Bachelor s Degree Special Problem. Title. Half-Life estimation of Tilletia contraversa Kühn (dwarf bunt of wheat) spore

Transcription

1 Bachelor s Degree Special Problem Program in Agricultural Biotechnology Title Half-Life estimation of Tilletia contraversa Kühn (dwarf bunt of wheat) spore By Miss Panpatchara Tongnual This special problem has been accepted Date... (Assoc. Prof. Dr. Sontichai Chanprame)

2 Half-Life estimation of Tilletia controversa Kühn (dwarf bunt of wheat) spore Panpatchara Tongnual Abstract Tilletia controversa Kühn (TCK) is the pathogen of Dwarf Bunt in wheat, which caused a lot of lose in both quality and yield of wheat. Its teliospores can remain viable for more than 20 years in dry storage room. This experiment was conducted to find halflife from 29 samples of TCK spore s sample that collected by The Utah State Agricultural Experiment Station from year Spore s samples were germinated on 2% soil extracted agar by aseptic technique at 4-5 ºC under low light and evaluate their germination percentage twice a week for 8 weeks. The result founded that spores stored less than 13 years germinated from week 3 by over 1 percent of germination and over percent on week 8, while those spores with more than 16 years of storage germinated on week 8 by under 0.1 percent of germination. Half-life calculated by Eight pharmacokinetic (PK) function for Microsoft Excels using germination percentage in week 8 is at 3 years. Key words Degree Advisor Year : 2011 Pages : 22 : Tilletia controversa Kühn, TCK, germination, teliospore, half-life : Bachelor of Science, Program in Agricultural Biotechnology, Faculty of Agriculture Kamphaeng Saen, Kasetsart University : Dr. Sontichai Chanprame

3 การประเม นค าคร งช ว ตของสปอร Tilletia controversa Kühn เช อราก อโรค dwarf bunt ในข าวสาล นางสาวปานพ ชร ทองนวล บทค ดย อ Tilletia controversa Kühn (TCK) เป นเช อราก อโรค dwarf bunt ในข าวสาล ซ งก อให เก ด ความเส ยหายต อผลผล ตข าวสาล ท งด านค ณภาพและปร มาณ สามารถอย ข ามป ได ด วย teliospore ซ งสามารถม ช ว ตอย ได นานมากกว า 20 ป ในโรงเก บท ม สภาพแห ง จ งต องการทดลองเพ อหาค าคร ง ช ว ตของต วอย างสปอร ท เก บร กษาในโรงเก บต วอย างของ Utah State Agricultural Experiment Station ซ งม ต วอย างรวงข าวสาล ท เป นโรค 29 ต วอย าง ท ม อาย ระหว าง 1-50 ป แล วเพาะเล ยง สปอร บนอาหาร 2% soil extract agar ในสภาพปลอดเช อ บ มในต ควบค มอ ณหภ ม ท 4-5 องศา เซลเซ ยส พร อมท งให แสงสว าง การเพาะเล ยงท านาน 8 ส ปดาห โดยบ นท กเปอร เซ นต การงอกของ สปอร ส ปดาห ละสองคร งท กส ปดาห ผลการทดลองพบว า ต วอย างสปอร ท อาย ต ากว า 13 ป พบการ งอกมากกว า 1 เปอร เซ นต ต งแต ส ปดาห ท 3 และม เปอร เซ นต การงอกในส ปดาห ท 8 มากกว า เปอร เซ นต แต ต วอย างอาย มากกว า 16 ป พบการงอกของสปอร ในส ปดาห ท 8 โดยม เปอร เซ นต การงอกต ากว า 0.1 เปอร เซ นต เม อน าเปอร เซ นต การงอกในส ปดาห ท 8 มาคานวณหาค า คร งช ว ตโดยใช ฟ งก ช นการหาค าคร งช ว ต Eight pharmacokinetic (PK) function for Microsoft Excle ได ค าคร งช ว ตท 3 ป คาส าค ญ : Tilletia controversa Kühn, TCK, germination, teliospore, ค าคร งช ว ต ป ญหาพ เศษ : ปร ญญาตร สาขาว ชาเทคโนโลย ช วภาพทางการเกษตร คณะเกษตร ก าแพงแสน มหาว ทยาล ยเกษตรศาสตร อาจารย ท ปร กษา: รศ. ดร. สนธ ช ย จ นทร เปรม ป ท พ มพ : 2554 จ านวนหน า : 22

4 Table of Contents Page Table of contents List of figures List of tables I II III Introduction 1 Objective of the Study 3 Reviews of Related Literatures 4 Materials and Methods 10 Results 14 Discussions 18 Conclusion 20 References 21

5 List of figures Figures Page 1 Germination of teliospores of Tilletia contraversa over time Germinating teliospores of Tilletia contraversa Modelled viability decline over years of storage with a 3-year 19 half-life estimation.

6 List of Table Table Page 1 Mean of 29 samples of Tilletia controversa teliospore 15 germination (shown as percentage), data collected between weeks 3-8 after spreading on soil extract agar.

7 Introduction Wheat (Triticum aestivum L. and other Triticum species) is the host for the pathogen, dwarf bunt (caused by Tilletia contraversa Kühn). The disease infects winter wheat because it requires a long period of snow cover during winter to stimulate germination of T. contraversa s teliospores (Pascoe et al., 2009). A Zero Tolerance quarantine of Dwarf Bunt by the People s Republic of China ended the exportation of wheat from northwestern of USA to China by 1974, the year that China released this policy to protect their wheat crops from dwarf bunt. In fact, dwarf bunt of wheat in USA normally occurs in five northwestern states; Oregon, Washington, Montana, Idaho and Utah, in a very limited area as a result of the germination preference of T. contraversa teliospores (Trione, 1982). Where it occurs regularly, the disease can cause severe yield losses, and reduces the quality of the grain due to the presence of a secondary metabolite, trimethyl amine, which gives the grain a strong fishy odor affecting its end use market. Dwarf bunt (Hoffmann 1982, Goates, 1996; Mathre, 1996; Pascoe et al., 2009) was reported since 1935 to be different from common bunt (Hoffmann, 1982; Trione, 1982) mainly due to the different requirements for teliospore germination, dwarf bunt requires low temperature for germination and a long snow cover period during the winter for infection of wheat seedling that are just starting to tiller. This means that dwarf bunt is only a problem in winter wheat in some production areas of western USA (Trione, 1982), though other temperate parts of the world outside the US also report the disease regularly.

8 Many researches and reviews have noted the longevity of T. contraversa teliospores, though reports vary from 1-10 years depending on the form of the teliospore; individual teliospore or teliospore in form of bunt ball (Trione,1982; Goates 1996) and bunt ball kept in ambient temperature in storage room could stay alive for over 20 years, but no research has identified the real age limit of teliospore viability (Goates, 1996). Only a single fungicide is labeled for use in controlling dwarf bunt, Difenoconazole. Reliance on a single chemical is unsustainable, and so continued breeding for resistance is important. To determine the viability of teliospores, using half-life estimation, is important for wheat breeders to improve the resistance to infection because races of the disease may vary from year to year. In addition, knowledge of spore longevity could help farmers to avoid contamination of dwarf bunt in their wheat by assisting them to manage the rotation system or land use planning.

9 Objective of The study - Evaluate spores of Tilletia contraversa for germination. - Determine speed of germination of spores of different ages. - Estimate the half-life of spore viability in a population of field-collected spores dating back over 50 years that have been stored in ambient outdoor temperatures and humidity as intact bunt sori. Place Utah State University Experimental Research Greenhouse Logan, UT USA Duration April May 2011

10 Reviews of related Literatures Dwarf Bunt Dwarf bunt (DB) originated in Near East but only in high elevation areas or in mountain regions which have persistent winter snow cover and the disease presents in very limited areas of winter wheat planting in the Americas, Europe and West Asia. Dwarf bunt is caused by Tilletia contraversa Kühn is closed to Common bunt (CB) caused by T. leavis Kuhn and T. tritica (Bjerk.) Wint. (Goates, 1996). Both are important wheat diseases that affect economic loss, especially in the Western USA. The impact from dwarf bunt is not because of the yield losses, though they can be severe, but by the prohibition of wheat importation into the People s Republic of China which reports no dwarf bunt in their country (Trione, 1982; Goates 1996). Before 1935, DB was thought to be a race of CB but it was finally distinguished by Young that DB cannot be controlled by seed treatments that effectively control CB (Young, 1935), and does not infect by seed inoculation (Goates, 1996). In addition, DB germinates at a much lower temperature than CB. Classification Kindom: Fungi Division: Basidiomycotina

11 Class: Ustilaginomycetes Order: Tilletiales Family: Tillertiaceae Genus: Tilletia Species: Tilletia contraversa (Pascoe et al., 2009) While the similarity to common bunt has caused controversies about the origin of dwarf bunt, even the scientific name of the disease is controversial. The disease was first identified by Kühn as Tilletia contraversa but this was likely a spelling error because Kühn used controversa in later publications, and the Latin derivation of controversa is more sensible than contraversa. It has often been spelled controversa since Recently, however, pathology taxonomists have reverted the accepted name back to the original contraversa (Goates 1996; Pascoe et al., 2009). Frequently, the disease is simple abbreviated TCK for Tilletia contraversa Kühn. Teliospore germination The process of germination is start by growing of promycelium or basidium through the spore s wall that extend to the outside in various lengths, long on agar or in high moisture condition, but short in soil; then the promycelium form filliform primary

12 sporidia (basidiospores) at the tip of the promycelium. Two mating types of primary sporidia pair to opposite mating types making up the H-body that then produce infection hyphae, vegetative hyphae or secondary sporidia of allantoids or filiform type. The secondary basidiospores then germinate to produce infection hyphae, vegetative hyphae or addition allantoids or filiform sporidia. Three to eight weeks of low temperature, about 3-8 ºC, and high humidity is required for teliospore germination and development of hyphae capable of infecting tillering seedlings. This stable condition can be found in high elevation areas with long snow cover in the end of winter, so this is the reason why dwarf bunt is only a problem on winter wheat in very limited areas. For laboratory experiments, incubating on soil extract agar at 5 ºC with continuous low light from fluorescent bulb is recommended (Goates, 1996). Infection and disease cycle Teliospores remaining near the soil surface will germinate if environmental conditions are appropriate and possibly infect winter wheat seedlings by infection hyphae when it starts to tiller. Fungus hyphae will invade through stomata and grow systemically inside the plant eventually reaching the ovary then forming a sorus filled with teliospores instead of producing a wheat kernel (Goates, 1996). The relationship between virulence genes and resistance genes during the signaling process of infection is not well understood.

13 Hosts The primary host plant of dwarf bunt is winter wheat (Triticum aestivum L.), but it also occurs on numerous grasses as determined after artificial inoculation (Goates, 1996). These native grasses that are alternative hosts may provide a source of inoculum even when resistant cultivars prevent teliospore production in wheat fields. Their role in development of new races of the disease is also unclear. Artificial inoculation of spring wheat cultivars, such as Chinese Spring, grown under winter sewing conditions indicates that they are generally susceptible to the disease and only normally escape under spring sowing conditions. Adventitious infection of native grasses is thought to minimal, however. Wheat Growth regions Although wheat is grown primarily as a temperate-zone, cool-season crop, it thrives in many regions around the world. Each types of wheat also requires specific conditions for the best growth and development. Wheat is a worldwide grain that has an importance as a major food consumed by people through the world. Many types of food are made from wheat including; breads, noodles, spaghetti and macaroni, crackers, cakes and cookies. Each kind of food made from the different market class of wheat that meets the need of product s texture.

14 Types of wheat The earliest domesticated wheat was einkorn which is a diploid wheat, but wheat that is commonly planted at present is tetraploid and hexaploid. Three species of wheat that commonly grown are: - Triticum aestivum (Hexaploid) consists of hard red winter, hard red spring, soft red winter, hard white and soft white. - T. aestivum ssp. compactum (Hexaploid) includes club wheat. - T. durum (Tetraploid) which includes durum and red durum. Three sets of words used in describing types of wheat by their characters are hard/soft, which refer to kernel hardness, or resistance to crushing; red/white, which refers to the presence of phenolic coloring in the pericarp; and winter/spring, which refers to the season of planting. Winter wheat is planted in the autumn and harvested the next summer, this type requires vernalization for the development of heads, while spring wheat is planted in spring and ready to harvest in late summer or autumn (Atwell, 2001). PK Functions for Microsoft Excel Eight pharmacokinetic (PK) functions developed to simplify pharmacokinetic calculations by Microsoft Excel worksheets consisted of Cmax, tmax, ElimRateConstant, Half_life AUC0_t, AUC0_inf, AUMC0_t and AUMC0_inf,. This helps user easily work on

15 their calculation without remember mathematical formulae for less human errors in working. Half-life calculation calculates by regression of the semi-logarithmic concentration versus time data. Half life tells how long it takes to reduce the concentration or amount by half of initial concentration (Usansky, n.d.). Half-life is usually used in pharmacokinetics to examine the elimination of a drug. It is also used in radiochemistry to explain the radioactive decay of isotopes. However, it frequently is useful in biological situations to explain the decay of viability of seeds or spores over time.

16 Materials and Methods Germination media For Tilletia contraversa spore germination, soil extract agar is superior to water agar or nutrient agar (Meiners and Waldher 1959). On water based agars, spore germination is low and on nutrient agars, contaminating organisms may be a problem. To prepare 2% soil extract agar, the following protocol was followed: Boiling water (500 ml) was filtered through 75 g of soil and the volume of the filtrate was brought up to 1 L with fresh water in a 2-L flask; 20 g of Difco bacto-agar was added and autoclaved. The agar was poured relatively deep (approximately 20 ml of medium per 9-cm dish) when preparing petri dishes for T. contraversa germination to reduce drying of the agar since spore germination can exceed 8 weeks at 4 C. (Goats, 1996) In this experiment, 375 g of powdered soil were used for 2500 ml of soil extract solution. After filtering, the soil extract was sent to the media preparation laboratory of Utah State University for making soil extract agar in petri dishes. From 2500 ml of soil extract solution, 5 L of soil extract agar (2% agar) was prepared allowing 250 petri dishes (20 ml per dish) to be prepared.

17 Spore preparing, plating and incubating Teliospores of T. contraversa were extracted as intact sori, bunt balls, from infected heads of wheat. Collections of infected head were stored in the storage room outside of laboratory in dry atmosphere with otherwise no climate control. Only a few heads of infected wheat were collected from field samples in each year and 29 samples representing individual years in the collection dating back to 1960 were selected for this study. Bunt balls were aseptically excised from heads, and sori were placed into sterile microcentrifuge tubes then crushed by a small sterile aluminum rod. Four separate bunt balls from each year were excised separately to provide four biological replications. Teliospores were suspended in distilled water and allowed to settle for one day at room temperature to allow other contaminating fungal spores to germinate before surface sterilization. Spore suspensions were then sieved through cheese cloth to remove any larger sori particles. The tubes were centrifuged at 600 g for 3 minutes then the supernatant was decanted leaving the teliospore pellet. For spore surface sterilization (and to kill any germinated contaminating spores), 15 ml of 0.25% sodium hypoclorite aqueous solution were added to each tube. These were mixed with a vortexer, and then allowed to stand for 1 minute. The tubes were then centrifuged as before and the disinfectant was poured out. The tubes were then rinsed twice with sterile water using centrifugation as before. Following the final rinse, the disinfected teliospore pellet was re-suspended in 15 ml of sterile water (personal communication, Blair Goates). Approximately 0.2 ml of spore suspension were aseptically spread on the surface of each soil extract agar dish using a flamed plastic rod in in the laminar flow hood. The plates were allowed to air dry in the hood for 3-5 minutes before replacing the lid to allow excess water to evaporate from agar surface. This helped to minimize condensation during incubation. The plates were

18 incubated upside down at 4-5 C with low light from fluorescent light bulb (Goates, 1996) for 8 weeks. Beginning at two weeks, the plates were inspected for spore germination. Germination counting Germination percentage was collected by counting random isolated individual spores per plate, without overlapping field, under an Olympus stereo microscope. For each plate at each counting date, the spores germinated were divided by the total counted to determine the percent germination. Spore germination was counted twice a week for 8 weeks. Areas of the plates that had excessive numbers of spores were avoided due to possible miscounting after primary sporidia began to develop. Since a different section of the plate was potentially examined each time, the percent germination could possibly decrease over time. Averages of replicates were used to minimize the errors associated with this. Half-Life estimation Half-life Half-life is defined as a time at which concentration reaches 50% of its initial value. The formula to calculate the half-life is as follows: t1/2 = ln(2) / ElimRateConst Where, t1/2 = Half-life ElimRateConst = Elimination rate constant

19 ln = Natural logarithm While it was hypothesized that the longevity of teliospores in storage would fit a half-life model, it was anticipated that the variability in germination, and the change in races over time, would complicate the model. For this reason, several methods were used to estimate spore half-life. Only the most recent years were utilized, since as spore germination rate dropped off, the data become much more variable. Several slices of the data set were used to estimate half-life including using germination from the 4th week, and the 6th week, as well as including small numbers of years. Ages of spores utilized for half-life estimation ranged from one year old spores to twelve year old spores. Since biological half-life estimation is inherently more difficult that radioactive half-life decay, a series of pharmicokinetic fuctions available for Microsoft Excel were utilized (Usansky, Desai, and Tang-Liu, Department of Pharmicokinetics and Drug Metabolism, Allergan Inc., Irvine CA 92606). The functions for Excel are freely available for download at

20 Results High levels of spore germination from younger spores (back to year 1996) were observed after the first 3 weeks of the experiment, while spores from older collections were much slower, and germinated in very low percentage. The patterns of mycelia in initial of germination were also different in each year. Germination of spores older than 20 years (year ) was observed in the last week with very low number of germinated spores. Table 1 illustrates the germination of each of the spore collections of the most recent years over the germination time from 15 days to 50 days. The age of the spores affected both the speed of germination and the final germination percentage are shown in figure 1. Spores that germinated earlier produce secondary sporidia, and released those from primary sporidia randomly. Secondary sporidia were found everywhere on germination media as in figure 2, and even on the cover lid of petri dish.

21 Table 1 Means of samples from 29 years (four biological replications, each) of Tilletia contraversa teliospore germination (shown as percentage), data collected between weeks 3-8 after spreading on soil extract agar The check sample was a composite of the most recent two years of collected spores. year week 3 week 4 week 5 week 6 week 7 week Check

22 Percent spore germination Days of Germination 1997 Figure 1 Germination of teliospores of Tilletia contraversa over time. Year of spore collection is identified on the graph. Each point is an average of four biological replicates. Final spore germination rates for all years prior to 1997 were below 1%. Spores were surface sterilized and germinated on 2% soil extract agar at 5 C with continuous low light.

23 Figure 2 Germinating teliospores of Tilletia contraversa. Black arrow indicates conidia from germinating spore; blue arrow indicates released secondary sporidia.

24 Discussion Half-life estimates ranged from 1.9 years to 5.9 years with a consensus value of about 3 years as shown in figure 3, From this graph that is the set of data calculated by Eight pharmacokinetic (PK) functions using excel spread sheet, the year gives 50 percent of germination is located at around 3 year. The expected germination percentage for spores that are 25 years old is estimated at 0.4%. While that may seem low, the millions of spores released into the soil from a single infected plant means that significant numbers of viable spore are still present after 25 years even if no disease has been present in the intervening years due the use of resistant cultivars or fungicidal treatment. It is noted that these spores were stored in unfavorable conditions. While this may have resulted in earlier death of spores than in soil conditions, it did keep spores from germinating in favorable conditions in the field which would preserve those spores for later germination. These two condition of this experiment effect opposite deviations on the half-life estimation. An estimate of about 3 years for half-life degradation of viability is consistent with anecdotal field evidence, and is useful as a general rule for grower purposes. In general, it is confirmed that this organism has an extraordinarily long viability compared to other soil fungal pathogens.

25 Expected germination % with 3 year half-life Age of Teliospores Figure 3 Modeled viability decline over years of storage with a 3-year half-life estimation.

26 Conclusion It is confirmed that teliospores of TCK are long lived. Spores stored for over 50 years were still alive but in very limited numbers. Spores stored in storage silos of the Utah Stage University Experiment Station in the form of intact heads under ambient temperature and humidity have an estimated half-life for spore viability of about 3 years. Under favorable field conditions, it is expected that germination of spores would reduce that number, but storage under controlled conditions or refrigerated storage with controlled humidity would result in much longer viability. This pool of long lived spores will impact the increase of new virulent races.

27 References Atwell, W.A An overview of wheat development, cultivation, and production. Cereal Foods World. 46 : Goates, B.J Dwarf bunt and common bunt, pp In Wilcoxson, R.D. and E.E. Saari (eds.). Bunt and Smut Diseases of Wheat: Concepts and Methods of Disease Management. Mexico. D.F.: CIIMYT Hoffmann, J.A Bunt of wheat. Plant Dis. 66 : Meiners, J.P., and J.T. Waldher Factors affecting spore germination of twelve species of Tilletia from cereals and grasses. Phytopathology 49 : Cited by Goates, B.J Dwarf bunt and common bunt, pp In Wilcoxson, R.D. and E.E. Saari (eds.). Bunt and Smut Diseases of Wheat: Concepts and Methods of Disease Management. Mexico. D.F.: CIIMYT Mathre, D.E Dwarf bunt: politics, identification, and biology. Annu. Rev. Phytopathol. 34 : Pascoe, J., N. Crump and R.H. Jones Diagnostic methods for dwarf bunt of wheat Tilletia contraversa. [online]. Available : hp?q=node/193&pbtid=163 [2012, Jauary 17].

28 Trione, E.J Dwarf bunt of wheat and its importance in international wheat trade. Plant Dis. 66 : Young P.A A new variety of Tilletia tritici in Montana. Phypath. 25 : 40