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BULLETIN 773

Black Shank of Tobacco
in Connecticut Fields

By G.S.Taylor, J.L.McIn tyre, RE .Waggoner

JUNE 1978 p

THE CONNECTICUT AGRICULTURAL EXPERIMENT STATION NEW HAVEN

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in 2011 with funding from

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Black Shank of Tobacco
in Connecticut Fields

By G.S.Taylor, JL.McIntyre, P.E.Waggoner

In mid-July, 1973, a grower of shade tobacco discovered
wilting plants with stems blackened near the soil. Although
the wilting plants were scattered, they were most frequent-
ly found in low places where water drained slowly (Fig. 1).
The grower, who had seen similar symptoms on black-
shank-diseased tobacco in North Carolina, brought the wilt-
ed plants to the attention of a Station scientist. Pieces of
diseased stem kept in water overnight developed sporangia

Fig. 1. Black shank disease on shade tobacco in Connecticut. Mid-
July-note dying plants on left and wilted plant among still
healthy plants in right hand row.

of the fungus Phytophthora parasitica, and we knew that
black shank, which had not been reported north of Pennsyl-
vania, had reached Connecticut (Walton and Rich, 1974).

The black shank pathogen, Phytophthora parasitica (Dast)
var. nicotianae (B. de Haan) Tucker, can grow throughout
the tobacco plant Nicotiana tabacum L., but generally
attacks the lower stem and roots. The typical blackening of
the lower stem gives the disease its name, and the decay of
the stem and roots causes the wilting of leaves. Sporangia
form rapidly in water in the upper soil. Each sporangium
germinates to form about eight zoospores that swim in
water; the zoospores are carried to other tobacco plants,
spreading the disease. The fungus is encouraged by moisture
and warmth. Since Phytophthora parasitica var. nicotianae
is found in warm climates around the world and has an op-
timum temperature for growth of about 30°C, we had
thought Phytophthora parasitica was restricted to the war-
mer, more southerly states by the Connecticut weather.

Our first question was, "How had this Southern pathogen
reached Connecticut?" It is known that it can be moved in
soil, and hence it could have reached Connecticut in many
ways: on the wheels of autos, on irrigation pipe, on plants,
or even on used shade cloth. No clear evidence of which
route was taken has been established.

The next question was, "Would the Southern pathogen
be killed by the first cold winter or had a new race arisen,
and we would have years of black shank?" We soon learned
that a specimen from Connecticut growing in oatmeal agar
survived quick freezing to — 23°C for 12 days or for a

Connecticut Experiment Station Bulletin 773

month at — 2°C. The fungus also survived in infested soil ex-
posed to four cycles of 6°C for 4 days and — 12°C for 4
days (Taylor, 1975). During the ensuing winter, soil out-
doors in the black-shank -infested field did not freeze deeper
than 10 cm.

During the summer of 1974, black shank reappeared on
three fields and appeared for the. first time on seven other
fields (Taylor, 1975). In 1975, the disease was found in 18
fields. The pattern of spread is mapped in Fig. 2. Planting
of resistant varieties and use of non-infested fields reduced
disease incidence drastically in 1976 and to only a few
fields in 1977. Black shank has appeared only on fields of
a cooperating group of growers who raise similar tobacco
cultivars and sometimes exchange equipment and workers.
Evidently, Phytophthora parasitica var. nicotianae had ar-
rived in Connecticut to stay, at least for a while, and a cold-
resistant strain was suspected.

The tasks were then to learn whether a cold-resistant
strain in fact existed, to observe its response to the field en-
vironment, and to learn how to control it with chemicals or
resistant varieties.

RESPONSE TO COLD OF RACES 0 AND 1, AND OF
CONNECTICUT ISOLATES.

Materials and methods.

The following isolates of/', parasitica var. nicotianae were
examined: race 0 (isolate 1587, from C. Litton, University
of Kentucky, Lexington KY; isolate 1189-A-2, from J.
Apple, North Carolina State University, Raleigh, NC; iso-
lates 1 189-A-3, A- 10-2, M-16 (9), and D-Barrow-1 , from G.
Lucas, North Carolina State University , Raleigh, NC ; isolates
42 and 230, from R. Flowers, Georgia Coastal Plains Ex-
periment Station, Tifton, GA; and isolates M-15E, M-15H,
and M-15I from unknown source); race 1 (isolate 1668-1
from C. Litton; isolate 1452-1-2-2 from J. Apple; isolate
85-B-1-75 from G. Lucas; isolates 43 and 228 from R.
Flowers; and isolates M-15F and M-15G from unknown
source); and Connecticut (isolates M-15J, K, M, O, P, S,
T, U, V, and W collected from infected tobacco plants
during the summers of 1973, 1974, and 1975). Soil was
infested with pooled race 0, pooled race 1 , and pooled Con-
necticut isolates.

Tolerances of these isolates to simulated winter tempera-
tures in Connecticut were determined in the following man-
ner. Cultures were grown in nutrient broth (100 ml per 500
ml Erlenmeyer flask, five flasks per isolate) at 25°C in the
dark. After seven days incubation, mycelium was harvested
by filtration, washed with sterile distilled water, and ground
with 50 ml of sterile distilled water in a Waring blender.
This suspension was poured into 500 g of steam-sterilized
soil. The soil was air-dried, and 5 g samples were enclosed in
10.5-x-l 1.5-cm plastic bags. The samples were maintained
at 22 C or — 10°C in the dark, or cycled between 22 and
-10°C (one cycle is seven days at 22°C and seven days at
— 10°C). At the beginning of the experiment and after each
cycle, 0.1 g of soil was removed from each bag and placed
in a petri dish, and another sample was inserted in an apple
(Campbell, 1949).

GRANBY 1
7 MILES I

Fig 2. Location of tobacco fields where at least some black shank
was found for the first time in 1973 (circles), 1974 (triangles), and
1975 (squares). Insets show locations other than Windsor and ap-
proximate road miles from Windsor.

The soil in the dish was flooded with 10 ml of sterile dis-
tilled water. A 1-cm disk was cut from a leaf of the highly
susceptible tobacco cultivar WS-117, the disk was floated
on the water, and the dish incubated in the dark at 30°C.
Two days later, the leaf disk was examined for sporangia.
This method resembles one introduced by Grimm &
Alexander (1973).

The inoculated apple was incubated at 25°C in the dark,
and if rotting occurred, tissue was removed from the apple,
placed on medium for isolation of Phytophthora (Tsao and
Ocana, 1969), and observed for the presence of the pathogen.

Results.

Both the floating leaves and the apples produced the same
results. Survival after a temperature treatment is shown by
+ in Table 1, and non-survival is shown by -. All isolates
survived at 22°C, but only the Connecticut one survived
— 10°C. All isolates survived one or two cycles of cold, but
only the Connecticut one survived three or four cycles.

Clearly, the Connecticut isolates are more tolerant of
cold than races 0 and 1 from the South. Thus, the races,
which are biochemically different and can be differentiated
by resistant varieties (Melntyre & Taylor, 1978), are also
different in susceptibility to cold. Because of these dif-
ferences, the pathogen labeled "Connecticut" in Table 1 is
now called "race 3" (Melntyre & Taylor, 1978).

Black Shank of Tobacco in Connecticut Fields

Table 1. Survival in soil of Connecticut isolates of Phytophthora
parasitica var. nicotianae after six temperature treatments. Survi-
vallisshovynby+andjTO^i-Sjjr^^

Isolates

Race 0

+

Temperature treatments

22 C (continuous)
— 10 C (continuous)
Cycled3 Once

Twice

3-Times

4-Times
a One cycle was 7 days at 22°C followed by 7 days at -10°C

Race 1

Connecticut

RESPONSE TO SOIL MOISTURE IN THE FIELD.

Materials and methods.

On May 22, 1976, 50 breeding lines of shade tobacco were
planted in Windsor on 10 "bents" of a field where black
shank had reappeared annually since 1974. A bent is about
10 x 10 meters or 1/100 hectare. The field, 20 x 50 m,
sloped gently from two opposite corners toward a broad de-
pression that crossed diagonally and drained surface water
gently toward the lowest corner of the field (Fig. 3). The
soil on the slopes was well-drained Hadley silt loam and the
soil in the depression was moderately well-drained Winow-
ski silt loam with a gray surface that grades into a mottled
gray and brown subsoil. Ten m of row were planted with
each variety in two randomized blocks. There were from 25
to 30 plants per row.

At about "weekly intervals during July and August the
incidence of black shank was observed, and diseased plants
were pulled and laid in the furrow. On October 8, the total
number of plants that had become diseased during the sea-
son was determined for each 10 meters of row. On Decem-
ber 1, elevations were measured by transit at 3.3 meter in-
tervals across the rows and 6.6 meter intervals along the
rows for each 10 x 10 m bent. Mean elevations were deter-
mined for each 10-meter row.

Results.

The greatest incidence of disease was in the depression that
passes diagonally through the field (Fig. 3). This confirms
the well-known favorability of water and moist soil for the
disease and its spread (Lucas, 1965). This heterogeneity of
elevation and moisture interferes with comparisons among
varieties to determine their resistance, and we shall later re-
move this confusing effect.

CONTROL USING CHEMICALS

Soil fumigants have been tested in Southern states where
black shank has long occurred (e.g., Todd, 1973). The ef-
fective treatments contain methyl isothiocyanates or
chlorpicrin.

In Connecticut, Taylor, et al. (1975) tested commercial
soil fumigants against the pathogen in the laboratory and
found that only those containing chloropicrin or methyl
isothiocyanate appeared effective. In their field tests, two
materials DD 30 and DD-MENCS at a rate of 30 gallons per

acre (280 1/ha) resulted in only 8% to 9% diseased plants
vs. 45% diseased plants on the untreated plots.

Generally, chloropicrin decreases the quality of wrapper
tobacco by producing darker, mottled leaves. Therefore, we
tested fumigants containing chloropicrin or methyl isothio-
cyanates for their effect upon quality.

Materials and methods.

A field that had not grown tobacco for 10 years was plow-
ed and harrowed in the fall of 1973 before fumigants were
applied. The fumigants were DD 15 (a mixture of 85%
dichloropropene-dichloropropane and related C3 hydrocar-
bons [DD] and 1 5% chloropicrin), DD 30 (70% DD plus 30%
chloropicrin), EDB 15 (a mixture of 85% ethylenedibromide
[EDB] and 15% chloropicrin), EDB 30 (70% EDB plus 30%
chloropicrin), and DD-MENCS (a mixture of 80% DD and
20% methyl isothiocyanate).

During October, 1973, the fumigants were injected into
the soil at a rate of 280 1/ha at a depth of 20 cm on 25 cm
centers. The experimental design was three randomized
blocks of six 10-by-50 m plots.

In the spring of 1974, the. field was fertilized with 4000
kg/ha of 6-3-6 cottonseed-meal-base tobacco fertilizer. In
late May, the highly susceptible cultivarWS-1 17 was planted
with rows in the same direction as the fumigation. On July
22, and about 2 week intervals thereafter until August 29,
samples of 88 leaves from each plot were picked, cured, and
processed by the grower. The color of the processed leaves
was evaluated by ranking the six samples from a date and a
block from 1 for darkest, to 6 for lightest color. Thus, for
each date and block (or replicate) there was a sample rated
1 and a sample rated 6. In addition, the individual leaves
from the August 1 harvest were sorted into the commercial
grades used by the grower. These were combined into
"best", "average", and "poor", and the percentage of the
weight (yield) in each class was determined. Plant height to
tip of tallest leaf was measured on June 24.

Results

Generally, height of plant was increased by fumigation,
especially by DD (Table 2). The plants grown on plots
treated with the four fumigants containing chloropicrin

Fig. 3. Incidence of black shank and elevation of soil in a field of
shade tobacco. Rows extend 50 m from left to right in the figure.
The field is 20 m wide. The depression that extends diagonally
across the field lies between the two 10-cm contours drawn on the
figure. A diseased plant is shown by a short, wide segment of a
row and a series of diseased plants is shown by a continuous, wide
segment.

Connecticut Experiment Station Bulletin 773

produced darker leaves on nearly every date and in every
replicate (Table 2). The treatment without chloropicrin
(DD-MENCS) did not cause darker leaves.

Table 2. Effect of soil fumigants upon plant height and color of to-
bacco leaves.

Height, cm

Rank of color

Fumigant

7/22

»<?//

8/ 14

8/29

A verage

untreated

30

c

5.3

5.0

4.3

4.3

4.7

DD-MENCS

32

be

5.0

5.0

5.0

5.7

5.2

EDB 15

37

a

2.7

2.7

4.0

2.0

2.8

DD 15

35

ab

2.7

3.7

3.0

4.7

3.5

EDB 30

34

abc

3.7

3.3

1.7

2.7

2.8

DD 30

38

a

1.7

1.3

1.7

1.7

1.6

Color was ranked within a date and replicate from 1 for darkest to
6 for lightest. Figure shown is mean rank of the three replicates.
Means with the same letter are not significantly different accord-
ing to Duncan's multiple range test.

The yield was also increased by fumigation (Table 3). The
commercial grading, however, gave different results from
color ranking. That is, DD-MENCS decreased commercial
quality (Table 3), although it did not darken the leaves
(Table 2). Therefore, one has to conclude that DD-MENCS
harmed the other components of commercial quality, such
as uniformity of color or flexibility.

Table 3. Effect of soil fumigants upon commercial quality of tobacco
leaves harvested on August 1.

Percentage of weight

Yield,

Fumigant

kg/lia/yr

Best

A verage

Poor

Untreated

1870

80

19

0.3

DD-MENCS

1990

66

34

0.6

EDB 15

2010

61

37

2.5

DD 15

2100

64

35

1.2

EDB 30

2060

60

35

2.6

DD30

2210

53

43

2.0

RESISTANCE IN SHADE TOBACCO.

Seedlings in the laboratory.

Tests of resistance of large plants in infested fields have the
disadvantage of variation among seasons and also of varia-
tion among places in the field caused by differences in
either inoculum or soil. A method for determining resist-
ance of small seedlings in the laboratory allows greater con-
trol of the environment and saves time and space. Tests for
resistance in seedlings have been devised (e.g., Litton, et al.,
1970). Here we modified the rapid method of Mclntyre and
Taylor (1976), tested seedlings of several tobacco lines and
will later compare the results with the performance of ma-
ture plants in Phytophthora-infested fields.

MATERIALS AND METHODS. Seedlings were grown in
pans containing sterilized sand or a sterilized mixture of
equal parts of peat, perlite, and sand. Seedlings with 4 to 5
leaves were prepared by gently rinsing in tapwater until the
roots were free of most particles. Ten seedlings were then
arranged in a circle with roots to the center and resting on a
moist paper in the bottom of a 100 mm x 20 mm deep plas-
tic petri dish.

Inoculum was prepared by the method of Taylor (1978).
A 45-mm diameter disk of rayon cloth (Handiwipe) was
placed on the surface of lima bean agar in a 60-mm petri
dish. Phytophthora parasitica var . nicotianae was transfer-
red to the center of the disk. After five days at 28- to 30°C,
the cloth and attached mycelium was removed, rinsed in
deionized water, and placed on 3% water agar in 100-mm
petri dishes. After three- to five days at 28- to 30°C in the
dark, these cultures had produced zoosporangia. Zoospores
were released by chilling the cultures to 4°C for 20 min,
adding water and holding at 20°C for an hour. Concentra-
tion of the resulting spore suspension was adjusted to 5000
zoospores per ml. Swimming zoospores congregate near the
water surface, thus reducing the uniformity of subsamples.
A more uniform inoculum was obtained by shaking the sus-
pension vigorously for a few minutes to stop the swimming
(Goode, 1956) and stirring the suspension of encysted zoo-
spores while samples were withdrawn. One ml of this sus-
pension (2 applications of 0.5 ml each) was dribbled over
the- roots of each unit of 10 plants. The seedlings were then
incubated at 26 C, and the number of diseased plants was
recorded after 6 days.

In several experiments we found that this method pro-
vided less variable results than when the entire seedling was
sprayed with a suspension of motile zoospores. Also, when
mortality was observed at 4 or 7 rather than 6 days, either
too few or too many seedlings had died to reveal differences
among lines. At 5 days the difference among varieties was
less and the variation within varieties was more than at 6
days. Thus, the inoculation of roots alone and 6-day incu-
bation are both important.

Fifty-nine tobacco lines of unknown susceptibility to
black shank were tested in three groups of about 20 lines
each. The first group, which was inoculated on March 23,
also included two standards of known performance in the
field: resistant commercial line L and susceptible commer-
cial line WS-1 17. On April 5, these standards plus highly re-
sistant Florida breeding line FI were included with two
other groups in the seedling tests. Since all unknown lines
were not in all groups, we calculated their susceptibility
relative to the standard susceptible line WS-1 17 within each
group. The number of diseased plants of an unknown line
times 100 was divided by the number of diseased WS-1 17
(See Column 2, Table 4).

RESULTS. Within all three groups of seedlings, fewer re-
sistant L than susceptible WS-1 17 were diseased, demon-
strating the reasonable outcome of the method. Further, in
the second and third groups, fewer of the highly resistant
FI were diseased than either resistant L or susceptible
WS-1 17. Significantly fewer L than WS-1 17 were diseased
in all three groups and significantly fewer FI and L were
diseased in the third group.

Turning to a practical question, we now ask whether
seedlings from some of the unknown lines appeared more
resistant than L, a line which provides useful resistance in
the field. The answer is "yes", because 10 of the 59 un-
known lines had fewer diseased plants than L, and two of
these, 77-15 and 77-53, had significantly fewer. Later we
report a field test that included the sixteen selected lines of
Table 4.

Black Shank of Tobacco in Connecticut Fields

Table 4. Seedling and field susceptibility. Seedling susceptibility is
100 times the diseased plants of an unknown line divided by the
diseased WS-117 plants in the same test. Field susceptibility is 100
times the diseased plants of an unknown line divided by the
diseased WS-117 in the same plot. The n is the number of plots
where WS-1 1 7 was diseased and hence field susceptibility could be
calculated. Seedling value for L is the mean of three groups.

planted susceptible
line of tobacco.

'witnesses" in every row of any test

Line

Seedling

Field

n

117

100

47

3

77-36

72

10

2

77^4

72

12

2

77-52

72

55

4

77^6

70

10

2

77-54

68

72

1

77-53

62

68

4

77^3

50

26

2

77-57

50

80

2

L

50

7

1

77-39

49

12

1

77-28

41

0

1

77-60

33

54

4

77-15

28

0

1

Fl

14

0

1

77-18

4

30

1

Large plants in the field, 1976.

Although the test of seedlings in the laboratory has advan-
tages of control and economy, the payoff comes in the field.

MATERIALS AND METHODS. On May 22, forty -five lines
of unknown response and five lines of known response to
black shank were planted in an infested field as described
under the section on "Response to soil moisture in the
field". The five lines of known response to black shank
were two selections of the highly susceptible WS-1 17, the
resistant L, the resistant WS-149b, and the highly resistant
FL

RESULTS. At season's end the number of diseased
plants in a row was related to mean elevation by regression
analysis. The equation, diseased plants per row= 17.0 - .99
x elevation (cm), says that the number of diseased plants
per row decreases about 1 for each cm of elevation. This
equation explains about a third of the variation in disease.
In Fig. 4, we have shown the regression line for all the
plants and the number of diseased plants for five lines of
known response. Susceptible plants should have much more
disease for elevation, i.e., be above the regression line, and
resistant ones should be below the line.

Four tests of susceptible WS-117 were performed, i.e.,
two selections in two replicates. In two cases, disease was
much more than predicted for the elevation, and the sym-
bol W in Fig. 4 appears above the regression line. In two
cases, WS-1 17 was not diseased because it was at high eleva-
tions. Resistant WS-1 49b, symbol B, had more disease in
one case and less in another than predicted for the elevation.
Finally, the resistant L and resistant FI had less disease than
predicted for the elevation.

The variable performance of WS-149b reduced our confi-
dence in the 1976 field experiment. If the experiment and
refinement of disease observations by measurements of ele-
vation could not consistently characterize the well-known
WS-1 49b as either resistant or susceptible, the results for
the 45 unknown lines were questionable. Therefore, the ef-
forts to develop the seedling method were increased, and
finally in 1977 we experimented in another field and inter-

Large plants in the field, 1977.

MATERIALS AND METHODS. On June 13, twenty-nine
lines of shade tobacco and one of broadleaf were planted in
Poquonock on twelve bents of a field where black shank
had attacked nearly every WS-1 17 plant two years before.
Line L had been grown on the field in the intervening year,
and little disease was seen in this resistant variety. The
thirty lines of tobacco included the sixteen listed in Table
4. The lines were arranged in four randomized blocks. Un-
like the 1976 experiment, the first and every sixth plant in
a varietal row were susceptible WS-117 plants, which pro-
vided the witnesses mentioned above. Thus, each plot had
21 to 23 plants of the variety under test and five WS-117
plants.

On July 18, black shank was first observed. The plants
were observed regularly, and on October 17, the diseased
plants were counted.

RESULTS WITH 30 VARIETIES. The mean percentage of
diseased plants in four plots as determined on October
17 was transformed to angles before analyzing the variance.
Fifteen lines were significantly less susceptible than suscep-
tible standard WS-1 17, and six lines had less disease than L,
but not significantly less.

Among the entries significantly more resistant, i.e., less
susceptible than WS-1 17, were several lines resistant in seed-
ling tests over a 3-year period. They were: FI; a selection of
FI (128); WS-149b; L; a flue-cured tobacco line resistant to
black shank in North Carolina (NC 2326); a broadleaf to-
bacco line resistant to black shank in Maryland (MD 609); a

ELEVATION CM

Fig. 4 Diseased plants per 10 m of row as a function of elevation
above lowest point in the field of 1976 experiment. The regres-
sion line drawn on the figure was estimated from 100 observations
in two replicates of 45 tobacco breeding lines of unknown and 5
tobacco breeding lines of known resistance. The observations of
the two replicates of 5 known tobacco breeding lines are shown
by W (WS-117), L, B (WS-149b), and F (FI).

Connecticut Experiment Station Bulletin 773

Table 5. The percentage of diseased plants among 16 lines in four
blocks in 1977. The numerator shows the disease among the test-
ed line, and the denominator shows the disease among the WS-1 17
witnesses in the same plot. The blocks are identified as A, B, C,
and P.

Disease is % in

tested/% in WS-1 17

Line

A

B

C

D

WS-1 17

0/0

17/60

27/40

37/80

77-60

21/40

4/20

5/20

48/40

77-43

0/20

23/50

0/0

4/60

77-57

0/0

32/40

4/0

48/60

L

0/0

0/0

0/0

4/60

77-18

0/0

0/0

15/50

4/0

77-39

19/0

5/40

0/0

0/0

77-28

0/0

0/0

12/0

0/80

77-36

0/0

9/80

0/0

4/40

77-44

0/20

5/20

0/0

0/0

77-52

17/25

29/40

9/20

14/40

77-15

10/0

0/20

0/0

0/0

77-46

0/20

4/20

0/0

0/40

Fl

0/0

0/0

0/20

0/0

77-54

18/25

5/0

10/0

5/0

77-53

21/0

22/20

4/17

54/80

highly resistant line from South America (Beinhart 1 000-1 );
and a line developed from WS-1 49b (ACC 75).

FIELD SUSCEPTIBILITY VS. SEEDLING SUSCEPTIBILITY.
Among the 30 lines were 16 whose seedling susceptibility
had been appraised as shown in Column 2 of Table 4. We
now examine their disease susceptibility in the field.

Black shank disease among the 16 lines can be judged
against disease in the WS-1 17 witnesses planted in the same
rows. In Table 5, we show the percentage of disease among
the 16 lines in the four plots as a numerator divided by the
percentage of disease among the five witness WS-1 1 7 plants
in each plot. Within the sixteen lines we asked whether a
WS-1 17 witness within each plot reduced variability and
whether seedling resistance clearly predicted susceptibility
in the field.

Witnesses. The disease among the WS-1 17 witnesses ex-
plains 35% of the variability in the tested lines (Fig. 5).
Disease in a tested line increased 31% as much as that in
WS-1 17. It is logical that the regression practically goes

60 -

50

40

50

20

0 -

X

20 30

40 50
WS-II7

J,

J.

1 '

60 70 80 90

Fig. 5 The percentage of diseased plants among sixteen lines in four
field plots in 1977 as a function of the disease among witness
WS-1 1 7 plants in the same plot. (Note 0 is moved to show obser-
vations of 0 disease). Fewer than 64 points show because of dup-
licates, especially (0, 0).

through the origin: i.e., no disease in WS-1 17. no disease in
tested line

A further examination of the witnesses, however, reveals
that their testimony cannot be taken on faith as an apprai-
sal of the initial inoculum in the soil. Planted among other
WS-1 1 7 in four plots the witness WS- 1 1 7 was 0, 40, 60, and
80% diseased, while among eight plots of resistant L and FI
lines it was only 0, 0, 0, 0, 0, 0, 20 and 60% diseased.
WS-1 17 had more disease among susceptible than among
resistant neighbors.

The influence of neighbors upon the amount of disease is
well-known. For example, when a "multiline" mixture of
resistant and susceptible varieties is grown, disease is de-
creased markedly (Browning and Frey, 1969). Evidently
the amount of disease in WS-1 17 could be held in check
somewhat by mixing it with a resistant line like L. This is a
practical result.

Our view of using WS-1 17 as an indicator, however, be-
came clouded as we wondered whether WS-1 1 7 was indicat-
ing the soil and inoculum as we hoped or the surrounding
variety. We reasoned through this problem.

Mathematical analysis of witnesses. The field was planted
to many u plants, i.e., test or unknown lines, and a few w
plants, i.e., WS-1 17. Disease (W) in the w plants and disease
(U) in the u plants increased the number (N) of pathogenic
propagules per u plant. Disease W and U are the diseased
plants per total w or u plants at time T.

After T days the disease W (T) is a sum, or integral, of in-
fections during time T if latency or time from infection to
symptom is short relative to T.

W(T) = K / Nw(t) dt

(1)

K is w plants diseased per propagule per day. Nw(t) is pro-
pagules per w plants at time t. The dimensions of equation
(l)are

Plants diseased = plants diseased x propagule x day
plants propagule day plant

Similarly, disease in the unknown lines of u plants is

U(T) = k / N (t) dt
o

(2)

where k is u plants diseased per propagule per day.

If the inoculum or propagules that cause the disease ob-
served are from the initial infestation in the soil plus an in-
crement contributed by neighboring plants and if re-infec-
tion of a plant by propagules from itself is insignificant,
analysis is simplified. Since the immediate neighbors of w
plants are all u plants and since five of six immediate neigh-
bors of u plants are other u plants, the inoculum Nu(t) and
Nw(t) around w and u are nearly the same.

Thus,

W(T)/K = U(T)/k
or simply
U(T)/W(T) = k/K

T

Nw(t) dt :

Nu(t)

dt

(3)

Black Shank of Tobacco in Connecticut Fields

Thus, W(T) is an honest witness in the sense that the ratio
of U to W where W is not zero shows the susceptibility k of
an unknown line relative to the standard susceptibility K of
WS-117. Accordingly, we have calculated 100 U(T)/W(T)
in the n plots of each line where W(T) was not zero, and
tabulated it as "Field susceptibility" in the third column of
Table 4.

Alternatively, the 64 observations of U can all be analyz-
ed, using disease in the witness plants as a covariate.

Prediction of susceptibility in the field by the test of
seedling susceptibility. The sixteen lines are arranged in
Table 4 from most to least seedling susceptibility. If seed-
ling susceptibility predicts field susceptibility, the field sus-
ceptibility in column 3 of Table 4 should decrease from top
to bottom in the table. In the case of the three standard
varieties this is true: WS-1 1 7 is more susceptible at 47 than
L at 7 and than FI at 0.

Unfortunately, the field susceptibility of the thirteen un-
known lines does not decrease regularly from top to bot-
tom in the table, and we must conclude that seedling resis-
tance is an imperfect predictor of resistance in the field.

Nevertheless, we remember that the seedling test did
show the known field resistance of Land FI, and it is profit-
able to look for other lines resistant in both seedling and
field tests. In fact, lines 77-15, 77-28, and 77-39 appeared
resistant in both tests, a promising clue to future resistant
varieties.

RESISTANCE IN BROADLEAF TOBACCO.

Generally, Connecticut farmers who grow broadleaf do not
grow shade tobacco and do not use fields used for shade to-
bacco. To date, black shank has not been found in commer-
cial plantings of broadleaf tobacco in Connecticut. Since,
however, the disease can be expected eventually to appear
in fields of broadleaf, the susceptibility of broadleaf tobac-
co needs to be known. Although broadleaf tobacco has no
clear varieties or cultivars, seed stocks maintained by various
growers do differ in such characteristics as earliness and
number and widthof leaves. Since the standard K broadleaf
seed was always highly susceptible in seedling tests, we test-
ed other selections of broadleaf seed in the field.

Materials and methods.

On June 14, 1977, broadleaf tobacco from seven lots of
seed from various growers was tested with two susceptible
shade lines and one resistant shade line. They were planted
in part of the field described in the section, "Large plants in
the field, 1977". Plots of each lot were rows 10 m long.
There were three randomized blocks. Plants were observed
regularly, and on August 19 diseased plants were finally
counted. _

RESULTS. Witness WS-1 17 plants were 25 to 75% diseased
in all but 4 of the 30 plots, showing the uniform distribu-
tion of the pathogen. The seven lots of broadleaf tobacco
and the two susceptible shade lines were 28 to 41% diseas-
ed, while the resistant shade line ACC-75 was only 2% dis-
eased. Evidently, broadleaf tobacco is generally susceptible

to black shank in Connecticut.

DISCUSSION AND CONCLUSIONS

Black shank of tobacco, caused by the fungus Phytoph-
thora parasitica Dast. var. nicotianae (B. de Haan) Tucker
has long been considered a warm climate disease of no con-
cern to Connecticut growers. However, in 1973, the disease
did appear in Connecticut fields. We have now determined
that the fungus present in Connecticut is a new race, race 3,
but we do not know its origin. Race 3 differs from the
Southern races 0 and 1 by a) tolerating colder soil tempera-
tures, b) differentially infecting standard tobacco cultivars,
and c) "displaying differences in metabolism (Mclntyre &
Taylor, 1978, Mclntyre & Hankin, 1977).

Since the disease has reappeared every year since its dis-
covery, some control measures are necessary. Fumigant
chemicals in the soil have been effective at high rates
(280 1/ha). Fumigants containing chloropicrin reduced dis-
ease in Connecticut as they have in Southern states, but
they reduced leaf quality to an unacceptable level for Con-
necticut cigar-wrapper tobacco. The fumigant DD-MENCS
(vorlex) does not contain chloropicrin, is often used as a
control for the tobacco cyst nematode, and did reduce
black shank when used at the higher than normal rate of
280 1/ha). A slight reduction in quality was noted. For
economic reasons, however, resistant varieties may be the
best approach to control.

Although the cigar-wrapper variety L, grown commercial-
ly for a decade, tolerates black shank in Connecticut fields,
the horticultural features of L do not suit the needs of all
growers. Other resistant varieties, therefore, are needed. To
develop such varieties, one must be able to test reliably for
resistance to black shank. Preferably an early test should be
as compact and as brief as possible, minimizing space and
time needed for the necessary field evaluation.

Various methods have been devised to determine resis-
tance or susceptibility in young plants in the laboratory
(Litton, et al. 1970). None are ideal. Results reported in
this bulletin suggest that the rapid method of Mclntyre and
Taylor works reasonably well when a) only the roots of
small seedlings are inoculated, b) the inoculum is kept as
uniform as possible by stirring encysted zoospores, and c)
one uses a standard period of time between inoculation and
recording of data. Lines with a long history of resistance or
susceptibility in the field or in several types of tests for
young plants were well distinguished in the seedling stage,
thus showing the method to be reasonable.

Some aspects of the test, however, are still not under-
stood. We were unable to get a high correlation between the
seedling and field response for all entries in thirteen lines
with unknown resistance to black shank. Among thirteen
unknowns, two in particular appeared resistant in the seed-
ling test, but not in the field test (Table 4). Only one ap-
peared susceptible as a seedling but resistant in the field.
Thus, one is reasonably confident that a line susceptible as
a seedling would be susceptible in the field and need not be
tested in the field. One is less confident that lines resistant
as a seedling will be resistant in the field.

Connecticut Experiment Station

Bulletin 773

Although field testing is certainly the final criterion,
many variables may interfere with a clear indication of re-
sistance. Results we report here indicate that variations in
elevations of the soil within a plot influence the outcome.
Low areas where water stands or runs after rains and high
areas with good drainage mask practical differences in re-
sistance. By measuring the elevations, some variability can
be accounted for, but other factors also operate in the field.

We attempted to account for variability of inoculum po-
tential from spot to spot in the field by including a known
susceptible line as a witness in the row of a line under test.
Disease in the witness did improve discrimination of resis-
tance even though disease in the witness was modified by
the resistance of its neighbors. A similar phenomenon for
other diseases is known as multi-line resistance, and mixing
resistant with susceptible plants evidently would decrease
black shank in the susceptible.

We conclude that the search for resistant germ plasm is
important since chemical control is expensive and may af-
fect quality of the crop. A successful field test requires a)
a level field with moist soil and irrigation when necessary,
b) infestation and previous cropping with a susceptible
variety, c) witnesses, and d) replications to reduce effects
of unknown variables. Highly susceptible lines have been
detected as seedlings, and confidence in the prediction of
field response would surely improve by repeating seedling
tests. Despite these difficulties, the promising line ACC-75
was developed by testing resistance in the seedling stage,
and it was grown commercially in 1977.

The results reported in this bulletin will, we hope, hasten
attainment of the goal of safely and economically control-
ling black shank in Connecticut.

Ack nowledgements

We wish to thank Drs. H.C. De Roo and George Stephens for the
measurements of soil surface contours in the 1976 field studies. We
also thank the Brown Tobacco Company and H.C. Thrall and Sons
for use of their fields, equipment, and personnel in growing and pro-
cessing tobacco used in these studies and Robert Borg, Jr. for apply-
ing the fumigants. Jill Morse and Lila DeRobertis provided excellent
technical assistance.

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