Improved bunt inoculation of wheat utilizing ...

Improved bunt inoculation of wheat utilizing adventitious shoots' L. M. KAWCHUK AND J. NIELSEN Agriculture Canada, Research Station, 195 Dafoe Road, Winnipeg, Man., Canada R3T 2M9 Received August 14, 1986

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KAWCHUK, L. M., and NIELSEN, J. 1987. Improved bunt inoculation of wheat utilizing adventitious shoots. Can. J. Bot. 65: 1284- 1285. A procedure is described of injecting inoculum of dwarf or common bunt at the bases of the internodes of wheat. Physical stimulation by wounding of the intercalary meristem located above the nodes resulted in the formation of adventitious shoots. If inoculum of dwarf or common bunt is injected at the time of stimulation, the spike of the resulting shoot is usually infected. This procedure shortened the period between teliospore germination and production of son, gave a high infection rate, and induced several infected spikes on one primary stem.

KAWCHUK, L. M., et NIELSEN, J. 1987. Improved bunt inoculation of wheat utilizing adventitious shoots. Can. J. Bot. 65 : 1284- 1285. Les auteurs dkcrivent une mCthode pour injecter l'inoculum de la carie naine ou commune du blC, la base des entre-noeuds de cette plante. Une stimulation physique par blessure du mCristBme intercallaire situC au dessus du noeud entraine la formation de racines adventives. Si l'inoculum de la carie naine ou commune du blC est inject6 au moment de la stimulation, les Cpis portCs sur la tige qui en provient sont habituellement infect&. Cette mkthode rCduit le dklai entre la germination des tkliospores et la productions des sores, conduit 2 un taux rapide d'infection et peut induire l'infection de plusieurs Cpis sur une m&metige primaire. [Traduit par la revue]

Introduction Artificial inoculation of wheat with the dwarf bunt fungus (Tilletia controversa Kiihn) requires very specific environmental conditions for spore germination and infection. To obtain infection, the products of teliospore germination (i.e., sporidia and hyphae) must be placed close to the growing regions of the host, probably active meristematic tissues. Such tissues are located in both germinating seeds and young seedlings, and if infected, mature son will form after 5 to 6 months from the start of teliospore incubation. Femindez and Durin (1978) developed a technique of injecting products of spore germination approximately 2.5 cm above the uppermost node near the developing spikes. This procedure reduces the time required to produce mature son, i.e., bunt balls, but only a few of these develop per spike. Bodine and Durrell (1930) had obtained similar results by inoculating wheat with Tilletia foetida (Wallr.) Liro near developing spikes at various growth stages. Our inoculations of wheat with i? controversa by the method of Femindez and Durin (1978) also resulted in very few sori on very few spikes, while Churchill and Mills (1985) failed altogether to get sporulation by this method. The following method of inoculation is similar to those cited above, but it consistently gives a high level of infected spikes in which all the kernels are bunted. The method is based on the fact that in wheat and some other cereals there is an intercalary meristem at the base of each internode (Percival 192l), and on our chance observation that the physical stimulus induced by wounding causes this intercalary meristem to form an apical meristem, which differentiates into an adventitious shoot with stem, leaves, a spike, and often with rootlets. Materials and methods Teliospores of six collections of T controversa (numbers 29, 295, 555,567,574, and 592), each differing in virulence, were germinated on 2% soil extlact agar prepared by filtering 500 mL boiling water through 50 g of soil and adding water to the filtlate to make 1 L. The 'Contribution no. 1243. Printed in Canada I lmprimC au Canada

plates were incubated at 4-7°C under approximately 2150 lx of continuous cool white fluorescent light. Teliospores of T caries (DC.) Tul. (race T28) and T foetida (race L16) were incubated on 1.5% water agar at 15°C without light. Inocula were prepared by adding 2 mL of water to each plate with germinating spores and scraping off the spores, sporidia, and hyphae. Alternatively, the germination products from the agar plates were transferred to Zscheiles MT2 medium (Zscheile 1951) and grown at room temperature on a shaker. The resulting mycelial cultures turned opaque after 7- 10 days and were then homogenized in a Sorvall blender for 30 s to facilitate injection with a syringe and a 20-gauge hypodermic needle. The spring wheat (Triticum aestivum L.) cultivars HY 320 and Red Bobs, which lack genes for bunt resistance, were used as hosts and grown in a growth cabinet under a 16 h light : 8 h dark cycle and at 17: 14°C (1ight:dark). To inoculate only plants with one culm, secondary tillers were clipped just above the soil throughout plant development; this appeared to increase infection levels. The plants were inoculated between growth stages 37 to 39 or 49 to 59 (Zadoks et al. 1974). For inoculation, the hypodermic needle was inserted approximately 2-3 mm above each node, at a 45" downward angle to the stem, and with the tip of the needle penetrating to slightly above the top center of the node before inoculum was injected as the needle was withdlawn. By slightly increasing the angle between needle and stem, the tip of the needle protruded on the opposite side of the stem at the top of the node. This allowed easier injection of inoculum during withdlawal of the needle. Each of the eight collections or laces was used to inoculate 10 plants of each cultivar and growth stage. For each of these combinations, 10 plants were injected with water as a control.

Results and discussion Injection of either water or bunt inoculum just above the nodes of the primary stem resulted in the production of an adventitious shoot at the base of an internode, indicating that these shoots formed as an effect of wounding the intercalary meristem. Of the stems inoculated at the lowest node above soil level 70- 100% formed adventitious shoots about 1 week after injection. Shoot formation was less frequent at higher nodes, where inoculum was more effective than water at inducing shoots. Almost all adventitious spikes that formed after inoculation with bunt were infected; moreover, all

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NOTES

kernels were converted into bunt balls. Infection could be confirmed about 35 days after inoculation by microscopic observation of immature teliospores within the young, dark green ovaries. Similar results were obtained in both 'HY 320' and 'Red Bobs' regardless of fungal species, race, or form of inoculum. Although a few adventitious shoots were formed following inoculation between growth stages 49 to 59, most of their spikes did not mature and hence did not produce sori. The highest number of infected shoots were formed if plants were inoculated between growth stages 37 to 39. Often several adventitious stems (Fig. 1) formed on one primary stem if all its detectable nodes had been inoculated. Earlier inoculation might also have been successful, but in younger plants the nodes were difficult to locate. Occasionally, inoculation close to the highest node resulted in local infection of only a few kernels, as described by Fernfindez and Durfin (1978). The method of artificial inoculation described has several advantages when compared with previously published methods. Like those, it shortens the period between inoculation and production of son, but the infection rate is much higher and several infected spikes, even bearing different races, can be produced on one primary stem. The procedure is reliable and has been used to increase dwarf bunt teliospores in a short period. It should also serve to rapidly screen for virulence and to study reactions between cultivars and races of the bunt fungi.

Acknowledgements The authors thank Dr. J. A. Hoffmann for supplying teliospores of six collections of dwarf bunt and two races of common bunt. This research is funded in part by a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship held by L. M. Kawchuk. BODINE, E. W., and DURRELL, L. W. 1930. Inoculation of wheat with Tilletia levis. Phytopathology, 20: 663 -668. . CHURCHILL, A. C. L., and MILLS, D. 1985. Heterokaryon formation

FIG.1. Inoculation above each node of a primary stem (P) of wheat with Tilletia controversa resulted in the formation of three adventitious shoots with infected spikes.

in planta by genetically marked strains of Tilletia caries. Can. J . Bot. 63: 1924- 1927. FERNANDEZ, J. A., and DURAN,R. 1978. Hypodermic inoculation, a rapid technique for producing bunt in wheat. Plant Dis. Rep. 62: 336-337. PERCIVAL, J. 1921. The wheat plant. Duckworth and Co., London. ZADOKS, J. C., CHANG, T. T., and KONZAK, C. F. 1974. A decimal code for the growth stages of cereals. Weed Res. 14: 415-421. ZSCHEILE, F. P. 1951. Nutrient studies with the wheat bunt fungus, Tilletia caries. Phvto~atholo~v. u- , 41: 1115 - 1124. a

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