**3. Results and discussion**

Different authors [7,8] reported that glucosinolate content differs in different plant species, this was confirmed also by our research. In the samples of the studied species of Brassicas we established the presence of only one glucosinolate, namely glucobrassicin, it was noticed in all plant species. We found out that the content of glucobrassicin in the samples of oilseed radish negatively influence the studied group of harmful organisms, both cabbage flea beetles and cabbage stink bugs. Despite the fact that the connection between the content of glucobrassicin and the extent of damage by cabbage flea beetles (*r* = −0.30) and cabbage stink bugs (*r* = −0.32) was negative, we cannot talk about identical influence throughout the growth period. This is consequently conditioned by the fact that glucosinolate content in Brassicas varied both during the growth period and between individual plant organs. It is also interesting that we found more glucobrassicin in the blossoms of oilseed radish than in the leaves.

Research has established [9] higher contents of five different glucosinolates in blossoms of black mustard (*Brassica nigra*) than in leaves of the said plant species. In our research this statement holds true for glucobrassicin in glucoraphanin in the samples of oilseed radish and for sinalbin and epiprogoitrin in the samples of white mustard. Based on research done by [10], glucosinolate content can vary also within individual genotypes of the same plant species, this was confirmed also in our research – between the selected hybrids of cabbage we discovered significant differences in the contents of progoitrin and glucobrassicin. In the samples of the hybrid 'Hinova' we discovered more progoitrin, while a higher share of glucobrassicin was present in the hybrid 'Tucana'. Of the nine glucosinolates which were analysed in the main crops, we confirmed the presence of four (sinalbin, gluconasturtiin, glucoraphanin and progoitrin) in both hybrids of cabbage, yet only in traces (below detection threshold).

High concentrations of aliphatic glucosinolates in Brassicas can be an important factor of resistance for the said plants against harmful organisms [11]. We also found out that progoitrin in oilseed radish and white mustard was below detection threshold, which enables wider spectrum of usefulness of these plant species, as the said aliphatic glucosinolate can cause negative effects for feeding of animals [12].

**2.5. Sowing mixture of Brassica trap crops is recommended to reduce** *Phyllotreta* **beetles**

We studied the extent of damage caused by cabbage flea beetles on four different *Brassica* species in a 2-year field experiment (2009–2010) at two locations in Slovenia. The entire experiment was based on testing oilseed rape, white mustard and oil radish as potential trap crops to protect cabbage from cabbage flea beetles [5,6]. A significant influence of the *Brassi‐ ca* species on the feeding by the flea beetles was confirmed at both the locations. The damage index on oil radish was the highest throughout most of the growth period, whereas oilseed rape and white mustard were preferred only during a certain growth period. The initial damage caused by the cabbage flea beetles occurred in the first half of May, whereas the heaviest damage occurred at the beginning of July. This research shows that the onset of cabbage flea beetle feeding can be controlled in a medium–late cabbage cultivar using trap cropping. However, since none of the tested trap crops strongly attracted the flea beetles throughout the entire growing period of the crop, we recommend sowing mixtures of crops for cabbage production such that each of three *Brassica* species would attract phytophagous

Different authors [7,8] reported that glucosinolate content differs in different plant species, this was confirmed also by our research. In the samples of the studied species of Brassicas we established the presence of only one glucosinolate, namely glucobrassicin, it was noticed in all plant species. We found out that the content of glucobrassicin in the samples of oilseed radish negatively influence the studied group of harmful organisms, both cabbage flea beetles and cabbage stink bugs. Despite the fact that the connection between the content of glucobrassicin and the extent of damage by cabbage flea beetles (*r* = −0.30) and cabbage stink bugs (*r* = −0.32) was negative, we cannot talk about identical influence throughout the growth period. This is consequently conditioned by the fact that glucosinolate content in Brassicas varied both during the growth period and between individual plant organs. It is also interesting that we found

Research has established [9] higher contents of five different glucosinolates in blossoms of black mustard (*Brassica nigra*) than in leaves of the said plant species. In our research this statement holds true for glucobrassicin in glucoraphanin in the samples of oilseed radish and for sinalbin and epiprogoitrin in the samples of white mustard. Based on research done by [10], glucosinolate content can vary also within individual genotypes of the same plant species, this was confirmed also in our research – between the selected hybrids of cabbage we discovered significant differences in the contents of progoitrin and glucobrassicin. In the samples of the hybrid 'Hinova' we discovered more progoitrin, while a higher share of glucobrassicin was present in the hybrid 'Tucana'. Of the nine glucosinolates which were analysed in the main crops, we confirmed the presence of four (sinalbin, gluconasturtiin, glucoraphanin and

progoitrin) in both hybrids of cabbage, yet only in traces (below detection threshold).

insect during a particular part of the cabbage growing season.

more glucobrassicin in the blossoms of oilseed radish than in the leaves.

**3. Results and discussion**

**injury to cabbage**

426 Insecticides Resistance

Despite previously proven nematicide effects of sinigrin [13], in our experiment this glucosi‐ nolate in the mid-late hybrid of cabbage stimulated the feeding of cabbage stink bugs, thus causing greater extent of damage. Because the content of progoitrin in the samples of oilseed rape was highest on the date of assessment (31st August), the authors maintain that this substance can potentially negatively influence cattle if oilseed rape is used as fodder [12]. Marked preference of different harmful pests for oilseed rape (in our case this holds true for both years of the experiment) is often caused by the fact that farmers do not choose to grow the said plant species [14].

On the basis of the results of our research, we can conclude that the content of progoitrin in the hybrids of cabbage could in future represent an important factor when selecting hybrids for cabbage production in our conditions. A research carried out in the Netherlands [15] found out that the content of sinigrin and progoitrin influences the taste of Brussels sprouts. Conse‐ quently, great efforts were made to reduce the content of the said glucosinolates or improve plants [15]. Despite the reports about negative influence of progoitrin in fodder for cattle, we cannot talk about negative influence of the said glucosinolate and other glucosinolates on human diet [16]. In the past, there were suspicions also about negative effects of glucosinolates on human diet [17], but they were later rejected [16, 18].

Potential negative influence of glucobrassicin on feeding of cabbage flea beetles was detected in all plant species except in oilseed rape. On one hand this glucosinolate has negative influence on the species from the genus *Phyllotreta*, on the other hand it has positive (anticarcinogenic) influence on human health [16]. These are thus »good« properties of this secondary metabolite. Despite this, glucobrassicin proved to be very susceptible to environmental factors, which will be dealt with later in our discussion.

We confirmed that glucoraphanin and sinalbin were significantly most present among aliphatic glucosinolates in the samples of oilseed radish, while among indole glucosinolates glucobrassicin was present. The content of sinalbin was among the studied plant species of Brassicas highest in the samples of white mustard (30.12±5.52 μmol/g mass of dry seed) and oilseed rape (11.16±6,50 μmol/g mass of dry seed). On the basis of the above finding that glucosinolate content differs between individual species of Brassicas, we believe that there are also differences between species of Brassicas in regard to their suitability for feeding of cabbage flea beetles and cabbage stink bugs. While in the hybrids 'Tucana' and 'Hinova' and in white mustard and oilseed radish, we noticed negative influence of glucobrassicin on the extent of damage by cabbage stink bugs, in the samples of oilseed rape we noticed positive influence of glucobrassicin on the extent of damage. Gluconapin is also one of the glucosinolates which in our research stimulated feeding of the species from the *Eurydema* spp.

The content of secondary metabolites [19] in plants is influenced also by environmental factors, which was noted by [20], this fact was confirmed also in our research. The knowledge about the influence of individual parameters of the environment on glucosinolate content in plants is thus gradually building up. Authors [21] report about positive influence of extreme temperatures on the content of the said secondary metabolites. Influence of temperatures is supposed to be more pronounced in indole glucosinolates [22], this was confirmed also in our research in which we found out that the content of glucobrassicin was conditioned primarily by the average daily and the highest daily temperature of air.

Yet very little is known about the influence of individual environmental factors on the content of aliphatic and aromatic glucosinolates. Our research has established that the influence of environment on individual glucosinolates from the said two groups differs greatly. Thus we cannot speak about some uniform effects of environment on types of glucosinolates. Experts suppose that the reason for different responses of glucosinolates to the environment's activity can be the fact that the genesis of specific glucosinolates involves specific enzymes, which differ from the enzymes which are required to produce other glucosinolates [23].

As we have already emphasised, the importance of alternative methods in plant protection is increasing, and in connection with this we are glad to report that also the trap crop method studied in our dissertation was successful at both locations of the field experiment. The trap crops were much more susceptible to damage by cabbage stink bugs (*Eurydema* spp.) and cabbage flea beetles (*Phyllotreta* spp.) than the cabbage. In Gorenjska, oilseed rape was most susceptible to feeding by cabbage stink bugs, preference of cabbage flea beetles was at the same location detected in oilseed radish. In Ljubljana in 2009, we established the significantly highest extent of damage by the species from the genus *Eurydema* on oilseed rape, while in 2010, we noticed no difference between the trap crops. We have nevertheless established that suscept‐ ibility of different Brassicas to attacks by the studied harmful insects varies during the growth period.

In the field in Gorenjska, we noticed in 2009 the highest extent of damage due to cabbage stink bugs on oilseed rape (the average index of damage 3.38±0.05), in the second year of the experiment, the most susceptible to damage by cabbage stink bugs was oilseed rape (3.58±0.02). In white mustard in the first (2.72±0.04) and the second (2.56±0.05) year of the experiment, we recorded the significantly lowest index of damage by cabbage stink bugs. In the second year of the experiment, we recorded the highest susceptibility to damage by cabbage flea beetles in oilseed radish (3.5±2.82), while the extent of damage on white mustard was significantly lowest (2.82±0.02).

Authors [15] report that glucosinolate content in Brassicas is conditioned also by feeding of harmful organisms. With their feeding – by damaging the cellular structure – they regulate glucosinolate content. In our research, we established that the connection between glucosino‐ late content and the content of damage by the species from the genera *Phyllotreta* or *Euryde‐ ma* differs between different glucosinolates, as well as that individual glucosinolates differently influence the extent of damage.

The plant species which were used as trap crops in our research are in Slovenia usually used as catch crops [24]. Oilseed rape is among the studied trap crops considered a plant species which is very susceptible to attacks by harmful organisms and is consequently less popular in ecological production [14]. If we compare indexes of damage by cabbage stink bugs on different species of Brassicas, we see that these were the highest on the said plant species. From the point of view of possible application of Brassicas as trap crop systems in the production of cabbage, white mustard represents the least suitable species, as it has been confirmed as the least susceptible to feeding of cabbage flea beetles. White mustard is in the research carried out by [6] and [25] mentioned as a plant species with very high natural resistance to attacks by flea beetles. It has been established that epidermis, thickly covered with trichomes, is the main reason for the resistance of plants, which is confirmed also by the research carried out by [26].

The content of secondary metabolites [19] in plants is influenced also by environmental factors, which was noted by [20], this fact was confirmed also in our research. The knowledge about the influence of individual parameters of the environment on glucosinolate content in plants is thus gradually building up. Authors [21] report about positive influence of extreme temperatures on the content of the said secondary metabolites. Influence of temperatures is supposed to be more pronounced in indole glucosinolates [22], this was confirmed also in our research in which we found out that the content of glucobrassicin was conditioned primarily

Yet very little is known about the influence of individual environmental factors on the content of aliphatic and aromatic glucosinolates. Our research has established that the influence of environment on individual glucosinolates from the said two groups differs greatly. Thus we cannot speak about some uniform effects of environment on types of glucosinolates. Experts suppose that the reason for different responses of glucosinolates to the environment's activity can be the fact that the genesis of specific glucosinolates involves specific enzymes, which differ

As we have already emphasised, the importance of alternative methods in plant protection is increasing, and in connection with this we are glad to report that also the trap crop method studied in our dissertation was successful at both locations of the field experiment. The trap crops were much more susceptible to damage by cabbage stink bugs (*Eurydema* spp.) and cabbage flea beetles (*Phyllotreta* spp.) than the cabbage. In Gorenjska, oilseed rape was most susceptible to feeding by cabbage stink bugs, preference of cabbage flea beetles was at the same location detected in oilseed radish. In Ljubljana in 2009, we established the significantly highest extent of damage by the species from the genus *Eurydema* on oilseed rape, while in 2010, we noticed no difference between the trap crops. We have nevertheless established that suscept‐ ibility of different Brassicas to attacks by the studied harmful insects varies during the growth

In the field in Gorenjska, we noticed in 2009 the highest extent of damage due to cabbage stink bugs on oilseed rape (the average index of damage 3.38±0.05), in the second year of the experiment, the most susceptible to damage by cabbage stink bugs was oilseed rape (3.58±0.02). In white mustard in the first (2.72±0.04) and the second (2.56±0.05) year of the experiment, we recorded the significantly lowest index of damage by cabbage stink bugs. In the second year of the experiment, we recorded the highest susceptibility to damage by cabbage flea beetles in oilseed radish (3.5±2.82), while the extent of damage on white mustard was significantly lowest

Authors [15] report that glucosinolate content in Brassicas is conditioned also by feeding of harmful organisms. With their feeding – by damaging the cellular structure – they regulate glucosinolate content. In our research, we established that the connection between glucosino‐ late content and the content of damage by the species from the genera *Phyllotreta* or *Euryde‐ ma* differs between different glucosinolates, as well as that individual glucosinolates differently

The plant species which were used as trap crops in our research are in Slovenia usually used as catch crops [24]. Oilseed rape is among the studied trap crops considered a plant species

by the average daily and the highest daily temperature of air.

period.

428 Insecticides Resistance

(2.82±0.02).

influence the extent of damage.

from the enzymes which are required to produce other glucosinolates [23].

By seeding trap crops among the main plant species, the latter (cabbage) was protected against early attacks by cabbage flea beetles and cabbage stink bugs. It is known that cabbage flea beetles which appear in the beginning of May are very harmful for young plants of Brassicas, which have at that time small surface of leaves [27]. The age of plants is among more important factors which influence the extent of damage by the species from the genus *Phyllotreta*. Authors [28] in their research report successful reduction of the extent of damage on cabbage done by the species from the genus *Phyllotreta* by different terms of seeding.

We have found out that there is a negative correlation between the plants of trap crops and the extent of damage by cabbage flea beetles in the beginning of the developmental stage in which leaves are developed (BBCH 12-14). On the basis of the results of our research, we can conclude that adult specimens of cabbage stink bugs in spring appear in the second half of May or in the beginning of June. In the said period we noticed the first damage interval [6]. Damage was first detected on the trap crops, only later on the main crops. This was the case at both locations in the experiment.

The hybrid cabbage 'Hinova' in all comparisons proved to be much more susceptible to damage by cabbage stink bugs and cabbage flea beetles [29] – in comparison with the hybrid 'Tucana'. We have nevertheless found out that susceptibility of Brassicas in individual parts of the growth period varies between genotypes of cabbage, in some parts of the growth period we thus noticed pronounced damage also on the hybrid 'Tucana'. The damage by cabbage flea beetles on the cultivar 'Tucana' were thus more extensive in the beginning of the growth period, in the phase when plants were developing leaves and opened from 4 to 7 proper leaves. Greater extent of damage was later recorded on the cultivar 'Hinova'. The mid-late hybrid cabbage was not so susceptible to damage by cabbage stink bugs. This finding was established already in the research by [30]. The connection can be found in the interaction between the length of the growth period of the hybrid and bionomy of the studied harmful pest. At the time when the species *Eurydema ventrale* and *Eurydema oleracea* only just began to appear massively, the crops of the early hybrid were already collected.

We have found out that usefulness of the selected alternative methods in plant protection is conditioned by the selection of an appropriate main crop and appropriate hybrid. The average crop of cabbage was influenced by the selection of a cultivar, while we did not notice any pronounced differences in the average crop between individual treatments (species of trap crops). We also compared the damage by the species from the genera *Phyllotreta* and *Euryde‐ ma* on the main crop according to the distance from the trap crop. We found out that cabbage stink bugs prefer to appear on plants of cabbage which are farthest from the trap crop, while the influence of a plant's distance on damage by cabbage flea beetles was not established. 'Hinova' in our conditions of production proved to be the hybrid which yielded a bigger average crop, the selection of the early hybrid 'Tucana' proved as unsuitable. According to the results of our research we can say this is congruent with the findings of the authors [31–33], who report that alternative methods (intermediate crops, green covers) did not reach the desired goals.

By more intensive care for the main crop (watering during the growth period, dressing with fertilisers...) we could provide bigger crops. It is well known that the trap crop method can be carried out in several ways. One of these is to collect the trap crops in the middle of the growth period where we carried out the experiment [34]. At the same time we would risk that the studied group of harmful pests massively moved to the main crop [34]. We could treat the trap crop with insecticides and thus provided the so-called dead-end trap crop. Yet by using insecticides we would bring more damage than benefits [35]. The species which were used as trap crops were for a part of their growth period also blossoming and thus attracting useful organisms. The reasons for different susceptibility in the studied species of Brassicas can be found in natural resistance of plants. Glucosinolates, which are considered by some as an important factor of plants' resistance against harmful organisms, and by others as negligible in this regard, can in the protection of plants act stimulatory or deterring. While gluconasturtiin in oilseed rape acts as a deterrent (*r* = −0.99) for feeding by cabbage flea beetles, we cannot attribute the said glucosinolate the same property in case of white mustard and oilseed radish. According to the research, we can conclude that the effects of these secondary metabolites on harmful insects are very complex. We thus cannot talk about some universal influence of the three groups of glucosinolates on harmful organisms, and the influence of each glucosinolate should be analysed separately.

Gluconasturtiin in oilseed rape acts negatively on feeding by cabbage flea beetles (*r* = −0.99) and cabbage stink bugs (*r* = −0.98). Feeding by cabbage flea beetles (*r* = −0.80) and cabbage stink bugs (*r* = −0.99) on the said plant species is strongly influenced by epiprogoitrin content. Glucoiberin content in the samples of oilseed rape negatively influenced the feeding by cabbage flea beetles (*r* = −1) and cabbage stink bugs (*r* = −1), progoitrin in the samples of oilseed rape stimulated the feeding by cabbage stink bugs (*r* = 0.51) and cabbage flea beetles (*r* = 0.51), while the activity in the samples of 'Tucana' negatively influenced the feeding by cabbage stink bugs (*r* = −1.0). Gluconapiin is the only glucosinolate which in the plants oilseed rape acts stimulatory on the feeding by cabbage stink bugs (*r* = 0.64) and cabbage flea beetles (*r* = 0.67). We detected no pronounced influence of the studied glucosinolates on feeding by the species from the genus *Phyllotreta* in the samples of oilseed radish. We can conclude that the barely perceptible presence of gluconasturtiin, glucoiberin and gluconapiin most probably contrib‐ uted to the higher index of damage by cabbage flea beetles on the studied species of Brassicas.

Because of the thus far collected findings that glucosinolate content changes during the growth period and depends on the plant species, we wanted to compare the extent of damage by cabbage flea beetles also in regard to the location of the experiments.

In Slovenia, cabbage flea beetles are most massively present in July [36], this was confirmed also in our research. The feeding of cabbage flea beetles is influenced also by the average daily temperature [37], which was also found out in our research; we noticed several such cases during the growth period in both years of the experiment. In 2010 we thus in the last ten days of May on the laboratory field of the Biotechnical Faculty in Ljubljana noticed higher average daily temperatures than on the location of the experiment in Gorenjska.

Indexes of damage due to both groups of harmful pests were in the second year of the experiment markedly higher, which confirms the fact that using crop rotation is also one of the principles of successful agricultural practice [38, 39, 40, 41, 42]. We thus in both years of the experiment detected a higher extent of damage at the laboratory field of the Biotechnical Faculty and in the field in Gorenjska. The importance of crop rotation was confirmed especially in Gorenjska, where some years before our experiment in the vicinity there were no larger field with Brassicas, so the attacks of both groups of harmful pests in the first year of the experiment were relatively weak. At the laboratory field of the Biotechnical Faculty, Brassicas have been grown every year for quite some time, consequently, the attacks of cabbage flea beetles and cabbage stink bugs were strong already in the first year of the experiment. The finding that a considerably lesser extent of damage by harmful pests is influenced also by crop rotation [42, 43] is confirmed also in our research.

The average daily temperature 17.4 °C at the laboratory field of the Biotechnical Faculty in Ljubljana thus stimulatory influenced the group of the studied harmful pests, while the average daily temperature in the same period in Gorenjska was 16.1 °C.

We have found out that white mustard was the first to reach the phase of maturing (BBCH 80– 89) [25], at the same time the susceptibility of this plant species to attacks by the studied harmful pests was reduced. The low susceptibility of white mustard to damage by the species from the genus *Phyllotreta* in the beginning of the growth period was still very pronounced in 2009 in Gorenjska. We can thus sum up that the developmental phase of plants is one of the more important factors of feeding by cabbage flea beetles and cabbage stink bugs. In Gorenjska, the trap crops for cabbage flea beetles were most susceptible at the time of blossoming (BBCH 60-67), at that time the cabbage was entering the phase of developing vegetative parts of plants suitable for crops (forming heads). Feeding by cabbage flea beetles at the plants' blossoms was recorded already in other studies [44].
