Phenology and suppression of stem weevils ( Ceutorhynchus napi Gyllenhal and Ceutorhynchus pallidactylus Marsham) in oilseed rape in northern Serbia

SUMMaRY Stem weevils ( Ceutorhynchus pallidactylus and Ceutorhynchus napi ) are significant pests of oilseed rape that farmers regularly suppress by insecticide treatments intended against Brassicogethes aeneus . Recent studies have shown that in some regions of northern Serbia C. napi is dominant, while C. pallidactylus prevails in others . Increased damage from stem weevils has elicited an exploration of the situation and a study of their phenology and effects of insecticides in three differently managed fields: conventional, organic and integrated. New findings regarding the phenology of stem weevils show that both species were sporadically present in our experimental oilseed rape fields as early as during autumn (from the end of October 2010, BBCH 17-18). The main period of stem weevil settlement is the end of winter and early spring. Maximum flight of both weevil species was recorded on March 23 at the beginning of oilseed rape (OSR) stem elongation (BBCH 22-25), and one week later in the organic field. There were no significant differences in the number of settled weevils among the three differently managed fields . The insecticide treatment against C. napi was applied two weeks earlier than the usual treatment against B. aeneus and C. pallidactylus . Thus, it is clear that insecticide treatment directed against B. aeneus and C. pallidactylus is not effective against C. napi. In this way, such a practice can contribute to an increase in OSR damage, and density of the next generation of C. napi . A new generation of C. pallidactylus emerged from OSR fields in June 2011, while the majority of C. napi emerged in March of the following year.


iNTRODUCTiON
Oilseed rape (OSR) stem weevils (Ceutorhynchus napi Gyllenhal and Ceutorhynchus pallidactylus Marsham) (Coleoptera, Curculionidae) are important pests throughout Europe. Their presence in OSR crops often motivates farmers to apply insecticides. Economic importance of OSR stem weevils is variable in Europe. In northern regions, C. pallidactylus is known as the main stem weevil pest, while C. napi dominates in southeastern and central Europe (Williams, 2010). Their phenology and abundance are useful to know in order to achieve effective insecticide application.
The published data on stem weevils in the Pannonian plains (Hungary and northern Serbia) indicate that C. pallidactylus is the most abundant and economically very important pest (Marczali et al., 2007;Milovac et al., 2010). However, recent findings suggest that C. napi is actually the dominant stem weevil species in some regions (Bozsik, 2010;Sivčev et al., 2015). Although their phenology is similar, their chemical control is sometimes three weeks apart (Büchs, 1998). Therefore, this research aimed to determine the possible cause of C. napi domination and provide evidence on potentially changed phenology of Ceutorhynchus weevils in commercial oilseed rape fields located in the plain of northern Serbia.

Experimental site
The trial was set up in northern Serbia (N45 57.280 E19 37.554) on a calcic chernozem soil of loamy texture. The climate is mild continental with total annual precipitation of 926.5 l/m 2 during 2010, 417.6 l/m 2 in 2011 and 434.1 l/m 2 during 2012. The mean annual temperature was 11.3°C in 2010, 11.8°C in 2011, and 12.3°C in 2012, according to data from Palić meteorological station (http://www.hidmet.gov.rs).

Experimental fields
Three commercial experimental fields (1 ha each) were sown with OSR on September 17, 2010, and with winter wheat (WW) on October 20, 2011 for the following season 2011/2012. These fields had never been planted with oilseed rape before. The OSR fields were managed according to conventional, integrated and organic production requirements. No herbicides, foliar fungicides or molluscicides were applied in the OSR crop. Monitoring of pests with yellow water traps revealed that only C. napi exceeded threshold for insecticide treatment (EPPO, 2003). On March 25, 2011, the conventionally managed OSR field was sprayed with chlorpyrifos + cypermethrin (500 g/ha a.i. + 50 g/ha) and the field with integrated management with cypermethrin (40 g/ha a.i.). The organic field was sprayed with spinosad (96 g/ha a.i) on April 6, 2011. Standard tractor sprayer with 300 l water/ha was used. The fallow period lasted from OSR harvest on June 21 until October 20, 2011 when WW was sown. The succeeding WW crops received no insecticide treatment and there were no differences in the management system applied in the three fields.

Sampling and identification
Stem weevils were sampled in the middle of each field, at points 50 m apart, using yellow water traps, emergence traps and plant dissection. The collected weevils were identified according to Freude et al. (1983) and Hoffmann (1954).

Yellow water traps
Yellow water traps (YWT) were used to monitor the autumn and spring immigration of adult weevils into the OSR fields. On October 2, 2010 (BBCH 12-13) three YWTs were set in each field and the traps were then checked 4 times until the end of November (BBCH 18-19) (October 20, November 1, November 10, November 30). On January 20 2011, four traps were placed in each field and sampling was performed at 2-4 weeks intervals as convenient. In the spring, sampling continued at 7 days interval until May 15, 2011. The YWTs were placed in the middle of each field, at 50 m distance from each other. The YWTs were mounted on metallic holders and were periodically raised to be always above crop level.
The repeated measures ANOVA was used to compare the mean counts of both weevils settled in each of three differently managed OSR fields, as recorded with YWTs. In order to fulfill the assumption of homogeneity of variances, data on C. pallidactylus were log transformed.

Plant dissection
To check the presence of stem weevil larvae, each leaf stalk, stem and plant root was dissected. Ten randomly selected OSR plants were sampled on 8 points from each field in 2010 and 2011. In 2010, OSR plants were sampled and dissected on November 10, 2010 (BBCH 17-18) and November 30, 2010 (BBCH 18-19). In 2011, OSR plants were sampled and dissected three times: on April 12 (BBCH 55-57), on April 19 at the full flowering stage of OSR (BBCH 63-65), and on May 3 at the end of flowering (BBCH 69-71).

Emergence traps
The emergence of a new generation of stem weevils was monitored using 8 emergence traps (www.ecotech-bonn. de) in each field. The traps were placed at 8 sampling points in the middle of each field at 50 m distance from each other. The sampling period in OSR crops was from the end of flowering until harvest, and countinued throughout fallow and the growing period of WW until its harvest. In OSR and WW, traps were checked every 7 to 14 days, while fallow traps were checked at about 4-week intervals.

Immigration of C. napi and C. pallidactylus into OSR fields
The flight pattern of immigrating C. napi and C. pallidactylus adults was similar (Table 1). Both species started to immigrate into the OSR fields at the end of October 2010 (BBCH 15-17). The number of autumn immigrating stem weevils caught in YWTs was small. We recorded 11 C. pallidactylus and 5 C. napi adults in all three fields. Next year, the activity of weevils was detected on February 12 . Later immigration of weevils was interrupted by a spell of cold weather until the second half of March, when mass flight of both species started and reached its maximum on March 23, i.e. at the beginning of stem elongation , and one week later in the organic field. By April 15 (BBCH 55-57), immigration of  C. pallidactylus into the OSR fields finished, while a few more specimens of C. napi were found by April 27, 2011 (BBCH 67-69). The counts of immigrating C. napi were 2.7 times higher than C. pallidactylus. Infestation of the three differently managed OSR fields with C. napi weevils was similar. Mean counts (± s.e.) of C. napi weevils settled in the conventional (174.75±13.0), integrated (181.75± 35.7) and organic (207.00± 17.2) fields did not differ significantly (F(2,6) =.512, p=.623). Infestation of the three differently managed OSR fields with C. pallidactylus showed a similar pattern. Mean counts (± s.e.) of C. pallidactylus weevils that settled in the conventional (35.7±12.1 ), integrated (50.75±14.0 ) and organic (123.75±28.2) fields were not significantly different (F(2,6) =2.737, p=.143).

Male/female ratio of immigrating stem weevils
At the beginning of stem weevil immigration in February  the percentage of C. napi females was 33.3%, while C. pallidactylus accounted for 22.5% (Table 2). As soon as stem elongation started, the immigrating weevil population reached its maximum. In the period preceding the peak immigration flight, the percent of C. napi females was higher than C. pallidactylus. The maximum percentage of C. napi females was recorded on March 10-23 (BBCH 22-25) at the peak of immigration flight. In contrast to C. napi, the maximum of C. pallidactylus female percentage did not coincide with maximum immigration activity of the population. The percentage of C. pallidactylus females was gradually increasing and becoming predominant with 78% of the total number of adults at the end of colonization of OSR fields. In contrast to C. napi, the share of C. pallidactylus females was much greater in the post-peak flight period. The ratio of male/female adults of C. pallidactylus collected in YWTs over the complete sampling period January 30-April 7, was 0.8:1, while that of C. napi was 1.3:1.

Assessment of stem weevil larvae by dissection of OSR stems and leaf petioles
Dissection of OSR plants sampled in the autumn of 2010 showed that weevil larvae were not present. Weevil larvae were found in OSR plants only in spring 2011, over the period from April 12 (BBCH 19-20) until May 3, 2011 (BBCH 55-71).
The number of weevil larvae in OSR stems (BBCH 63-65) was clearly different from their counts after insecticide treatment (Figure 1). The effective insecticides chlorpyrifos+cypermethrin and cypermethrin significantly reduced the number of stem weevil larvae. However, treatment of the organically managed OSR field with spinosad insecticide was not efficient, since it resulted in infestation of 96.8% of all OSR test plants with an average of 18 larvae/stem (ranging 0-130 larvae/ stem). In contrast, the conventionally managed OSR field, which was sprayed with chlorpyrifos+cypermethrin only had 6% of all test OSR plants infested with an average of 1 larva/stem. In the field with integrated management, which was sprayed with cypermethrin, only 2% of OSR stems were infested with one larva/plant on average.

Emergence of new stem weevil generation
The emergence of a new generation of C. pallidactylus beetles occurred during the OSR ripening stage at the beginning of June (BBCH 79-80), and ended by June 21 (BBCH 87-88) (Figure 2). A total of 177.0 weevils/m 2 emerged during that period. Over the fallow period in July and August, there were no weevils recorded in the emergence traps. However, one imago of C. pallidactylus was recorded on September 9, 2011 and another one on November 14, 2011 in one of the fields with subsequent winter wheat grown at that time. In the spring of 2012 (March 26), one adult was found in an emergence trap again. The results infer that a great majority of C. pallidactylus adults emerged from the OSR fields before harvesting. However, a very small number of adults aestivated in soil of the OSR fields, so that only 3 adults were found in emergence traps in the autumn of 2011 and spring of 2012.
Emergence of the next generation of C. napi weevils was recorded in emergence traps located in the subsequent winter wheat crop in the spring of 2012. First adults were recorded in the emergence traps on March 5, 2012 (Figure 3).    Maximum emergence was recorded over the sampling period from March 12 until March 19, 2012. In total, 146.44 weevils/m 2 emerged in that period. No hatching adults of C. napi were noted during the summer and autumn of 2011. The male-to-female ratio of rape stem weevils which emerged during spring 2012 was 1:1 .

DiSCUSSiON
The dominance of C. napi found in the study is associated with specific conditions in the region where our research was conducted which allowed such abundance. Insecticides, climate and alternative host plants were the factors potentially responsible for such development.
OSR fields are sprayed with insecticides as soon as in early spring. This treatment primarily targets B. aeneus and Ceutorhynchus stem weevils, and one treatment is usually sufficient for these most important pests (Williams, 2010). However, C. napi is more destructive and insecticides must be applied immediately after its high density is observed. When no monitoring with YWTs is conducted, a delayed insecticide application can miss a significant portion of C. napi weevils and the ensuing infestation of OSR fields can therefore increase dramatically. This is probably the most important reason for an increased damage observed in OSR production. Therefore, it is necessary to monitor stem weevil immigration into OSR fields in springtime very carefully in order to enable proper timing of insecticide treatments when the population density exceeds action threshold. In our trial, a single insecticide treatment was sufficient to control Ceutorhynchus stem weevils when infestation of OSR buds by B. aeneus was not high. However, pollen beetles usually do require a later insecticide treatment at the OSR young bud stage, the period when they are most harmful. Therefore, a shift regarding the dominance and pest status of stem weevils is significant since the timing of C. napi and B. aeneus control with insecticides can be very different. In our trial the difference in proper timing of control treatment of these two pest species was about 2 weeks. Application of the "biological" insecticide spinosad under organic management has shown to be ineffective and in this study resulted in 96.8% of infested OSR plants. This is a very high potential for subsequent infestations, and organic production of OSR should consider more effective ways of stem weevil control. Therefore, the use of ineffective or weakly effective insecticides can results in a buildup of stem weevil population density. Timely application of highly efficient insecticides, such chlorpyrifos+cypermethrin and cypermethrin (Milovac et al., 2017) therefore has a strong impact on the abundance of these two OSR stem weevils.
Wild and especially cultivated plants of the family Brassicaceae are widely distributed in Europe, and oilseed rape is its most prominent crop plant with a production area of about 6 million hectares. Cultivation of oilseed rape in large areas induces mass occurrence of stem weevil populations (Günthart, 1949). In addition, a large number of different brassicaceous weeds are widespread on roadsides, in ruderal habitats or on arable land and they support weevil populations as alternative host plants. Ceutorhynchinae are showing preference for brassicaceous ruderal species. Preference for these hosts may explain a considerable number of Ceutorhynchinae species damaging cultivated plants (Korotyaev, 2006).
The role of weeds as alternative host plants is frequently neglected in phenology and population studies. C. napi and other Ceutorhynchus weevils are associated with brassicaceous weeds or arable crops. In years when OSR area declined, Lepidium draba L. was the preferred host plant responsible for the survival of weevil populations (Günthart, 1949). In the region in which our research was conducted, L. draba is a common and abundant weed. Other widespread brassicaeous weeds in this area are: Sinapis arvensis L., Thlaspi arvense L. and Capsella bursa pastoris (L.) Medik., the preferred plant species for adult feeding.
C. napi was found to be an exceptionally abundant weevil species whose dominance had not been previously recognized in the region. Recently, similar reports have come from climatically similar regions in Hungary and Romania (Bozsik, 2010;Bucur & Roşca, 2011) which caused us to consider the climate as one of the responsible factors for a shift in what we know about weevil abundance. Warmer regions of Central Europe are known as suitable for C. napi (Günthart, 1949).
Our findings that first adults of C. napi and C. pallidactylus immigrated into OSR fields as early as in October/November 2010, when OSR was at the growth stage BBCH 17-19, confirmed the projected earlier weevil emergence and their settlement in OSR fields (Junk et al., 2012). In northern countries, such as Latvia, the first stem weevils are usually recorded at the beginning of April. However, it also coincided with the oilseed rape growth stage BBCH 18 and onwards (Grantiņa et al., 2011). So far, there have been no records of weevil migration into OSR fields during autumn. In this paper we provided evidence that first migration to OSR fields under warmer climate conditions may happen already during autumn. Considering the aspect of plant pest control, it is important that adaptation to climate changes may vary among different species regarding their range from becoming rare to disproportionally abundant (Putten et al., 2010). Climate change may therefore be expected to lead to a shift regarding the status of pests due to their changing abundance and dispersal.

aCKNOwLeDGeMeNTS
This study was supported by SEEER A NET. PLUS project 051, grant ON 173038, and by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant III 46008. We owe special gratitude to Predrag Kulundžić, B.Sc. for his priceless help and to Zelic Ivica, who kindly accepted this experiment to be conducted in his fields.