopic inspection consisted of

cutting off the stem ends of all the tubers from each hill and

squeezing the m as they were cut to see if the "ooze" of bacteria

would result. The yellow discoloration was not used as a basis

for discarding a hill in this test as it was not thought to be con­

clusive enough. As soon as an oozing potato was found, that hill

was eliminated and tabulated in the "Macroscopic positive" column.

If none of the tubers showed this symptom, they were "apparently

free" and a representative tuber looking the most suspicious with

regard to ring color was used to make a stain test. These stains

were made when ten "Apparently Free" hills had accumulated, A

tuber found to have the disease on microscopic examination was

classed "stain positive" and the remainder which had successfully

passed both the macroscopic and microscopic tests were classed

"Ringrot Free," See Table 1.The Michigan State selections of seedling varieties were

handled in the same manner except that one hill showing a ringrot

tuber served to eliminate all hills of that selection. See Table 2.

-17-Results of 19^2 Experiments

Emergence

Emergence was fairly low in the inoculated groups. Of the

797 tubers of Pedigree No. B185 planted, ij-5̂ or 57 Per cent emerged. Of the 77^ tubers of Pedigree No. Blg6 planted, 501 or 65 per cent emerged. Seventy-three of the inoculated Chippewa checks

emerged.

Fifty tubers of each pedigree number, selected at random, and

fifty of the Chippewa variety were planted uninoculated to check

for the presence of ringrot so that the efficiency of the method of inoculation might be determined. All of these emerged and remained

free of ringrot as determined by Gram-stain tests.

Undoubtedly, much of the poor emergence in the inoculated groups

was due to ringrot because in addition to the 27 per cent failure in the Chippewa check, h3 per cent of Pedigree No. Bl?5 and 35 per cent of Pedigree No. Blg6 failed to emerge or died before setting tubers as there was no evidence of them at digging time.

Per cent of Disease-free Tubers Present.

Michigan State Seedling Varieties.

The selections of Michigan State seedling varieties are, re­

ported without percentages being figured as each selecti on number

represents a variety. Those that were ringrot free were a four-

hill lot of Selection 1 of the cross Eindenburg x 627-618, a two- hill lot of Hindenburg x 8-1 selection unknown, a six-hill lot of the selection K38-8, a six-hill lot of selection 3 of the cross 627-618 x Katahdin, an eigat-hill lot of selection 2 of cross

- 18-336-l^U x Ostragis, a six-hill lot of the variety K38-7 and a three-hill lot of 528-118. (See Table ?)U. S, D. A, Seedling Varieties

There is a noticeable difference in the ease with which the

presence of ringrot may be detected macroscopically in the two

U. S. D. A, groups. All but 11 of the 301 hills showing ringrot

positive tubers were recognizable macroscopically in Pedigree

Ho. B185, In contrast, 77 of the 33& positive for ringrot hills

of Pedigree No. B186 passed the macroscopic test but showed in­

fection by ringrot organisms in the stain tests.

The percentage of hills from emerging tubers free of the

ringrot organism was the same in both of the U. S. D. A. groups

being 33 P©r cent. Of the ^Vbills of Pedigree No* B185 evident at the time of digging, 153 were free of the disease and of the 501 hills of Pedigree No. B186, 165 were also free of the disease.

Since 35 Per cent of the inoculated check yielded negative

Gram-stain reactions, it seems necessary to attribute at least a

third of the ringrot free survivors, in the hybrid groups, to

the failure of inoculati on. This would give 102 tubers of Pedi gree

No. BI85 and 110 tubers from Pedigree No. Blg6 free of ringrot.In an effort to determine why such a high per cent of the

check should give rise to apparently ringrot-free tubers, twenty-

five Chippewa tubers used whole, cut, and in some cases as "hearts"

(center portions containing no eyes which result when large tubers

are cut for seed) were inoculated and allowed to stand in an un­

covered jar on the laboratory table until they had sprouted.

- 19-It was interesting to note that of the three "hearts" included,

one developed two sprouts. These were either from very inconspic­

uous eyes or adventitious buds which developed during the incubation

period. These sprouts were free from the disease as the ringrot

infection had not had sufficient time to progress from the point

of inoculation to these buds before the shoots were big enough to

have become firmly established. It might be possible, therefore,

to have healthy plants, where all eyes were inoculated, through

the agency of these very small insignificant eyes or adventitious

buds.

In a few cases, the sprout appeared to be healthy even though

the eye had been inoculated and it thus seems possible that sprouts

may become established and then softrot, or some other rots, would

destroy the seed piece before the slow-growing ringrot organism

could get into the daughter plant’s vascular system, again yield­

ing an "escape" plant.In some cases, sprouts were produced from uninoculated eyes of

the inoculated tuber. Thus it may be seen that there are several

possible ways in which healthy plants may arise from inoculated

tubers and, considering these, the number of escapes does not

seem too high.That ringrot may reduce emergence was shown by some inocu­

lated tubers in that they did not seen to be able to nroduce a

viable sprout. The sprouts that attempted to grow were weak and

the tips shrivelled and blackened and death occurred when they

were about two inches in length.

- 20-It was necessary to make further inoculations of the surviv­

ing material in order to determine if the survivors were escapes

and to eliminate these from the material being tested. Four tubers

of each Michigan State variety and one tuber from each ringrot-free

hill of the U. D, A. seedlings were saved for testing in 19^3»

Varieties Used in I9U3In addition to the tubers just mentioned above, a number of

seedling varieties from 15 U. S. D, A. crosses and selfed lines

were included in the tests for the first time this year. See

Table 3« Since all tubers, including those of the new accessions,

were larger this year than last when two eyes were inoculated,

they were inoculated into each of three eyes, incubated for a

short time at room temperature, and planted on May 10, 19^3* The

method of planting and cultural practices were essentially the

same as in 19h2.

The weather was unseasonably cold and rainy and very little

growth was made early in the season. The ground was extremely

moist, and, a.fter each rain, large puddles remained for consider­

able lengths of time at various points in the field. As a con­

sequence, there was a large reduction in stand, many tubers fail­

ing to emerge, and any attempts to compare most of the varieties

according to the per cent of infection would necessarily lead to

erroneous conclusions. However, even though the stand was re­

duced, where considerable numbers were present, the results give

a fair indication of that variety's resistance because the sample

is sufficiently large. That the inoculations were quite successful

*•21-is shown by the high proportion of tubers showing ringrot infection;

there was a total of J20 out of the 753 plants giving that reading.

The severe reduction in stand is shown in that, excluding the checks,

of the 2631+ tubers inoculated and planted, only 753 hills or 29 per cent were evident at time of digging, The 50 Chippewa inoculated

checks were very hard hit being on the low side of the field and

only eighteen plants survived. Of these, seven were positive and

eleven negative. The Michigan State varieties and selections were

in this same portion of the field and it was only possible to

identify and harvest a selection of Green Mountain and the cross

627-6I8 x Katabdin Selection No, 3» ^he selection from the

variety Green Mountain was obtained from H. C. Moore of the Farm

Crops Department, as a suspected ringrot carrier which had been

elimiated from bushel lots by the use of ultra violet light. It

was included because it had failed to show a positive staining re­action.

The Chippewa uninoculated checks were favored in locati on,

being on the high side of the field and J2 per cent or 36 out of

50 tubers survived.The results obtained with each different cross or varietal

selection are shown in Table 3*Results

The highest per cent of tubers apparently free from ringrot

was from Cross B272 (President x 336-1M O which had 67.7 per cent showing negative Gram-stain reaction followed by Cross ®355 (96-56 x 336-IHU) with 66.9 per cent and the Cross B27I (President x Earlaine)

- 22-with 63.0 per cent. An intermediate group consisted of Cross BP91

(Katahdin x 1+6952) with 57*1 per cent obtained from 7 tubers, Cross

B269 (Earlaine x 33^"^^) with 5^»7 per cent, and Cross B?7l+ (Presi­dent x H715-6) with 55*1 Per cent of negatives. Crosses B303 (Houma x ^6952), U6000 x U6952) and B3IK) (C. S. 1608 x Earlaine 2)had 27.7 per cent, 26.3 per cent and 21.U per cent tubers showing negative Gram-stain reaction respectively.

The selfed line B1103 (President selfed) and the Cross B316 (Friso x Katahdin) had too few survivors to warrant placing much

confidence in their relatively high per cent of gram-negative re­

actions. This was also true of the Michigan State seedling variety

and the varietal selection, and, to a certain extent, of the inocu­

lated check. Cross B?6h (Irish Cobbler x Earlaine) had a very low survival rate and all of these plants gave positive Gram-stain re­

action for ringrot.

Discussion and Conclusions

The progenies of the two crosses in their second year of test­

ing have moved up from 33 Per cent showing negative gram-stain re­

action to 87.0 for Pedigree No. B185, and 8U.7 for Pedigree No. Blg'6. These are well above the averages of the other strains, which are

present in sufficient numbers to warrant direct comparison, so it

seems reasonable to attribute a large share of this rise to the

elimination of susceptibles in the first year test. The high per­

centage of individuals negative for ringrot in the Blg6 cross, being tested for the first time this year, is probahLy due to the fact

that infected tubers were unable to survive in the field and so

there was a preponderance of the disease-free tubers present at

time of harvest. Probably the general tendency to a higher per

cent of tubers showing negative Gram-stain reaction in all strains

as compared to the rate of the first season is also due in part

to the same response to adverse conditions, i. e., a low per

cent survival due to disease and a correspondingly higher per

cent of apparently ringrot-free tubers in the surviving plants.

The frequency with which President appears as one parent, in

those crosses having a high per cent of tubers testing negative

for ringrot, shows it to possess some resistance and to be capable

of transmitting this when used as a parent in making hybrids.

Earlaine and the selections ?Bd also appear in

crosses having a fair degree of resistance.

On the other hand, Irish Cobbler, Strain C. S. 1608, and the

varieties with numbers in the U6000rs seem to be low or lacking in resistance.

The results obtained on the tubers inoculated the second year

strongly support the idea that resistance to ringrot is hetero­

zygous (3) and that progeny more resistant than either parent can be obtained from hybrids with resistant material.

A final conclusion is that persons working with such material

should examine their survivors microscopically because it is im­

possible to diagnose the disease by the plant symptoms or macro-

scopically in the ring of the tuber with the degree of accuracy

that is possible with the stein test. This is shown by the number

of stain nositives obtained from apparently ringrot-free material

as .judged by the macroscopic test.

—2H—Summary

The results obtained from two years* work on ringrot identi­

fication at Michigan State College are discussed with regard to

the diagnostic technics used with recommendations regarding their

use and showing a modification of the stain technic to give more

certain results. The uncertainty of diagnosis of the presence of

ringrot from symptoms on the tops or in the tubers forces the use

of some other technic. In spite of its shortcomings, due to

variability of rates of dye absorption under different conditions

and in different strains of the bacteria, the Gram-stain seems to

be the method of choice. A modification of this technic using

stains intended for a fast technic but with more conventional

periods of application is given. The importance of familiarity

with the technic is discussed and the fact that improper readings

may be made because of irregularities in dye absorption is

stressed.

The needle technic of inoculation was used and gave a high

percentage of ringrot positives in the two years that this study

was in progress. The survival rate was always lower in the

inoculated groups, as compared to the uninoculated checks, and so

a considerable portion of the failure is assigned to rotting by

tie ringrot organism before the plants emerged or soon enough

afterwards so that there were no tubers present at digging time.

Cold wet weather in 19^3 materially reduced the stand, , but

large enough numbers of several of the lines remained so that

comparisons may be made.

-25-The seedling varieties used were from selections of hybrids

carried at Michigan State College for scab resistance and from

several crosses and selfed lines of U. S, D. A. material.

Tables showing the results obtained from the inoculation of

crosses and selfed lines of potatoes are given. The varieties

President, Earlaine, and 336-lUU appeared frequently in resistant crosses. Materiel being tested the second year showed a tendency

toward greater resistance because of the discard of susceptibles

the preceding year.

A comparison of macroscopic and microscopic readings showed

a fairly high number of apparently ringrot-free tubers to give a

positive stain test.

Maps of Michigan and the United States of America are included

and show the prevalence of ringrot by counties and states respec­

tively,

- 26-Literature Cited

1. Bonde, Reiner. A Bacterial Wilt and Soft Rot of the Potatoin Maine. Phytopath. 27:106-108. 1937*

2,_______________ Ring Rot in Volunteer Plants. Amer. PotatoJour. 19:131-133, 19U2.

3* , P. J. Stevenson, C. I*. Clark, and R. V, Akeley.Resistance of Certain Potato Varieties and Seedling Progenies to Ring Rot. Phytopath.32:813-819. 19U2.

4. Brentzel, W. E. and J. A. Munro, Bacterial Ringrot of thePotato: Investigation on Possible Dissemi­nation by Grasshoppers. N, Dak. Agr. Exp.Stat. Bull. ?95, 19UO.

5. Burkholder, W. H. The Occurrence in the United States of theTuber Ring Rot and Wilt of the Potato. Amer. Potato Jour, 15:2^3-2^5. 1938.

6. Diagnosis of the Bacterial Ring Rot of thePotato. Amer. Potato Jour. 19:208-212. 19^2.

7. Committee on Bacteriological Technic of the Soc. of Amer. Bac­teriologists. Staining Procedures. Pure Culture Study of Bacteria, 2(2): I V .-3 to IV , -2U. 101U. ^3>+

8. Conners, I* L. Canad, Plant Disease Survey Ann. Report 11:HP.1932.

9. Dowson, W. J. On the Generic Name of the Gram-Positive Bac­terial Plant Pathogens. Transactions of the British Mycologies! Society19U1-U2.

10. Dykstra, T. P. Results of Experiments in Control of BacterialRingrot in IPUO. (A compilation).Amer. Potato Jour. 18:27-55* 19^1.

11. Iverson, V. B. and H. C, Kelly. A New Method of IdentifyingPotato Tubers Free from Bacterial Ringrot and Other Types of Tuber Decay. Mont, Agr. Exp, Stat. Mimeo, Cir. 20, 19^0.

12. Kreutzer, W. A., D. P. Glick and J. G. McLean. Bacterial RingRot of Potato. Colo. Exp, Stat, Press Bull.9U. 19U1.

17. and J. G. McLean. Location and Movement of the~ ’ Causal Agent of Ring Rot in the Potato Plant,

Col. Agr. Exp. Stat. Tech. Bull. 30* 19^3*

- 27-lh, Leach, J, G, et al. Report of the Committee to Coordinate

Research on New and Unusual Potato Diseases. Amer. Potato Jour. 17:81-88. 19h0.

15- List, S, M. and W, A, Kreutzer. Transmission of the CausalAgent of the Ring-Rot Disease of Potatoes "by Insects, Jour, of Scon. Ent. 35:^55-^56* 19^2.

16. Marten, E. A,, C. V. Lowther, and J, G. Leach, A DifferentialMedium for the Isolation of Phytomonas sene- donica. Phytopath. 33: h0S-h07. 19̂ -3 •

17. Moore, H, C. Better Potatoes for Michigan. Mich, Ext. Bull.U9» 1939.

18. Muncie, J. H, Bacterial Ringrot of Potato. Mich. Ext. Bull.227. 19^1.

19. Rscicot, H. N.t D. B, 0, Savile, end I, L, Connors. BacterialWilt and Rot of Potatoes — Some Suggestions for Its Detection, Verification, andControl. Amer. Potato Jour. 15:312-318. 1938.

20. Rupert, J. A. Investigations on Bacterial Ringrot of Potatoescaused by Phytomonas sepedonica (Spieckermann and KotthoffJ Bergey et al. Master's Thesis (Unpublished.) Michigan State College. 19hl.

21. Salley A. J. fundament al Princ iples of Bacteriology, p. 6l.McGraw-Hill Book Company. Hew York, 19h3»

22. Savile, D. B. 0., and H. N, Racicot. Bacterial %lt and Rot ofPotatoes. Sci. Agric. 17:518-522. 1937.

23. Skaptason, J. B, and W. H. Burkholder. Classification andNomenclature of the Pathogen Causing Bacterial Ring Rot of Potatoes. Phytopath. 32:h39-UUl. 19^2.

2h. Spieckermann, A, Zur Kenntnis der in Deutschland auftretendenGefSsskrankheiten der Kartoffelpflanze.Illus. Landw. Zeitung 33:380-382. 1913*

25. Stafseth, H, J. A Laboratory Guide in Pathogenic Bacteriology.p. 6. University Lithoprinters. Ypsilanti, Michigan. 19h2.

26 Starr, G. H. and. W. A, Riedl. Bacterial Rina-Bot of Potatoes.Wyo, Agr. Exp. Stat. Bull. 2hh. IP hi.

?7* stapp, C,

28. Stuart, W.

- 28-Beitrage zur Kenntnis des Bacterium sepedoni- cum Spieckerm. et Kotth. Zeitschr. f. Para- sitenk. 2:756-822. 1929-30.

The Potato, pp. 3“ »̂ BippincottCompany. pp. (New York.) 1937*

29. The Plant Disease Survey. Plant Disease Beporter 255 130.19^1.

- 29-

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- 30-

Tab le 2

Presence or absence of ringrot infection in 3^ selections from IS hybrid varieties and selections carried at Michigan State College inoculated and tested at East Lansing, Michigan, in 19^2.

Ringrot Positive

Cross or Selection Selection Ho, ■ Ho, of hills

Chippewa X Ostragis 5 7m 11 6 2Chippewa X 8-1 7 16ii X Katahdin 5 12ii X 627-618 3 8Ogtragis X Katahdin 1 .— .

ii 11 2 7627-618 X Ostragis 1 6ii if 2 19ii X Katahdin 1 12ii X 11 6 12

Katahdin X 627-618 3 8Hindenburg x Katahdin 5 1

ii 11 9 —ii X 8-1 2 11

Russet Rural x Katahdin 6 2n II 11 — 5528-102 3c 11 1 5336-lU^ :ic Ogtragis 1 11.336-lHU 2 6528-118 6 7K38-7 H 15AKY-5 - 1 211 2 2

Ringrot Negative

K38-8 . inr 6Hindenburg x 627—1618 1 U

" x 8-1 — 2627-618 x Katahdin 3 6336-1UU x Ostragis 2 8528-118 1 3K38-7 1

Table

3, Emergence

and

type

of response

to ringrot

inocxilation

of seve

ntee

n crosses

or varietal

selections,

mostly

o"bta,

ired

from

the United

States

Department of

Agriculture, as

determined

in field

trials at

East Lansing,

Michigan,

in 19^3*

- 31-

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Table

3 (continued)

^mergence

and

type of

response

to ringrot

inoculation

of

seventeen

crosses

or varietal

selections,

mostly

obtained

from

the United

States Department of

Agriculture, as

determined

in field

trials at

East

Lansing, Michigan,

in 19^3*

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Figure

1. Brevalence

of ringrot

in the

United

States

of America

in I9

U0

□ Disease in field

H Diseased potatoes on sale for seed

Figure 2. Ringrot in Michigan including I9I& reports — hy counties.