WWI WWNHINIIIIWHIHJHIIIWJ H ill RES‘STM‘ICE C“: 321,,er ' ' WE}; L LU‘EF EEEVLE 22:22 22.. 222222222222 22222.; rfl n 3 afPh.3. F2'ZECHK‘2522‘2 EST ATE ‘ 222222.222: SANG KIHAHN 1967 THESIS ’J This is to certify that the thesis entitled RESISTANCE OF HARLEY 'ro CEREAL IEAF BEE'FLE («mam MELANOPUSL.) presented by SangKiHahn has been accepted towards fulfillment of the requirements for __PL‘P_degree in_Cr92 30161100 0-169 LIBRARY Michigan State University ABSTRACT RESISTANCE OF BARLEY TO CEREAL LEAF BEETLE (OULEMA MELANOPUS L.) by Sang Ki Hahn The cereal leaf beetle (Oulema melanopus L.), a new insect pest, was first identified in North America at Galien, Michigan in 1962, and since then has shown a great potential to damage the small grains. Shortly after the identification of the cereal leaf beetle in 1962, the search for and identification of host resistance of barley was initiated. On the basis of the field and laboratory screening results, eight parental lines were selected to make a diallel cross.set in order to investigate the genetic basis of resistance. In addition, a back-cross with Larker2 X CI 6671 was made. The laboratory larval test for resistance was con- ducted for the F1 from the six parental diallel cross set and the field test was carried out for the F2 progenies of the eight parental diallel cross and of the back—cross. The resistance to cereal leaf beetle in barley appears to be recessive. The resistance mechanism associated with cereal leaf beetle in barley seems to be both due to nonpreference of the barley plant by feeding larvae and differential egg laying. Sang Ki Hahn The most resistant combination from the eight parental diallel cross was CI 667l.XCI 6U69. Trans- gressive inheritance was found in the cross of the two lines which indicates the possibility of obtaining higher resistance. RESISTANCE OF BARLEY TO CEREAL LEAF BEETLE (OULEMA MELANOPUS L.) By Sang Ki Hahn A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop Science 1967 ACKNOWLEDGMENTS The author wishes to express his sincere appreci- ation to Dr. John E. Grafius for his encouragement, ad- vice, and guidance in this research and for his helpful advice in the preparation of the manuscript. The author is also very grateful to Dr. Carter M. Harrison for his kindness in thoroughly reading the manuscript and for his deep understanding. Further adknowledgment is ex- pressed to Dr. David H. Smith and Dr. John A. Schillinger for their assistance in the laboratory and field studies. 11 TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . 11 LIST OF TABLES . . . . . . . . . . . . iv LIST OF FIGURES . . . . . . . . . . . . v1 INTRODUCTION . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . 2 MATERIALS AND METHODS . . . . . . . . . . 7 RESULTS . . . . . . . . . . . . . . . l 12 DISCUSSION. . . . . . . . . . . . . . 33 SUMMARY. . . . . . . . . . . . . . . 37 LITERATURE CITED. . . . . . . . . . . . 39 iii Table 10. 11. LIST OF TABLES Parents Used for the Diallel Cross . Analysis of Variance for Larval Feeding Damage from the Laboratory Test; Six Parental Diallel Cross, Fl Larval Feeding Damage Score from the Laboratory Test, Total of Three Repli- cations; Six Parental Diallel Cross, F Analysis of Variance for Larval Feeding Damage Prior to Heading from the Field Test; Eight Parental Diallel Cross, F2 Larval Feeding Damage Score Prior to Head— ing from the Field Test, Total of Two Replications; Eight Parental Diallel Cross, F2. . . . . . . Analysis of Variance for Larval Feeding Damage After Heading from the Field Test; Eight Parental Diallel Cross, F2 Larval Feeding Damage Score After Heading from the Field Test, Total of Two Replications; Eight Parental Diallel Cross, F2. . . . . . . Analysis of Variance for Larval Feeding Damage Prior to and After Headin from the Field Test; Backcross Larker X CI 6671, F2.. . . . . . . . . . Mean Larval Feeding Damage Between Glossy and Normal Progenies . . . . . Xz-Test for Normal to Glossy, 3:1. Number of Larvae per Plant and Larval Feeding Damage Score; Eight Parental Diallel Cross, F2 . . . . iv 1' Page 1a 16 18 2O 22 23 25 29 29 31 Table Page 12. Analysis of Variance for Number of Larvae on Ten Plants Prior to Heading from the Field Test; Eight Parental Diallel Cross, F2 . 32 LIST OF FIGURES Figure l. A Diallel Graph for Larval Feeding Damage in the Laboratory Test; Six Parental Diallel Cross, Fl . . . . . 2. A Diallel Graph for Larval Feeding Damage Prior to Heading in the Field Test; Eight Parental Diallel Cross, F2 3. A Diallel Graph for Larval Feeding Damage After Heading in the Field Test; Eight Parental Diallel Cross, F2 A. The Most Resistant F2 Progeny Resulting from the Cross, CI 6671 X CI 6469. 5. A Histogram of Distribution of Feeding“ Damage Scores for She F Backcross Pro- genies from Larker X C 6671; Before Heading and After Heading . . . . vi Page 17 21 2M 26 28 INTRODUCTION The cereal leaf beetle, Oulema melanopus L., a newly introduced insect pest, has shown a great potential to damage small grain production in the United States. This insect was first identified in North America at Galien, Michigan in 1962 and since then, has spread throughout most of Michigan, Indiana, and Ohio (2, 3). Both adults and larvae of the beetle greatly damage the small grains, with the greatest damage to cats follow- ed by barley and wheat. Shortly after the identification of this insect, an extensive screening program was initiated to search for host plant resistance in the small grains. World col- lections of small grains have been tested both in the field nursery and in the laboratory by the United States Department of Agriculture, Michigan State University and Purdue University. Highly resistant wheat strains were found but only some moderately resistant barley strains were obtained. There have been no studies on the genetics of resis- tance to this new insect pest in barley. This study is an attempt to find the genetic basis of resistance of the barley plant to the cereal leaf beetle. REVIEW OF LITERATURE The cereal leaf beetle has been a pest of small grains in Europe for many years, occurring throughout the humid‘and subhumid areas of the entire Western Paleo- arctic region including Norway, Central Siberia, North Africa, the entire Mediterranean Basin and the Canary and Madeira Islands (cf. 2, 3). The biology regarding the life cycle, seasonal appearance and habits of the cereal leaf beetle has been discussed by Ruppel (11) and Castro, Ruppel and Gomulinski (2). Gallun and Ruppel (4) reported the field screening results of adult and larval feeding damage in Michigan in 1962-1963 for small grains including wheat, oats and barley. The wheats with highly pubescent leaves were largely avoided for oviposition. Gallun, Everson, Ruppel and Craddock (5) continued the field studies to evaluate host plant resis— tance of cereal crops in 1963-1964. They reported the re- sults of larval feeding damage for 16,095 wheat, 5,h23 cat and 8,63u barley strains totaling 30,152 of which, approximately 12 percent of the wheats, A percent of oats and less than 1 percent of the barleys had zero to a trace of feeding and rated as resistant. The report of the continued studies in 1964-1965 were made by Schillinger, Gallun, Everson, Smith and Cradock (12). No cats or barley entries were found to possess as high a level of resistance as wheat but some were less preferred as hosts. After four years of successive field tests under various conditions, Schillinger, Smith and Cradock (15) selected-many of the highly resistant lines of wheat and only a few of spring barley. Among the several resistant spring barley lines, two of them, CI 6671 and CI 6A69, have shown the most resistance with 15 to no percent of foliage damage. No winter barley lines were found with more resistance than the spring types. Resistance is a relative aspect which arises from the relationships between insect and plant. Beck (1) divided the relationships into two principal aspects: (1) host selection by the insect; and (ii) resistance to the insect by the plant. Painter (10) defined the resis- tance of plant to insect as "the relative amount of heritable qualities possessed by a plant which influence the utlimate degree of damage done by the insect." Recently Beck (1) employed a slightly different definition that "plant resistance is the collective heritable character- istics by which a plant species, race, clone, or indivi— dual may reduce the probability of successful utilization of that plant as a host by an insect species." Painter (9,10) divided plant resistance mechanisms into three main categories: (i) preference and nonpreference: in which a plant displays a degree of resistance by exerting an adverse effect on an insects behavior; (11) antibiosis: in which a plant is resistant by exerting an adverse influence on the growth and survival of the insect; and (iii) toler- ance: in which a plant is capable of supporting an insect population without loss of vigor and without reduction of crop yield. While, Beck (1) dropped the tolerance from Painter's three main categories and classified the mechan- isms into Just "non-preference and antibiosis," because tolerance is an important agronomic plant characteristic and it implies a biological relationship between insect. and plant that is quite different from resistance in the strict sense. According to Gallun and Ruppel (A) and Schillinger (14), plant resistance of wheat to the cereal leaf beetle is primarily associated with nonpreference for oviposition by the adult due to the hairness of the leaf surface. Schillinger (14), working with wheat, has shown that all highly resistant Triticum dicoccum accessions were pubescent but pubescent leaf surface per se, is not the sole factor in determining resistance. However, a barley with pubes- cence like that in wheat is not known and resistance in barley involves a different mechanism from that of pubes- cence (15). Schillinger, Gallun, Everson, Smith and Craddock (l2) pointed out that resistance to the cereal leaf beetle in small grains seems to be complicated by the stage of physio- logical development, type of vegetative growth and disease susceptibility of the plant. Wilson and Shade (16) showed that preference for oviposition differed with the advances of growth stages of the host plant. Schillinger, Gallun, Everson, Smith and Craddock (12) reported that when pubescent winter wheats were grown in the spring nursery, the resistance was dissipated. Schillinger (13) stated that winter wheat varieties which appeared immune to beetle attack in a fall-planted field nursery were susceptible when tested as non-vernalized seedlings in the laboratory. He also stated that the resistance was greatly influenced by environmental vari- ations and by instability of the adult beetle population under field nursery conditions. Gallun, Ruppel and Everson (6) pointed out that damage from adult feeding is very little compared to that from larval feeding in the spring unless the number of beetles is extremely high. Everson, Gallun, Schillinger, Smith and Craddock (3) also reported that the most severe feeding damage in the field is influenced by the larval stage. Schillinger (14), working with wheat, has shown that the resistance reaction in-the field nursery was highly consistent with the resistance to larval damage in the laboratory. Thus, the larval test clarified and substantiated the leaf damage ratings of resistant re- actions to the cereal leaf beetle that were obtained in the field nursery. Gallun, Ruppel and Everson (6) stated that larval preference will influence the amount of damage per plant because larvae tend to migrate from leaf to leaf, seek- ing a preferred food, and the resulting feeding on any one plant could be slight. Everson, Gallun, Schillinger, Smith and Craddock (3) proposed that the primary center of resistant germ plasm of wheat seemed to be the large continuous area of Asia, Asia Minor and Eurasia. MATERIALS AND METHODS Shortly after the identification of the cereal leaf beetle in 1962, the search for and identification of host resistance of barley was initiated. These tests have been conducted cooperatively by entomologists, plant geneticists, and agronomists of Michigan State University, Purdue Uni- versity and the United States Department of Agriculture. On the basis of the field and laboratory screening results, eight parental lines were selected to make a diallel cross set. They are given in Table 1. Among the eight lines, CI 6U69 and CI 6671 were used as resistant parents and All—l, CI 10001 and CI 10968 were used as susceptible ones. The materials for study consisted of 28 F1 and F2 plants from all possible crosses of eight barley lines. Crosses were made in the growth chamber during the summer of 1965. Part of the F1 seeds from each combination were grown in a growth chamber to obtain F2 seeds for field examination. The remaining Fl seeds of each combination were saved for laboratory tests. In addition a back- cross with Larker2 X CI 6671 was made in the field and the F2 progenies from each Fl plant were run in a field test. cam nwson .309 m “mama manfipamomsm wcfinam .¢ .m V: mmmoa Ho m cam spoosm .309 m .manmm manfipaoomsm mafinam mpmcmo Hoooa Ho n can nmson asap .309 m .manmm_pmoz imammm.pmoz wcfindm cmnH Hmmm Ho 0 czm nuoosm 30p 0 .mpmHUmEnoch pampmfimmm mcfipam pcmaom mozm Ho m czm Beacon .onwc .SOE m .zasmm opmnmooz wcfimdm manowa< pmom Ho 3 23m nwzoa .3on m “mung mpmnopoz wcfipam mfiamnpmz< Hmoa Ho m can zwzop .zop m .zammm mpmnmooz weandm macammmn< mmm Ho m can zwson . .zop m .mpfipspme CH coma oanaugmomsm nopqfiz .Hmm .noaz HIHH: H mpmpomsmno maummm on panwm mopsom pcopmm nonssz mocmpmamom £93090 Hmpcmnmm .mmomo HmHHMHp one how omms mpcmnmmln.a mqm D) it intersects on the negative side. In the absence of non—allelic interaction the array vari- ance (Vr) and array covariances (wr) are: ll‘ili l2 s: ' 2 Vr z ui vi (di : hi) wr = z 291 vi d1 (d1 1 h 1) Where negative the signs correspond to positive alleles th in the r parent and vice versa. The points on the (Wr, Vr) graph lie in order of dominance along the straight line from the complete dominant with minimum Vr = z ”i 1 (d1 1 V1 d1 (d1 ' hi) . = 2 to complete receSSive with maximum Vr Z ui vi (di + hi) v - hi)2 and WP = 2 2u and Wr = 22hfi.vi d1 (d1 + hi>’ where ui and vi are the frequency of dominant genes and recessive genes, re— spectively. And di is the additive genetic effect and hi is the dominant effect. On the (Wr, Vr) graph, array points of the regression line depict the dominance order of the parents, and the distance between points provides a measure of the effective— ness of the dominant and recessive alleles of the extreme genotypes. The above conclusions are reliable, provided the following restrictions hold for the material under study: (1) homozygous parents, (ii) no multiple allelism, (iii) genes independently distributed in the parents, and (iv) no genic interaction. Failure of the hypotheses is indicated by a non- significant regression or when the regression is signifi- cantly different from a slope of unity. Non-significance l3 of regression may also arise if all h1 = 0. Test of significance of regression of Wr on Vr was done by the t test using the formulae: (a) t = 9 Sb Sb Where, 82b = Eiléi, and the apprOpriate degree of freedom is (r — 2), r Eging the number of arrays. When regression coefficient is significantly different from zero, dominance is present and when b is significantly different from 1, it indicates-that gene interaction plays a part in determining the control of the characters examined. RESULTS The intent of this study was to obtain genetic information on the resistance to the cereal leaf beetle among selected barley lines. These lines are discussed first on the basis of the results obtained from the laboratory and field larval feeding tests for the F1 and F diallel cross and secondly on the basis of the field 2 feeding test for the F2backcross progenies. The first step in the diallel analysis was to test the variability of parents and hybrids for larval feeding damage scores. The results of analysis of variance for larval feeding scores from the laboratory test on F1 of six parental diallel cross is given in Table 2. There TABLE 2.-—Analysis of variance for larval feeding damage from the laboratory test; six parental diallel cross, F 1' Degree of Mean Source Freedom~ Square F Total 62 -- --— Reps. 2 5.32_ l6.63** Lins 20 9.72 30.38** Error 40 0.32 **Significant at the 1 percent level. 14 15 are highly significant differences among hybrids. The means of larval feeding damage in the F1 larval test range from 2.5 to 5.3. This indicates that among barley strains there may be some difference in resistance to cereal leaf beetle which might be due to differences in genetic background. Having found significant differences among parents and hybrids in resistance to larval feeding, the genetic relationship among parents and their diallel cross was investigated using the Jinks-Hayman's diallel analysis and graphical analysis based on array variances and co- variances. In the analysis of the F1’ laboratory data of three replications were pooled and subjected to the diallel analysis. The pooled Fl data of laboratory larval feeding scores from the 6 x 6 diallel cross set are given in Table 3, where each figure is a total of F1 values of three replicates for each combination. The variances and covariances of arrays are presented in the right hand column of the table. Using the statistics V Wr, and: r, Vp, the (Wr, Vr) graph was drawn and the limiting parabola. was constructed using the formula Wr2 = Vr V The p' graphical analysis provides the degree of dominance, domi- nance order of parents, and additional information about the genetic relationship among the parents. The graphical analysis is shown in Figure 1. Inspection of Figure 1 shows that analysis of the data for all arrays gives an l6 mmmo.o maeo.o m.:H o.mH 0.:H o.mH o.mH 0.:H mowed Ho m mmma.o ommo.o 0.:H o.ma 0.:H 0.:H o.mH HOOOH Ho A mmme.o mmme.o m.e o.MH m.mH o.mH Heme Ho m meH.o mama.o 0.:H o.mfl 0.0H @030 HO m :omm.o mmma.o o.mH o.mH emom Ho : oooa.ou mmma.o 0.:H Hmoa Ho m p3 E> m w m m z m unmhmm HMNMMMMm .H m .mmono Hoaamfiv Hancosma xfim mmcoapmofiaaop oopcp mo HmpOp .pmmp mechanonmd one 809% opoom omemo mcfipmmm Hm>pmq22.m mqm Hmpcmhmm AH m .mmono Hoaamfin Hmpconma xfim meOHmeHadms omen» mo HmpOB .pmop >g0pmponma on» 509% whoom mmemp mcflvmom Hm>pmqll.m mqm m A m m z m m a pemnmm Hmwmwmmm .mm .mmopo Hoaamfip Hmpcopmd pnwfio meOHpmoaaamp 03p mo Hence .pmmp vamam on» 80pm wzfipmmn o» EOAEQ whoom mwmemp wcfipmmm Hm>pmqnl.m mqm A m m a m m H Esteem Hmwmwmmm .mm “30.3 HmHHMHp Hmpcmnma pnwfio meOHmeHaaon 03» Mo HMpOp .pmmp pHmHm on» Eonm wzfipmmh Empmm choom ommsmp mcfipmom Hm>pmq22.A mqm<9 24 9-0.3060 + o.5923** 1 0.2256 Figure 3.-—A diallel graph for larval feeding damage after heading in the field test; 8 parental diallel cross, F2. 25 cross CI 6671 x CI 6469. This combination resulted in the most highly resistant strain among all the barley entries tested in the field nursery in 1966. The picture is presented in Figure 4. The 60 F2 progenies from the backcross Larker2 X CI 6671 were tested in the field nursery with two replications. Strain CI 6671 was used as the resistant parent. In the field test, two entries for each parent and one entry for each progeny were included. Notes concerning feeding damage were taken at two different times, before heading and after heading. The analysis for the data of larval feeding scores from the F2 backcross progenies are given in Table 8. TABLE 8. Analysis of variance for larval feeding damage prior to and after heading from the field test; backcross Larker2 x CI 6671, F 2. Mean Square F Source Degree of Freedom Prior to After Prior to After Heading Heading Heading Heading Total 127 Blocks 1 46.32 36.120 54.49** 70.34** Lines 63 1.506 1.194 1.77* 2.34* Error 63 0.850 0.510 *Significant at 5 percent level. **Significant at 1 percent level. llll lllll2lflhl‘ f IliI‘II‘ [I l 26 Figure 4.——The most resistant F progeny resulting from the cross, CI 6671 X CI 6469. 27 There were significant differences among the progenies in resistance to larval feeding under field conditions. Comparing the feeding damage of progenies with that of their parents, there was remarkable segregation in resistance to the cereal leaf beetle. Even the parental difference was not as distinctive. In the observations made prior to heading, the progenies showed variation in resistance ranging from the least damage 4.5 to the most 8.5, when compared to their parents: 5.8 for Larker and 4.5 for CI 6671. For the observation made after heading, the variation was from 6.8 to 9.5, compared to 8.4 and 6.4 for their parents, Larker and CI 6671. The histogram of the distribution of the feeding damage scores is shown in Figure 5. When the backcross is made, there is a tendency to move to the side of the recurrent parent Larker which is susceptible to the beetle. The tendency is much more noticeable after heading when a great number of older larvae have migrated into the nursery. This again indicates that the resistance to cereal leaf beetle is recessive. Resistant strain CI 6671 has a glossy leaf character. The problem is, whether or not the glossy character is related to cereal leaf beetle resistance. The comparisons of means of larval feeding between the progenies segregating with glossy character and the homozygous normal ones are given in Table 9. 28 15 "E —( 10 ~- CI 6671 = 4.5 Larker = 5.8 Mean = 6.2 5 V r _—_——F O 15 2— C1 6671 = 6.4 Larker = 8.4 Mean = 8.1 10 ~ 51" 0 L—#~ ! i 5 E 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 .0 Damage Figure 5.-—A histogram of distribution of feeding damage scores for the F2 backcross progenies from Larker2 Upper: before heading. Lower: x CI 6671. after heading. 29 TABLE 9.——Mean larval feeding damage between glossy and normal progenies. Mean Damage Number of Character Lines Before Heading After Heading Glossy 25 12.42 16.04 Normal 35 12.24 16.14 Before and after heading, there were no differences in feeding damage between the progenies segregating with the glossy character and the homozygous normal ones. Hence, it could be concluded that the glossy character is not associated with resistance to cereal leaf beetle in the cross. The segregation ratio of normal to glossy was checked as 3 to 1, suggesting that a single recessive gene is governing the glossy character. The test result is given in Table 10. TABLE 10.-—X2-test for normal to glossy, 3:1. Number of Seedlings 2* Character From Segregating Lines X Glossy 113 l.08n.s. Normal 380 * 2 = X 005,1 3.84. n.s.: nonsignificant 30 From this backcross progeny test, it was observed that the lines 201-2-11 and 201-2-18 were most resistant to feeding damage from the cereal leaf beetle. Correlations between damage scores from the laboratory test and those from the field test failed to show signifi- cance prior to or after heading. The correlation coeffici- ent for before heading was r = 0.32 and that for after heading was r = 0.36. These correlations were calculated using the progeny values of the larval feeding scores from six parental diallel cross. In order to examine whether there was any relation between the number of larvae and larval feeding damage, correlation coefficients were calculated for both obser— vations. The observed data of number of larvae per plant and larval feeding damage for each combination are compared in Table 11. The correlation coefficient for before head- ing was r = 0.64 and that for after heading was r = 0.71. Both correlation coefficients are highly significant. These correlation coefficients were calculated from the field nursery data before and after heading of eight parental diallel cross. This indicates that damage in the barley line is proportional to the number of 1arvae--pro- viding there are no significant differences among the cause and further suggests that there are differences in preference of larval feeding in barley. 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