A third type of resistance of codling moth against Cydia pomonella granulovirus (CpGV) shows a mixture of a Z-linked and autosomal inheritance pattern

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1 EM ccepted Manuscript Posted Online 30 June 2017 ppl. Environ. Microbiol. doi: /em Copyright 2017 merican Society for Microbiology. ll Rights Reserved. 1 2 third type of resistance of codling moth against Cydia pomonella granulovirus (CpGV) shows a mixture of a -linked and autosomal inheritance pattern 3 4. J. Sauer a, S. Schulze-Bopp a,b, E. Fritsch a, K. Undorf-Spahn a, J.. Jehle a* Institute for Biological Control, Julius Kühn Institute Darmstadt, Germany a ; Institute for Phytopathology, gricultural Service Center Palatinate (DLR Rheinpfalz), Neustadt/Wstr., Germany b *Correspondence to J.. Jehle, Institute for Biological Control, Julius Kühn Institute (JKI), Federal Research Centre for Cultivated Plants, Darmstadt, Germany Phone: ; Johannes.Jehle@julius-kuehn.de 13 1

2 bstract Different isolates of Cydia pomonella granulovirus (CpGV) are worldwide used to control codling moth larvae (Cydia pomonella; CM) in pome fruit production. Two types of dominantly inherited field resistances of CM to CpGV have been recently identified: - chromosomal type I resistance and autosomal type II resistance. In the present study, a CpGV-resistant CM field population (termed S-GO) from North-East Germany was investigated. S-GO individuals showed cross-resistance to CpGV isolates of genome group (CpGV-M) and genome group E (CpGV-S), whereas genome group B (CpGV-E2) was still infective. Crossing experiments between individuals of S-GO and the susceptible CM strain CpS indicated the presence of a dominant autosomal inheritance factor. By single-pair inbreeding of S-GO individuals for two generations, the genetically more homogenous strain CpRGO was generated. Resistance testing of CpRGO neonates with different CpGV isolates revealed that isolate CpGV-E2 and isolates, namely CpGV-I07 and I12, were resistance-breaking. When progeny of hybrid crosses and backcrosses between individuals of resistant CpRGO and susceptible CpS was infected with CpGV-M and CpGV-S, resistance to CpGV-S appeared to be autosomal and dominant for larval survivorship but recessive when success of pupation of the hybrids was considered. Resistance to CpGV-M, however, is proposed to be both autosomal and -linked inherited, since -linkage of resistance was needed for pupation. Hence we propose a further type III resistance to CpGV in CM, which differs from type I and type II resistance in its mode of inheritance and response to CpGV isolates from different genome groups

3 Importance The baculovirus Cydia pomonella granulovirus (CpGV) is registered and applied as biocontrol agent in nearly all pome fruit growing countries worldwide to control codling moth caterpillars in an environmentally friendly manner. It is therefore the most widely used commercial baculovirus biocontrol agent. Since 2005, field resistance of codling moth to CpGV products has been observed in more than 40 field plantations in Europe, threatening organic and integrated apple production. Knowledge about inheritance and mechanism(s) of resistance is indispensible for the understanding of host response to baculovirus infection on population level, the co-evolutionary arms race between virus and host, as well as for the development of appropriate resistance management strategies. Here, we report a codling moth field population with a new type of resistance, which appears to follow a highly complex inheritance to different CpGV isolates Introduction The codling moth (CM; Cydia pomonella) is a Lepidopteran (family: Totricidae), which larvae cause serious damage in pome fruit production, mainly in apple and pear (1). CM is worldwide distributed mostly through the temperate fruit growing regions (2). In conventional plant protection this pest has been controlled with chemical insecticides 56 entailing a variety of environmental and safety detriments (3). Thus, the demand for environmentally safe control agents of CM increased. Cydia pomonella granulovirus (CpGV), first described from diseased CM larvae found in Mexico (4), has a very narrow host range and is highly virulent for CM larvae. CpGV belongs to the genus Betabaculovirus of the Baculoviridae family. Its genome comprises double-stranded circular DN of about kb (5, 6). CpGV isolates are phylogenetically classified into five genome groups to E 3

4 (6). Because CpGV is highly effective against early larval stages and harmless to non-target insects, mammals and the environment, biocontrol agents based on CpGV have been developed for CM control (1). Since its first registration in 1987 in Switzerland, CpGV products have been used as biopesticide in virtually all apple growing areas worldwide (7). The CpGV occlusion bodies (OB) can be applied in their natural form; CM larvae taking up the OB during feeding die within a few days after ingestion (1). Most of the commercial CpGV products of the first generation contained the Mexican isolate, termed CpGV-M (4, 7). In 2005, after nearly two decades of field use in Europe, several organic apple plantations, where CpGV treatment had failed to control CM satisfactorily, were identified in South-West Germany (8). When laboratory bioassays were performed with progeny of these populations, a 1000 to 100,000-fold reduced susceptibility to CpGV-M was confirmed (9). This was the first time that resistance of insect populations against commercial baculovirus products had been reported. In the following years, observations of field resistance of CM populations against CpGV products were observed in about 40 apple plantations in ustria, Czech Republic, France, Germany, Italy, the Netherlands and Switzerland (10-13). cquired resistance of insects to baculoviruses has been also described for several insect/baculovirus associations, such as Phthorimaea operculella/phopgv (14), nticarsia gemmatalis/gmnpv (15), Trichoplusia ni/tnsnpv (16, 17), and doxophyes honmai/dhonpv (18, 19). However, these incidental laboratory selections never reached the wide geographic distribution and the economic meaning of CpGV field resistance in CM. The codling moth exhibits a karyotype of 2n = 56 chromosomes, with 54 autosomes () and a W/ (female/male) sex chromosome system (20). For the laboratory selected strain CpRR1, which derived from the resistant field population CpR, a dominant, monogenic and - 4

5 linked inheritance of CpGV resistance was demonstrated (9, 21). Genetic analyses of the resistant CM strains RGV (France) and CpR-C (Czech Republic) revealed a -linked and dominant inheritance mode also for these geographically distant populations (11, 22). Hence, a single main inheritance type has been initially assumed in European CM populations. This so-called type I resistance appeared to be targeted only against CpGV-M (group ), whereas other CpGV isolates (genome groups B-E) were able to overcome resistance (6, 23). For CpRR1, it was demonstrated that resistance-breaking properties of the CpGV isolates depended on the lack of an insertion of 24 bp in the ORF pe38 in CpGV-M (group ), which is missing in all resistance-breaking isolates of genome group B-E CpGVs (6). By registration of some of these isolates, a successful commercial control of the type I resistance in CM was eventually re-established in Europe (23-28). More recently, CM field populations which could not be controlled by the use of novel resistant-breaking CpGV products were identified. Resistance of one of these populations, termed NRW-WE, is targeted not only against CpGV-M but also to isolates belonging to the genome groups C-E (13), suggesting an alternative mode (= type II) of resistance in NRW-WE (13). Indeed, when the laboratory strains CpR5M and CpR5S were generated by selection experiments starting with NRW-WE individuals, it was demonstrated that type II resistance followed a dominant, monogenic but autosomal inheritance pattern. Furthermore, a crossresistance to at least two CpGV isolates, CpGV-M and CpGV-S, was observed (29). further CM field population from North-East Germany, called S-GO, showed also a reduced susceptibility to both CpGV-M and CpGV-S. S-GO was first reported in a study concerning the distribution of CpGV resistance in Europe (12). Its lethal concentration (LC 50) was about 1,000,000-fold increased, compared to that of susceptible CM representing a CM population with one of the highest resistance levels ever observed in the field (12). The aim 5

6 of the current study was to investigate the genetic basis and inheritance mode of CpGV resistance in S-GO. Resistance tests were conducted to identify CpGV isolates with resistant-breaking properties for S-GO. Single-pair crosses between field collected individuals of S-GO and the susceptible CM (strain CpS) were carried out to obtain first indications on the inheritance mode and the frequency of resistance in this field population. two-step inbreeding of S-GO individuals combined with simultaneous resistance testing of the offspring resulted in a genetically homogenous strain CpRGO with very low susceptibility to CpGV-M and CpGV-S. CpRGO was tested against further resistance-breaking CpGV genome groups to find suitable candidate CpGV isolates to be used as control agents of this type of resistance, whereas hybrid crosses and backcrosses between CpRGO and CpS unveiled the inheritance mechanism of this CpGV resistance. These investigations did not allow allocating resistance of CpRGO either to the type I or type II resistance but suggested a further resistance type with a highly complex mode of inheritance, which we termed type III resistance RESULTS Resistance testing of S-GO with different CpGV isolates Resistance of neonates of the field derived CM population S-GO to the isolates CpGV-M, -S, and -E2 was tested at a discriminating concentration of 5.8 x 10 4 OB/ml, which would cause >95% virus induced mortality in susceptible CM strains after seven days (9); the susceptible laboratory strain CpS was included as a control (Fig. 1). Whereas virus-induced mortality of CpS larvae exposed to different CpGV isolates varied between 87% and 97% after seven days and was more than 99% after 14 days, mortality of S-GO neonates on CpGV-M and CpGV-S was very low and did not exceed 9% for both viruses after 14 days. Significant mortality of 6

7 S-GO was only observed on CpGV-E2 with 28% mortality after seven days and 81% mortality after 14 days. The differences of mortality were significant between S-GO and CpS for all viruses after seven and 14 days, except for CpGV-E2 (NOV, pairwise t-test, P < 0.05) (Fig. 1). These results indicated resistance of S-GO against both CpGV-M and CpGV-S, whereas CpGV-E2 expressed full resistance-breaking characteristics in S-GO only after 14 days Resistance testing of F 1 progeny of the reciprocal single-pair crosses between field-derived S-GO moths and susceptible CpS moths To obtain immediate information on the character of the resistance inheritance in S-GO, reciprocal hybrid crosses between CpS moths and adults that had emerged from diapausing field larvae of S-GO were performed. Neonates of the F 1 progeny were then subjected to resistance testing with a discriminating concentration of 5.8 x 10 4 OB/ml. Virus-induced mortality of F1 progeny of nine female crosses (S-GO x CpS ) was between 0% and 34% after seven days of exposure to CpGV-M, with a median of 4% (Fig. 2a). On CpGV-S, mortality ranged between 0% and 87%, with a median mortality of 20%. Similar mortality rates were observed for 24 single-pairs of the male crosses (S-GO x CpS ) (Fig. 2a). fter 14 days, the F 1 progeny of the female crosses showed CpGV-M-induced mortality between 0% and 75%, with a median of 33%. On CpGV-S, mortality of female crosses ranged 50% and 100% with a median of 62% (Fig. 2b). Mortality of the progeny of the male crosses were again similar to those of the female crosses. Full details of the crossing experiments are given in Fig. S1. The results of the single-pair crosses between CpS and S-GO suggested a dominant resistance against both CpGV-M and CpGV-S in S-GO, otherwise, a higher mortality close to 100% would have been expected after seven or and 14 days of virus 7

8 exposure. Four female crosses showed 0% mortality and no difference in mortality was observed between male and female crosses; both findings contradict the assumption of a - linkage of resistance when mortality after seven and 14 days was considered Establishing of CpRGO To further evaluate the inheritance of CpGV resistance in S-GO a genetically more homogeneous strain was established by two consecutive rounds of inbreeding using singlepair crosses (families) of S-GO individuals combined with resistance testing of the offspring of the single families. Starting with 20 single-pair crosses, seven families rendered sufficient F 1 offspring (20-60 neonates) to be further used for resistance testing and breeding. Mortality of the F 1 larvae, exposed to CpGV-M or CpGV-S for seven days, was between 0% and 33%. The mean mortality of all tested siblings was 7% on CpGV-M and 9% on CpGV-S. fter 14 days, the mean mortality increased to 19% for CpGV-M and to 11% for CpGV-S (Table S1). ll control groups of the different families were reared to pupae and separated by sex to undertake a second round of single-pair inbreeding. Only one family (#12) produced enough F 1 moths to perform further four single-pair crosses (#12.1, #12.2, #12.3 and #12.4). When offspring of these crosses was subjected to resistance testing, mortality ranged from 0-14% on CpGV-M and from 0-10% on CpGV-S at seven days post infection (Table 1); all offspring of the family #12.2 and #12.4 survived the treatment with CpGV-M, whereas mortality on CpGV-S was 0% and 6%, respectively, did not increase to more than 18% for both viruses after 14 days. The offspring of the two families #12.2 and #12.4 was combined, further massreared and termed CpRGO. Resistance testing of CpRGO progeny revealed similar mortality to that of the parental families #12.2 and 12.4 (Table 1). Eventually, CpRGO was reared in 8

9 the laboratory without any selection pressure and further used for crossing and backcrossing experiments with CpS (see below). In addition, CpRGO individuals were frequently tested for their resistance level during three years of rearing; in ten independent assays mean mortality caused by CpGV-M and CpGV-S after seven days was 11% and 20%, respectively, and increased further until 14 days (Table 1) Genetic inheritance studies Female and male single-pair crosses and backcrosses between CpRGO and CpS individuals were performed to determine (i) the mode of inheritance of the resistance against CpGV-M and CpGV-S and (ii) the sex ratio of the surviving pupae. Following an autosomal or -linked, dominant or recessive inheritance hypothesis, differences in the mortality rates in the crosses and backcrosses were expected following Fig. 4. Mortality was recorded after seven days to assess larval resistance and after 21 days to measure pupation success. Female crosses: In total, 38 single female crosses (CpRGO x CpS ) were performed, of which nine crosses rendered sufficient offspring for resistance testing. Seven of these female crosses (cluster ) showed a similar response; their mortality was below 50% (mean 31%) after seven days and increased to 47-76% (mean 60%) after 21 days (Tab. 2). Two families did not follow this response pattern and were not considered further. It was found that 40% of the progeny exposed to CpGV-M successfully pupated until day 21, all of them were males (Tab.2). This observation did not fully support autosomal inheritance of CpGV-M resistance, especially when pupation success is considered. Female crosses tested with CpGV-S drew a slightly different picture, though only three families rendered enough individuals to complete these tests. Whereas mortality after 7 days was 43% and thus in the range of CpGV-M treatment, only 3 out of 60 tested individuals pupated, two of them were female. 9

10 Hence, in contrast to the CpGV-M treatment the rate of surviving pupae on CpGV-S was much lower, an influence of the sex on the pupation success was not given. Male crosses: In total, 19 single-pair male crosses (CpRGO x CpS ) were performed. Eight single pairs produced sufficient offspring for resistance testing. Their mortality response to CpGV-M and CpGV-S was highly heterogenic and ranged from 2-50% and 4-91%, respectively, after seven days. ccording to their response to CpGV-M we grouped these crosses into three clusters (B-D). Cluster B exhibited a very low mortality of 7% and 10% after 7 and 21 days, respectively, whereas cluster C and D showed increased mortality but very high variation on both CpGV-M and CpGV-S. In contrast to the female crosses, the sex ratio of the surviving pupae on CpGV-M and CpGV-S was nearly equal (Tab. 2). Backcross : In total, 37 families of the backcrosses (BC ; F1 x CpS ) were set-up, of which 19 single pairs with F1 originating from cluster, C and D could be used for resistance testing (Tab. 3). gain, a considerable variation in response to CpGV-M was observable, which could not be explained by either autosomal or -linked inheritance. ll F 2 families derived from F 1 cluster and half of cluster C (termed Ca in Tab. 3) showed a similar mortality of 51% and 59%, respectively, after seven days, and a slight increase of mortality until pupation. In contrast, the other three families derived from F1 of cluster C (termed Cb in Tab. 3) and progeny of cluster D showed 100% mortality already after seven days, without any surviving pupae. When cohorts of neonates of the same BC backcrosses were subjected to CpGV-S the 7-day mortality was 43% for progeny derived from F1 cluster and 66-75% for those from cluster Ca, Cb, and D (Tab. 3). Here, pupation was only observed in cluster, which showed a balanced sex ratio of pupae. Backcross B: Six out of 18 single-pair families set-up in backcross B (BC B; F1 x CpRSO ) could be used for resistance testing (Tab. 3). The BC B families derived from cluster and B 10

11 showed similar responses to each other, which were far below 50% mortality after seven days exposure to CpGV-M, a rate that would have been expected if resistance in CpRGO followed a classic monogenic -linked or autosomal inheritance. On CpGV-S, only progeny from cluster showed mortality of 50%; however, all individuals died until pupation. Progeny from cluster B showed low mortality of 23% Comparative resistance testing with different CpGV isolates in CM strains To obtain a more comprehensive picture, how CpRGO differs in its resistance from other resistant CM strains, neonate larvae of susceptible CpS and the four resistant strains CpRR1 (type I resistance), CpR5M and CpR5S (type II resistance), and CpRGO (proposed type III resistance) were systematically tested for their response to five different CpGV isolates, representing the CpGV genome groups -E. ll tests were carried out at the discriminating concentration of 5.8 x 10 4 OB/ml and were evaluated after seven and 14 days (Fig. 4). In CpS mortality ranged from 76% (CpGV-E2) to 97% (CpGV-I07) after seven days and was higher than 97% for all CpGV isolates after 14 days (Fig. 4a and Fig. 4b). Thus, all tested CpGV isolates were infective for CpS. For CpRR1 (type I resistance) low mortality of 5% and 11% was only observed on CpGV-M after seven and 14 days respectively. In contrast, treatments with CpGV-S, -E2, -I12 and -I07 caused significantly higher larval mortality after seven and after 14 days (NOV, post-hoc Tukey HSD test P < 0.05), corroborating previous findings that type I resistance is targeted exclusively against CpGV-M (6). In CpR5M and CpR5S (type II resistance) mortality was low for CpGV-M, -S and -I12 and did not exceed 23% after seven and 50% after 14 days. Only isolates CpGV-E2 and -I07 caused significant mortality of >80% after 7 days and of >95% after 14 days. 11

12 For CpRGO, the virus-induced mortality caused by CpGV-M and CpGV-S was 12% and 19%, respectively, after seven days, and increased to a maximum of 37% (CpGV-S) after 14 days. These mortality rates were very similar to the mean mortality observed for CpRGO during a three-year survey (Tab. 1). Treatment with CpGV-E2, -I12 and I07 resulted in mortality of CpRGO larvae between 64% and 91% after seven days and increased to 87-99% until 14 days. The mortality in the treatments with CpGV-M and CpGV-S differed significantly from that in the treatments with CpGV-E2, -I12 and -I07 (NOV, pairwise t-test, P < 0.05). In summary, CpRGO showed a response that was again different from CM strains representing type I and type II resistance DISCUSSION In the present study, resistance of the German field population S-GO exhibiting an extreme level of resistance against CpGV-M (12) was analyzed. Infection experiments showed that S-GO larvae were not susceptible to both CpGV-M and CpGV-S, concluding that the resistance in S-GO is directed at least against two different genome groups and E of CpGV (6, 23). Hence, resistance in S-GO appears to be different from type I resistance reported previously for CM in Europe (9, 11, 12, 26) and seems to resemble more to type II resistance, which is described as a cross-resistance against CpGV-M and CpGV-S (13, 29). The results of the single-pair crosses between individuals of S-GO and susceptible CpS provided first evidence of a dominant and autosomal inheritance (Fig. 2, Fig. S1). Otherwise higher mortality of the hybrid progeny should have occurred and reciprocal male and female hybrid crosses should have rendered different mortality rates, which was not the case. In addition, zero mortality was observed in several S-GO x CpS crosses (Fig. S1), which is also not compatible with a -linkage of the resistance gene(s). 12

13 To gain more insight into the resistance of S-GO, the genetically more homogenous strain CpRGO was selected from S-GO by a two-step inbreeding process with single-pair crosses. t first, mortality of CpRGO and its parental families #12.2 and #12.4 was close to zero on both CpGV-M and CpGV-S, indicating strain homogeneity and success of the selection process; during three years of rearing of CpRGO without selection pressure an increase of susceptibility was noted, suggesting some plasticity of resistance within CpRGO. Hybrid crossing experiments and backcrossing experiments with CpRGO and CpS individuals confirmed some of our initial predictions of a dominant and autosomal inheritance that was already made with parental S-GO. However, extended observation of infected animals for 21 days and thorough analyses of the mortality of siblings from single-pair crosses generated a much more complex picture of CpGV resistance in CpRGO (Tab. 2, Tab. 3): (i) Strikingly, female crosses resulted in significant mortality after seven days on both CpGV- M and CpGV-S. This observation would be compatible with either -linkage or an autosomal inheritance of heterozygous individuals. -linkage was supported for resistance to CpGV-M because only males pupated. This finding could not be confirmed for the resistance to CpGV- S, because a very few male and female pupae survived in these experiments. The outcome of the male crosses was even more puzzling because different response clusters (B-D) were observed showing a highly heterogenic reaction among the different families. (ii) The backcrossing experiments again did not match with a -linked or an autosomal inheritance hypothesis. Even more confusing, the -linkage signal observed for CpGV-M resistance of the female cross in cluster fully disappeared in BC and evidence for an autosomal inheritance was noted. Within BC, the progeny of the male cross cluster C split into two response groups (Ca, Cb) for CpGV-M but not for CpGV-S, supporting different modes of inheritance for CpGV-M and CpGV-S resistances. 13

14 (iii) In general, only a few males and female pupae developed on CpGV-S, indicating a variation in the efficacy of CpGV-M and CpGV-S in both infection process and sex selection. No or a very low correlation in the mortality response between treatments with CpGV-M and CpGV-S were detected in the hybrid crosses and backcrosses, also suggesting a somehow different and heterogeneous action of resistance in CpRGO for these two CpGV genome groups. To summarize, the results of the different hybrid crossing and back crossing experiments did not support classic monogenic -linked nor autosomal inheritance, as it was clearly observed for type I and type II resistance (9, 13,29). In addition, cross-resistance of CpRGO to CpGV-M and CpGV-S followed different inheritance patterns, which was also not the case for type II resistance (29). Considering that CpRGO originated from only a single pair (#12) of CM individuals, the heterogeneity observed in the F 1 crosses and the backcrosses is unexpected, especially after mortality of early generations of CpRGO was very low. However, by assuming the combined action of a -linked and an autosomal genetic factor, most of the observed crossing results can be interpreted. We hypothesize, therefore, that resistance against CpGV-M is based on at least two loci on an autosome and the chromosome and depends on a sex and dose effect (two-gene hypothesis). By taking all results together it is proposed that survivorship after seven days of the crosses needs at least one resistance gene either on an autosome ( R ) or on the chromosome ( R ) (Fig. 5). Individuals with only one resistance gene on the autosome but missing a second resistance gene on the chromosome are able to survive seven days but do not develop to pupae. This is the case for F 1 females from the female crosses, which obtained their S chromosome from a susceptible CpS male. Our hypothesis further includes a certain increase of the number of dead larvae until day 21 caused by longterm virus exposure (Fig. 5). n increase of larval mortality with extended virus exposure 14

15 appears to be typical for CpGV resistance; it was also observed for type I and type II resistance (9, 13, 21). Our two-gene hypothesis is also compatible with the hypothetical mortality of the F 1 progeny of homogeneous resistant females crossed with susceptible CpS males ( R W R R x S S S S = S W S R, R S R S ) which fits to all observed mortality of cluster. For the male crosses, F 1 offspring were clustered in three groups (B-D). The results for cluster B can be explained by selecting a homogeneous male moth ( R R R R ) and C and D for two different genotypes of heterogeneous individuals ( R S R R or R S R S ). Because clear chromosomal genotypes were generated for the F 1 progeny of the crosses, most of the backcrossing results are explainable: The mortality of BC, F 2 offspring originating from clusters, B and Ca, fits to the expected mortality because the selected F 1 male moth had a homogeneous genotype ( R S R S ). If a F 1 male with one resistant gene ( S S R S ) from cluster Cb was randomly selected, mortality up to 100% was determined as shown in Fig. 5. The hypothesis also matched for the most of the observed results for the BC B. These results suggest that the original #12 pairs used to establish CpRGO did not only consist of fully genetically homogeneous resistant individuals ( R W R R, R R R R ), but were heterozygous in their -linked and/or autosomal component. Such heterogeneity also explains the observed decline of resistance during further breeding without selection pressure of CpRGO, which might have been caused by fitness costs or genetic drift. Therefore, the two-step inbreeding clearly helped to identify some genetic traits of the resistance in CpRGO, but it was not successful to generate a genetically fully homozygous line. For the CpGV-S treatments low mortality after seven days but a strong increase in mortality until 21 days was detected for nearly all crosses and backcrosses. These findings were 15

16 independent of the number of resistance allele(s) suggesting a more recessive nature of inheritance of the resistance in CpRGO against CpGV-S, when pupation success of the hybrid offspring is considered. No sex-linkage was observable for CpGV-S. In heterozygous offspring, CpGV-S appeared to be infective though the death of the larvae was highly delayed compared to susceptible larvae. Taking together, neither a -linked nor an autosomal inheritance alone can explain the highly complex mortality patterns of the crosses and backcrosses observed in this study. Indeed statistical tests supported a polygenic inheritance pattern in the majority of the backcrosses for the resistance against both viruses (data not shown). These observations are in clear contrast to the monogenic mode of inheritance suggested for type I resistance against CpGV-M in CpRR1 (9, 11, 26) and for type II resistance in CpR5M/CpR5S (29). Interestingly, some circumstantial evidence for an autosomal and -linked inheritance was previously suggested for French strain field population St-ndiol, though this trait was also lost during selection experiments rendering only -linkage of the descendent strain RGV (22). The observed combination of autosomal and -linked inheritance observed for CpRGO is unique. So far, reports on genetically-based baculovirus resistance referred mainly to laboratory selections (14-18). Inheritance patterns had been determined for baculovirus resistance in CM (9, 11, 22, 29) and P. opercullela (14, 30); it was generally monogenic and dominant, and either -linked (CM) or autosomal (CM and P. opercullela). Recently, a laboratory strain of. honmai highly resistant to dhonpv was selected (18, 19) and a midgut-based mode of resistance to oral infection by dhonpv OB was identified. It is thus obvious that baculovirus resistance does not follow a common mode but varies among different baculovirus-host systems. 16

17 CpRGO shows a new and highly complex inheritance pattern that is different from type I and type II resistance in CM. Regarding its inheritance, it is not fully clear whether this new type of resistance, to which we refer as type III resistance, is functionally independent from type I and type II resistance or whether it is a combination of both. systematic comparison of all available resistant CM strains with CpGV isolates belonging to different genome groups revealed susceptibility of CpRGO to CpGV-E2 (genome group B), -I12 (D) and -I07 (C) (Fig. 3). Thus, in addition to the differing inheritance also the response of CpRGO to different CpGV isolates does not comply with that of type I and type II resistant CM strains and lacks the strong correlation of cross-resistance to CpGV-M and CpGV-S that was found with type II resistance. These variations might be an effect of differences in the resistance mechanism(s) and/or a consequence of quantitative effects based on the numbers of expressed resistance genes in the hosts. Such quantitative effects could also explain why the level of resistance against CpGV-M observed in the original field population S-GO (12) was much higher than in other tested CM populations. In any case, the startling complexity of CpGV resistance amounting to CM populations with highly different susceptibilities and inheritance of resistance provides a unique model to study the potential of host adaptation to baculovirus infections in the field. Understanding the underlying genetic factors of CpGV resistance will be crucial to sustain the efficacy of CpGV isolates in the field

18 MTERILS ND METHODS Insects The codling moth (CM) strain CpS is susceptible to all known CpGV isolates and has been reared at the Julius Kühn Institute (JKI), Institute for Biological Control in Darmstadt (Germany), for many years (9). The CM field population S-GO derived from an organic orchard in Saxony, Germany, where CpGV-M products had been applied in the past. In autumn 2008, cardboard stripes were stripped around tree trunks to collect diapausing larvae, which were reared in the laboratory henceforward. Resistance to CpGV-M in this orchard was already demonstrated by full range bioassays (12). CpRR1 carries type I resistance against CpGV-M and arose from the resistant field population CpR (BW-FI-03, Sudbaden ) by single-pair crosses (9). The two CM strains CpR5M and CpR5S originated from the field population NRW-WE, which was selected on either CpGV-M or CpGV-S for five generations; CpR5M and CpR5S show cross-resistance against both CpGV-M and CpGV-S and showed a second type of resistance (type II resistance) (13, 29). ll CM strains were reared under laboratory conditions at 26 C with 16/8 h light/dark photoperiod and 60% relative humidity on modified artificial diet (31) Viruses Five different Cydia pomonella granulovirus (CpGV) isolates were used in this study: CpGV-M (4), belonging to the genome group, isolate CpGV-E2 (genome group B) (32), CpGV-S (genome group E) (6). Both Isolates CpGV-I07 (genome group C) and CpGV-I12 (genome group D) originated from Iran (23, 33). CpGV occlusion bodies (OB) were purified as described previously (34, 35) and all samples were stored at -20 C. Quantitation of virus 18

19 stocks was performed by OB counting with a light microscope (Leica DMRBE) using dark-field optics with the Petroff-Hauser counting chamber (depth 0.02 mm) Resistance testing To differentiate between resistant and susceptible individuals, first instar larvae were tested for resistance as described elsewhere (36). For resistance testing of different CpGV isolates, purified OBs were mixed with modified diet of Ivaldi-Sender (31) to obtain a final discriminating concentration of 5.8 x 10 4 OB/ml, which causes > 95% mortality in susceptible CpS neonates after seven days (9). For untreated control, larvae were reared on diet without virus. Mortality of larvae was determined seven, 14 and 21 days post infection (p.i.). For resistance testing in the crosses and backcrosses, all larval offspring were separated into three cohorts (i-iii); (i) neonate larvae were tested for resistance on artificial diet containing CpGV-M, (ii) larvae were exposed to diet with CpGV-S, and (iii) for untreated control, larvae were reared on diet without virus. For determinate the rate of surviving male individuals, surviving pupae were sexed after 21 days according to the number of their abdominal segments described previously (37) Single-pair crosses For single-pair crosses (families) pupae of S-GO were sexed as described before (37) and one virgin female moth was mated with a male moth and the obtained eggs were collected daily and stored at 4 C until end of oviposition. Then all eggs of a family were incubated at 26 C until hatching of the neonates. Single-pair families producing less than 20 neonates were excluded from the resistance testing. For details of single-pair crosses see sser-kaiser et al. (9). 19

20 Reciprocal single-pair crosses In order to obtain information about the mode of inheritance in both, S-GO or CpRGO, reciprocal single-pair crosses between one of the two resistant strains and the susceptible strain CpS were conducted. Two different crosses were performed; female crosses represent the mating of one virgin female ( ) moth of S-GO or CpRGO with a male ( ) moth of CpS (S-GO or CpRGO x CpS ). Male crosses means the crosses of a male moth of S-GO or CpRGO mated with a virgin female moth of CpS (S-GO or CpRSO x CpS ). The resulting offspring of the reciprocal male or female crosses (F 1) were divided in three cohorts and subjected to resistance tests on CpGV-M, CpGV-S and untreated control as described above. If the number of F 1 neonates was lower than 90 individuals only one virus (CpGV-M or CpGV- S) treatment and the untreated control group were included Backcrossing experiments The F 1 control group of the male or female crosses were reared to pupae and separated by sex. The hatching male moths were individually backcrossed with female CpS moths (backcross ; BC ; CpRGO x CpS ) or with a virgin female moth of CpRGO (backcross B; BC B; CpRGO x CpS ). The resulting offspring (F 2) of BC or BC B were tested for resistance as described above Statistical analysis The observed virus-induced mortality in the resistance testing was corrected by the mortality of the untreated control group according to bbott s Formula (38). Statistical differences in the resistance testing with the field resistance strain S-GO and CpS were 20

21 analysed using NOV pairwise t-test. Differences between different CM strains and treatments in the resistance testing were statistically evaluated using NOV and post-hoc Tukey HSD test of the means (RStudio edition ) CKNOWLEDGEMENT The author thanks Sarah Schilling, Doris El Mazouar and Birgit Weihrauch for excellent technical assistance in insect rearing. We thank Sabine sser-kaiser for advice in hybrid single-pair crossing experiments. This work was supported by the Federal Organic Farming Scheme (grant O50E023/1) and by the Deutsche Forschungsgemeinschaft (DFG) (grant Je245/14-1) SUPPLEMENTRY MTERIL (TBLE S1, FIG S1) REFERENCES 1. Lacey L, Thomson D, Vincent C, rthurs SP Codling moth granulovirus: a comprehensive review. Biocontrol Science and Technology 18: Barnes MM Codling moth occurrence, host race formation, and damage. Tortricid pests: their biology, natural enemies and control. By Van der Geest LPS, Evenhuis HH, Elsevier, msterdam, The Netherlands: Lacey L, Shapiro-Ilan DI Microbial control of insect pests in temperate orchard systems: Potential for incorporation into IPM. nnu Rev Entomol 53: Tanada Y Granulosis virus of codling moth, Carpocapsa pomenella (Linnaeus) (Olethreutidae, Lepidoptera) Journal of Insect Pathology 6:

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26 Table 1 Mortality of first instar larvae of F1 family #12 after the single-pair inbreeding of S- GO, tested for resistance on artificial diet containing CpGV-M or CpGV-S at the discriminating concentration of 5.8 x 10 4 OB/ml for seven and 14 days post infection (p.i.). Individuals of the untreated control cohort of family #12 were crossed to obtain F 2 offspring (families # ). Untreated control cohorts of #12.2 and #12.4 were pooled to obtain strain CpRGO. SD, standard deviation; n, number of tested larvae; N, number of independent replicates. Mortality data are corrected following bbott Formula with mortality <6% (7 days) and <16% (14 days) in untreated control cohorts (36) Family/ strain n [N] % mortality on CpGV-M % mortality on CpGV-S Mean 14 days Mean Mean (SD) p.i. (SD) n 7 days p.i. (SD) 7 days p.i. 14 days p.i. # # # (4.4) # (6.3) 11.1 (4.2) # CpRGO CpRGO ( ) 273 [10] 11.3 (17.2) 28.0 (25.7) 330 [10] 19.6 (23.8) 20.7 Mean (SD) 13.4 (8.0) 33.9 (35.7) 26

27 Table 2 Mortality of neonate larvae subjected to CpGV-M and CpGV-S at the discriminating concentration of 5.8 x 10 4 OB/ml determined after seven and 21 days post infection (p.i.). Single-pair crosses were grouped in Cluster (-D) according to their mortality response against CpGV-M. The rates of surviving male pupae were assessed after 21 days; n, number; SD, standard deviation. Cluster n crosses per cluster (n tested individuals) Treatment with CpGV-M % mean mortality and [SD] (7 days) % mean mortality and [SD] (21 days) % male pupae [n males: n females] n crosses per cluster (n tested individuals) Treatment with CpGV-S % mean mortality and [SD] (7 days) %mean mortality and [SD] (21 days) % male pupae [n males: n females] Female crosses CpRGO x CpS 7 (244) 31 [12] 60 [9] 100 [92:0] 3 (61) 43 [22] 95 [6] 33 [1:2] 602 Male crosses CpRGO x CpS B 4 (179) 7 [3] 10 [7] 43 [33:44] 4 (151) 23 [10] 81 [17] 48 [10:11] C 3 (143) 16 [23] 48 [3] 38 [27:45] 3 (138) 41 [45] 94 [7] 50 [4:4] D 1 (32) 50 [-] 88 [-] 75 [3:1] 1 (33) 13 [-] 76 [-] 38 [3:5]

28 Table 3 Mortality of the offspring of the back crosses BC and BC B exposed to diet containing CpGV-M and CpGV-S at the discriminating concentration of 5.8 x 10 4 OB/ml seven and 21 days post infection (p.i.). The F 1 male ( ) moth used for backcrosses originated of the control group of the female ( ) or male single-pair crosses between CpRGO and CpS (see Table 1). Resulting F 1 males were reared to adulthood and moths were backcrossed by single-pair crosses with female CpS moths (BC ; F 1 x CpS ) or CpRGO female moths (BC B; F 1 x CpRGO ). The rates of surviving male pupae were assessed after 21 days; n, number; SD, standard deviation. F 1 from cluster n crosses per cluster (n tested individuals) Treatment with CpGV-M % mean mortality and [SD] (7 days) % mean mortality and [SD] (21 days) % male pupae [n males: n females] n crosses per cluster (n tested individuals) Treatment with CpGV-S % mean mortality and [SD] (7 days) %mean mortality and [SD] (21 days) % male pupae [n males: n females] BC ; F1 x CpS 11 (406) 51 [13] 77 [10] 38 8 (242) 43 [24] 82 [21] 45 Ca 3 (111) 59 [14] 66 [9] 33 3 (119) 66 [17] 100 [0] n.a. Cb 3 (126) 100 [0] 100 [0] n.a. 3 (138) 72 [12] 98 [2] n.a. D 2 (93) 100 [0] 100 [0] n.a. 2 (90) 75 [2] 100 [0] n.a. 612 BC B; F1 x CpRSO 4 (141) 31 [21] 58 [17] 63 3 (48) 48 [28] 100 [0] n.a B 2 (32) 19 [15] 39 [13] 50 2 (29) 23 [21] n.d. n.a