”um LL; ”I... n I. A .r'r_~.’ "”8 51's I t.:_ _-.-‘-ul :‘e-‘B C, '—l--— .r‘. L.OI'A :~1'-- 2......9I1.‘ 'IIIV"; ‘ 'V This is to certify that the thesis entitled Effects of the oat cyst nematode on small grains in Michigan presented by Beth Burnett has been accepted towards fulfillment of the requirements for M.S. degree in Crop and Soil Sciences -. ,- /’ 1/" (197‘ a o{;({$£fl’/G( pkflbkz Major professor Date Rikki-1531’ 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution )V1SSI_J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from _:_. your record. FINES will be charged if book is returned after the date stamped below. EFFECTS OF THE CAT CYST NEMATODE ON SMALL GRAINS IN MICHIGAN By Beth Burnett A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1986 ABSTRACT EFFECTS OF THE OAT CYST NEMATODE ON SMALL GRAINS IN MICHIGAN By Beth Burnett The oat cyst nematode, Heterodera gvenae Wollenweber 1924 is a small grain pathogen with world-wide distribution. In 1983, nematologists at Michigan State University identified it in a soil sample from Tuscola County, Michigan, its initial discovery in Michigan. In 1985, field trials at two sites on the farm thus identified gave information used in evaluating the nematode’s effects on oats, wheat and barley. Population density counts from all plots indicated varying degrees of infestation within the trial locations. Height, yield, and test weight measurements suggested that the nematode was causing loss of yield. Growth chamber trials using cyst infested soil corroborated field results. Height and yield were reduced by the nematode. Cyst reproduction was increased by uniform cool temperature. Of the two oat cultivars in the growth chamber study, Ogle seemed a more efficient host but yield and test weight were not proportionately affected. This work is dedicated to my spouse, David Eichinger, who maintained his patience, humor and understanding throughout. ii ACKNOWLEDGEMENTS I gratefully acknowledge the help, advice and support of my major advisor, Dr. Russ Freed, in seeing this thesis to completion. The study was conceived in the nematology lab overseen by Dr. George Bird, and would never have become a reality without his willing help and the unstinting efforts of Lorraine Graney and John Davenport. Dr. Everett Everson and the wheat crew under the direction of Dave Glenn provided valuable technical assistance and the use of much of their time and equipment. Dimon Wolfe, Dave Livingston and Rex Alocilja of the oat and barley breeding project cheerfully did more than their share of planting, harvesting, soil-collecting, and hauling dirt, in addition to being excellent information sources. Dr. Carter Harrison skillfully edited the first draft. For over two years, my fellow graduate students provided information, advice, camaraderie, and many pleasant if hazily remembered social occasions. Lastly, I am indebted to David, my spouse, who helped out in innumerable ways, and made sure that when the going got tough, I kept going. iii TABLE OF CONTENTS PAGE LIST OF TABLES.........................................v LIST or FIGURES......................................viii INTRODUCTION..........................................1 Objectives.....................................2 LITERATURE REVIEW.....................................4 CONTROL..............................................13 MATERIALS AND METHODS................................19 RESULTS AND DISCUSSION...............................27 Field Study Nematode population density.................27 Pathogen effectSOOOOOOOOOOOOOOO00,000.00000034 Controlled temperature study Nematode population density.................43 Pathogen effects............................46 SUMMARY AND CONCLUSIONS 0 O O O O O O O O O O O 0 O I O O O O O 0 O O O O O O O O O 50 LIST OF REFERENCES 0 O O O O O O O O O O O O O O O O O O O O O C O O O O O O O O O I O O 54 APPENDIX A. O O O O O O O I O O O O O O O O O O O O O O O O O O O 0 O O O O O O 0 I O O O O O .59 APPENDIX 300......IOOOOOOOOOOOOOOO0.0.0.000000000000064 iv LIST OF TABLES TABLE PAGE 1. Nematode population counts from five experiments on the Kosik farm, Tuscola County, 19850000.....0OO0..OCOOOOOOOOOOOOOOOOOOOOO0.00.00.00.28 Significant differences in total cyst counts from bean field trials, 1985 growing season, Kosik farm, Tuscola County................................30 Significant differences in total cyst counts from corn field trials, 1985 growing season, Tuscola countyOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.32 Comparison of viable cyst counts throughout the growing season in the presence and absence of aldicarb, corn field trial, Kosik farm, Tuscola County, 1985.........................................33 Significant differences in yield, height and test weight from field trials in Tuscola County, 1985.....35 Average oat yields in 1985 vs. experimental yields obtained from a cereal cyst nematode infested field in Tuscola County, 1985...............37 Average oat test weights in 1985 vs. experimental test weights obtained from cereal cyst nematode infested fields in Tuscola County, 1985.00.0.0.0.0000...O...OO0.000000IOOOOOOOOOOOOOOOO.38 Significant differences in yield, height and test weight from combined analyses of cat and wheat trials in two locations on the Kosik farm, Tuscola County, 1985.................................40 A comparison of adjusted with unadjusted treatment means for presence or absence of aldicarb from analyses of yield, height and test weight of cats and wheat in Tuscola County, 1985.00000000000000000000000...00.00.00.000000000000042 10. 11. 12. 13. 10. Comparison of E; avenae population density between 2 experiments: Field trials in Tuscola County, 1985, and growth chamber trials, 1986 ...... .................. ..... ....................44 Significant differences from analysis of cyst counts taken from unsterilized soil in growth chamber experiment, 1986............................45 Comparison of young cyst counts between soil from two sites used in a growth chamber experiment, 1986..COO...OOOOIOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOCOO..45 Significant differences in yield and dry weight analyses from 1986 growth chamber experiment using 2 cat cultivars and 2 soil treatments..........47 APPENDIX A AOV for test weight bean field oat trial.............59 AOV for yield bean field wheat trial........ ..... ....59 AOV for yield corn field oat tria1...................60 AOV for height corn field oat trial..................60 AOV for test weight corn field oat trial.............60 AOV for yield corn field wheat trial.................61 Combined AOV for oat yield over two locations (bean andcorn field)OOOOOOOOOOOOO00.0.0000000000000061 Combined AOV for wheat test weight over two locations (bean and corn field)......................62 ANCOVA for yield corn field oat trial adjusted for uniform viable cyst distribution at planting....62 AOV for total number of cysts per 100 cc soil at planting for bean field oat tria1....................63 APPENDIX B AOV for yield, two oat cultivars............. AOV for dry weight, two oat cultivars............ AOV for recent cysts in unsterilized rhizosphere soil.......... AOV for empty cysts in unsterilized rhizosphere soil............................................. vii .64 .64 .65 .65 LIST OF FIGURES PAGE 1. The Kosik farm, with experimental sites for Q; avenae studies indicated........................22 viii INTRODUCTION The cereal cyst nematode, Heterodera avenae, is also known as the oat cyst nematode and the cereal root eelworm. Usually considered a native of Northern Europe, it was first recognized as a pathogen affecting cereals in 1874 in Germany. Since then, it has been found worldwide. Information here is principally from studies done in Australia, Denmark, Britain, the Netherlands and Canada. In the past twenty years, increased concern has accompanied awareness of E; avenae as a disease-causing agent in wheat (Triticum aestivum), oats (Avena sativa), barley (Hordeum vulgare) and rye (Secale cereale). Cereal cyst nematode is the most important cereal pathogen in Australia’s southern wheat belt. Meagher (1972) estimated losses of 73-89%. In Europe and Canada, it is more important as a pest of barley and oats. For many years, resistance has been used as a control of E; avenae. Andersen (1961) first showed the existence of biotypes, i.e. morphologically identical strains of the nematode that varied in their response to cereal cultivars. Different biotypes occur wherever the cereal cyst nematode has been studied. Resistant cereals from Australia may not be resistant in Europe or elsewhere and l vice versa. Resistance must be found for each proven biotype. A single dominant gene is responsible for resistance in wheat and barley, while three dominant genes may confer resistance in cats (Clamot & Rivoal, 1984). The nematode was first found in the United States in 1975, in Oregon. Eight years later, nematologists at Michigan State University identified it in a soil sample from Tuscola County, Michigan. A 1984 Michigan Department of Agriculture survey identified the extent of the infestation in Michigan. Its potential as a serious pest is great, since under favorable climatic conditions, it will attack wheat, oats and barley. Objectives How serious a pest is the cat cyst nematode in Michigan? We planned to look at its effects on small grains, in the field and under controlled conditions. In what circumstances and with which cultivars could it prove the most damaging? How was it being disseminated and what are some feasible ways to control it? Originally, the study’s objectives were to find resistance to Heterodera avenae in oat, wheat and barley cultivars grown in Michigan and to determine the type of damage it caused. Maintenance of a homogenous culture of the nematode was critical in order to determine the effects of an infestation. Several months into the study it became obvious that finding viable cysts and rearing populations of Heteroderg avenae in the greenhouse were more difficult than originally supposed. Although the cysts found in Tuscola County may be the same biotype discovered and described in Ontario, Canada, in 1933 (Putnam & Chapman), this has not yet been proven. Because this pathogen is relatively unknown in the United States, the following section describes its morphology, distribution, life cycle, and the host range and reaction. A discussion of resistance follows descriptions of three other control methods: cultural, chemical and biological. LITERATURE REVIEW Nomenclature and morphology The nematode Heterodera avenae was first observed as a pest on cereals in Germany in 1874 (Kuhn). Originally, it was thought to be the "oat strain" of Heterodera schachtii, the sugar-beet nematode. Wollenweber wrote a brief statement in 1924 on the form of the cysts, which, when combined with host range knowledge, made identification possible. The nematode was then known as 3; schachtii, var. avenae. O. Schmidt (1950) observed that juveniles from oat-infesting nematodes were larger than sugar beet nematodes, and named the former fl; schachtii subsp. major. Unaware of Wollenweber's contribution, Franklin (1957), raised the subspecies to a species and gave it the name Heteroderg major (O. Schmidt 1930) Franklin 1940. Since Wollenweber's description and the name avenae antedated Schmidt's and Franklin’s work, the latter names were eventually set aside by a ruling in 1959 in favor of Heterodera avenae Wollenweber 1924 (Thorne, 1961). EL avenae is a sexually dimorphic species, producing one generation per year. Like other members of the genus Heterodera, cereal cyst nematode females form a tough, brown sac from their cuticles. The protection this gives eggs within the female’s body is "one of the most outstanding instances of defensive adaptation found among the plant parasitic nematodes.” (Thorne, 1961). Typically, cysts are dark brown and lemon-shaped, between 0.55 and 4 0.75 mm in length. Most cysts contain 200-250 eggs, but large cysts may contain more than 600 (Andersen, 1961). Cyst nematodes pass through five developmental stages, from unsegmented eggs to adults. The egg, formed inside the cyst, becomes a first-stage juvenile. Molting of the juvenile’s first cuticle results in the infective second stage. In this stage, the nematode emerges from the cyst and invades its host. It becomes sedentary, passing through third and fourth juvenile stages until reaching sexual maturity. The female becomes lemon shaped, with her neck region embedded in root tissues. The vermiform adult male emerges from its juvenile cuticle and moves through the soil in search of the female. Males probably do not feed on root tissue. Johnson and Fushtey (1966) examined, the parasitic development of E; avenae on oat roots. They found that, upon entering the root, the nematodes concentrated in undifferentiated tissue behind the root cap. By the fourth day after infection, they had oriented themselves parallel to the root with their heads toward the root tip. Giant cells (synctia) were evident after three days. The synctium, in nourishing the nematode, causes considerable damage to the vascular tissue of the root. After twenty- four days and the final molt, mature males had left the root. Females were visible soon after near or on the root surface. By the thirtieth day, developing eggs were visible. Other diagnostic characters for Q; avenae include a conspicuous vulval cone with a spheroid vaginal structure and a very short vulvar slit. Bullae (knoblike, dark brown objects just inside the cone) are prominent and crowded into the vulval cone. The underbridge is inconspicuous, and the cyst is ambifenestrate. In addition, g; avenae normally infests only graminaceous plants (Thorne, 1961; Williams & Siddiqi, 1972). Some authors (Thorne, 1961; Hesling, 1965) have suggested that the cereal cyst nematode in Australia may be a species distinct from Q; avenae. Meagher (1974b) refutes this and believes the confusion between European, Canadian and Australian biotypes may have been caused by environmental influences, different fixation methods and reporting errors. Graney (1985) indicated that the morphometrics of Q; avenae juveniles and cysts found in Michigan correspond to previously published descriptions of the pathogen. World distribution and dissemination Known for years as the oat cyst nematode, H.avenae was important in Europe because of its daprsdstions on oats. Recently, more attention has been directed towards its effects on barley. In Australia, it was first identified on wheat in 1930 but was later discovered on herbarium specimens dated 1904 (Meagher, 1972). Putnam and Chapman (1935) recognized it in a greenhouse study on oats in Ontario, Canada, in 1933. The first recorded infestation 7 in the United States was identified in Washington County, Oregon, in 1975 (Jensen, et al. 1975). In 1984, Hafez and Golden reported finding the cyst in two barley fields in Idaho. A 1984 statewide survey of 349 small grain fields in Michigan, conducted jointly by Michigan State University and the Michigan Department of Agriculture, found the cyst limited to an area adjacent to the site of the original infestation in Gilford Township, Tuscola County . To date, two surveys, in addition to the work done in 1984, have located 10 farms known to harbor fl; avenae. It is an interesting note on the dissemination of cyst nematodes that the owners of five of those farms are related (Bird and Graney, 1986). The cereal cyst nematode was once described as a parasite of cool-to-temperate zone crops (Fushtey & Johnson, 1966). While this may be true, it has also been found in the Mediterranean-type climates of Italy, Tunisia, Spain, Portugal, Greece, Yugoslavia, and Israel as well as in India, Australia and New Zealand (Meagher, 1977). The wheat and barley disease "Molya" in India is caused by the cereal cyst nematode (Gill & Swarup, 1971). The species is common in Japan and the USSR but has not been reported in China (Meagher, 1977). The cereal cyst nematode is widespread in Northern Europe where different biotypes have evolved, making Europe a "center of diversity". This suggests that it is indigenous to Europe and has spread to other areas of the world. Dissemination can occur through accidental or deliberate soil transport, cultivation, or water movement. (Meagher, 1977). Field experiments in Australia show that desiccated, viable eggs may be wind dispersed over large areas (Meagher, 1974a,1982). Swarup and Swarup (1985) found that cyst dormancy is induced by high temperatures and may be facultative, another mechanism used to maintain viability under unfavorable conditions. Host range Heterodegg avenae is primarily a parasite of grasses, although it is difficult to generalize host efficiency because cyst production varies within a cereal species (Spaull & Hague, 1978). Kort (1972), however, says host efficiency is highest in oats and lists the following cereals in order of increasing susceptibility: maize, rye, barley, wheat and oats. Research in India found that, although the pathogen was more damaging to barley than to wheat, cysts multiplied more efficiently on wheat (Hands, at al. 1985). Different reports exist regarding maize host efficiency to H; avenae. Johnson and Fushtey (1966), in Canada, found that although the nematode completes its life cycle in maize, many larvae die after entry because of root necrosis. Also, mature females do not break through the root cortex and are not fertilized. Johnston and Fushtey concluded that maize was an inefficient host. On the other hand, Saefkow and Lucke (1979) reported in Germany that the nematode can develop normally and form a new generation on maize. These different reactions to maize could be due to differences in cultivar or nematode biotype. Although oats, wheat, and barley are economically important hosts for H; avenae, it is also found on a variety of wild and cultivated grasses from the genera Avena, Bromug, Festuca,Hordeum‘L Lolium, PhalarisL Secglg and Triticum (Thorne,1961). Gill a Swarup (1971) found a non-graminaceous host, Senebiera pinnatifida, a member of the Cruciferae and a common weed in wheat fields of Rajasthan, India. Host reaction As with many nematode-caused infections, primary symptoms of cereal cyst infestations are non-specific and readily confused with other diseases or deficiencies. Plants may have an overall stunted appearance and reduced tillering. Discolored leaves may resemble those affected by nitrogen or phosphorus deficiency, aluminum toxicity or drought conditions. Cereal heads produced by stunted plants are usually small and poorly filled. Infected roots divide near the invasion point, and roots may bear small, gall-like formations. Distribution within a field is often uneven; an infested field may show patches of stunted plants which do not recover and allow for weed development. Plants on fertile soil with abundant moisture may show few 10 or no symptoms, even under heavy infestation. One sure indication of H; avenae's presence is the occurrence of white cysts on roots (Thorne, 1961; Griffin, 1984; Kort, 1972). The cysts are easily dislodged and, once they darken, difficult to see with the naked eye. Their presence may be overlooked when plants are removed from soil, so Meagher (1972) suggests that the condition is most easily diagnosed after cysts first appear in early spring. Kubler (1980) concluded that 10 - ll cysts per 200 grams of soil were sufficient to cause visible damage in the field. Meagher et al. (1978) found that cereal cyst nematode infection was affected by the nematode’s association with the fungus Rhizoctonia solani. Studies showed that yield reductions in wheat caused by H; gyggg and R.solani were greater when the pathogens were together than with each alone. Life cycle Heterodegg avenae completes one generation a year except where it overwinters on autumn sown cereals. It completes its life cycle within 9-14 weeks of juvenile invasion (Griffin, 1984). In Europe, juveniles emerge from cysts between mid—March and mid-July, and infect spring- sown. cereals. Beginning in mid-June, young females are found on roots. Autumn-sown cereals are invaded by second- stage juveniles in autumn, but their development stops over the winter (Kort, 1972). European autumn-sown cereals suffer more from juvenile invasion than spring-sown crops 11 because their roots are at a later stage of development at the time of invasion than the roots of spring-sown crops (Duggan, 1961a). Different climate and cropping conditions in Australia result in a slightly different time table. Cysts are usually seen in August through October (Griffin, 1984). Juvenile development is favored during the comparatively mild winter because plants grow slowly (Meagher, 1972). Regardless of seasonal and geographic variations, the largest number of cysts occurs three-and-a- half months after planting. (Kort, 1972). Factors affecting juvenile emergence from cysts include temperature, soil moisture, rainfall, root exudates, desiccation and soil type, as well as season. Low temperature encourages nematode reproduction. Cotten (1963) found that a period of low temperature is necessary to stimulate hatching. A minimum dormancy of eight weeks at low (0 — 7 degrees C) temperature substantially increased hatching in Ontario (Fushtey and Johnson, 1966). Below-freezing temperature decreased hatching rates. Emergence occurs at a minimum temperature of approximately 2 degrees C and a maximum between 23 and 30 degrees C. Working in England, Williams and Beane (1979) corroborated these results. Fidler and Bevan (1963) found that soil pores must become filled with water before the larvae can hatch and move freely through the soil. High spring rainfall is generally associated with increased nematode damage. In one 12 British study, lower rainfall in April resulted in a larger root system and fewer larvae per plant than high rainfall. (Dixon, 1963). Duggan (1961b) found that parasitized plants showed heaviest damage after high spring rainfall followed by a late-season drought, because already damaged root systems were less able to take up water. In Australia, severe symptoms occur after high autumn and early winter rainfall, corresponding to high spring rainfall in Europe and Canada (Meagher, 1972). Root exudates stimulate hatching in certain species, including the sugarbeet nematode, H; schachtii, and the potato cyst nematode, Globodera rostochiensis (Thorne, 1961). Hesling (1957), Kort (1974) and Griffin (1984) state that H; avenae hatching is not affected by root leachates. William and Beane (1979) found that root exudates of wheat, barley and oats stimulate hatching. They specify that exudate activity should be studied in hatching tests of relatively short duration. Cereal cyst nematode infestations are not restricted to any particular soil type. Under continuous cropping, the nematode will cause economic damage regardless of soil conditions (Kort, 1972). Outbreaks of damage in cereals are more common on light soils in Australia and there is some evidence that disease severity is increased on light soils. (Meagher,1972). Putnam and Chapman (1935), however, found that the nematode, although present in both light and heavy soils, was more common and troublesome in heavy soils 13 in Canada. Soil pH and fertility affect the degree of plant damage because they affect plant growth. Hesling (1959) found that fertilizer stimulated cyst production in cats, barley, wheat and rye. Generally, a healthy plant will produce more cysts. CONTROL Cultural control Intensive cereal production favors multiplication of H; avenae. Meagher and Rooney (1966) found that disease severity was greatest with fallow-wheat or fallow-wheat- oats cropping. Crop rotation, using a non-host crop, is an effective way to reduce nematode populations. A bioassay has been developed in Australia that is used before planting and indicates the degree of infestation and what potential damage there may be to specific crops (Brown, 1982a). Sowing date can also affect population levels. Earlier-sown crops avoid high juvenile concentrations and can establish vigorous seedlings with less infestation. Brown (1984) found that sowing a month late can result in a yield loss of 1 ton/ha. Resowing diseased crops, additional fertilizer, and herbicide applications to reduce weed competition are not effective in reducing yield loss caused by the cereal cyst nematode (Brown, 1984; Barry et al. 1974). 14 Chemical control Most published work on chemical control comes from Australia. There are five registered nematicides for control of H.avenae in Australia: Ethylene dibromide (EDB), terbufos, oxamyl, aldicarb and carbofuran. Although EDB is no longer registered in the United States, it is used in Australia (Brown, 1984). Chemical control on small grains, although effective, is usually too expensive. Aldicarb, used to eliminate the nematode in the present study, is an organo-carbamate or oxime carbamate. It is a broad spectrum pesticide, used as an insecticide, acaricide and nematicide. A systemic, aldicarb is thought to act by retarding nematode development for several months after application (Steele, 1984). Biological control Fungal parasitism of cysts has been studied in England. Kerry (1980) asserts that, although total eradication of H; avenae under field conditions by fungi is unlikely, fungi have potential as control agents. Kerry maintains that Nematophthorg gynophila Kerry and Crump, widespread in England, is an agent in reducing cereal cyst nematode populations to non-damaging levels. Females fail to form egg-containing cysts when parasitized by H; gygophila and Verticilligg'chlggydosporiug, another fungus identified by Kerry et al. (1982). These two fungi were responsible for 60% of a population of non-egg bearing cysts in this study. 15 Resistance Genetic resistance is an effective form of control. Nilsson-Ehle began looking for resistance in barley in 1920. Andersen contributed the pioneering work he did in Denmark in 1961. The first resistant Danish barleys were Drost, Fero and Kron (Andersen, 1959). Investigating H; avenae populations from four different fields in 1956, Andersen found that the populations differed in their reactions to Stal oats and Maja, Drost and Alfa barley. Drost and Alfa were resistant to two nematode populations but susceptible to two others. Andersen concluded that there were two "races" or biotypes. He also identified resistance in wild oats, Hyggg sterilis, to both biotypes (Andersen, 1961). Further studies in England and Wales established the existence of biotypes varying in pathogenicity (Gair et al, 1962; Cotten, 1963; Fiddian and Kimber, 1964.) In the Netherlands, Kort, et al, (1964) recognized four biotypes of H; avenae using Andersen’s series of test varieties. Biotypes have also been recorded in Germany (Neubert, 1967) and India (Mathur et al, 1974). In Australia, one biotype has been found but it is unlike any of the European biotypes. (Brown, 1969, 1974). A resistant plant has no (or very few) cysts on its roots (Cotten, 1963). Holm-Nielson (1984), working on oats, suggests the following definitions of resistance: specific resistance, involving a gene-for-gene system; 16 intermediate resistance, involving specific and/or partial resistance at a not very high level; and partial resistance, determined by many additive genes each of slight effect, with no corresponding virulence genes. As Andersen demonstrated, resistance varies with biotype. In Europe, resistance in barley can be traced to Drost, LP-191 or Moroccan strains. Several resistant oat varieties have been developed: CI 2094, 2154, and 3444; PI 175022,175024, 185775, and "Silva". (Griffin, 1984). The Australian biotype attacks cat and barley cultivars resistant to infestation in Europe. Few European sources of resistance in barley and oats have been used in Australian breeding programs. However, breeders have found and use resistance from AUS 10894, a spring wheat from Afghanistan; Marocaine 079, a barley; and wild cat, H; sterilis (Brown,1982b). Galleon, a resistant barley, and Katyil, the world’s first wheat cultivar bred specifically for resistance to H; avenae, are available in Australia. Nematode resistance may be explained by 1) the nematode’s failure to produce a synctium, 2) an excess or deficiency of chemicals in the plant, 3) differentiation of plant root tissue or "woody" roots and 4) hypersensitivity or necrosis. (Andersen, 1961). O’Brien and Fisher (1978) found that the resistance mechanism was probably induced following development of a resistance factor by the host in response to an invasion by larvae. They postulated that the actual mechanism may be inadequate synctia or synctia 17 degeneration. Inheritance of resistance to H; avenae is attributed to a single dominant gene in wheat and barley. Nielson (1966) found complete dominance for resistance in wheat crosses. Brown and Ellis (1976) described a backcross breeding program incorporating resistance into three wheat parents. O’Brien et al. (1979) found that inheritance of resistance in four barley cultivars was due to a single dominant gene. They could not eliminate the possibility of 2-gene inheritance in a fifth cultivar, however. Sparrow and Dube (1981) found, by analyzing crosses of susceptible and resistant barley varieties, that at least three and possibly five major resistance genes were present in the cultivars studied. Resistance breeding programs for cats usually use H; sterilis, because of its resistance to all European and Australian biotypes. It appears to involve three dominant genes. Two of the genes, when present, confer high resistance. A third, alone, confers intermediate resistance. Only the completely recessive genotype allows normal nematode development (Clamot & Rivoal, 1984). Chew (1983) found that resistance in some oat cultivars was monogenic dominant at one locus, but that partial resistance in other cultivars is recessive, controlled by a single gene, and involves at least three loci. Another cultivar ("Mortgage Lifter") seemed to have a pair of double recessive genes which confer resistance. Monogenic 18 dominance was found to be the most effective resistance system, followed by monogenic recessive, and the double recessive. MATERIALS AND METHODS Most data are from a field study in 1985 and a controlled temperature experiment in 1986. Two kinds of measurements were taken: cyst counts from soil samples in order to estimate nematode population density; and agronomic data including height, heading date, and dry weight as well as yield and test weight (in order to determine the pathogen’s effects). Nematode population density counts Soil for population density counts was processed using a direct sieve technique. In the field, 500 to 1000 g of soil was collected in plastic bags from each plot, using standard soil samplers. In most cases, 100 cc of moist soil containing roots was taken directly from the plastic bag. The soil and roots were mixed in a bucket with water. Two sieves were used for the technique: a Number 25 screen (opening in micrometers = 710, and in inches = .0278) to catch roots, small stones and detritus, over a Number 60 screen (opening in micrometers = 250 and in inches = .0098) to capture the cysts. Roots from the sample were laid on the No. 25 screen while the well-mixed soil and water solution was poured through the two sieves. Silty soil at the bottom of the bucket did not go through the sieves. The contents of the top sieve were rinsed well, and then discarded. Everything in the No. 60 sieve was washed into a small beaker, to be examined and the cysts counted under 19 20 a dissecting scope. Counts were taken of all cysts of whatever age as well as new cysts (presumably from the present season) and the number of eggs and J-2 juveniles they contained. New cysts, in addition to being whole and for the most part not parasitized by fungi, were distinguishable by the presence of a milky-white coating called the sub-crystalline layer. This layer disappears after one season. For certain samples, 500 cc of soil was processed using a Fenwick can. The Fenwick can is equipped with the same two sieve sizes as used in the direct sieve technique. It is modified to allow a greater volume of soil to be processed. Five hundred cc of soil from each sample was air dried for several weeks. The cloddy soil was broken into smaller particles by pressing it through a sieve-like greenhouse bench. This soil was mixed with running water in the can. The water-soil solution was spilled into two sieves for four minutes. The larger 25 mesh screen caught debris while the contents of the 60 mesh screen were rinsed into a beaker and screened for cysts. Because the soil is thoroughly dried before it is processed, this technique usually kills J-2 juveniles and eggs inside the cyst. Agronomic data Agronomic data were recorded from the field and greenhouse. Height was measured at physiological maturity. Heading date (from the greenhouse study) was measured when 21 the head was half out of the boot. Dry weight was taken after the plants harvested from the greenhouse were dried for one week. Test weight was measured in grams per pint. Experimental site selection In April of 1985, four sites were sampled on the Kosik farm, where the cereal cyst nematode was found in 1984. The farm is located in Section 16 of Gilford Township, Tuscola County. The dominant soils are wet and loamy. nematode cysts were found in all four areas. A field to be planted to corn that showed high H; avenae levels in 1984, and a field to be planted in beans were chosen for the experiments. Relative positions of the fields are shown in Figure 1. Both fields were in Tappan loam, a nearly level, poorly drained soil usually found on broad flats and in depressions. Field Study The plots were large enough to accommodate five experiments: two oat trials, two spring wheat trials, and one barley trial. CULTIVARS USED Oats Wheat Barley Ogle *3 Sinton it Robust 8* Heritage Max *3 Bowers Mariner Porter Menominee Mackinaw Korwood T879112 ‘* Cultivars used as borders in the experiments 22 DITCH '-"l ROM) :1 SITE A . 180' x 80' a El ______ “‘r““‘““‘ l l l 3 l “ l n I 1 SITE B I 180' x 60' . l L Figure 1. The Kosik farm with experimental sites for H. avenae studies indicated. 23 One oat, one wheat, and the barley trial were planted in the bean field. Due to space limitations, the barley trial was not planted twice. An oat and a wheat trial were in the corn field. The cat and barley cultivars represented varieties grown in Michigan. T379112 is a potential release from Canada. Morex is a malting barley and Bowers, a feed barley. The two spring wheat varieties were obtained from Cornell University in Ithaca, New York. The five trials were split-plot randomized block designs with four replications and two factors. The factors were varieties and presence or absence of aldicarb, a nematicide not registered for use on small grains. Aldicarb was applied at 3 lbs. active ingredient per acre or 20 lbs. in formula. This is a standard‘application for potatoes. Because the corn field had already been fertilized, only the bean field required fertilization, applied at the rate of 200 lbs per acre of 19-19-19. Planting date was May 7, 1985. Aldicarb was distributed by an applicator pulled behind the planter. Plots were 18 ft. x 4 ft., although, after alleys were cut, the harvested area was reduced to 12 ft. x 4 ft., or 48sq. ft. The plots were harvested on August 8, 1985, using a Hege, a small plot harvester-combine. Data were taken on height of the tallest head, yield, and test weight. The plots in the bean field were heavily attacked by birds in late July. Sections of the experiments in the corn field 24 had tractor and herbicide damage. Field experiments planned for spring, 1986, were not planted because growers with cereal cyst nematode infested fields were unwilling to rent land for the trials. Soil samples taken from each of 176 individual plots at planting (May 7), midseason (July 10) and harvest (August 8) were screened for cysts. The planting, 48 of the midseason, and all of the harvest samples were processed using direct sieve technique. One hundred and twenty-eight of the midseason samples were processed in the Fenwick can. In an attempt to recover vermiform Heteroderg avenae juveniles and males, roots from plants taken at midseason were processed using the shaker technique. The washed roots, cut into 1-2 cm. segments, were covered with incubating solution and incubated for 48 hours on a gyratory shaker at 100 rpm. The incubating solution is a mixture of 10 ppm mercuric chloride and 50 ppm dihydrostreptomycin sulfate. All 48 samples were screened without finding any vermiform H; avenae. Controlled temperature study A growth chamber experiment was planted in soil collected from the summer 1985 field plots in Tuscola County. The soil was collected in November and held at 60 degrees F or lower until planting in February. Ogle and Mackinaw were chosen for the experiment based 25 on information from the previous field trials. Their reactions were as different from each other as any of the eight oat cultivars from the field study. Soil sterilization rather than nematicide application was used to eliminate the nematode in the greenhouse. Half of the pots were autoclaved at 15 psi and 240 degrees F for 1 hour and 45 minutes. This change in technique was mandated by cost and safety factors as well as the possibility that aldicarb could have had growth regulating effects on the field study plots. Sand was mixed with the two soils from the farm at a ratio of one part sand to four parts soil. In general, soil from the corn field was heavier than soil from the bean field. After addition of sand, four-inch clay pots were filled with the soil. The experiment was designed as a 2 x 3 factor factorial in fourteen replications. The three factors, each at two levels, were cultivar, source of soil (bean or cornfield), and sterilized or non-sterilized soil. It was planted on February 16, 1986, in a growth chamber set at 60 degrees F. The seeds germinated after 5 to 7 days. Each pot was thinned to two oat plants. The pots were fertilized every two weeks with an N—P-K solution, and twice with micronutrients. Due to three growth chamber malfunctions, the temperature did not remain constant; once the chamber went below freezing, and twice above 90 degrees F. 26 Population counts, using the direct sieve technique, were done on the rhizosphere soil from each pot. The roots, and soil clinging to them, were submerged in a graduated flask containing 400 cc of water. Additional soil from the pot was added until the volume of~ the solution in the flask reached 500 cc. The 100 cc of soil thus obtained was sieved normally. The roots were carefully washed into the solution, in order to recover as many clinging cysts as possible. Heading date, height of the topmost node, height of the tallest tiller, yield, and dry weight were measured for each plant in each pot. The heads were harvested on. June 9, 1986. Dry weight was taken on each plant after harvest and a one-week drying period. MSTAT, a microcomputer statistical package, was used for design of the experiments and analysis of the results, except for covariance analyses, which were hand calculated. RESULTS AND DISCUSSION Field study NEMATODE POPULATION DENSITY Analyses run on the population counts indicated few significant differences and little change over the season. Counts were taken, not only of total cysts, but of cysts (presumably recent) containing "viable units" - eggs and J-2 juveniles. Pre-plant, midseason, and harvest samples were taken from all five experiments (totalling 176 plots); out of 528 (3 x 176) 100-cc samples thus screened, only one had as many as 4 cysts with viable units per 100 cc of soil. Five had three viable cysts, and the rest had 2, 1 or none. In general, the cysts were older than one season, as evidenced by their lack of a sub - crystalline layer, found on only two cysts during the counts. No white females were found. However, almost all of the samples had older, usually empty cysts, sometimes parasitized by fungi. Samples contained as many as 27 to as few as zero. So few viable cysts were found in the planting and part of the midseason samples that, in an attempt to increase efficiency, larger (500 cc) samples processed with the Fenwick can replaced direct-sieved 100 cc samples. However, after completion of the midseason samples using the Fenwick can, it was obvious that the new technique was not recovering any more cysts than less-time-consuming direct sieving. The counts obtained from 500 cc samples were divided by five in order to run the analyses. 27 28 Table 1. Nematode population counts from five experiments, on the Kosik farm, Tuscola County, 1985. Total cysts Viable cysts (w/ viable units) SITE A Pre-plant 448 8 OATS Mid-season 451 6 (64 plots) Harvest 478 2 TOTALS 1377 26 Mean no. cysts/sample! 21.5 0.41 Pre-plant 115 3 WHEAT Mid-season 117 4 (16 plots) Harvest 124 4 TOTALS 356 14 Mean no. cysts/sample 22.2 0.87 Pre-plant 140 3 BARLEY Mid-season 142 1 (16 plots) Harvest 127 1 TOTALS 409 5 Mean no. cysts/sample 25.5 0.31 SITE B Pre-plant 276 30 OATS Mid-season 184 15 (64 plots) Harvest 318 34 TOTALS 502 79 Mean no. cysts/sample 7.8 1.2 Pre-plant 77 19 WHEAT Mid-season 43 4 (16 plots) Harvest 100 19 TOTALS 220 42 Mean no. cysts/sample 13.75 2.62 —; 100 cc of soil screened from 48 sq. ft. plot 29 Table 1 indicates that, while the Site A (bean field) samples contained more total cysts, the Site B (corn field) samples had more viable cysts and therefore, more nematode activity. This supports the conclusion that significant differences for yield, height and test weight in the corn field over aldicarb application were caused by the cereal cyst nematode. It is interesting that, over the season, nematode counts and especially the all-important viable cysts, remained more or less uniform; they did not show expected reproduction increases. Dry weather at crucial hatching times or too-high temperatures could have limited the nematodes’ reproduction. Sample size was probably insufficient to obtain a thorough cyst count. Height and test weight of wheat planted in the corn field were not significantly affected by aldicarb application, even though those plots had the highest mean number of viable cysts per sample (2.62/ 100 cc. of soil) of the five experiments. See Table l. A lower average count in the cat plots from the corn field (1.2), where height, yield and test weight were significantly affected, suggests that cats are more susceptible than spring wheat to H; avenae. Site A - H; avenae population density The following table summarizes significant differences from total cyst count analysis at Site A, not including 30 viable cysts. Table 2. Significant differences in total cyst counts from bean field trials, 1985 growing season, Kosik farm, Tuscola County. Bean field OATS WHEAT BARLEY R A C R A C R A C Pre-plant x t Midseason x t x Harvest * x 3 indicates significance of at least .05. R = Replication, A = Aldicarb application C = Cultivar The replication differences probably reflect uneven distribution of the nematode. The only significant response noted to the presence or absence of aldicarb occurs in wheat for pre-plant samples. These samples were taken before aldicarb was applied, and probably reflect distribution, since“ aldicarb was applied to half of the field, not on a plot-by-plot basis. The one cultivar difference, for barley in the bean field at harvest, indicates that Bowers had more cysts (9.625 per 100 cc of soil) than Robust (6.250). This is probably a chance association since 1) these are old cysts, not newly reproduced nematodes that may have been affected by the cultivar and 2) the barley experiment was small, involving 31 16 plots. Error degrees of freedom for the analysis was only 9. For the number of cysts with viable units at harvest, the oat AOV table indicated a difference among replications. This was the only significant difference in fifteen analyses of cysts containing viable units. This is not surprising given their scarcity in the samples and homogeneity of the data (mostly 0’3, 1’3 and 2’s). The differences found in replication probably reflect non-uniform distribution of the cysts throughout the fields. Site B - H; avenae population density As at Site A, there were few significant differences, summarized in Table 3, in total cyst analyses. The information was inconclusive, because the significant differences found here in the presence or absence of aldicarb do not in all probability reflect a true reaction to aldicarb but rather a clustering of cysts in that half of the field where aldicarb was applied. As in the bean field analysis, a significant difference in pre-plant samples for aldicarb application does not offer information on the activity of the chemical because it had not 1 been applied when the samples were taken. Analyses of variance done on viable cysts in the corn field did not indicate any significant differences over replication, aldicarb application, or cultivar. 32 Table 3. Significant differences in total cyst counts from corn field trials, 1985 growing season, Tuscola County, 1985 CORN FIELD OATS WHEAT R A C R A C Pre—plant 4 Mid-season Harvest t * * indicates significance of at least .05. Summary and discussion Environmental factors and late planting probably limited nematode reproduction in the field. There was little change in recovery of new cysts over the season, while total cyst counts remained static. At Site B, cyst recovery did not decrease over the season where aldicarb had been applied, but was less at midseason. (See Table 4). Aldicarb application should have reduced the numbers of cysts in the areas where it was applied. Instead, slightly higher numbers of viable cysts were recovered where the chemical had been applied. By chance, cysts probably were more abundant to begin with in the area that received aldicarb. Recovery techniques for viable cysts may have been inefficient, and sample size too small. All of the Site B midseason samples were processed with the 33 Fenwick can; the change in technique could have decreased viable cyst recovery. Table 4. Comparison of viable cyst counts throughout the growing season in the presence and absence of aldicarb, corn field trial, Kosik farm, Tuscola County, 1985. No aldicarb Aldicarb OATS Pre-plant 13 17 (64 plots) Midseason* 7 8 Harvest 15 19 BARLEY Pre-plant 7 12 (16 plots) Midseason! l 2 Harvest 6 l3 * Midseason samples were processed with Fenwick can. Analysis of numbers of cysts resulted in little information. Total cyst counts indicated that, although the bean field had many more old cysts, the nematode was reproducing better in the corn field. Paradoxically, the reverse occurred in the growth chamber, where reproduction in bean field soil was greater than in corn field soil. Knowledge of previous crops in the fields would have helped to explain this anomaly. Significant differences found over presence or absence of aldicarb probably reflect cyst distribution in the field because the cysts were old when 34 aldicarb was applied. Even with little recovery of viable cysts, the agronomic data gives clear indication that the cyst nematode was affecting these cereals. PATHOGEN EFFECTS Significant differences were found in yield, height and test weight in field trials of oats, barley and wheat at two locations in Tuscola County in 1985. Significant reactions in cats to aldicarb indicate that yield, height and test weight were increased by its use. In wheat, aldicarb increased yield. There were no significant differences to aldicarb application in barley. The analyses suggest that cats, as indicated in the literature, are more susceptible to the cereal cyst nematode than the other cereals. Table 5 summarizes significance found in the five experiments (two cats, two spring wheat and one barley). Sample analyses of variance are in Appendix A. Site A (Bean field) Qggg Test weight in cats showed highly significant differences for presence or absence of aldicarb. The mean test weight for aldicarb application was 26.5 lbs/bu while plots without aldicarb averaged 24.8 lbs/bu test weights. Either the presence of aldicarb or the absence of the organisms it eliminated appeared to affect test weight. 35 Table 5. Significant differences in yield, height and test weight from field trials in Tuscola County, 1985. Site A Site B Bean field Corn field oats wheat oats wheat R A C R A C R A C R A C YIELD ** * ** * ** ** ** HEIGHT ** ** * ** ** TEST WT. * ** ‘** * ** ** ** * Indicates significance of at least .05. 3 Indicates significance of at least .01. replication, A 2 presence or absence of aldicarb, x R C cultivar Some grain from the plots planted in the bean field was lost to birds, increasing the error in yield data. It is not surprising that cat yield in this field showed significant differences only for different cultivars. Height of cats, wheat and barley did not appear to be affected by the aldicarb application. Oat heights correlated approximately to heights measured in other locations in Michigan (Freed, et al. 1986). Elia; In the 16 wheat plots in the bean field, yield was significantly increased by aldicarb, being 750.78 g (25 bu/acre) and, without aldicarb 688.844 g (22.9 bu/acre). Both cultivars were higher yielding with the aldicarb 36 application. Interactions The following aldicarb x cultivar interactions were significant in the bean field: height in wheat and test weight in barley. No interactions were significant in oats or in the cornfield. In the wheat interaction, Sinton seems to be shorter with the application of aldicarb; Max seems shorter without aldicarb. With barley. Robust’s test weight appears lower with the aldicarb application, while Bowers seems to have a reverse pattern. Both wheat and barley results may not be definitive since the experiments were limited in size to sixteen plots. Site B (Corn field) Qggg Yield, height and test weight all showed significant differences over cultivars and significant increases in the presence of aldicarb in the cat plots. Mean yield with aldicarb was 1435.125 g (90 bu/acre) while in the absence of aldicarb, it was 1219.78 g (76 bu/acre). The analysis of variance for oat yield in the corn field is reproduced in Appendix A. Yields were higher than in the bean field study because birds were less of a problem. The mean yield in the corn field without aldicarb was 1219.7 g. (76.2 bu/acre), for example, while the corresponding yield in the bean field was 688.84 g (43.05 bu/acre). 37 In comparison with "normal" oat yields in Tuscola County in 1985, yields obtained from the corn field on the Kosik farm are low. The average yields in Table 6 were obtained from Michigan State University Extension Bulletin E-899 (Revised), April, 1986 (Freed, et al). Table 6. Average oat yields in 1985 vs. experimental yields obtained from a cereal cyst nematode infested field in Tuscola County, 1985. CORN FIELD 1985 CULTIVARS Average No Aldicarb Aldicarb (bu/acre) (bu/acre) (bu/acre) Heritage 134 79.2 95.5 Ogle 146 79.0 88.5 Mariner 136 70.3 91.4 Mackinaw 111 77.3 99.1 Korwood 119 61.1 77.7 Poor yields could have been due to climatic factors, aggravated by late planting. Yield in the corn field, moreso than test weight, was probably reduced by herbicide damage and soil compaction. The plots with aldicarb more closely approached a "normal" yield, indicating that something eliminated by the chemical was reducing yield. Height in cats was significantly lower in the untreated plots. Mean height in the corn field without aldicarb was 78.026 cm. and with aldicarb 81.041 cm . 38 Oats in the bean field also reflected this, although at .304 probability. (Mean height without aldicarb = 88.266 cm. and with aldicarb = 89.53 cm.). The analysis of variance for height in the corn field is reproduced in Appendix A. Oat test weight analysis showed significance in aldicarb application, replication, and cultivar. The presence of aldicarb resulted in a higher test weight (33.3 bu/acre) than where aldicarb was not applied (32.06 bu/acre). Again, this mirrored the situation in the other field, although test weights in the bean field were lower than the corn field (grand means for the two fields were 25.6 lbs/bu and 32.7 lbs/bu, respectively). Average test weights in Tuscola County for 1985 were considerably higher than test weights obtained from experimental plots, as indicated in Table 7. Table 7. Average oat test weights in 1985 vs. experimental test weights obtained from cereal cyst nematode infested fields in Tuscola County, 1985. CORN FIELD BEAN FIELD 1985 CULTIVARS Average No Aldicarb/Aldicarb No Aldicarb/Aldicarb (lbs/bu)* (lbs/bu) (lbs/bu) Heritage 39.6 32.4 33.7 25.4 26.0 Ogle 35.0 31.3 31.6 25.4 28.0 Mariner 36.6 32.3 34.8 25.4 28.0 Mackinaw 36.7 33.9 35.6 23.4 25.1 Korwood 36.6 31.8 33.6 27.0 26.7 * Average test weights are from MSU Extension Bulletin E-899 39 The highest test weights were obtained in the corn field with aldicarb. Aldicarb prevented a reduction in cat test weight, by eliminating the nematode (or other soil microorganisms). Lhasa Wheat grown in the corn field, as in the bean field, showed significant differences in yield over aldicarb application and in yield and height over cultivar . Mean wheat yield without aldicarb was 786.75 g. (26.19 bu/acre) and with aldicarb, 970.375 g. (32.3 bu/acre). Both Sinton and Max yielded significantly better after aldicarb application, indicating that aldicarb prevented the nematode from reducing wheat yields. Combined site interpretation Aldicarb significantly increased yield, height and test weight in oats and yield in wheat, in a combined analysis. This corroborated information found in the previous analyses. In addition, the analysis indicated an increase in wheat test weight with aldicarb application (mean test weight with aldicarb was 57.514 bu/acre compared 53.906 bu/acre without aldicarb). Oat and wheat data were combined over location for these analyses. Barley was only planted in one location, due to lack of space. Results are summarized in Table 8, below. Locations were different, as indicated by highly 40 significant differences for yield, height and test weight in both crops. Table 8. Significant differences in yield, height and test weight from combined analyses of cat and wheat trials in two locations on the Kosik farm, Tuscola County, 1985. OATS WHEAT L R(L) A LXA C L R(L) A LXA C YIELD xx xx x xx xx x xx x xx HEIGHT xx ‘ x xx xx x xx TEST WEIGHT xx xx xx xx xx x xx * Indicates significance at .05 ** Indicates significance at .01 L=Location, R(L)=reps nested in location, A=Aldicarb, C = Cultivar Adjustment to uniform initial cyst count Covariance analyses are used to increase precision by error reduction and adjustment of treatment means. The analysis adjusts for the covariate, an independent variable, and its effects on the dependent variable. One assumption of the covariance linear model is that the covariate is not affected by treatments. In this case, the independent variable, X, is the initial number of viable cysts (those containing eggs and juveniles) from each plot. Pre-plant viable cyst counts 41 were taken from both fields. However, covariance analyses were only run on corn field counts, because viable cyst levels in the bean field were very low. The purpose of the analysis was to adjust dependent variable Y (yield, height and test weight) measurements to a uniform initial viable cyst count. This resulted in standardizing the data, so that analyses of aldicarb effects would be more precise. Essentially, results supported the earlier analyses. F-tests of adjusted treatment means indicated significant increases after for aldicarb treatment in yield, height and test weight in oats and yield and test weight in wheat. Test weight in wheat was significant at .05 probability; all others were significant at .01. Height in wheat, as in the other analyses, was not significant. Covariance, as a combination of analysis of variance with regression analysis, allows calculation of adjusted treatment means using b, the estimate of a common slope, in the equation Adj. Yj = unadj. Yj - b(Xj - X). Table 9 compares the adjusted treatment means for yield, height and test weight in the presence and absence of aldicarb, with unadjusted treatment means. In many cases, the adjustments are slight, indicating that the covariance procedure, while increasing precision, did not result in large changes. A uniform H; avenae infestation, at the low recovery levels from the Kosik farm, would not have changed field results from summer, 1985, that much. Adjustments in wheat were larger than in oats. There 42 were, on average, more cysts per sample in the wheat plots than in the cat plots (2.62 to 1.2, respectively; see Table 1), resulting in a larger covariate adjustment. Table 9. A comparison of adjusted with unadjusted treatment means for presence or absence of aldicarb from analyses of yield, height and test weight of cats and wheat in Tuscola County, 1985. NO ALDICARB ALDICARB Means Means Unadj. Adj. Unadj. Adj. OATS Yield (3) 1219.78 1219.40 1435.12 1435.40 Height (cm) 78.02 77.97 81.04 81.07 Test wt. (8) 227.49 227.47 236.22 236.18 WHEAT Yield (3) 786.75 782.15 970.37 974.95 Test wt. (g) 370.00 368.80 381.50 392.80 Summary and discussion Site B (corn field) data, at higher viable cyst levels, was more valuable and showed significant reduCtions in yield, height and test weight for cats and yield for wheat in the presence of the cereal cyst nematode. An analysis combined over the two locations indicated an increase in test weight in wheat after aldicarb application. A covariance analysis gave the same 43 significant differences as the combined analysis, adding an increase in test weight in wheat after aldicarb application to the differences obtained from the original analyses. This preliminary test indicated that H; avenae could significantly reduce cereal yields in Michigan and resulted in information on the nematode’s effects on small grains. Differences between results from the two fields were striking. For the most part, yields were better, test weights higher and plants shorter in the corn field. More significant differences were obtained from analyses on the corn field plots. Although bird damage precluded getting some information from the bean field., a record of the two fields’ cropping history would have helped to explain some of the differences. Controlled temperature study NEMATODE POPULATION DENSITY Population counts taken from non-sterilized pots in the growth chamber experiment gave a lower old cyst/young cyst ratio than in the field study, indicating more reproduction under continuously cool temperature. (See Table 10.) Numbers of recent cysts with sub-crystalline layers 44 were analyzed as well as numbers of older, empty cysts. In total, from 56 pots (14 replications x 4 unsterilized pots per replication), 427 recent cysts were recovered. From the same number of samples, 192 older cysts were found. No recent cysts were recovered from sterilized soil, although some old cyst remains were present. Table 10. Comparison of H; avenae population density between two experiments: Field trials in Tuscola County, 1985, and growth chamber trials, 1986. FIELD GROWTH CHAMBER TOTAL CYSTS 3030 619 No. of samples 528 56 x per sample 5.74 11.05 OLD CYSTS 2864 192 x per sample 5.42 3.42 YOUNG CYSTS 166 427 x per sample .314 7.62 Old cysts/young cysts 17.25/1 .44/1 The difference between the two experiments is probably due to temperature control in the growth chamber. The cereal cyst nematode reproduces more readily in cool situations. Consistently moist soil probably also favored reproduction. V Analyses of recent cysts are summarized in Table 11 and suggest that cysts were concentrated in bean field 45 soil (350 were found as compared to 77 in corn field soil), and Ogle was a better host than Mackinaw (399 reproduced on Ogle to 28 on Mackinaw). See Table 12. The significant location x cultivar interaction indicates that, while soil from the bean field supported the largest population, it was concentrated on Ogle. In yield and dry weight analysis, Ogle was not more affected than Mackinaw. Mackinaw may be resistant to reproduction by the cyst nematode, and Ogle may be resistant to effects produced by it. Table 11. Significant differences from analysis of cyst counts taken from unsterilized soil in growth chamber experiment, 1986. L C L X C RECENT CYSTS ** ** 8* OLDER CYSTS ** 3* Indicates significance at .00 L = Location C = Cultivar Table 12. Comparison of young cyst counts between soil from two sites used in a growth chamber experiment, 1986. Site A BEAN FIELD Ogle 328 Mackinaw 22 Site B CORN FIELD Ogle 71 Mackinaw 6 46 One hundred thirty-six older cysts were found in bean field soil as compared to 56 in corn field soil. Since these cysts did not reproduce in the growth chamber, it is not surprising that the analysis did not indicate significant differences for cultivar. The bean field soil had more older cysts than the corn field soil, corresponding to field counts. In the summer, however, more reproduction occurred in the corn field, while the Opposite is true of the growth chamber trial. Summary and discussion The growth chamber experiment was useful in order to study the cyst under optimal conditions. Reproduction is favored by cool temperatures. Analysis of young cysts indicated that Ogle may be a better host than Mackinaw, although Ogle’s yield and dry weight were not reduced more than Mackinaw’s. Differences between the two fields were highlighted again by a puzzling production of new cysts in bean field soil. In field trials, more reproduction occurred in the corn field. PATHOGEN EFFECTS Yield and dry weight analyses Soil sterility and location x sterility interactions were both significant for yield and dry weight analyses, as summarized in Table 13. The mean yield of plants in 47 sterilized soil was 1.920 g. while plants in non-sterilized soil averaged a reduced mean yield of 1.158 g. The location x sterility interaction appears to indicate that the same trend is apparent but the difference is more striking in corn field soil. In the yield analysis, location significance indicates that soil from the bean field gave higher yields. A significant location x cultivar interaction seems to indicate that while Ogle yielded better in the bean field, Mackinaw had a slightly better yield in the corn field. Table 13. Significant differences in yield and dry weight analyses from 1986 growth chamber experiment using 2 oat cultivars and 2 soil treatments. YIELD DRY WEIGHT ** ** ** ** 3 indicates significance of at least .05. 3* indicates significance of at least .01 R = Replication, L = Location (field) 8 = Sterile vs. non-sterile soil When dry weight was analyzed, sterile vs. non-sterile soil location x sterility interactions, soil location, and cultivar all showed significant differences. (See Table 13). Dry weight from sterilized soil was 1.614 g. and from non-sterilized soil, 1.224 g, indicating a significant increase in dry matter when the nematode was eliminated. 48 This was true as well for the location x sterility interaction. As with yield, the difference in dry weight between sterilized and non-sterilized soil is more striking in soil from the corn field. Dry weight was higher in soil from the bean field (1.547 g. vs. 1.291 g. for corn field soil). and Mackinaw had more dry matter than Ogle (1.737 g. compared to 1.101 g.) The analyses of variance for dry weight and yield in the growth chamber are in Appendix B. Height analyses No significant differences were found for height data taken after two months. At harvest, height was not affected by soil sterility or lack of sterility. Both tallest head and tallest node height analyses indicated a significant interaction between location and soil sterility. The results are opposite in that non-sterilized soil from the bean field resulted in taller plants while non-sterilized soil from the corn field gave shorter plants. Heading date Heading date was also measured. Soil sterilily or lack of sterility did not seem to affect days to heading in the growth chamber. Summary Results from agronomic data in the growth chamber experiment strongly suggest a relationship between presence 49 of the cereal cyst nematode and reduction in yield and dry weight. Although height and heading date did not show significant differences due to soil sterilization, several location x sterility interactions were significant. Conceivably, differences in cropping history, structure, texture and fertility between the two soils may mask effects of nematode infestation when comparing the two. SUMMARY AND CONCLUSIONS One objective of the study was to look at the cereal cyst nematode’s effects on small grains. We found that significant reductions in yield, test weight, dry weight and height on oats and wheat can be attributed to the nematode. Oats were more susceptible to damage by H; avenae. Barley was unaffected. At Site A, some grain yield was lost to birds. The growth chamber experiment indicated that this was the higher yielding of the two soils, although it was impossible to correlate this with the field study. Test weight in oats in the bean field was less affected by feeding birds, and elimination of the nematode resulted in higher test weights. Wheat yield in the bean field was significantly increased in the absence of H; avenae. At Site B, yield, height and test weight in cats were significantly increased where the nematode had been eliminated. Wheat yield in the corn field also benefited from control of the pathogen. Additionally, in combined and covariance analyses, test weight in wheat was significantly increased where chemical treatment eliminated the nematode. Cyst count analyses indicated few significant differences. More viable cysts were found in the corn field during the summer, but soil taken from the bean field had more nematode reproduction in the growth chamber. 50 51 Factors including late planting, and a dry, warm spring probably limited cyst production in the field. Results from the growth chamber strongly suggest that the cereal cyst nematode reduced yield and dry weight in oats. Height and heading date did not show significant differences due to the nematode. Cysts were concentrated in bean field soil. Ogle was a more effective host than Mackinaw, although cultivar x soil sterility interactions were not significant. H; avenae may reproduce better on Ogle without an increased effect on height, yield, dry weight or heading date. In what circumstances and with which cultivars could the cereal cyst nematode prove the most damaging? The nematode, sensitive to greenhouse culture, is also sensitive to climatic conditions in the field. Cool, wet springs and early summers may significantly increase populations of the cereal cyst nematode. Resistance is probably not present in most commonly grown oat and barley cultivars in Michigan. Kubler (1980) indicates that 10-11 cysts per 200 grams of soil sufficed to cause visible damage. The infestations in Tuscola County are probably at least that serious, and even in the Kosik bean field, where reproduction was limited during the growing season, the nematode decreased test weight in oats and yield in wheat. How is the nematode being disseminated? Survey results indicate that it may be spread on equipment shared by members of the same family in Tuscola County. In other 52 parts of the state, it could arrive on equipment, via wind or water, or on vehicle tires from Canada. Growers in Canada who were plagued by the nematode fifteen years ago controlled it with crop rotation. Rotation into non-cereal crops for several years eliminated Heterodera avenae inoculum in the fields, now planted in wheat and corn. The study was not without its problems. Uneven infestation in the two fields made it difficult to differentiate between true significant differences and cyst distribution. Cropping history was unknown and might have clarified some differences between the two fields. Interactions were especially hard to interpret with only one year’s field data. Two years’ worth could have justified or changed conclusions. Viable cyst counts over the growing season did not decrease in the areas treated with aldicarb, although yield, test weight and height were positively affected by its application. Cyst lrecovery techniques should be improved. The organism proved so difficult to culture that some aspects of the study had to be changed. Biotype identification and discovery of resistance and susceptibility were not possible, nor was establishment of a homogenous culture. In the field, positive effects attributed to elimination of the nematode by aldicarb could have been caused by elimination of some other organism or growth regulating effects of the chemical itself. By using soil sterilization as an alternate means of nematode control in 53 the second part of the study, and producing positive results in the absence of the pathogen, an argument against secondary effects of aldicarb was obtained. LIST OF REFERENCES LIST OF REFERENCES Andersen, S. 1959. 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Kerry, Brian. 1980. Biocontrol: Fungal parasites of female cyst nematodes. J. Nematology 12:253-259. Kerry, B.R., D.H. Crump, and L.A. Mullen. 1982. Studies of the cereal cyst nematode, Heteroderg avenae, under continuous cereals 1975-1978. II Fungal parasitism of nematode females and eggs. Ann. Appol. Biol. 100:489-499. Kort, J. 1972. Cereal cyst nematode, Heterodera avenae. lg John M. Webster (ed.), Economic nematology. Academic Press, Orlando, Florida. 57 Kort, J., G. Dantuma, and A. Van Essen. 1964. On biotypes of the cereal root eelworm (Heterodera avenae) and resistance in cats and barley. Neth. J. Plant Path. 70:9-17. Kubler, E. 1980 [Occurence and damage produced by Heterodera avenae depending on locality and crop rotation] Ger. Abhangig keit von Standort und Fruchfolgestellung. Kali- Briefe 15: 228-231. Kuhn, J. 1874. Uber das vorkomman von ruben-nematoden and der wurzeln der halmfruchte. Landwirts. Jahrb. 3:47-50. Mathur, B.N., H.C. Aiya, R.L. Mathur, and D.K. Handa. 1974. The occurence of biotypes of the cereal cyst nematode (Heterodera avenae) in the light soils of Rajasthan and Haryana, India. Nematologica 20:19-26. Meagher, J.W. 1972. Cereal cyst nematode (Heterodera avenae Woll.) studies on ecology and control in Victoria. Tech. Bull. 24, Dept. Agric. Vict. Meagher, J.W. 1974 a. Cryptobiosis of the cereal cyst nematode (Heterodera avenae) and effects of temperature and relative humidity on survival of eggs in storage. Nematologica 20:323-336. Meagher, J.W. 1974 b. The morphology of the cereal cyst nematode (Heterodera avenae) in Australia. Nematologica Meagher, J.W. 1977. World dissemination of the cereal cyst nematode (Heterodera avenae) and its potential as a pathogen of wheat. J. of Nematology 9:9-15. Meagher, J.W. 1982. The effect of environment on survival and hatching of Heterodera avenae. EPPO Bull. 12:361- 369. Meagher, J.W., R.H. Brown, and A.D. Rovira. 1978. The effects of the cereal cyst nematode (Heterodera avenae) and Rhizoctonia solani on the growth and yield of wheat. Aust. J. Agric. Res. 29:1127-37. Meagher, J.W. and D.R. Rooney. 1966. The effect of crop rotations in the Victorian Wimmera on the cereal cyst nematode (Heterodera avenae), nitrogen fertility and wheat yield. Aust. J. Plant Path. 6:425-431. Neubert, E. 1967. Uber das vorkommen von biotypen des Haferzystenaichens (Heteroderg avenae) im norden der DDR. Nbl. Dtsch. Pflazenschitzd (Berlin) 21:66-68. 58 Nielson, C.H. 1966. Untersuchungen uber die vererbung des resistanz gegen den getreidenematoden (Heterodera avenae) beim weizen. Nematologica 12:575-578. O’Brien, P.C. and J.M. Fisher. 1978. Studies on the mechanism of resistance of wheat to Heterodera avenae. Nematologica 24:463-471. O’Brien, P.C., D.H.B. Sparrow, and J.M. Fisher. 1979. Inheritance of resistance to Heterodera avenae in barley. Nematologica 25:348-352. Putnam, D.F. and L.D. Chapman. 1935. Oat seedling diseases in Ontario. I. The cat nematode Heterodera schachtii Schm. Can. J. Ag. Sci. 15:633-651. Saefkow, M. and E. Lucke. 1979. Entwicklung von Heterodera avenae in maiswurzeln. Nematologica 25:312. Schmidt, 0. 1930. Sind ruben und hafernematoden identisch? Wiss. Archiv. fur Landwirts.:Abteilung A:Archiv. fur Pflanzenbau 3:420-46. Sparrow, D.H.B. and A.J. Dube. 1981. Breeding barley cultivars resistant to cereal cyst nematode in Australia. Barley Genetics IV, Proceedings of the Fourth Annual Barley Genetics Symposium, July,1981. Spaull, A.M. and N.G.M. Hague. 1978. Influence of cereal cultivar on the population dynamics of the cereal cyst nematode, Heterodera avenae. Nematologica 24:376-383. Steele, A. E. 1984. Nematode parasites of sugar beet. Ch. 14 Lg William R. Nickle (ed.), Plant and insect nematodes. Marcel Dekker, Inc. Swarup, R. and G. Swarup. 1985. Dormancy in cereal cyst nematode, Heterodera avenae. Indian Journal of Nematology 14:118-120. Thorne, G. 1961. Principles of nematology. McGraw-Hill, Williams, T.D. and J. Beane. 1979. Temperature and root exudates on the cereal cyst nematode Heteroderg avenae. Nematologica 25: 397-405. Williams, T.D. and M.R. Siddiqi. 1972. Heteroderg avenae. C.I.H. descriptions of plant-parasitic nematodes. Set. 1,No. 2, Ed. Bur. Helminth. St. Albans, England. Wollenweber, H.W. 1924. Zur Kenntnis der Kartoffel Heterogeren. Illus. Landwirts. Zeit. 44: 100-101. APPENDICES APPENDIX A Sample analyses of variance from the field study 1. AOV for test weight bean field oat trial SOURCE DF MEAN SQUARE F PROB Rep 3 615.266 9.51 .048 Aldicarb appn. 1 2173.891 33.61 .010 Error 3 64.682 Cultivar 7 546.712 5.85 .000 Aldicarb x cult. 7 180.462 1.93 .088 Error 42 93.403 x with aldicarb = 187.87 g. x without aldicarb = 176.219 g 2. AOV for yield bean field wheat trial SOURCE DF MEAN SQUARE F PROB Rep 3 9551.563 8.05 .060 Aldicarb appn. 1 19251.563 16.23 .027 Error 3 1186.229 Cultivar 1 175351.563 19.93 .004 Aldicarb x cult. 1 1870.563 0.21 Error 6 8797.563 x with aldicarb = 1132.5 g. x without aldicarb = 1063.125 g. 59 6O 3. AOV for yield corn field oat trial SOURCE DF MEAN SQUARE F PROB Rep 3 5637.682 0.21 Aldicarb appn. l 741966.891 28.01 .013 Error 3 26486.766 Cultivar 7 74283.712 3.41 .005 Aldicarb x cult. 7 24158.533 1.11 .374 Error 42 21764.903 x with aldicarb = 1435.125 g. x without aldicarb = 1219.781 g. 4. AOV for height corn field oat trial SOURCE DF MEAN SQUARE F PROB Rep 3 1.875 1.17 .450 Aldicarb appn. 1 22.563 14.06 .033 Error 3 1.604 Cultivar 7 42.643 34.40 .000 Aldicarb x cult. 7 0.884 0.71 Error 42 1.240 x with aldicarb = 31.9 in. x without aldicarb = 30.72 in. 5. AOV for test weight corn field oat trial SOURCE DF MEAN SQUARE F PROB Rep 3 378.807 15.50 .024 Aldicarb appn. 1 1233.766 50.50 .005 Error 3 24.432 Cultivar 7 835.426 22.34 .000 Aldicarb x cult. 7 53.016 1.42 .223 Error 42 37.394 x with aldicarb = 236.2 g. x without aldicarb = 227.4 g. 61 6. AOV for yield corn field wheat trial SOURCE DF MEAN SQUARE F PROB Rep 3 29076.396 7.79 .062 Aldicarb appn. 1 134872.563 36.11 .009 Error 3 3734.563 Cultivar 1 237412.563 56.55 .000 Aldicarb x cult. 6 10764.063 2.56 .160 Error 6 4198.313 x with aldicarb = 970.375 g. x without aldicarb = 786.750 g. 7. Combined AOV for cat yield over two locations (bean and corn field). SOURCE DF MEAN SQUARE F PROB Location 1 11815268.133 505.40 .000 Rep(location) 6 61642.654 2.64 .131 Aldicarb 1 615079.133 26.31 .002 Loc x aldicarb 1 188267.820 8.05 .029 Error 6 23377.852 Cultivar 7 114899.026 4.50 .000 Loc x cultivar 7 79037.615 3.10 .005 Aldicarb x cult 7 33198.151 1.30 .260 Loc x ald x cult 7 42256.195 1.66 .131 Error 84 25531.366 x with aldicarb = 1092.953 g. x without aldicarb = 954.313 g. 62 8. Combined AOV for wheat test weight over two locations (bean and corn field). SOURCE DF MEAN SQUARE F PROB Location 1 7170.031 24.19 .002 Rep(location) 6 637.865 2.15 .186 Aldicarb 1 2261.281 7.63 .032 Loc x aldicarb 1 225.781 0.76 Error 6 296.448 Cultivar 1 10989.031 22.70 .000 Loc x cultivar 1 2432.531 5.02 .044 Ald x cultivar 1 913.781 1.89 .194 Loc x ald x cul 1 2502.781 5.17 .042 Error 12 484.115 x with aldicarb = 399.125 g. x without aldicarb = 382.313 g. 9. ANCOVA for yield corn field oat trial adjusted for uniform viable cyst distribution at planting. Y adjusted for X SOURCE DF XX XY YY DF SS MS F Total 491.4 Rep 2.56 -77.9 16913 3 Ald/No l .25 430.7 741967 Cult 7 3.94 444.4 74283 A X C 7 1.75 -167.2 24158 Error 45 29.44 -l38.5 48251 44 47588.7 1081.8 A + E 46 29.69 292.1 790218 45 787344 Adj. for test of adj. tmt means 1 739744.3 683.83! 63 10. AOV for total number of cysts per 100 cc. soil at planting for bean field oat trial. SOURCE DF MEAN SQUARE F PROB Replication 3 47.417 4.04 .013 Aldicarb 1 9.00 0.77 Cultivar 7 7.429 0.63 Aldicarb x cult 7 9.857 0.84 Error 37 11.723 Sample analyses of variance from the APPENDIX B controlled temperature study 1. AOV for yield, two oat cultivars SOURCE DF MEAN SQUARE F PROB Replication 13 0.413 1.01 .449 Location 1 1.695 4.14 .044 Cultivar 1 1.082 2.64 .107 Location x cult 1 1.063 2.60 .110 Sterile vs. non 1 16.257 39.70 .000 Loc x sterility 1 4.146 10.13 .002 Var x sterility 1 0.155 0.38 Loc x var x ster 1 0.282 0.69 Error 91 0.410 x for sterilized soil = 1.920 g. x for non-sterilized soil = 1.158 g. 2. AOV for dry weight, two oat cultivars SOURCE DF MEAN SQUARE F PROB Replication 13 0.217 0.83 Location 1 1.827 7.01 .009 Cultivar 1 11.306 43.38 .000 Location x cult 1 0.016 0.06 Sterile vs. mom 1 4.245 16.29 .000 Loc x sterility 1 3.290 12.62 .000 Cult x sterility 1 0.013 0.05 Loc x cult x ster 1 0.755 2.90 .092 Error 91 0.261 x for sterilized soil = 1.614 g. x for non-sterilized soil 1.224 g. 65 3. AOV for recent cysts in unsterilized rhizosphere soil SOURCE DF MEAN SQUARE F PROB Replication 13 67.067 0.99 Location 1 1330.875 19.74 .000 Cultivar 1 2457.875 36.46 .000 Location x cult 1 1037.161 15.38 .000 Error 39 67.419 4. AOV for empty cysts in unsterilized rhizosphere soil SOURCE DF MEAN SQUARE F PROB Replication 13 4.632 0.85 Location 1 114.286 21.07 .000 Cultivar 1 1.143 ' 0.21 Location x cult 1 4.571 0.84 Error 39 5.423 Mllilllellilrlllllllilil HillilfilllilliIlilllsllilllilfis 3 1293 030613750