Freising, Germany
Bayerische Landesanstalt für Bodenkultur und Pflanzenbau
Evaluation of international resistance genes against the European corn borer under Central European conditions --Papst, C, Götze, S, Eder, J The European corn borer (ECB, Ostrinia nubilalis) is a worldwide major pest of maize. Populations with up to four generations per year are observed ( 2001) in the U.S. corn belt, in contrast to Central Europe, where only one generation per year is usual. Depending on the stage of development of the plant at infestation time, more or less extensive yield reductions are reported. The first generation mainly feeds on the leaves of young plants, the following generations cause stalk breakage and dropped ears. If only one generation appears, in case of shorter growing seasons like in Germany, the plants are mostly damaged by tunneling in stalks and ears. Here the neonate larvae feed only a short time during the first two stages of development (early to mid-July) on the leaves before they begin tunneling. In most cases, the larvae feed from the tassel down to the stem base, where they overwinter as the fifth larval stage (L5) and in spring they pupate. The flight of the moths and egg-laying is observed from mid-June to mid-July.

In the most important maize growing regions, like in the Middle West of the U.S.A. and in Mexico, material with increased resistance to ECB is being developed. Particularly in the case of a resistance to the second generation, this material could be useful for European maize breeding as a source of resistance. But in most cases this material needs far longer growing-seasons than central European material. To determine the resistance of international material under German conditions, experiments with populations and inbred lines were carried out for the present study.

Germplasm. For resistance examination, 18 (1999) to 34 (2000) lines and populations from the U.S.A., Mexico and South Africa were cultivated (Table 1). To compare the resistance and agronomic traits with adapted European material, the two inbred lines D06 und D408 were evaluated in 2000. D06 is regarded as resistant, and D408 as susceptible (Melchinger, AE et al., Euphytica 99:115-125, 1998). The populations 590, 590B, 591 and G1 IST were provided by CIMMYT (Mexico). They have been selected for resistance to the first generation of ECB, and in the case of population 591, also for resistance to the second generation of larvae (Bergvinson, D, unpublished, 1999). In 2000, selfings from these populations were tested and signed with "S" and the respective population number. Diverse MBR/MDR lines (multiple borer resistance/multiple disease resistance) also derive from the populations described above by self-pollination. Material from South Africa has come out of a backcross program of various selections with one experimental line from North American material (verbal information, van Rensburg 1999). The lines Mo45, Mo46 and Mo47 (Barry, BD et al., Crop Science 35:1232-1233, 1995), as well as the Mo-2 ECB population from Missouri is adapted to the climatic conditions in the southern part of the U.S. "Corn Belt" and carries resistance genes to the second generation of ECB. The Mo-2 ECB-2 population is derived from the "Nigerian Composite B" material by a recurrent selection for resistance to the second generation of ECB. Material from Iowa (Hallauer, AR, Crop Science 37:1405-1406, 1997) is reported to be resistant to the first generation of ECB.

In the year 1999, the lines and populations were tested in one-row plots of 23 plants and without replication. The plot size was 4.0 m x 0.75 m with a planting density of 8 plants/m2. In 2000, the variants were evaluated in a lattice design with two replications in one-row plots of 4.0 m x 0.75 m and 25 plants. The trials were overplanted and later thinned to the necessary plant density of 8 plants/m2. Because of extremely late maturation of the foreign genotypes in 1999, the trial was covered with a perforated transparent polyethylene foliage for three weeks after sowing in 2000.

For evaluating the level of resistance against ECB, every plant was manually infested at three times in weekly intervals with 20 neonate ECB larvae. The date of application was synchronized with the natural flight of the ECB. For the infestation freshly hatched larvae were mixed with maize-cob grits and placed into the whorl or leaf collar of maize plants with a dispenser (Mihm, JA, CIMMYT, Mexico, D.F., Mexico, 1983). In both years of experimentation, the plants were split longitudinally from the stem base to the tassel at harvesting time in late October to record infestation level and the number of larvae alive (LA). From the number of infested plants per plot, the relative infestation frequency (RIF) was calculated referring to 100 plants. Additionally, in 2000, the stalk damage rating (SDR) was evaluated based on a 1 to 9 scale (1 for intact plant, 9 for dropped ears or breakage below the ear, Hudon, M and Chang, MS, Maydica 36:69-70, 1991).

As agronomic traits, date of anthesis in days after sowing (DA) and plant height (PH) were recorded in both years, and dry matter content of the ears (DME) was determined from 3 ears per genotype in the year 2000.

The two European dent lines had an RIF of 100%, whereas the non-European genotypes were infested in 1999 between 0 and 94%, and in 2000 between 8.3 and 98%. The highest RIF of more than 64% showed the genotypes from Missouri and Iowa, except Mo47 with a relatively low RIF in the year 2000 (Fig. 1). The populations, their progenies as well as the lines from Mexico and South Africa achieved a RIF of less than 64%. The analysis of variance showed in 2000 highly significant differences among the genotypes (p * 0.01; r2 = 15.2%).

In the established European dent lines D06 and D408, 155 and 77, respectively larvae could survive per 100 plants in 2000. Whereas in 1999 only between 0 and 29 larvae and in 2000 between 0 and 78 larvae per 100 plants could survive in the international genotypes. The analysis of variance showed highly significant differences (p * 0.01; r2 = 17.6 %) in 2000. The highest amounts of larvae (52 - 78 larvae/100 plants) were found in two genotypes from Iowa and Missouri, the lowest number in the genotypes from Mexico. The only exception was Mo47 from Missouri with 6 larvae per 100 plants.

The European lines D06 and D408 showed a SDR of 2.2 and 3.8, respectively, whereas the SDR of the foreign genotypes varied between 1.0 and 3.0 (p * 0.05; r2 = 0.4). Populations from Mexico and lines from South Africa showed no stalk damage, whereas only lines from Iowa and Missouri had higher SDR levels (Fig. 2).

For the European dent lines D06 and D408, the DA was after 78 and 76 days, using a foliage for three weeks after sowing. In contrast the DA of the foreign genotypes was much later between 99 and 116 days after sowing (Table 1).

In the year 2000, the DME was 57.1% and 55.5 %, respectively, for lines D06 and D408. In contrast to this, the DME of 19 genotypes mostly originating from Mexico and South Africa was below 40 %. Ten genotypes mainly from Missouri and Iowa - reached between 40 % and 50 % and only four lines (SM42-7; Mo2-ECB, Mo2-ECB2 and B106) had a DME of 50 % or more (Table 1) and, thus, were comparable to the adapted European lines.

PH of the European lines D06 and D408 was 169 cm and 166 cm, respectively, whereas the foreign genotypes had a PH between 140 cm and 328 cm.

Highly significant correlations among the resistance characteristics were observed in the present study (Table 2). The resistance traits RIF, LA, and SDR showed a positive correlation (r = 0.5 - 0.9; p * 0.01) in both years. In 2000 the correlation between RIF, LA, and SDR was highly significant (r = 0.5 and 0.6), so that in the future probably the resistance could only be evaluated by SDR instead of the more labour intensive traits RIF and LA.

A high RIF could be observed in genotypes of Iowa and Missouri. These lines also showed a higher SDR in our evaluation and had the highest number of LA. In contrast the populations and lines from Mexico, had a very low RIF and in most cases no or only very small SDR. The European lines had a substantially higher RIF than all the other genotypes. For these, a very high number of LA and a relatively high SDR were found. Even the line D06, classified as resistant, had a SDR of 2.2, and was with one exception (Line B102 from Iowa) not exceeded by the other genotypes. Already in an earlier study with European material (Melchinger et al., Euphytica 99:115-125, 1998), relatively high SDRs between 2.5 and 7.3 were observed. Many of the genotypes examined in the present study showed an essentially lower infestation level than the established material, and thus, are very promising as probable sources of resistance genes.

A positive relationship between resistance traits and DA as well as maturity (DME) could be observed. Also in earlier studies (Russel, Crop Science 14:725-727, 1974; Kreps, Vortr. Pflanzenzüchtung 38:171-186, 1997), a positive correlation between late flowering time and increased resistance (lower SDR, AL, and RIF) to ECB has been found. Hudon, M and Chang, MS (Maydica 36:69-70, 1991) explained this relationship by an improved stalk stability of late maturing germplasm at harvesting time. For the non-adapted genotypes the DA was on average about 30 days later than for the European dent lines D06 and D408. Because of the late flowering also a bad maturation and therefore low DME, sometimes below 50 %, was observed. As the correlation analysis showed, the more susceptible lines from Iowa and Missouri were quite early in flowering (DA: 99 to 109 days after sowing), whereas the resistant populations from Mexico were later (DA: 110 to 118 days after sowing). So it could be supposed that the extraordinary "resistance potential" of the populations is not only based on genetic tolerance and antibiosis, but also is a consequence of late maturity. To study the influence of late maturity on the ECB resistance, and to use these genotypes commercially the material has to be adapted to Central European climatic conditions. Only a few of the genotypes examined (B106, Mo2-ECB2, Mo2-ECB and SM42.P28-7) reached a DME between 50 and 60% in late October and, thus, are near to the European breeding material in maturity. These genotypes appear to be the most suitable for further studies and practical breeding efforts.

Besides maturity, the PH as a further agronomic trait could also play a role in resistance. The negative correlation, observed between the PH and resistance, has already been described by Schulz, B et al. (Plant Breeding 116:415-422, 1997) and by Magg, T et al. (Plant Breeding 120:397-403, 2000). They showed that bigger genotypes mostly had a lower SDR and RIF. Possibly an increased stem stability of big plants is responsible for a smaller SDR. Additionally high lignin or phenol levels in higher plants are reported (Bergvinson, D et al., Insect resistant maize, CIMMYT, 1994) to have on the one hand an influence on the plant stability, and on the other hand a negative effect on the development and survival rate of larvae. Kreps, R et al. (Vortr. Pflanzenzüchtung 38:171-186, 1997) describe a "diluting effect" on the larvae by the increased PH, whereby the damage is minimized. But even if a better stalk stability is desirable for a lower SDR, for silage use the digestability should not be disregarded. As t is shown in Bohn, M et al. (TAG 101:907-917, 2000) the in vitro digestability of the organic matter (IVDOM) is negatively correlated with resistance. By splitting the stems for the resistance traits, we observed that the very tall and resistant populations of Mexico just had a great stalk stability in contrast to other lines. Therefore the digestibility of the plants is also to be evaluated for further ECB trials.

The non-European genetic material showed a very low susceptibility to the ECB that could not be reached by most of the Central European lines (see Melchinger, AE et al., Euphytica 99:115-125, 1998). Especially with regard to controversial discussions about transgenic maize and to the increasing importance of the European corn borer in Central Europe, the use of conventional sources of resistance genes is of great importance. Therefore marker assisted selection is promising to avoid high costs for manual infection and evaluation. Because breeding in Central Europe requires early material, above all the earlier flowering genotypes from Missouri and Iowa are of great interest. In these lines the high level of resistance is not only a consequence of late maturity. Nevertheless further breeding processes are necessary to improve the yield characteristics of this genetical material and to eliminate the effect of late maturity on the ECB resistance. The next step will be the selection for early-flowering individuals from the most promising sources, as well as a backcross program with established German material. Because ECB resistance is a quantitative trait, a lot of QTL (quantitative trait loci) studies for certain adapted populations have already been done (Groh S et al., Plant Breeding 117:193-202, 1998; Jampatong, C, Maize Genetics Conference Abstracts 40 (, 1998; Bohn M et al., J Econ Entomol 92:1892-1902, 2000), so the combination of different gene pools and their QTL could be of great importance for further genetic analyses and the improvement of the European breeding material.

Acknowledgement. Thanks to our colleague M. Walser for her excellent assistance in the course of the implementation of the experiments and the working-up of the data, as well as L. Schleicher for the helpful translation of the manuscript. We also thank our colleagues from the University Hohenheim-Stuttgart for the provision of know-how and equipment for raising the larvae and their application.

Figure 1. Comparison of relative infestation frequency in % of 1999 and 2000.

Figure 2. Stalk damage rating in 2000, according to a 1-9 rating scale of Hudon et al. (Maydica 36:69-70, 1991)(1=intact plant, 9=stalk damage below the ear or dropped ears).

Table 1: Overview of the evaluated genotypes and results of our study

Table 2: Correlations among agronomic and resistance traits

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