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INFLUENCE OF TEMPERATURE AND SPORE STAGE
ON PRODUCTION OF TELIOSPORES BY
SINGLE AECIOSPORE LINES OF CRONARTIUM RIBICOLA

Geral |. McDonald and Duane S. Andrews

USDA Forest Service Research Paper INT-256
Intermountain Forest and Range Experiment Station

U.S. Department of Agriculture, Forest Service

THE AUTHORS

GERAL I. McDONALD is principal plant pathologist with the Intermountain
Station's genetics and pest resistance research work unit in Moscow,
Idaho. Dr. McDonald received his B.S. in Forest Management (1963) and
Ph.D. in Plant Pathology (1969) from Washington State University.
Since he joined the Station in 1966, he has investigated the genetic
interaction of the blister rust organism and its hosts as well as
epidemiology of blister rust.

DUANE S. ANDREWS is biological technician for the Station's genetics and
pest resistance work unit in Moscow, Idaho. He has a B.S.
in Range Management from Washington State University (1965) and an
M.S. in Range Management from the University of Idaho (1967). He
joined the Station in 1969.

USDA Forest Service
Research Paper INT-256
July 1980

INFLUENCE OF TEMPERATURE AND SPORE STAGE
ON PRODUCTION OF TELIOSPORES BY
SINGLE AECIOSPORE LINES OF CRONARTIUM RIBICOLA

Geral |. McDonald and Duane S. Andrews

Intermountain Forest and Range Experiment Station
U. S. Department of Agriculture
Forest Service
Ogden, Utah 84401

RESEARCH SUMMARY

A major problem facing those who wish to inoculate white pines with
Cronartium ribicola is the reliable production of teliospores. Also, a
better understanding of the nature of the various spore stages will add to
our ability to develop workable integrated rust management plans.

The results reported in this paper will increase the reliability of
teliospore production in both whole plant and detached-leaf-culture of
Cronartium ribicola on Ribes plants, and add to our understanding of the
interaction between genes and the environment in the functioning of
epidemiological fitness traits.

CONTENTS

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INTRODUCTION

Over the last few years, attempts have been made to establish single aeciospores lines
of Cronartium ribicola to facilitate the investigation of the genetics of this important
conifer rust. A principal objective was to complete the life cycle in isolation so that
known homozygous aeciospores could be produced. A modified version (McDonald 1978) of Clinton
and McCormick's (1924) Petri dish method has worked well for the ribes portion of the life
cycle. But lack of consistent and profuse production of teliospores has been an unexpected
problem. Past greenhouse studies show that a constant 61 F (16 C) from inoculation to spore
production led to abundant telia (Riker and others 1947 and Van Arsdel and others 1956).
Also, telia would not form if the day and night temperature was above 68°F (20°C) (Van
Arsdel and others 956)).

Our first problem arose when a group of single aeciospore lines isolated at a constant
70°F (21-6) were transferred to a constant 55°F Geme One culture out of 69 produced telia
within 60 days (author's unpublished data). On the other hand, other single aeciospore cultures
held at 70°F+2 (21 C+1) produced teliospores that would germinate as they formed (McDonald and
Andrews, in preparation). So, a reliable supply of ungerminated teliospores to use for inocula-
tion back to Pinus monticola proved difficult to obtain. A series of experiments were initiated
to determine the best procedure for producing teliospores on the detached leaf cultures.

Two pieces of information were used to formulate a hypothesis. First, we observed that
when inoculations were made with urediospores instead of aeciospores, we could obtain teliospores
in as little as 14 “days at 70. F eileen and 16 hr days (author's unpublished data). Whereas,
aeciospore inoculations would nearly always require 28 to 30 days for earliest appearance of
telia, regardless of temperature and day length. Second, Spaulding (1922) reported that
Pennington and Snell followed generations of urediospore production in the field. Teliospores
were not produced with the first generation but were found with all later generations. We
hypothesized that aeciosnore and urediospore infections differed in their ability to produce
teliospores and that a temperature of less than 68 F (20°C) was needed to stimulate teliospore
production by infection resulting from urediospores. The objective of this paper is to report
the results of initial experiments designed to test the above hypothesis by growing both
urediospore and aeciospore stages of identical genetic background on one clone of ribes in one
growth chamber (urediospore lines compared to the aeciospore line from which they decended).
Thus, as near as is currently possible, only spore stage varied.

MATERIALS AND METHODS

We used two sets of materials, one isolated in 1976 and the other in 1979. Both consisted
of single aeciospore lines isolated according to McDonald and Andrews. (in preparation). Upon
production of urediospores, subcultures were established. All cultures of both spore types
were grown on Ribes hudsonianum var. petiolare clone INT-1. The 1976 material has already
been described (McDonald and Andrews, in preparation) and was included in this paper to provide
some comparisons with other sources of aeciospores. The 1979 material was produced and the
experiment conducted as outlined in fig. 1. Two cankered P. monticola seedlings growing in
the greenhouse were producing aeciospores. Spores from one blister on each tree were used as
follows: About half of the 1,058 attempted isolations were made from each blister, and isolated
spores were randomly placed in a cool 55 F+2 (13 C41). and a warm 70° F+2 (21 CHI), chamber as
they were isolated (16 hr day 8 hr night). a 7 7

The aeciospore cultures were inspected every 7 days for 65 days, and presence of uredio-
spores and teliospores was recorded. Six subcultures were attempted from all the single
aeciospore cultures that had produced sufficient urediospores by 21 days. These cultures are
a random selection of possible cultures. One-half of these subcultures were placed in the
warm chamber and one-half in the cool chamber from each of the warm and cool cultures from
which they were isolated (fig. 1). These cultures were then inspected every 7 days for 49
days and presence of urediospores and teliospores was recorded.

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We used chi-square analysis (Snedecor 1956) to test for independence of selected comparisons
of spore type, spore source, and temperature for inoculation success and presence of teliospores.
In addition, the pattern of subculture age at teliospore appearance was recorded for all cool
chamber aeciospore lines that produced three subcultures.

RESULTS

As expected, the single aeciospore isolates placed in the cool chamber provided a higher
success rate than those placed in the warm chamber (table 1). But the warm chamber isolates
developed slightly faster. All the infections that appeared by 65 days were evident in 14
days in the warm chamber; but cool chamber infections were not evident until the 21 day inspectior

There was a significant difference due to temperature in teliospore production by 49 days
by aeciospore cultures (table 2). The cool chamber isolates showed a significant increase in
percent producing telial columns.

Table 1.--Percent of single aeciospores of Cronartium ribicola that produced infections
on leaf disks of Ribes hudsonianum var. petiolare within 21 days when grown
in a cool and a warm chamber, both under 16 hr days

Infections
Chamber temperature Yes No Total Percent
) fe)
So) yh (15 C41) 86 443 529 16
fe) fe)
WO F+2° (21 C+1) 35 494 529 if
Total neal 937 1058 11

Ae DADS Pot larger x = 0.0005

Table 2.--Percent of single aeciospore cultures of Cronartium ribicola that produced
teliospores by 49 days after inoculation when grown on Ribes hudsonianum
var. petiolare INT Clone-1 at cool and warm temperatures under 16 hr days

Teliospores

Chamber temperature Yes No Total Percent

Saree? (13-C-1) 43 39 82 52

70°F+2 (21°C+1) 2 13 15 13
Total 45 54 07. 46

ye 7.75 P larger x2 = 0.01

d.f. "51

Regarding urediospore inoculation success, the cool chamber was best regardless of tempera-
ture of parent culture's chamber (tables 3 and 4).

Table 3.--Percent of Cronartium ribicola urediospcre inoculations that produced
infection in 21 days. Urediospores obtained from a population of single
aeciospore cultures grown on Ribes hudsonianum var. petiolare INT Clone-1
leaf disks in, a 70 Ft2 (22 Ctl) chamber and inoculated back to INT Clone-1
in 55° R42 hag” Ctl) and FO" i (21 °C#1) chambers with all chambers at 16 hr days

Infections
Treatment Yes No Total Percent
Aeciospore Urediospore
culture to subculture
Warm to cool 25 16 41 Gill
Warm to warm 14 25 39 36
Total 39 41 80 49
x2 = 5.01 P of a larger x? = 0.025
dvtis.= 1

Table 4.--Percent of Cronartium ribicola urediospore inoculations that produced
infections in 21 days. Urediospores obtained from a population of single
aeciospore cultures grown on Ribes hudsonianum var. petiolare INT Clone-1l
leaf disks in a 55 ia Cig C+1) growth chamber and inoculated back to INT
Clone-1l in 55 Or+2 aoe Ctl) and 70 Or+2 (224. °c+1) chambers with all chambers
at 16 hr days

Infections
Treatment Yes No Total Percent
Aeciospore Urediospore
culture to subculture
Cool to cool 54 2 56 96
Cool to warm 30 23 53 57
Total 84 25 109 7
2 = 24.41 P of larger yx? = 0.0005

In the case of teliospore production by urediospore cultures, the cool chamber produced a
higher percentage of telial supporting cultures regardless of the temperature at which the
urediospores were produced (tables 5 and 6). A feature of importance to us was culture age at
teliospore appearance. The cool chamber produced teliospores more quickly than the warm
chamber (tables 7 and 8), but.a statistical test could not be performed because of the low
level of teliospore production in the warm chamber. The temperature of the chamber that
produced the urediospores had no apparent influence (table 9).

Table 5.--Percent of Cronartium ribicola urediospore cultures derived from population
of single aeciospore cultures grown in warm chamber on Ribes hudsonianum
var. petiolare INT Clone-1 that produced teliospores by 49 days after
inoculation when grown on Clone-1l at 55 Or+2 (13° CH) and 70 OF+2 (Zi CrI)
and 16 hr days

Teliospores

Treatment Yes No Total Percent

Aeciospore Urediospore
culture to subculture

Warm to cool 22 1 23 96

Warm to warm 2 LZ 14 14
Total 24 NS a 65

CS PerT P of larger x? = 0.0005

Geter

Table 6.--Percent of Cronartium ribicola urediospore cultures derived from population
of single aeciospore cultures grown in cool chamber on Ribes hudsonianum
var. petiolare Clone INT-1 that produced teliospores by 49 days after
inoculation when grown on Clone-1l at 55 Or+2 (13 Ctl) and 70 OF +2 (27: °C+1)
and 16 hr days

Teliospores
Treatment Yes No otal Percent
Aeciospore Urediospore
culture to subculture
Cool to cool 41 9 50 82
Cool to warm 5 24 29 iz
Total 46 35 79 58
Se A67, Probability of larger y* = 0.0005

X
dies, = ll

Table 7.--Number of Cronartium ribicola urediospore cultures derived from a population
of single aeciospore cultures grown in cool chamber on Ribes hudsonanium
var. petiolare INT Clone-l that produced teliospores at various times after
inoculation when grown at two different temperatures on INT Clone-1

Days after inoculation
Treatment 21 28 55 49 Total

Aeciospore Urediospore
culture to subculture

Cool «to. cool 19 18 0 4 41
Cool to warm 0) 0) i 4 5
Total 19 18 i 8 46
Table 8.--Number of Cronartium ribicola urediospore cultures derived from a population

of single aeciospore cultures grown in warm chamber on Ribes hudsonianum
var. petiolare INT Clone-1l that produced teliospores at various times after
inoculation when grown at two different temperatures on INT Clone-1

Days after inoculation
Treatment 21 28 55 49 Total

Aeciospore Urediospore
culture. to subculture

Warm to cool 7 12 1 2 22
Warm to warm 1 0 ) 1 2
Total 8 12 1 3 24

Table 9.--Number of Cronartium ribicola urediospore cultures derived from two
populations of single aeciospore cultures, one grown in a 70°F +2 (21°C#1)
chamber, the other grown in a 55 Ft2 (13 Ctl) chamber and both | grown on
Ribes hudsonianum var. petiolare, that produced teliospores at various times
after inoculation when grown at 55 Or+2 (is Ctl) on INT Clone-1

Days after inoculation

Treatment Z1 28 35 49 Total
Aeciospore Urediospore
culture to subculture
Warm to cool 7 12 i Zz 22
Cool. to..cool 19 18 0 4 41
Total 26 30 1 6 63
y = 2,95 P of larger y* = 0.40
Ger eS 5

Both initial aeciospore cultures and their derived urediospore cultures yielded a high
proportion of cultures producing teliospores when grown in a cool temperature, but a most
important difference showed up when culture ages were compared (table 10). Few aeciospore
cultures produced teliospores in less than 28 days, whereas nearly all the genetically related
urediospore subcultures had produced their teliospores by 28 days.

We conducted our first test of temperature effects on urediospore subcultures (1/single
aeciospore culture) for another population of aeciospore cultures isolated in 1976. When we
compared the 1976 and 1979 subcultures, a highly significant difference existed between the
two populations (table 11), but both populations tended toward the early production pattern.

The 1976 and 1979 aeciospore cultures were also compared because we had no previous data
on aeciospore cultures grown in both warm and cool chambers. The result was consistent (table
12) in that thevaeciospore cultures in the cool chamber produced telia before those in the
warm chamber. Also, both were consistent in not producing teliospores at an early age.

Finally, most urediospore subcultures within a single aeciospore line showed little
variation in age at teliospore appearance, and 2 (C-9 and C-70) of the 18 lines that had
complete sets of 3 subcultures failed to produce any teliospores by 49 days (table 13).

Tabie 10.--Number of single aeciospore cultures and urediospore subcultures of
Cronartium ribicola that produced teliospores at various times after
inoculation when grown on Ribes hudsonianum var. petiolare clone INT-1
at 55°F+2 (13°C+1)

Days after inoculation

Inoculation spore type PAA 28 35 49 Total

Aeciospores 0 Z iy 45 58

Urediospores 19 18 0 4 41
Total 19 20 11 49 99

MO = 76.52 Pot larger x2 = 0.0005

disfi.. = 3

Table 11.--Number of Cronartium ribicola urediospore subcultures from two populations
of single aeciospore cultures that produced teliospores at various times
after inoculation when growing at 55° F+2 (is C41) (16 hr days) on
Ribes hudsonianum var. petiolare INT Clone-1

Days after inoculation

Subculture source 21 28 55 Total

1976. Single aeciospore culture 39 83 49 171

1979 single aeciospore, culture 19 18 0 oy
Total 58 101 49 208

¥- = 19.49 P of larger yx? = 0.0005

dhe = 2

Table 12.--Number of single aeciospore cultures of Cronartium ribicola growing on
INT Clone-1 of Ribes hudsonianum var. petiolare that produced telial
columns at various times after inoculation contrasting 1976 isolates
grown at 70°F+2 (21°C+1) with 1979 isolates grown at 55°F+2 (13°C+1)

Days after inoculation

Isolate population 21 28 55 65 Total

1976 warm aeciospores 0) i) 20 202 225

1979..cool aeciospores 0 2 1l 45 58
Total 0 S Sir 2A, 283

x2 = 11.48 P of larger x? = 0.01

distin: = 5

Table 13.--Patterns of variation within 18 single aeciospore lines and among subcultures within lines in days to
appearance of teliospores. Cultures were growing on detached leaves of Ribes hudsonianum var. petiolare
at 55 Ete, (L3°C#EL) (16 hr days) for 49 days

Single Aeciospore Lines (C = cool; W = warm)

Urediospore 1/ 2/
Subculture C-1 C-26 W-2 W-3 C-42 C-44 C-35 C-18 W-4 C-50 W-34 C-31 C-63 C-22 C-71 W-7 C-9=— C-70—
1 21 21 21 Zu 21 2 21 21 21 21 28 28 28 28 28 28 0 0
2 21 21 21 21 ZL 21 Zu 21 28 49 20 20 20 20 20 20 0 0
3 Zi. 21 28 28 28 28 49 28 0 28 28 49 9 0 0 0 0

1 this parent aeciospore culture produced T in 56 days.

— This parent aeciospore culture produced T in 48 days.

DISCUSSION AND CONCLUSION

Cultures of C. ribicola obtained from aeciospores or urediospores differ in patterns of
teliospores production. However, one cannot say if most aeciospore-derived infections are
unable to produce the teliospore stage or if it just takes longer. The answer could come from
experiments designed to allow the aeciospore derived cultures to grow but to prevent all
possibility of urediospores reinfecting the leaves on which they were produced.

. . fo) Oo : :

Another conclusion is that temperatures near 55 F (13°C) stimulate both infection success

and teliospores production for both aeciospore- and urediospore-derived detached leaf cultures
Of (Cs. PIDTESIS,

The scenario suggested here may explain the pattern of teliospore production obtained by
Clinton and McCormick (1924). They made several hundred aeciospore and urediospore inoculations
over 3 years on detached leaves of many different ribes species. Their cultures were grown in
a greenhouse, so they were not able to exercise much control over temperature. Over years
they attempted 131 inoculations with aeciospores (not single spores) during April, May, and
Wune and obtained 83 infections (63 percent success). Of the 83 infections, 2 (2 percent)
produced teliospores within 3 weeks (their cultures apparently did not live much beyond 3
weeks although they did not explicitly state any average age). In addition, they obtained
about 47 infections from urediospores. Of these, 34 were inoculated between September 13 and
17, 1918, of which 22 (65 percent) produced teliospores. Of 49 infections obtained from
urediospore inoculation during April, May, June, July, and August, only 14 percent produced
teliospores. If we assume that their cultures lasted an average of 21 days and that only the
September 13 to 17, 1918 period was cool enough, our results could be interpreted as being in
total agreement with theirs.

New_attempts at isolation of single aeciospores should be conducted at a cool temperature,
So) F Gis. Gi. Time required to complete the life cycle in isolation can be reduced by reinocu-
lating with urediospores as soon as they are produced. Also, early teliospore production by
certain single aeciospore-derived infections may indicate the existence of yet another
C. ribicola marker gene as well as having possible important significance to the epidemiolo-
gical relationships of C. ribicola. Other rust species may have responded in a like manner
(Savile 1953) in their adaptation to short seasons. Genetic control of a trait such as
teliospore production by infections arising from different spore stages as well as variation
among individual spores could indicate the operation of a fundamental adaptive device that
might have its ultimate expression in the formation of microcyclic races.

PUBLICATIONS CITED

Clinton, G. P., and F. A. McCormick.
924. Rust infection of leaves in Petri dishes. Conn, Agr. Exp. Sta. Bull. 260, p. 475-
501. New Haven, Conn.
McDonald, G. I.
1978. Segregation of "red" and "yellow'' needle lesion types among monoaeciospore lines of
Cronartium ribicola. Can. J. Genet. Cytol, 20:313-324,
McDonald, G. I., and D. S. Andrews.
In preparation. Variation patterns of single aeciospore cultures of Cronartium ribicola
(J. C. Fisher en Rabenh).
Raker, A. J., T. F. Kauba, and B. W. Henry.
1947. The influence of temperature and humidity on the development of white pine blister
rust on Ribes leaves. Abs. Phytopath. 37:19.
Savale, D. B. O.
1953. Short-season adaptation in the rust fungi. Mycologia 45:75-87.
Snedecor, G. W.
1956. Statistical methods applied to experiments in agriculture and biology. Laura State
Press, Ames.
Spaulding, P.
1922. Investigations of the white-pine-blister rust. USDA Bull. 957.
wan Arsdel, E. P., A. J. Riker, and R. F. Patton.
1956. The effects of temperature and moisture on the spread of white pine blister rust.
Phytopath. 46:307-318.

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McDonald, Geral I., and D. S. Andrews.

1980. Influence of temperature and spore stage on produc-
tion of teliospores by single aeciospore lines of
Cronartium ribicola. USDA For. Serv. Res. Pap. INT-256,
9p. Intermt. For. and Range Exp. Stn., Ogden, Utah
84401.

Genetically identical aeciospore and urediospore popula-
tions grown on a single ribes clone varied in time required
to produce teliospores. Infection developing from uredio-
spores usually produced teliospores in less than 28 days when
grown at 55°F (13°C); whereas, infections developing from
aeciospore generally required more than 35 days at the same
temperature.

KEYWORDS: Cronartium ribicola, epidemiological fitness,
inherited traits, detached leaf culture of rust

McDonald, Geral I., and D. S. Andrews.

1980. Influence of temperature and spore stage on produc-
tion of teliospores by single aeciospore lines of
Cronartium ribicola. USDA For. Serv. Res. Pap. INT-256,
9p. Intermt. For. and Range Exp. Stn., Ogden, Utah
84401.

Genetically identical aeciospore and urediospore popula-
tions grown on a single ribes clone varied in time required
to produce teliospores. Infection developing from uredio-
spores usually produced teliospores in less than 28 days when
grown at 55°F (13°C); whereas, infections developing from
aeciospore generally required more than 35 days at the same
temperature.

KEYWORDS: Cronartium ribicola, epidemiological fitness,
inherited traits, detached leaf culture of rust

The Intermountain Station, headquartered in Ogden,
Utah, is one of eight regional experiment stations charged
with providing scientific knowledge to help resource
managers meet human needs and protect forest and range
ecosystems.

The Intermountain Station includes the States of
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About 231 million acres, or 85 percent, of the land area in the
Station territory are classified as forest and rangeland. These
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year.

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are maintained in:

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sity of Idaho)

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