NOTES

Inoculation of Euphorbia lathyris with cotton strings infested with Macrophomina phaseolina, causal agent of charcoal rot disease

PHYLLIS T . H I M M E L ' Department of Platlt Pathology, University of Arizona, Tucson, AZ 85721, U.S.A

Received April 24, 1990

HIMMEL, P. T. 1991. Inoculation of Euphorbia lathyris with cotton strings infested with Macrophomina phaseolina, causal agent of charcoal rot disease. Can. J . Bot. 69: 682-685.

Root infections caused by Macrophotnina phaseolina were initiated under optimal conditions for the host, Euphorbia lathyris. Two-week-old Euphorbia lathyris seedlings were inoculated by tying roots with cotton strings infested with Macro- phominn phaseolina. Ninety-three per cent of the inoculated roots developed infections after 2 weeks incubation in silica sand at 25'C. By using infested strings, differences in the incidence of lesion development were detected when infected roots were subjected to differing temperature regimes. After approximately 6 weeks, there was a significantly (P S 0.01) greater incidence of lesion development at 34OC than at 25"C, whereas there was no difference in the incidence of infection. Aerial symptoms indicative of charcoal rot were not observed during the course of these studies.

Key words: infested strings, charcoal rot.

HIMMEL, P. T. 1991. Inoculation of Euphorbia lathyris with cotton strings infested with Macrophotnina phaseolina, causal agent of charcoal rot disease. Can. J . Bot. 69 : 682-685.

L'auteur a provoque I'infection racinaire par le Macrophotnina phaseolina sous des conditions optimales pour 11h6te Euphorbia lathyris. Des plantules d'E. lathyris agees de 2 semaines ont CtC inoculCes en attachant les racines avec des cordes de cotton envahies par le M. phaseolina. Quatre-vingt-treize pourcent des racines inoculCes ont dCveloppC des infections apres 2 semaines d'incubation, sur sable de silice, a 25°C. En utilisant les cordes infectees, il a CtC possible d'obtenir diverses incidences de la maladie lorsque les racines ont et& soumises B diffirents regimes de temperature. Apres environ 6 semaines, on observe une incidence significativement plus grande (P < 0,Ol) du developpement des lesions a 34°C qu'a 25"C, alors qu'il n'y a pas d'effet sur l'incidence de I'infection. I1 n'a pas tte possible d'observer les sympt6me caracteristiques de la maladie sur les parties akriennes de la plante au cours de ces experiences.

Mots clks : cordes infestees, pourriture-charbon. [Traduit par la redaction]

Introduction to varied environmental conditions. An inoculation techniaue

Macrophomina phaseolina infects over 500 plant species worldwide ( 3 , 9 , 21). While disease development generally occurs under conditions of high temperature and low soil mois- ture ( 1 , 2 , 4 , 5 , l o ) , root infections can occur independently of these conditions ( 2 , 4 , 8 , 9 , 10, 14, 17, 18). In the case of Euphorbia lathyris, aerial symptoms associated with early stages of host infection are not always apparent (20). A path- ogen such as M. phaseolina that initially invades rootlets, root hairs, or superficial cortical cells-may seem to cause little vis- ual damage ( 2 , 18, 20) but could have a detrimental affect on water and nutrient uptake and movement (13) . As a conse- quence, such damage may not be recognized until infected but seemingly healthy plants are stressed by high temperatures, low moisture, flowering, or fruit set.

Inoculation studies using M. phaseolina involve planting into soils infested with known propagule populations, adding soil drenches of known propagule concentrations, or inserting infested toothpicks into stems of susceptible hosts (19 , 21). Using these methods, it has been possible to study infection and disease development in host populations, but not to follow the spread of this pathogen within an infected host subjected

'Present address: U.S. Department of Agriculture, Agricultural Research Service, Crop Protection Research Unit and Department of Plant Pathology, University of Illinois, Urbana, IL 61801, U.S.A.

is needed to initiate infection at known points along host roots to facilitate investigations of the effects of environment on infection and spread of this pathogen and to aid in understand- ing the etiology of the disease for many cropping systems. The objectives of this study were (i) to develop an inoculation tech- nique to ensure point infections at known positions along a root of E. lathyris, and ( i i ) to ascertain how useful this inoc- ulation technique would be in detecting differences in symp- tom expression when infected roots are subjected to different temperature regimes.

Materials and methods lnoculum development and storage

Cultures of M. phaseolina were established from collections of dis- eased, field-grown E. lathyris. Root sections containing lesions were rinsed in running tap water for 5 min, placed in 0.05% sodium hypo- chlorite for 1 min, and blotted dry. Sections were incubated for 3-4 days on a modified selective medium (mPDA, 12) at 34'C: Difco potato dextrose agar amended with 375 pg active ingredient (a.i.)/mL streptomycin sulfate, 375 pg a.i./mL penicillin G, and 150 pg/mL chloroneb (l,4-dichloro-2,5-dimethoxybenzene; E. I. Du Pont de Nemours & Co., Inc., Wilmington, DE).

Cotton (100%) strings infested with M. phaseolina were used to inoculate roots by modifying a technique used by Stutz and Leath (15). Strings were cut into 6-cm pieces and autoclaved 60 min on a wire mesh pedestal in covered beakers. A 5-mm diameter plug of

Printed in Canada I Imprim6 au Canada

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FIGS. I . Euphorbia lathyris roots were inoculated with M. phaseoli approximately 4 weeks. (A) No lesion. (B) Lesion present. Scale bars

agar with M. phaseolina hyphae was taken from the advancing margin of a 4-day-old culture growing on Difco potato dextrose agar and placed on mPDA adjacent to approximately 50 sterile strings. Plates were incubated in the dark for 2 weeks at 34OC. During the incubation period, hyphae grew through the strings and sclerotia formed. Infested strings were then transferred to sterile culture plates and allowed to dry at 34°C for 24 h, after which time they were stored at room tem- perature. Strings could be stored for up to 4 weeks before a loss in the viability of microsclerotia occurred (6). Sterile strings served as controls.

Inoculation of E. lathyris Seeds of E. lathyris plants used in all studies were collected from

field-grown plants. Seeds were sown into 8.6-cm diameter styrofoam pots containing premoistened, 20-grade silica sand. Pots were placed in growth chambers at 25°C with a 12-h photoperiod (19 355 Ix), then watered every other day with tap water and once a week with half strength Hoagland's solution (7). Unless otherwise stated, all growth chamber experiments utilized the same light intensities and diurnal cycles. After 2 weeks, the seedlings were removed from pots and the roots were rinsed with tap water for 2 min and inoculated by tying the tap root with one infested string within 2-5 cm of the root tip. If more than one root was to be inoculated, additional strings were tied at the same distance from the root tip. Ends of tied strings were trimmed to 0.5 cm, and the severed pieces incubated on mPDA in the dark at 34°C to confirm viability of M. phaseolina. An equal number of control plants were tied in the same fashion using sterile strings.

Incubatior~ of E. lathyris in rolled sterile towels Inoculation experiments were set up using the conditions described

above to learn if root infections could be initiated while the host was growing under optimal conditions. The tap roots of at least fifteen 2- to 8-week-old seedlings were inoculated. Seedlings were placed in sterile paper towels, rolled in plastic wrap, and set upright in glass beakers. A maximum of 10 seedlings were placed in each paper towel. Beakers of inoculated and control seedlings were placed side by side in growth chambers set at 25OC. All plants were watered as needed, at which time they were examined for aerial symptoms. After approx- imately 6-8 weeks, roots of all plants were examined for lesion development, and isolations were made from inoculation sites and the crown of each plant. This experiment was conducted three times. A total of 54 seedlings were inoculated.

Incubation of E. lathyris in sand Tap roots of thirteen 2-week-old seedlings were inoculated as

described above. Two inoculated seedlings were planted into each pot of silica sand. Pots of inoculated and control seedlings were placed side by side in growth chambers set at 25OC and watered as needed. Root infection was confirmed by isolation from inoculated sites on mPDA at the 2nd week of incubation. At this time all infested and

'na infested strings and incubated for 2 weeks at 25OC then 34°C for = I mm.

control strings were removed, and each inoculation site was marked by tying a sterile red string around the root at that position. All seed- lings were incubated at 34°C and watered as needed. After 3 weeks, the roots of all plants were examined for lesion development and isolations made onto mPDA from initial inoculation sites. This exper- iment was conducted twice to confirm that infection occurred during the 2-week incubation period at 25°C.

Effect of increased temperature on infected plants Tap roots of 2-week-old seedlings were inoculated and incubated

in pots of silica sand at 25OC as described. In a preliminary study, one site on four roots of each plant was inoculated. This was repeated once, and a total of 30 plants were inoculated. In the main experi- ment, a single site on one root was inoculated. Four trials were run, with at least 12 plants per temperature treatment inoculated in each trial. After the 2nd week of incubation, half of the inoculated plants were transferred to 34°C. All plants were watered as needed and examined for aerial symptoms at the same time. Roots of all plants were examined approximately 6 weeks after inoculation.

Results Itzcubation of E . lathyris in rolled sterile towels

After 6-8 weeks incubation at 25"C, lesions were found at all 54 inoculation sites, and M. phaseoliiza was recovered from 53 of these sites but not from the crown of any plants. Aerial symptoms indicative of infection by M. phaseolina were not observed. After the incubation periods there was considerable root growth, with subsequent secondary infections resulting as new roots grew over infested strings.

Incubation of E . lathyris in silica sand All seedlings with infested strings removed prior to incu-

bation at 34°C developed lesions at the original inoculation sites, and M. phaseolina was recovered from 92.3% of these sites. These tests verified that infection took place during the 2-week incubation period at 25°C.

Effect of increased temperature on infected plaizts In these experiments, evaluating infection and lesion for-

mation under conditions optimal for the pathogen (3) and less than optimal for the host (12) was aided by knowing the loca- tion of the original inoculation site (Fig. l). The effects of incubation temperature on infected seedlings are shown in Table 1. At 25"C, M. phaseolina was recovered from 75.0% of the single-site inoculations and 78.6% of the four-site inoc- ulations. At 34"C, M. phaseolit~a was recovered from 81.4% of the single-site inoculations and 86.1% of the four-site inoc- ulations. Root lesions developed in 13.2% of the single-site

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684 CAN. J . BOT. \. 'OL. 69, 1991

TABLE 1. Results of root inoculations of E. lathyris with cotton strings infested with M. phaseolina

% aerial % root 70 symptoms lesions successful (per plant) (per site) reisolation

Inoculation" 25°C 34°C 25°C 34°C 25°C 34°C - Four sites 0 0 62.5 100.0 78.6 86.1 One site 0 0 13.2* 48.2* 75.0 81.4

NOTE: TWO seedlings per 8.6-cn~ cup of silica sand were incubated for 2 weeks with a 12-h photoperiod at 25°C. at which time one-half the infectcd plants and their controls were moved to 34°C and watered as needed. Readings were taken approximately 6 wceks after inoculation. *, valucs were significantly different at P -S 0.01 based on ANOVA (df = 1 , F = 16.18).

"An infested string was tied around either one of four roots per seedling. For four-sitc inoculations. results are from two trials with 14-16 inoculated seedlings. For single-site inoculations, results are from four trials with at lcast 12 inoculated sccdlings incubated at cach tcrnperature for cach trial.

inoculations and 62% of the four-site inoculations incubated at 25°C. Root lesions developed in 48.2% of the single-site inoculations and 100.0% of the four-site inoculations incu- bated at 34°C. Preliminary results from the four-site inocula- tions showed a greater degree of lesion development at 34°C than at 25"C, whereas the rate of infection remained the same at both temperatures. When the single root sites were ana- lyzed, there was still no difference in the incidence of infection by M. phaseolina at 2 5 or 34"C, but the degree of lesion for- mation at 34°C was significantly greater (P a 0.01), indicating a greater degree of colonization subsequent to infection by this pathogen at the higher temperature.

Discussion

Delayed symptom expression is a crucial part of this path- ogen-host system. T o understand how environmental stresses play a role i n pathogenesis, it is important to duplicate this early phase of infection in controlled environments. Infested cotton strings tied to roots were successful in initiating infec- tions at known points on roots of nonstressed E. lathy& seed- lings. When infected and nonstressed plants were subjected to a higher temperature, not only was it possible to determine that M. phaseolitza could penetrate roots with equal efficiency at 25 and 34"C, but lesion development was significantly (P a 0.01) greater at 34°C (Table 1).

Unlike inoculating plants with infested toothpicks o r plant- ing into infested soil, this technique allows for the assessment of disease development based on the ability of the pathogen to invade the host. This is a reliable means of initiating root infections at discrete sites and should facilitate the study of factors affecting lesion development by M. phaseolina from a known point of inoculation.

Asymptomatic infections caused by M. phaseolina have been well documented. Though the pathogen has been recovered from roots of asymptomatic corn (8, la ) , cantaloupe (2), and E. lathyris (20), these reports were based only on field studies. This report of asymptomatic root infection in plants grown under environmentally controlled conditions supports reported field results. Further studies examining the individual and combined effects of high-temperature and low-moisture stress on symptomless root infections would help to determine if these infections should be considered asymptomatic o r latent as defined by Verhoeff (16). Given the importance of this early phase of infection in this host-pathogen system, a better understanding of this part of the disease cycle and how envi-

ronmental events affect pathogenesis is likely to contribute greatly to efforts to minimize the damaging effects of this unique pathogen.

Acknowledgments

I would like to thank R. L. Gilbertson, D. A. Glawe, A. D. Hewings, and M. E. Stanghellini for their expert advice and review of this manuscript.

1. BLANCO-LOPEZ, M. A., and JIMENEZ-DIAZ, R. M. 1983. Effect of irrigation on susceptibility of sunflower to Macrophornina phaseoli. Plant Dis. 67: 1214-1217.

2. BRUTON, B. T. , JEGER, M. J. , and REUVEN~, R. 1987. Macro- photnitla phaseolir~a infection and vine decline in cantaloupe in relation to planting date, soil environment, and plant maturation. Plant Dis. 71: 259-563.

3. DHINGRA, 0 . D., and SINCLAIR, J. B. 1978. Biology and pathol- ogy of Macrophornirza phaseolirza. Universida de Federal de Vis- cosa, Minas Gerais, Brasil.

4. EDMUNDS, L. K . 1964. Combined relation of plant maturity, temperature, and soil moisture to charcoal stalk rot development in grain sorghum. Phytopathology, 54: 5 14-5 17.

5. GHAFFAR, A., and ERWIN, D. C. 1969. Effect of soil water stress on root rot of cotton caused by Macrophotnirza phaseolincc. Phy- topathology, 59: 795-797.

6. HIMMEL, P. T. 1988. Asymptomatic infections of Euphorbia lathyris by Macrophomina phaseolina. Ph .D. dissertation, Uni- versity of Arizona, Tucson.

7. HOACLAND, D. R., and ARNON, D. 1. 1938. The water culture method for growing plants without soil. Univ. Calif. Coll. Agric. Agric. Exp. Stn. Circ. No. 347.

8. KOMMEDAHL, T., WINDELS, C. E., and STUCKER, R. E. 1979. Occurrence of Fusariutn species in roots and stalks of symptom- less corn plants during the growing season. Phytopathology, 69: 861 -966.

9. LIVINGSTON, J. E. 1945. Charcoal rot of corn and sorghum. Univ. Nebr. Lincoln Agric. Exp. Stn. Res. Bull. No. 136.

10. MEYER, W. A, , SINCLAIR, J. B., and KHARE, M. N. 1974. Fac- tors affecting charcoal rot of soybean seedlings. Phytopathology, 64: 845-849.

11. MIHAIL, J. D., and ALCORN, S. M. 1982. Quantitative recovery of Macrophomit~a phaseolina sclerotia from soil. Plant Dis. 66: 662-663.

12. PEOPLES, T. R., ALCORN, S. M., BLOSS, H. E., CLAY, W. F., FLUGG, M., HOFFMAN, J. J . , LEE, C. W., LUNA, S. , MCLAUCHLIN, S. P., STEINBERG, M., and YOUNG, D. 1981. Euphorbia lathyris L.: A future source of extractable liquid fuels. Biosources Dig. 3: 1 17-123.

13. SALT, G . A. 1979. The increasing interest in 'minor pathogens'. In Soilborne plant pathogens. Edited by B. Schippers and W. Gams. Academic Press, London. pp. 289-3 12.

14. SINGH, D. 1983. Histopathology of some seed-borne infections: A review of recent investigations. Seed Sci. Technol. 11: 651- 663.

15. STUTZ, J. C., and LEATII, K. T. 1983. Virulence differences between Fusariutn roseutn 'Acuminatum' and F. roseum 'Aven- aceum' in red clover. Phytopathology, 73: 1648-165 1.

16. VERHOEFF, K. 1974. Latent infections by fungi. Annu. Rev. Phytopathol. 12: 99-1 10.

17. WEIMER, J. L. 1944. Macrophomina root and stem rot and anthrhcnose of chamaecrista. Phytopathology, 34: 1077-1085.

18. WINDHAM, M. T., and KING, S. B. 1983. Mycoflora of roots of maize plants at seedling and silking stages in Mississippi. Plant Dis. 67: 1366- 1368.

19. YOUNG, D. J . , and ALCORN, S. M. 1982. Soilborne pathogens of Euphorbia lathyris: Macrophomina phaseolina, Pythium

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NOTES 685

aphatzidermaturn, and Rhizoctonia solani. Plant Dis. 66: 236- propagule populations in Arizona field soils. Plant Dis. 68: 587- 238. 589.

20. YOUNG, D. J . , and ALCORN, S. M. 1984. Latent infection of 21. YOUNG, P. A. 1949. Charcoal rot of plants in East Texas. Texas E~cphorbia lathyris and weeds by Macrophornina phaseolirza and Agric. Exp. Stn. Bull. No. 712.

Germination of pollen of the dwarf mistletoe Arceuthobium americanum

JEANNIE GILBERT AND DAVID PUNTER Departmetzt of Botatty, University of Manitoba, Winnipeg, Man., Canada R3T 2N2

Received July 18, 1990

GILBERT, J . , and PUNTER, D. 1991. Germination of pollen of the dwarf mistletoe Arceuthobiurn arnericunum. Can. J. Bot. 69: 685-688.

Pollen of Arceuthobium arnericanum Nuttall ex Engelmann, a parasitic dwarf mistletoe of Pinus banksiancr Lamb. in Manitoba, was germinated on media containing a range of concentrations of sucrose and salts. Highest germination levels occurred on 20% sucrose agar; addition of salts significantly depressed germination. Pollen germinability was below 5% at first but increased to near 50% as the season advanced. Mean germination differed significantly between flowers from 10 staminate plants but peaked at approximately the same times in association with increasing temperatures. Freezing night temperatures were followed by reduced germinability. The optimum temperature for in vitro pollen germination is close to 30°C, whereas 40°C impairs germination.

Key words: parasitic angiosperms, microclimate, jack pine, reproductive biology.

GILBERT, J . , et PUNTER, D. 1991. Germination of pollen of the dwarf mistletoe Arceuthobiurn otner-icor~~cm. Can. J. Bot. 69 : 685-688.

Afin d'en obtenir la germination, les auteurs ont soumis les pollens de 1'Arceuthobium americarzum Nuttall ex Engelmann, un petit gui parasite sur le Pinus banksiana au Manitoba, des milieux contenant diverses concentrations de sucrose et de sels mineraux. Les pourcentages les plus ClevCs de germination ont Cti obtenus sur gilose contenant 20% de sucrose; ['addition de sels riduit la germination. Au dipart, la germinabiliti du pollen est de 570 mais peut atteindre 50% i mesure que la saison avance. La germination moyenne diffkre significativement entre les fleurs de dix plantes staminees mais le maximum est atteint a peu prks au mCme moment, i mesure que les temperatures se rkchauffent. Des tempiratures de congilation nocturnes entrainent une reduction de la germinabilitk. La tempirature optimale qui permet d'obtenir la germination des pollens in vitro est prks de 30°C alors que celles de 40°C empCchent la germination.

Mots cle's : angiospermes parasites, microclimat, pin gris, biologie de la reproduction. [Traduit par la rCdaction]

Arceuthobiurn americanum Nuttall ex Engelmann is an important pathogen in the pine forests of Manitoba. A com- prehensive study of its reproductive biology was initiated as a basis for epidemiological and predictive modelling of potential damage. Arceuthobium americanum flowers early in the sea- son at a time when climatic conditions in Manitoba are variable and inconsistent from year to year. A s germinability of the pollen must be a key factor in the success of fertilization, and as the literature provided no information on the subject, the germination of A. americanum pollen and the effects of micro- climate on its maturation were investigated as part of the study.

All pollen samples were freshly collected from a site in Grand Beach Provincial Park (50°35'N, 96'35'W) consisting of a heavily mistletoe-infested, even-aged stand of jack pine (Pinus banksiana Lambert). Temperature and other environ- mental parameters were continuously monitored and recorded, both before and during the period in which male dwarf mis- tletoe flowers were open. Details of site and microclimatic monitoring procedures have been reported elsewhere (Gilbert and Punter 1990). In all cases, pollen grains were transferred from anthers to the germination medium by means of a fine camel-hair brush. They were considered to have germinated if after incubation for 24 h , the germ tube had reached a length at least equal to the diameter of the grain. The mean percent germination was derived from the examination of a minimum

of 100 grains per sample. Data were subjected to analysis of variance using the general linear models procedure and to Duncan's multiple range test. Programs of S A S Institute Inc. (1985) were used for all statistical tests.

Preliminary attempts to germinate pollen using conventional methods were unsatisfactory. Less than 5 % of pollen collected early in the 1985 flowering period germinated on 1.5% agar supplemented with sucrose (up to 20% w/v), boric acid (H,BO, up to 0 . I%), o r a macerate of female flowers, individually o r in combination. Placement of pollen together with receptive female flowers on water o r sucrose agar in the field gave sim- ilarly low levels of germination. By contrast, pollen collected at the beginning of May, approximately 3 weeks after the flow- ers opened, germinated well (up to 50%) on agar with various concentrations of sucrose and boric acid (Gilbert 1988). In the hope of establishing optimum conditions for in vitro germi- nation, pollen was incubated in 1986 under many combina- tions of conditions in liquid media in van Tieghem cells and on 1.5% agar in Petri dishes. The ranges of the parameters were 10-25% w/v sucrose, 5.7-8.3 initial pH (with and with- out buffer), and 10-25°C. Some media were also amended with boric acid (final concn. 0.01%) or a salts solution (Hodgkin 1983). In no case did the pollen in van Tieghem cells or the early season pollen on agar media achieve more than 1 % germination (Gilbert 1988).

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