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Summer and winter survival of Puccinia Recondita and infection by soilborne urediniospores
Trans. Br. mycol. Soc. 86 (3), 365-372 (1986)
Primed in Great Britain
SUMMER AND WINTER SURVIVAL OF PUCCINIA RECONDITA AND INFECTION BY SOILBORNE UREDINIOSPORES By Z. M . HASSAN*, C. L. KRAMER Division of Biology, Kansas State University, Manhattan, Kansas, 66506 U.S.A. AND M . G . EVERSMEYER U.S. Department of Agriculture, Agricultural Research Service, Department of Plant Pathology , Kansas State University , Manhattan, Kansas, 66506 U.S.A.
Emerging wheat seedlings readily trapped urediniospores of Puccinia recondita from soil or host plant debris. Soilborne spores survived simulated summer temp. of 34/22 °C day/night for up to 22 d on moist soil, 13 d on dry soil and 4-7 d when covered with a 5 mm layer of soil. Trapped spores survived 8 d on dry leaves at 34/22°. Spores in pustules on excised leaves placed on soil during simulated summer (30-33°) conditions survived up to 15 d. Spores floating on flood waters were able to cause infection when brought in contact with partially submerged host leaves . Dormant mycelium survived all conditions that host tissue was capable of surviving. Changes from 21 to + 1, or - 10 caused host tissues to remain chlorotic and, in some cases, dormant mycelium was able to sporulate, but reduction to -4° caused host tissues to become necrotic, prohibiting sporulation.
°
In the central Great Plains of the U .S.A., volunteer wheat (Triticum aestivum L.) occurs regularly and often serves as host for oversummering Puccinia recondita Rob. f.sp, tritici Eriks. The circumstances under which volunteer plants become inoculated during the summer in the central Great Plains have not, however, been clearl y understood. Large numbers of spores could become deposited on the soil before and during harvest and serve as an inoculum source for volunteer wheat. The fungus is able to reinfect volunteer plants, oversummer, and serve as a source for primary infection of autumn sown wheat. As autumn progresses, latent periods become longer because of lower temperatures (Eversmeyer, Kramer & Browder, 1980; Tomerlin et al., 1983). When infection occurs very late in the season, the mycelium may become dormant before it can sporulate. If the infected host tissue survives, the dormant mycelium may resume growth and sporulate to serve as a source of primary infection in the spring (Burleigh, Schulze & Eversmeyer, 1969). The objectives of this study were to determine the role of soilborne urediniospores and spores contained in old pustules on plant debris as an inoculum source for volunteer wheat and to investigate the conditions under which dormant mycelium overwinters in host tissues and serves as an inoculum source for the establishment of the disease the following year.
t Present address : P.O . Box 1482, Bur ydah, Ga ssim , Saudia Arabia.
MA TERIALS AND METHODS
Unless otherwise stated, the host used throughout the following experiments was Triticum aestivum (cv. Trison) and the parasite was Puccinia recondita f.sp. tritici (PRT U S 3).
Trapping of soilborne spores by emerging wheat seedlings Two days after planting 50 wheat seeds in pots, and before shoot emergence, freshly harvested spores were dispersed on the soil surface (1 mg /3 ern"). At the time of seedling emergence, soil surfaces were kept at three levels of moisture: approx, 50,75 and 100 % holding capacity (w.h.c.). Although the soil had to be watered to initiate seed germination, a layer of soil (approx. 5 mm thick) with a moisture content at 50 % w.h .c. was placed on the surface of the dry-treatment pots just prior to dispersing spores. Pots at 75 % w.h.c. were subirrigated every other day in order to maintain a moist soil surface. Pots with soil at 100 % w.h.c. were kept in tra ys of water to maintain a constant film of water on the surface. Two days after seedling emergence, impressions of 10 leaves were made by spreading a thin layer of Duco (D uPont) or similar household cement over th e leaf surface and allowing it to dry. The dried film was peeled off and mounted on microscope slides in lactophenol-cotton blue to make direct counts of trapped spores. Seedlings were placed in
366
Survival of Puccinia recondita
a dew chamber overnight and a second set of leaf impressions was made . the following day to determine spore viability. Seedlings were placed in the greenhouse to allow pustules to develop on the remaining leaves. Numbers of pustules per leaf were counted 10 d later. Effects of simulated summer temperatures and soil moisture on viability and longevity of spores
Spores (1'5 mg crn") were dispersed over the soil in each pot . Soil moisture was maintained at approx. either 50 or 100% w.h .c. in both field and environmental chamber studies. Spores in one set of pots were covered with 5 mm of soil and left uncovered in a second set. Pots were then placed in an environmental chamber at 34°, 8000 Ix for a 14 h day and at 22° for a 10 h night, or in the experimental field near Manhattan, Kansas during periods with daily max. temp. of 3cr-33°. At 1- or z-d intervals, soil samples containing spores were taken from the appropriate pots and suspended in a solution of 5 % Tween-So and 43'1 % glycerin in tap water (Mehta & Zadoks, 1970). After allowing the suspension to settle, supernatant containing spores was dispersed on l ' 5 % water agar and incubated in the dark at 21° for 6 h and the percentages of germinated spores determined. Ten 5 cm plastic straws were place at angles with one end approx. 1 em below the soil surface in pots. Soil at 50 or 100 % w.h.c. was covered with freshly harvested spores (3 mg cm") and placed in the controlled environment chamber as above. Three pots from each treatment and one control lacking spores were seeded on the first day and at 2 d intervals thereafter for 24 d. One seed was dropped through each straw which then was removed from the pot so as not to disturb the layer of spores directly above the seeds. On the second day after emergence, seedlings were placed in a 21° dew chamber for 16 h incubation and then moved to a 21 ± 5° greenhouse. Numbers of pustules per leaf were counted 10 d later as a measure of longevity of soilborne spores. Trapping of sporesfrom old pustules on host debris by emerging wheat seedlings
Leaves bearing mature pustules were excised, air-dried for 48 hat 30° and placed 1-2 em deep on the surface of soil in pots previously sown with wheat. Beginning on the first day after seedling emergence, one pot was transferred daily to a dew chamber to allow infection by viable spores that may have become trapped on emerging seedling leaves. Pots were moved to a greenhouse at 21° and the number of pustules per plant were counted 8-10 d later.
Longevity of spores in old pustules on dead host tissue Leaves of 2'5 wk-old seedlings with newly ruptured pustules were clipped and placed in nylon mesh bags. Bags were either placed on the soil surface or covered with 0"5 em soil during periods with daily max. temp. of3cr-33°. At 2 d intervals samples were collected from all treatments and spores were removed from the tissue, placed on water agar, incubated overnight in the dark at 21°, and percentage germination determined. Pots of senesced, infected seedlings were kept in the field during daily max. temp. of 25-30°. Longevity of spores trapped on host leaves Prior to seedling emergence, spores were dispersed (1'5 mg em -2) on the soil in pots; another set of pots was kept as controls. Half of each set was placed in a greenhouse (approx. 24° d, 20° night), while the other half was placed in an environmental chamber (34° d, 22° night and 16 h photoperiod of 8000 Ix). At 2 d intervals, one pot with spores and one control from each temp. treatment were placed in a dew chamber for 16 h then transferred to a greenhouse where all plants were subirrigated to prevent infection from contaminating airborne spores. The percentage of infected plants and number of pustules per plant served as criteria for spore survival. Infection from sporesfloating on flood waters Freshly harvested spores were dusted (1"5 mg cm" ) onto the surface of each of two pots of soil, without plants. The pots were placed in a large aquarium along with three pots of z-wk old wheat seedlings. The aquarium was then slowly filled, in a manner simulating rising flood waters, to a level covering about three-fourths the height of the seedlings. The water level was then lowered in increments of3 em by siphoning at 4-, 8-, 12-and 24-h intervals to determine how long floating spores might remain capable of causing infection . Plants were placed in a 21° greenhouse and examined for pustules after 10 d. Survival of mycelium in wheat tissues subjected to cold Wheat (cv. Eagle) was planted in a 6 x 50 m plot near Manhattan, Kansas in Sept. 1982. The plot was inoculated in Oct. when plants were at the 5-6 leaf stage. Leaf rust severity in the plot was estimated at 40 % (Peterson, Campbell & Hannah,
Z . M. Hassan, C. L. Kramer and M. G. Eversmeyer 1948) on the two lowest leaves by Nov . 1. Average temp. for Nov. and Dec . 1982 were 6 and 2°, respectively. Plants were protected by continuous snow cover from early Dec . to late jan., but after the snow melted they were exposed to the cold temp . of Feb. At 1- and z-wk intervals from Dec. 1982 to Mar. 1983,20 wheat plants were randomly selected from the plot , transplanted into pots and placed in a 21° greenhouse. The pots were sub irrigated in tra ys to prevent infection from airborne spores while in the greenhouse. Field survival of mycelium in host tissues was assessed by the occurrence of uredinia 10--14 dafter transplanting. Seedlings were transplanted into pots from the plot in Feb. and Mar. and after 10 d inoculated by dusting with spores . Inoculated plants were kept in a 21 o dew chamber for 8 h then transferred to a 21 o environmental chamber. In order to obtain mycelial colonies of different stages of development for testing, a set of pots was removed from the 21 c chamber at 8,16,24, and 72 h after inoculation and transferred to environmental chambers set at 5/0, 2/-2, -1/-4 and -4/-8° day/night temp. regimes with 8000 lx 16 h photoperiod. One-half of the plants with each mycelium growth stage was removed from the cold treatments after 4 and 8 h. Plants were examined after 10 d to determine if mycelium and/or host tissue was able to survive. A third experiment was designed to study the effects of immediate changes from warm to cold temp. on the survival of both host tissue and mycelium. Seedlings were inoculated and placed in a 21° dew chamber for 16 h to allow for spore germination and infection. Seedlings were transferred to a 21 o greenhouse for 24, 48, or 72 h to allow for the development of different amounts of mycelium. They were then transferred to cold temperatures of either + 1, 0, -1, or -4° in environmental chambers for periods of 2-7 h before returning to the greenhouse to allow for sporulation.
Table
1.
RESUL TS
Trapping of soilborne spores by emerging wheat seedlings Soilborne spores were readily picked up by emerging wheat seedlings from soils at all moisture contents (T able 1), and the capture rate did not appear to be correlated with soil moisture. Germination did not occur while spores were in contact with the soil, even when they were in contact with both the emerging leaf and film of free moisture present on the soil surface. Germination and infection did occur on leaves which were allowed to elongate and carry spores away from contact with the soil before being placed in a dew chamber (T able 1). Effects of simulated summer temperatures and soil moisture on viability and longevity of spores
Viability of spores decreased more rapidly on drier soil (50 w.h .c.) than on wet soil (100 % w.h.c.) (T able 2 ). The decline was even more rapid when the spores were covered with approx. 5 min of soil (T able 2). Spores under field conditions during Aug . and Sept. survived for 18 d but not for 22 d (T able 2) . The infectivity of soilborne spores also declined under controlled simulated summer temperatures. After 22 d exposure to those conditions, infection no longer occurred (T able 3). Trapping of sporesfrom old pustules on host debris by emerging wheat seedlings Emerging seedlings readily picked up spores from the debris on the soil surface, as indicated by the development of pustules. Because of the extreme non-uniform distribution of spores on the debris, there was a corresponding non-uniformity in the distribution of pustules on the seedlings (Table 4).
Comparative numbers of soilborne urediniospores trapped and resulting infection of wheat seedlings Spore germ ination after placing in dew chamber ('Yo )'"
Soil moisture ('Yo w.h.c.)
Spores trapped (mean/ leaf)
On leaves
On soil
5° 75
207 85 434
32'S 43'S 33'9
°
100
0
°
Plants tested
Plants with pustules
75 82 27
14
'" No germination before placement in dew chambers. t Nine plants also had pustules on leaf sheaths.
l1t °
Survival of Puccinia recondita Table 2. Germination of P, recondita spores subjected to various soil treatments under summer field conditions and controlled simulated summer temperatures Spore germination (%) Spores on soil surface
Length of treatment (d) 0 1 2 3 4 5 6 7 8 9 11 13 15 16 17 18 22
Spores covered with soil
50 *
100
50
100
88 66
88 71
88
88 34
14
71
12
73
11
57
0
12 8 2 1
54 31 34 21
0
23
Field conditions spores on soil surface approx. 50 88 64
7 0 8 0
59 30 18 30 42 9
16 0
3 0
5
-, Samples not taken. * Soil moisture content - % w.h.c. We observed an increase in the number of pustules corresponding to the length of time seedlings were allowed to develop before being placed in the dew chamber.
Longevity of spores in old pustules on dead host tissue The germination rate of spores from dead leaves declined from 59 % to 0 % after 15 and 8 d
respectively, when placed on or in soil in Aug . (T able 5). When infected seedlings were allowed to senesce, and then placed outside intact in the pots in which they were growing, the germination rate dropped from 65 % to 14 % in 30 d (T able 5). This test was done in Sept., however, when temp, were somewhat lower.
Longevity of spores trapped on host leaves Soilborne spores trapped by emerging whea t seedlings must survive until free moisture becomes
Tabl e 3. Longevity of soilborne spores under simulated summer temperatures Mean no. pustules per infected plant
Length of treatment (d)
Plants tested (no.)
Plants with pustules
4 6 8 10 12 14 16 18 20 22 24
27 28 29 27 31 29 28 24 26 24 27
17 9 21 6
3'8 3'0 2'3
11
3'1 1"4 1'0 3'0
1.5
8 7 2 1
2 '0
0 0
0 0
Table 4. Infectio n of emerging wheat seedlings fro m spores obtained f rom pustules on plant debris distributed over the soil surface Day placed in dew chamber* 1 2 3 4 5 6
7
Seedlings with pustules']0
5 1 5 20 20 25
Range in no. pustules/seedling 0 1-7 2 1-3 1-18 1-21 1-1 7
* Number of days following emergence that pot was placed in dew chamber. t Total plants = 30.
Z. M. Hassan, C. L. Kramer and M. G. Eversmeyer Infection from spores floating on flood waters When spores were deposited on soil which was later flooded, many spores were picked up and carried on the surface of the rising water. Floating spores had not germinated, but infection and pustule development readily occurred where the spores came in contact with partially submerged host plant leaves. When the water level was reduced stepwise to four lower levels over a total period of 48 h, additional pustules developed at the new positions where the floating spores came in contact with the wheat leaves. This indicates that at least some spores remained infective after floating on the water surface for as long as 48 h. On several occasions, large air bubbles with spores at the water/air interface were trapped on leaves below the water surface. Apparently these situations were sufficient to allow for infec tion to occur, since pustules developed in these areas after those portions of the leaves became exposed above the water level.
Table 5. Longevity of spores in pustules on clipped leaves and senescedseedlings kept outside during Aug. and Sept.* Spore germination ( % ) Senesced
Clipped leaves (Aug.) Leaves exposed on soil surface
Spore exposure time (d)
seedlings (kept intact in pots in
Leaves covered with 0'5 ern soil
Sept.)
65 59 59 ° 1 59 34 79 7 33 3 1 50 4 70 5 6 15 41 66 8 29 7 64 10 14 ° 12 12 47 ° 0 15 ° 16 43 23 3° 14 30 - , Samples not taken. * Av. daily max. temp. 30° and 25° respectively .
Survival of mycelium in wheat tissue subjected to cold
Wheat plants (cv. Eagle) p lanted from the field prior to loss of snow cover and incubated in the greenhouse in Dec. 1982 and Jan. 1983, developed at least one sporulating uredinium on 85 % of the plants. In Feb., after air temp. reached -15 0 and snow cover had disappeared from the plot, uredinia developed on less than 5 % ofthe plants. By the first wk of Mar., sporulating uredinia did not develop on any plants brought into the greenhouse. At tha t time, most of the older leaves had senesced ,
available on the leaf surface in order for infection to occur. Soilborne spores trapped and maintained on dry leaf surfaces were able to cause infections for as long as 13 d at the lower field temp. (T able 6). At the higher temp. pustules did not develop after 8 d.
Table 6. Long evity of spores on dry foliage and at two day/night temperature regimes Environmental chamber (34/22°)
Greenhouse (24/ 20°) Spore exposure time (d) ° 2 3 4 5 6 7 8 10 13 16
Plants tested (no.)
Plants with pustules
Mean no. pustules infected plant
29
10
3"3
26
7
2'9
25
17
3"7
27 29 28
10 21 0
2"3 2'7 0
-, Samplesnot taken.
Total no. of plants 25 22
Plants with pustules 4 18
Mean no. pustules infected plant 1·8 4"1
27
11
2,8
26
15
3'1
26 22 28
9 0
3'1 0
°
0
Survival of Puccinia recondita
37°
Table 7. Survival of vernalized seedling wheat leaves and Puccinia recondita mycelium that had been allowed to develop at 21 ° for 8-72 h after infection and prior to extended periods of low temperature Mycelial age before exposure to cold (h) Day /night temp. (Oe )
Exposure (h)
5/ 0
4 8 12
16 4 8 4
2/-2 -1/ -4
8
16
24
48
72
++* ++ ++ ++
++ ++ ++ ++
++ ++ ++ ++ - +
++ ++ ++ ++ ++
++ ++ ++ ++ ++
- +
++
8
-4/- 8
4 8
* + +, Survival of fungus and host tissue; - -, No survival of fungus or host tissue; producing uredinia on necrotic host tissue. Under controlled conditions, host plant tissue survived at least 16 h of exposure to 5/0° and supported development of uredinia when the mycelia had been allowed to develop for as little as 8 h prior to cold treatment (T able 7). Neither mycelium nor host tissue were able to survive 8 h exposure to 2/ - 2° regardless of the length of incubation period, but mycelium that had developed at least 24 h at 21 ° before cold exposure was able to survive 4 h (T able 7). In studying the effect of rapid reductions in temp., all plants subjected to chilling from 21 to -4° became necrotic and were unable to support sporulation of the fungus . Those chilled to -1 , a and + 1 ° remained chlorotic for 8-10 d, and in some cases allowed pustules to develop and sporulate (T able 8).
to less than 5 % within 22 d. The decline was more rapid when spores were on drier soils and when covered with soil. The viability of spores contained on host plant debris declined at a similar rate.
Table 8. Survival of Puccinia recondita mycelium when the mycelium was allowed to develop in vernalized Trison wheat seedling leaves at 21° and then subjected to chilling Hours of mycelium developmen t at 21° before host tissue exposed to low temp erature treatments Chilling (oq
DISCUSSION
During several years, when wheat was heavily infected in Kansas, volunteer plants in plots also became heavily infected following harvest. The amount of infection on the volunteer plants was heavier than thought attributable to airborne spores alone, and these results show that spores deposited directly on the soil or contained on wheat plant debris readily adhere to emerging seedlings when contact is made . Infection then depends on the viability of the spores and the availability of free moisture to initiate the germination process. Hwang ( 194 2) reponed germination of (50) 70-100 % for spores from newly ruptured pustules. We found similar variations to occur (H assan, 1983). The initial germination percentage, howe ver, was found to decrease at a rate dependent on several factors, with perhaps temp. being the most important. On wet, warm soil germination dropped
+, Fungus survived,
0
-1
Exposure time (h)
24
48
72
2 3 4 5
+* +
+ 0
+ +
0 0
+ +
0 0
6 7
0
0
0
+
0 0
2 3 4
+
0
0
0
5
+ +
+ +
6 7
0 0
+
+ + + + + +
2 3 4 5
+ + +
0 0
+ +
0
0 0
0 0 0
0
+ + +
6 + + 7 * + . Uredinia present on chloroti c host tissue ; 0 , No uredinia produced.
Z. M. Hassan, C. L. Kramer and M. G. Eversmeyer Germination of soilborne spores did not occur while the spores were in actual contact with soil. This was true even of spores trapped by emerging leaves. Since wheat leaves elongate by a basal meristern, however, trapped spores are carried away from contact with the soil. This also allows spores to be trapped along the length of an emerging leaf. Under field conditions, moisture may not be available immediately following seedling ernergence. Thus, trapped spores may have to survive several days on leaves before germination and infection becomes possible. Eversmeyer & Burleigh (1968) found spores to survive 45 d at 5-8° but only 9 d at 18-39° on dry foliage. Hwang (1942) reported foliage to reduce sunlight intensity and temp. resulting in prolonged germinability of spores trapped on lower leaves. Fulton & Coblentz (1929) found that spores in the centre of dumps retain viability longer than those on the outside. This apparently is due to a protective shading effect. Rehydrated spores were found by Leathers (1961) to maintain 70-80 % viability for up to 40 d on dry leaf surfaces, while viability of non-rehydrated spores declined to less than 5 % by 32 d. Swaebly (1955) reported a difference in survival of P. graminis race 1SB on cvs Little Club and Kentane S1A, while Mohamed (1960) found a difference in subrace 139B on the leaves of Marquis and Little Club. Although many factors may be involved in the survival of urediniospores before infection can occur, it seems apparent that, in some situations, spores are able to retain viability long enough to function as a primary source of infection of volunteer wheat following harvest. In autumn, spores often accumulate in pustules on rosette plants and eventually fall to the ground in large clumps. Tests of simulated flood conditions indicate that soilborne spores can be dispersed on the surface of rising water and cause infection if brought in contact with partially submerged wheat plants. Although field tests have not been made, observations of flood irrigated areas indicate that infections have occurred in that way. Burleigh, Schulze & Eversmeyer (1969) reported the occurrence of dormant mycelium from plants collected in Jan. to Mar. from numerous locations in Kansas, Colorado, Oklahoma, and Texas. We have shown that tillering plants removed from the field in late winter, often developed pustules when brought into a greenhouse. In early spring when temperatures become warm enough, i.e. 10-50°, to initiate renewed growth in the host plants, temp. may drop to near freezing or below within 12 h . Such changes are more destructive to host tissues than changes from 0 to
371
5°, to sub-zero temp. If mycelium is allowed to develop for at least 24 h following infection before chilling to below 0°, that mycelium apparently is capable of surviving all conditions that the host can. On several occasions when infected plants were subjected to frost, the mycelium was able to resume growth and develop sporulating uredinia when returned to the greenhouse even though host tissues were beginning to senesce, Presence or absence of mycelium made no detectable difference in the ability of the host tissues to survive sustained low temp. or abrupt changes in temp. as discussed above. The older or lower leaves of tillered plants are more easily affected by low temp. and often become necrotic before dormant mycelia are able to develop sporulating pustules. In two of the last five years, however, host tissues with dormant mycelia were able to survive long enough in Kansas for development of spores which served as primary inoculum for spring infections. Contribution of the Kansas State University in cooperation with the United States Department of Agriculture, Agricultural Research Service, no. 84- 114 J. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the USDA-ARS, and does not imply approval over other products that also may be suitable. This article is in the public domain and is not copyrightable. It may be freely reprinted with customary crediting of source. The work reported is part of the Ph.D. Thesis of the senior author.
REFERENCES
BURLEIGH, J. R., SCHULZE, A. A. & EVERSMEYER, M . G. (19 69). Some aspects of the summer and winter ecology of wheat rust fungi. Plant Disease Reporter 53,648-651. EVERSMEYER, M. G. & BURLEIGH, J. R. (1968). Effect of temperature on the longevity of Puccinia recondita f.sp. tritici urediospores on dry wheat foliage. Plant Disease Reporter 5z, 186-188. EVERSMEYER, M. G., KRAMER, C. L. & BROWDER, L. E. (1980 ). Effect of temperature and host: parasite combination on the latent period of Puccinia recondita in seedling wheat plants. Phytopathology 70, 938--941. FULTON, H. R. & CoBLENTZ, W. W. (1929). The fungicidal action of ultra-violet radiation. Journal of Agriculture Research 38, 159-168. HAsSAN, Z. (1983). Epidemiological studies ofleaf rust of wheat caused by Puccinia recondita f.sp. tritici. Ph.D . Thesis, Kansas State University, U.S.A. HWANG, L. (1942). The effect of light and temperature on the viability of urediospores of certain cereal rusts. Phytopathology 31, 699-711. LEATHERS, C. R. (1961). Comparative survival of rehydrated and nonrehydrated wheat stem rust uredospores on dry leaf surfaces. Phytopathology 51, 410-411.
37 2
Survival of Puccinia recondita
MEHTA, Y. R. & ZADOKS, J. c. (1970). Uredospore production and sporulation period of Puccinia recondita f.sp. tritici on primary wheat leaves. Netherlands Journal of Plant Pathology 76, 267-276. MOHAMED,H. A.(1960). Survival of stem rust uredospores on dry foliage of wheat. Phytopathology 50, 400-401. PETERSON, R. F., CAMPBELL, A. B. & HANNAH, A. E. (1948). A diagrammatic scale for estimating rust intensity of leaves and stems of cereals. Canadian Journal of Research C 26, 496-500.
SWAEBLY, M. A. (1955). Variability in the infection of wheat by Puccinia graminis tritici and studies on extracts from rust urediospores. Ph.D. Thesis, University of Minnesota, U.S.A. TOMERLIN, J. R., EVERSMEYER, M. G., KRAMER, C. L. & BROWDER, L. E. (1983). Temperature and host effects on latent and infectious periods and on urediniospore production of Puccinia recondita f.sp. tritici. Phytopathology 73, 414-419.
(Received for publication 7 February 1985)
Primed in Great Britain
SUMMER AND WINTER SURVIVAL OF PUCCINIA RECONDITA AND INFECTION BY SOILBORNE UREDINIOSPORES By Z. M . HASSAN*, C. L. KRAMER Division of Biology, Kansas State University, Manhattan, Kansas, 66506 U.S.A. AND M . G . EVERSMEYER U.S. Department of Agriculture, Agricultural Research Service, Department of Plant Pathology , Kansas State University , Manhattan, Kansas, 66506 U.S.A.
Emerging wheat seedlings readily trapped urediniospores of Puccinia recondita from soil or host plant debris. Soilborne spores survived simulated summer temp. of 34/22 °C day/night for up to 22 d on moist soil, 13 d on dry soil and 4-7 d when covered with a 5 mm layer of soil. Trapped spores survived 8 d on dry leaves at 34/22°. Spores in pustules on excised leaves placed on soil during simulated summer (30-33°) conditions survived up to 15 d. Spores floating on flood waters were able to cause infection when brought in contact with partially submerged host leaves . Dormant mycelium survived all conditions that host tissue was capable of surviving. Changes from 21 to + 1, or - 10 caused host tissues to remain chlorotic and, in some cases, dormant mycelium was able to sporulate, but reduction to -4° caused host tissues to become necrotic, prohibiting sporulation.
°
In the central Great Plains of the U .S.A., volunteer wheat (Triticum aestivum L.) occurs regularly and often serves as host for oversummering Puccinia recondita Rob. f.sp, tritici Eriks. The circumstances under which volunteer plants become inoculated during the summer in the central Great Plains have not, however, been clearl y understood. Large numbers of spores could become deposited on the soil before and during harvest and serve as an inoculum source for volunteer wheat. The fungus is able to reinfect volunteer plants, oversummer, and serve as a source for primary infection of autumn sown wheat. As autumn progresses, latent periods become longer because of lower temperatures (Eversmeyer, Kramer & Browder, 1980; Tomerlin et al., 1983). When infection occurs very late in the season, the mycelium may become dormant before it can sporulate. If the infected host tissue survives, the dormant mycelium may resume growth and sporulate to serve as a source of primary infection in the spring (Burleigh, Schulze & Eversmeyer, 1969). The objectives of this study were to determine the role of soilborne urediniospores and spores contained in old pustules on plant debris as an inoculum source for volunteer wheat and to investigate the conditions under which dormant mycelium overwinters in host tissues and serves as an inoculum source for the establishment of the disease the following year.
t Present address : P.O . Box 1482, Bur ydah, Ga ssim , Saudia Arabia.
MA TERIALS AND METHODS
Unless otherwise stated, the host used throughout the following experiments was Triticum aestivum (cv. Trison) and the parasite was Puccinia recondita f.sp. tritici (PRT U S 3).
Trapping of soilborne spores by emerging wheat seedlings Two days after planting 50 wheat seeds in pots, and before shoot emergence, freshly harvested spores were dispersed on the soil surface (1 mg /3 ern"). At the time of seedling emergence, soil surfaces were kept at three levels of moisture: approx, 50,75 and 100 % holding capacity (w.h.c.). Although the soil had to be watered to initiate seed germination, a layer of soil (approx. 5 mm thick) with a moisture content at 50 % w.h .c. was placed on the surface of the dry-treatment pots just prior to dispersing spores. Pots at 75 % w.h.c. were subirrigated every other day in order to maintain a moist soil surface. Pots with soil at 100 % w.h.c. were kept in tra ys of water to maintain a constant film of water on the surface. Two days after seedling emergence, impressions of 10 leaves were made by spreading a thin layer of Duco (D uPont) or similar household cement over th e leaf surface and allowing it to dry. The dried film was peeled off and mounted on microscope slides in lactophenol-cotton blue to make direct counts of trapped spores. Seedlings were placed in
366
Survival of Puccinia recondita
a dew chamber overnight and a second set of leaf impressions was made . the following day to determine spore viability. Seedlings were placed in the greenhouse to allow pustules to develop on the remaining leaves. Numbers of pustules per leaf were counted 10 d later. Effects of simulated summer temperatures and soil moisture on viability and longevity of spores
Spores (1'5 mg crn") were dispersed over the soil in each pot . Soil moisture was maintained at approx. either 50 or 100% w.h .c. in both field and environmental chamber studies. Spores in one set of pots were covered with 5 mm of soil and left uncovered in a second set. Pots were then placed in an environmental chamber at 34°, 8000 Ix for a 14 h day and at 22° for a 10 h night, or in the experimental field near Manhattan, Kansas during periods with daily max. temp. of 3cr-33°. At 1- or z-d intervals, soil samples containing spores were taken from the appropriate pots and suspended in a solution of 5 % Tween-So and 43'1 % glycerin in tap water (Mehta & Zadoks, 1970). After allowing the suspension to settle, supernatant containing spores was dispersed on l ' 5 % water agar and incubated in the dark at 21° for 6 h and the percentages of germinated spores determined. Ten 5 cm plastic straws were place at angles with one end approx. 1 em below the soil surface in pots. Soil at 50 or 100 % w.h.c. was covered with freshly harvested spores (3 mg cm") and placed in the controlled environment chamber as above. Three pots from each treatment and one control lacking spores were seeded on the first day and at 2 d intervals thereafter for 24 d. One seed was dropped through each straw which then was removed from the pot so as not to disturb the layer of spores directly above the seeds. On the second day after emergence, seedlings were placed in a 21° dew chamber for 16 h incubation and then moved to a 21 ± 5° greenhouse. Numbers of pustules per leaf were counted 10 d later as a measure of longevity of soilborne spores. Trapping of sporesfrom old pustules on host debris by emerging wheat seedlings
Leaves bearing mature pustules were excised, air-dried for 48 hat 30° and placed 1-2 em deep on the surface of soil in pots previously sown with wheat. Beginning on the first day after seedling emergence, one pot was transferred daily to a dew chamber to allow infection by viable spores that may have become trapped on emerging seedling leaves. Pots were moved to a greenhouse at 21° and the number of pustules per plant were counted 8-10 d later.
Longevity of spores in old pustules on dead host tissue Leaves of 2'5 wk-old seedlings with newly ruptured pustules were clipped and placed in nylon mesh bags. Bags were either placed on the soil surface or covered with 0"5 em soil during periods with daily max. temp. of3cr-33°. At 2 d intervals samples were collected from all treatments and spores were removed from the tissue, placed on water agar, incubated overnight in the dark at 21°, and percentage germination determined. Pots of senesced, infected seedlings were kept in the field during daily max. temp. of 25-30°. Longevity of spores trapped on host leaves Prior to seedling emergence, spores were dispersed (1'5 mg em -2) on the soil in pots; another set of pots was kept as controls. Half of each set was placed in a greenhouse (approx. 24° d, 20° night), while the other half was placed in an environmental chamber (34° d, 22° night and 16 h photoperiod of 8000 Ix). At 2 d intervals, one pot with spores and one control from each temp. treatment were placed in a dew chamber for 16 h then transferred to a greenhouse where all plants were subirrigated to prevent infection from contaminating airborne spores. The percentage of infected plants and number of pustules per plant served as criteria for spore survival. Infection from sporesfloating on flood waters Freshly harvested spores were dusted (1"5 mg cm" ) onto the surface of each of two pots of soil, without plants. The pots were placed in a large aquarium along with three pots of z-wk old wheat seedlings. The aquarium was then slowly filled, in a manner simulating rising flood waters, to a level covering about three-fourths the height of the seedlings. The water level was then lowered in increments of3 em by siphoning at 4-, 8-, 12-and 24-h intervals to determine how long floating spores might remain capable of causing infection . Plants were placed in a 21° greenhouse and examined for pustules after 10 d. Survival of mycelium in wheat tissues subjected to cold Wheat (cv. Eagle) was planted in a 6 x 50 m plot near Manhattan, Kansas in Sept. 1982. The plot was inoculated in Oct. when plants were at the 5-6 leaf stage. Leaf rust severity in the plot was estimated at 40 % (Peterson, Campbell & Hannah,
Z . M. Hassan, C. L. Kramer and M. G. Eversmeyer 1948) on the two lowest leaves by Nov . 1. Average temp. for Nov. and Dec . 1982 were 6 and 2°, respectively. Plants were protected by continuous snow cover from early Dec . to late jan., but after the snow melted they were exposed to the cold temp . of Feb. At 1- and z-wk intervals from Dec. 1982 to Mar. 1983,20 wheat plants were randomly selected from the plot , transplanted into pots and placed in a 21° greenhouse. The pots were sub irrigated in tra ys to prevent infection from airborne spores while in the greenhouse. Field survival of mycelium in host tissues was assessed by the occurrence of uredinia 10--14 dafter transplanting. Seedlings were transplanted into pots from the plot in Feb. and Mar. and after 10 d inoculated by dusting with spores . Inoculated plants were kept in a 21 o dew chamber for 8 h then transferred to a 21 o environmental chamber. In order to obtain mycelial colonies of different stages of development for testing, a set of pots was removed from the 21 c chamber at 8,16,24, and 72 h after inoculation and transferred to environmental chambers set at 5/0, 2/-2, -1/-4 and -4/-8° day/night temp. regimes with 8000 lx 16 h photoperiod. One-half of the plants with each mycelium growth stage was removed from the cold treatments after 4 and 8 h. Plants were examined after 10 d to determine if mycelium and/or host tissue was able to survive. A third experiment was designed to study the effects of immediate changes from warm to cold temp. on the survival of both host tissue and mycelium. Seedlings were inoculated and placed in a 21° dew chamber for 16 h to allow for spore germination and infection. Seedlings were transferred to a 21 o greenhouse for 24, 48, or 72 h to allow for the development of different amounts of mycelium. They were then transferred to cold temperatures of either + 1, 0, -1, or -4° in environmental chambers for periods of 2-7 h before returning to the greenhouse to allow for sporulation.
Table
1.
RESUL TS
Trapping of soilborne spores by emerging wheat seedlings Soilborne spores were readily picked up by emerging wheat seedlings from soils at all moisture contents (T able 1), and the capture rate did not appear to be correlated with soil moisture. Germination did not occur while spores were in contact with the soil, even when they were in contact with both the emerging leaf and film of free moisture present on the soil surface. Germination and infection did occur on leaves which were allowed to elongate and carry spores away from contact with the soil before being placed in a dew chamber (T able 1). Effects of simulated summer temperatures and soil moisture on viability and longevity of spores
Viability of spores decreased more rapidly on drier soil (50 w.h .c.) than on wet soil (100 % w.h.c.) (T able 2 ). The decline was even more rapid when the spores were covered with approx. 5 min of soil (T able 2). Spores under field conditions during Aug . and Sept. survived for 18 d but not for 22 d (T able 2) . The infectivity of soilborne spores also declined under controlled simulated summer temperatures. After 22 d exposure to those conditions, infection no longer occurred (T able 3). Trapping of sporesfrom old pustules on host debris by emerging wheat seedlings Emerging seedlings readily picked up spores from the debris on the soil surface, as indicated by the development of pustules. Because of the extreme non-uniform distribution of spores on the debris, there was a corresponding non-uniformity in the distribution of pustules on the seedlings (Table 4).
Comparative numbers of soilborne urediniospores trapped and resulting infection of wheat seedlings Spore germ ination after placing in dew chamber ('Yo )'"
Soil moisture ('Yo w.h.c.)
Spores trapped (mean/ leaf)
On leaves
On soil
5° 75
207 85 434
32'S 43'S 33'9
°
100
0
°
Plants tested
Plants with pustules
75 82 27
14
'" No germination before placement in dew chambers. t Nine plants also had pustules on leaf sheaths.
l1t °
Survival of Puccinia recondita Table 2. Germination of P, recondita spores subjected to various soil treatments under summer field conditions and controlled simulated summer temperatures Spore germination (%) Spores on soil surface
Length of treatment (d) 0 1 2 3 4 5 6 7 8 9 11 13 15 16 17 18 22
Spores covered with soil
50 *
100
50
100
88 66
88 71
88
88 34
14
71
12
73
11
57
0
12 8 2 1
54 31 34 21
0
23
Field conditions spores on soil surface approx. 50 88 64
7 0 8 0
59 30 18 30 42 9
16 0
3 0
5
-, Samples not taken. * Soil moisture content - % w.h.c. We observed an increase in the number of pustules corresponding to the length of time seedlings were allowed to develop before being placed in the dew chamber.
Longevity of spores in old pustules on dead host tissue The germination rate of spores from dead leaves declined from 59 % to 0 % after 15 and 8 d
respectively, when placed on or in soil in Aug . (T able 5). When infected seedlings were allowed to senesce, and then placed outside intact in the pots in which they were growing, the germination rate dropped from 65 % to 14 % in 30 d (T able 5). This test was done in Sept., however, when temp, were somewhat lower.
Longevity of spores trapped on host leaves Soilborne spores trapped by emerging whea t seedlings must survive until free moisture becomes
Tabl e 3. Longevity of soilborne spores under simulated summer temperatures Mean no. pustules per infected plant
Length of treatment (d)
Plants tested (no.)
Plants with pustules
4 6 8 10 12 14 16 18 20 22 24
27 28 29 27 31 29 28 24 26 24 27
17 9 21 6
3'8 3'0 2'3
11
3'1 1"4 1'0 3'0
1.5
8 7 2 1
2 '0
0 0
0 0
Table 4. Infectio n of emerging wheat seedlings fro m spores obtained f rom pustules on plant debris distributed over the soil surface Day placed in dew chamber* 1 2 3 4 5 6
7
Seedlings with pustules']0
5 1 5 20 20 25
Range in no. pustules/seedling 0 1-7 2 1-3 1-18 1-21 1-1 7
* Number of days following emergence that pot was placed in dew chamber. t Total plants = 30.
Z. M. Hassan, C. L. Kramer and M. G. Eversmeyer Infection from spores floating on flood waters When spores were deposited on soil which was later flooded, many spores were picked up and carried on the surface of the rising water. Floating spores had not germinated, but infection and pustule development readily occurred where the spores came in contact with partially submerged host plant leaves. When the water level was reduced stepwise to four lower levels over a total period of 48 h, additional pustules developed at the new positions where the floating spores came in contact with the wheat leaves. This indicates that at least some spores remained infective after floating on the water surface for as long as 48 h. On several occasions, large air bubbles with spores at the water/air interface were trapped on leaves below the water surface. Apparently these situations were sufficient to allow for infec tion to occur, since pustules developed in these areas after those portions of the leaves became exposed above the water level.
Table 5. Longevity of spores in pustules on clipped leaves and senescedseedlings kept outside during Aug. and Sept.* Spore germination ( % ) Senesced
Clipped leaves (Aug.) Leaves exposed on soil surface
Spore exposure time (d)
seedlings (kept intact in pots in
Leaves covered with 0'5 ern soil
Sept.)
65 59 59 ° 1 59 34 79 7 33 3 1 50 4 70 5 6 15 41 66 8 29 7 64 10 14 ° 12 12 47 ° 0 15 ° 16 43 23 3° 14 30 - , Samples not taken. * Av. daily max. temp. 30° and 25° respectively .
Survival of mycelium in wheat tissue subjected to cold
Wheat plants (cv. Eagle) p lanted from the field prior to loss of snow cover and incubated in the greenhouse in Dec. 1982 and Jan. 1983, developed at least one sporulating uredinium on 85 % of the plants. In Feb., after air temp. reached -15 0 and snow cover had disappeared from the plot, uredinia developed on less than 5 % ofthe plants. By the first wk of Mar., sporulating uredinia did not develop on any plants brought into the greenhouse. At tha t time, most of the older leaves had senesced ,
available on the leaf surface in order for infection to occur. Soilborne spores trapped and maintained on dry leaf surfaces were able to cause infections for as long as 13 d at the lower field temp. (T able 6). At the higher temp. pustules did not develop after 8 d.
Table 6. Long evity of spores on dry foliage and at two day/night temperature regimes Environmental chamber (34/22°)
Greenhouse (24/ 20°) Spore exposure time (d) ° 2 3 4 5 6 7 8 10 13 16
Plants tested (no.)
Plants with pustules
Mean no. pustules infected plant
29
10
3"3
26
7
2'9
25
17
3"7
27 29 28
10 21 0
2"3 2'7 0
-, Samplesnot taken.
Total no. of plants 25 22
Plants with pustules 4 18
Mean no. pustules infected plant 1·8 4"1
27
11
2,8
26
15
3'1
26 22 28
9 0
3'1 0
°
0
Survival of Puccinia recondita
37°
Table 7. Survival of vernalized seedling wheat leaves and Puccinia recondita mycelium that had been allowed to develop at 21 ° for 8-72 h after infection and prior to extended periods of low temperature Mycelial age before exposure to cold (h) Day /night temp. (Oe )
Exposure (h)
5/ 0
4 8 12
16 4 8 4
2/-2 -1/ -4
8
16
24
48
72
++* ++ ++ ++
++ ++ ++ ++
++ ++ ++ ++ - +
++ ++ ++ ++ ++
++ ++ ++ ++ ++
- +
++
8
-4/- 8
4 8
* + +, Survival of fungus and host tissue; - -, No survival of fungus or host tissue; producing uredinia on necrotic host tissue. Under controlled conditions, host plant tissue survived at least 16 h of exposure to 5/0° and supported development of uredinia when the mycelia had been allowed to develop for as little as 8 h prior to cold treatment (T able 7). Neither mycelium nor host tissue were able to survive 8 h exposure to 2/ - 2° regardless of the length of incubation period, but mycelium that had developed at least 24 h at 21 ° before cold exposure was able to survive 4 h (T able 7). In studying the effect of rapid reductions in temp., all plants subjected to chilling from 21 to -4° became necrotic and were unable to support sporulation of the fungus . Those chilled to -1 , a and + 1 ° remained chlorotic for 8-10 d, and in some cases allowed pustules to develop and sporulate (T able 8).
to less than 5 % within 22 d. The decline was more rapid when spores were on drier soils and when covered with soil. The viability of spores contained on host plant debris declined at a similar rate.
Table 8. Survival of Puccinia recondita mycelium when the mycelium was allowed to develop in vernalized Trison wheat seedling leaves at 21° and then subjected to chilling Hours of mycelium developmen t at 21° before host tissue exposed to low temp erature treatments Chilling (oq
DISCUSSION
During several years, when wheat was heavily infected in Kansas, volunteer plants in plots also became heavily infected following harvest. The amount of infection on the volunteer plants was heavier than thought attributable to airborne spores alone, and these results show that spores deposited directly on the soil or contained on wheat plant debris readily adhere to emerging seedlings when contact is made . Infection then depends on the viability of the spores and the availability of free moisture to initiate the germination process. Hwang ( 194 2) reponed germination of (50) 70-100 % for spores from newly ruptured pustules. We found similar variations to occur (H assan, 1983). The initial germination percentage, howe ver, was found to decrease at a rate dependent on several factors, with perhaps temp. being the most important. On wet, warm soil germination dropped
+, Fungus survived,
0
-1
Exposure time (h)
24
48
72
2 3 4 5
+* +
+ 0
+ +
0 0
+ +
0 0
6 7
0
0
0
+
0 0
2 3 4
+
0
0
0
5
+ +
+ +
6 7
0 0
+
+ + + + + +
2 3 4 5
+ + +
0 0
+ +
0
0 0
0 0 0
0
+ + +
6 + + 7 * + . Uredinia present on chloroti c host tissue ; 0 , No uredinia produced.
Z. M. Hassan, C. L. Kramer and M. G. Eversmeyer Germination of soilborne spores did not occur while the spores were in actual contact with soil. This was true even of spores trapped by emerging leaves. Since wheat leaves elongate by a basal meristern, however, trapped spores are carried away from contact with the soil. This also allows spores to be trapped along the length of an emerging leaf. Under field conditions, moisture may not be available immediately following seedling ernergence. Thus, trapped spores may have to survive several days on leaves before germination and infection becomes possible. Eversmeyer & Burleigh (1968) found spores to survive 45 d at 5-8° but only 9 d at 18-39° on dry foliage. Hwang (1942) reported foliage to reduce sunlight intensity and temp. resulting in prolonged germinability of spores trapped on lower leaves. Fulton & Coblentz (1929) found that spores in the centre of dumps retain viability longer than those on the outside. This apparently is due to a protective shading effect. Rehydrated spores were found by Leathers (1961) to maintain 70-80 % viability for up to 40 d on dry leaf surfaces, while viability of non-rehydrated spores declined to less than 5 % by 32 d. Swaebly (1955) reported a difference in survival of P. graminis race 1SB on cvs Little Club and Kentane S1A, while Mohamed (1960) found a difference in subrace 139B on the leaves of Marquis and Little Club. Although many factors may be involved in the survival of urediniospores before infection can occur, it seems apparent that, in some situations, spores are able to retain viability long enough to function as a primary source of infection of volunteer wheat following harvest. In autumn, spores often accumulate in pustules on rosette plants and eventually fall to the ground in large clumps. Tests of simulated flood conditions indicate that soilborne spores can be dispersed on the surface of rising water and cause infection if brought in contact with partially submerged wheat plants. Although field tests have not been made, observations of flood irrigated areas indicate that infections have occurred in that way. Burleigh, Schulze & Eversmeyer (1969) reported the occurrence of dormant mycelium from plants collected in Jan. to Mar. from numerous locations in Kansas, Colorado, Oklahoma, and Texas. We have shown that tillering plants removed from the field in late winter, often developed pustules when brought into a greenhouse. In early spring when temperatures become warm enough, i.e. 10-50°, to initiate renewed growth in the host plants, temp. may drop to near freezing or below within 12 h . Such changes are more destructive to host tissues than changes from 0 to
371
5°, to sub-zero temp. If mycelium is allowed to develop for at least 24 h following infection before chilling to below 0°, that mycelium apparently is capable of surviving all conditions that the host can. On several occasions when infected plants were subjected to frost, the mycelium was able to resume growth and develop sporulating uredinia when returned to the greenhouse even though host tissues were beginning to senesce, Presence or absence of mycelium made no detectable difference in the ability of the host tissues to survive sustained low temp. or abrupt changes in temp. as discussed above. The older or lower leaves of tillered plants are more easily affected by low temp. and often become necrotic before dormant mycelia are able to develop sporulating pustules. In two of the last five years, however, host tissues with dormant mycelia were able to survive long enough in Kansas for development of spores which served as primary inoculum for spring infections. Contribution of the Kansas State University in cooperation with the United States Department of Agriculture, Agricultural Research Service, no. 84- 114 J. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the USDA-ARS, and does not imply approval over other products that also may be suitable. This article is in the public domain and is not copyrightable. It may be freely reprinted with customary crediting of source. The work reported is part of the Ph.D. Thesis of the senior author.
REFERENCES
BURLEIGH, J. R., SCHULZE, A. A. & EVERSMEYER, M . G. (19 69). Some aspects of the summer and winter ecology of wheat rust fungi. Plant Disease Reporter 53,648-651. EVERSMEYER, M. G. & BURLEIGH, J. R. (1968). Effect of temperature on the longevity of Puccinia recondita f.sp. tritici urediospores on dry wheat foliage. Plant Disease Reporter 5z, 186-188. EVERSMEYER, M. G., KRAMER, C. L. & BROWDER, L. E. (1980 ). Effect of temperature and host: parasite combination on the latent period of Puccinia recondita in seedling wheat plants. Phytopathology 70, 938--941. FULTON, H. R. & CoBLENTZ, W. W. (1929). The fungicidal action of ultra-violet radiation. Journal of Agriculture Research 38, 159-168. HAsSAN, Z. (1983). Epidemiological studies ofleaf rust of wheat caused by Puccinia recondita f.sp. tritici. Ph.D . Thesis, Kansas State University, U.S.A. HWANG, L. (1942). The effect of light and temperature on the viability of urediospores of certain cereal rusts. Phytopathology 31, 699-711. LEATHERS, C. R. (1961). Comparative survival of rehydrated and nonrehydrated wheat stem rust uredospores on dry leaf surfaces. Phytopathology 51, 410-411.
37 2
Survival of Puccinia recondita
MEHTA, Y. R. & ZADOKS, J. c. (1970). Uredospore production and sporulation period of Puccinia recondita f.sp. tritici on primary wheat leaves. Netherlands Journal of Plant Pathology 76, 267-276. MOHAMED,H. A.(1960). Survival of stem rust uredospores on dry foliage of wheat. Phytopathology 50, 400-401. PETERSON, R. F., CAMPBELL, A. B. & HANNAH, A. E. (1948). A diagrammatic scale for estimating rust intensity of leaves and stems of cereals. Canadian Journal of Research C 26, 496-500.
SWAEBLY, M. A. (1955). Variability in the infection of wheat by Puccinia graminis tritici and studies on extracts from rust urediospores. Ph.D. Thesis, University of Minnesota, U.S.A. TOMERLIN, J. R., EVERSMEYER, M. G., KRAMER, C. L. & BROWDER, L. E. (1983). Temperature and host effects on latent and infectious periods and on urediniospore production of Puccinia recondita f.sp. tritici. Phytopathology 73, 414-419.
(Received for publication 7 February 1985)