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Dual action of phosphonate herbicides in plants affected by herbivore—Model study on black bean aphid Aphis fabae rearing on broad bean Vicia faba plants
ARTICLE IN PRESS Ecotoxicology and Environmental Safety 72 (2009) 1701–1706
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Dual action of phosphonate herbicides in plants affected by herbivore—Model study on black bean aphid Aphis fabae rearing on broad bean Vicia faba plants Jacek Lipok Faculty of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
a r t i c l e in fo
abstract
Article history: Received 15 October 2008 Received in revised form 24 February 2009 Accepted 16 March 2009 Available online 17 April 2009
The interactions between plants, herbicides and herbivore insects were studied as an aspect of possible side effect of the using of phosphonate herbicides. The experimental system was composed of phosphonate herbicides, broad bean Vicia faba (L.) plants and black bean aphid Aphis fabae (Scopoli). Two means of herbicide application, namely standard spraying and direct introduction of the herbicide into stem via glass capillary, were examined. The results obtained for N-2-piridylaminomethylene bisphosphonic acid and its derivatives show 10 times higher inhibition of the plant growth if glass capillary mode was used. When plants were infested by aphids 24 h after the use of herbicide, a significant decrease in plant growth rate was observed in relation to plants treated with herbicides alone. Moreover, the sensitivity of aphids towards glyphosate, N-2-piridylaminomethylene bisphosphonic acid and its 3-methyl derivative introduced to artificial diet indicated that these herbicidal phosphonates possessed also insecticidal activity if applied in a systemic manner. Additionally, olfactometer measurements revealed that aphids preferred intact V. faba leaves over those that had been treated with sublethal doses of herbicides. The results achieved in these experiments indicate that the use of phosphonate herbicides decreases plant resistance and influences the number of aphids accompanied with treated plants. Regarding these facts it can be concluded that the combined effect of herbicide-induced stress and insect herbivory reduced plant fitness and thus should be considered as also a factor enabling the reduction of herbicide doses. & 2009 Elsevier Inc. All rights reserved.
Keywords: Phosphonate herbicides Side effect Aphis fabae Vicia faba Additive action
1. Introduction Chemical treatments of crops are today the most effective way to increase the effectiveness of plant breeding through quantity of harvests. However, both chemical fertilisation and plant protection with pesticides result in the introduction of substantial quantities of agrochemicals into ecosystems. Although the research is focused on the required action of these substances, unexpected side effects on non-target organisms are also of great environmental concern (Croft, 1990; Desneux et al., 2007). Many examples show that the objective of high yields of desirable crops led to increased use of pesticides, which were in the past often applied even prophylactically (Fabre et al., 2003; Pemsl et al., 2004; Bortoluzzi et al., 2007). It is one amongst the important reasons as to why there is a renewed support for studies on cultural and biological control accompanying pesticide use (Wijnands, 1997), especially considering the integrated pest management (IPM), in which even sublethal doses of pesticides
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could affect the insect behaviour (Wei and Du, 2004; Medrzycki et al., 2003) and the attractiveness of crops for herbivorous insects (Norris and Kogan, 2005). Similarly the implementation of glyphosate-ready and gluphosinate-ready crops into agricultural practise promotes the incorporation of this commonly used herbicides to IPM strategies. Considering the non-selective mode of action of phosphonate herbicides and their predicted negative effect on non-crop plants (Riemens et al., 2008), there is a requirement for better understanding of some aspects of the environmental impact of these chemicals and thus the possibility to utilise or alleviate the observed side effects in common practise. Glyphosate (N-phosphonomethyl glycine) is now one of the most widely used broad-spectrum, systemic herbicides for vegetation control. A number of examples of side effects of glyphosate and related compounds have been reported so far. They result from over 30 years of its non-target impact on microorganisms, aquatic invertebrates and algae, terrestrial invertebrates, birds, fishes, mammals and finally on humans (Austin et al., 1991; Eijsackers, 1985; MacKinnon and Freedman, 1993; Folmar et al., 1979; Bronstein and Sullivan, 1992). There is, however, surprisingly little information on the direct or
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indirect impact of N-phosphonomethyl glycine on insects, which feed on treated plants. The field experiments on cereal aphid (Rhopalosiphum padi L.) showed that they were rapidly killed by herbicides containing paraquat or paraquat+diquat (1/4 of the recommended field dose of this herbicide) when sprayed either on or off plants, whereas glyphosate had no effect on this insect (Wright et al., 1985). The same results were obtained by cited authors in laboratory tests carried out on aphids fed on sucrose solutions containing herbicides. In the case of glyphosate and other aminophosphonates, potential disruption of aphid population might be of particular interest because of the mode of action of these compounds (Forlani et al., 1996). Representatives of the animal kingdom lack the shikimic acid pathway, a target pathway for the action of glyphosate, and thus should be unaffected by glyphosate. Similar situation should be achieved in the case of pyridyl derivatives of aminomethylenebisphosphonates, which most probably inhibit the first enzyme of shikimic pathway-3-dioxyarabino heptulosonate 7-P synthase (DAHP synthase) (Kafarski et al., 1997). However, the influence of these substances on the shikimic pathway, which provide the chemicals indispensable for plantdefence system and thus change the interactions with pathogens and herbivore insects (De Moraes et al., 2004), might have great importance in agriculture, especially in IPM strategies. The objective of this paper is to find out whether aminophosphonate herbicides are toxic only for host plants or whether, through decreasing the quality of plants as the nutrient source, tested compounds also affect herbivore insects. This effect might be of special interest considering the influence of the nutritional quality of host plants for herbivore insects’ feeding and behaviour (Behmer, 2009) and the combined effect of herbicide-induced stress and insect herbivory to reduce weed fitness (Williams et al., 2004). Thus, this paper describes the sensitivity of black aphid A. fabae towards tested herbicides as the nutrients and the influence of herbicide-treated plants of broad bean V. faba L. on the host plant choice and population development of black aphid A. fabae. The combined effect of sublethal doses of herbicides and presence of aphids on the growth of broad bean plants was also investigated.
2. Materials and methods 2.1. Broad bean Vicia faba culture Dry seeds of broad (fava) bean V. faba cv. White Windsor, a common crop in Opole region, Southwest part of Poland, were used. Seeds were germinated for 5 days between two 0.5-cm-thick wet cotton layers in plastic pots, with 12 seeds in each pot (20 cm 30 cm, 5 cm height). Obtained seedlings having 2 cm root and 2 cm hypocotyl were transferred separately to glass jars, where they were grown hydroponically on cotton gauze over the surface of tap water at 2071 1C, 65% relative humidity (RH), 1200 lx and 14:10 h light:dark. Plants (about 10 cm height) were infested by aphids as soon as the first pair of leaves had unfolded.
2.2. Aphids culture (breading) The culture of A. fabae was established from a single virginoparous apterous female collected from V. faba plant near Opole in May 2000. Aphids were reared on preflowering V. faba (cv. White Windsor) plants in a growth chamber at 2071 1C, 65% relative humidity, 1200 lx in a long-day LD 16:8 h photocycle.
2.3. Tested herbicides In order to study the real effects of pure compounds, which possessed herbicidal activity, all experiments were arranged using active substances only. Pure glyphosate (N-phosphonomethylglycine) was obtained by the author via laboratory procedure: from commercial Roundups 360 SL (Monsanto, MO, USA) formulation by dissolving in water and maintaining the pH of the solution to 1.5–2.0 with hydrochloric acid. This resulted in crystallisation of the pure
herbicide. Its structure and purity were confirmed using 1H, 13C and 31P NMR spectroscopy. The retention time of this substance in capillary electrophoresis was the same as the retention time of glyphosate standard obtained from Monsanto. The bisphosphonic compounds N-(2-piridylamino)methylene bisphosphonic acid (PMBA), N-[2-(3-methylpiridylamino)]methylene bisphosphonic acid (3MPMBA) and N-[2-(6-methylpiridylamino)]methylene bisphosphonic acid (6MPMBA) were gifts from Prof. P. Kafarski (Wroc"aw University of Technology). The chemicals were diluted in water to reach the final concentrations ranging from 150 to 0.015 mM depending on the experiment.
2.4. Olfactometer bioassay The two-chamber diffusion olfactometer was used to measure responses of mating females to herbicide-treated V. faba plants. The olfactometer consisted of the glass tube (4 cm diameter, 25 cm length) with the airflow window located over the arena in the middle of its length and also two connected glass bulb chambers, ending with glass filters. The airflow inside the olfactometer was established by air circulation based on the diffusion process. The lack of constrained airflow distinguished this olfactometer from standard ones (Vet et al., 1983; Nazzi et al., 2001); however, its simplicity and subtle gradient of volatiles concentration allowed one to examine the responses of delicate, wingless aphids. Odour fields were arranged by adding the plants to the glass bulb chambers. All plants used in the experiments were logged immediately after the groups of 20 virgin adult females were introduced into the olfactometer arena, and then the choice test was started. The volume of 0.1 cm3 of tested herbicides at the concentration of 1.5 mM, which is adequate to a sublethal field dose per plant, was applied to the plant tissues via glass capillary. In each experiment 1 g of plant tissue (stems and leaves) treated with appropriate herbicide was placed into one bulb chamber (experiment), while 1 g of V. faba tissue treated in the same manner with water was placed in the opposite bulb chamber (control). The positions of aphids were recorded every 5 min for 60 min. To protect against positional bias, the olfactometer was shielded with white cards and rotated by 1801 between the replications of experiments. Each experiment was carried out in 15 applications. The numbers of aphids recorded after appropriate time in each experiment – each herbicide treatment, respectively, in experimental and control chambers – were collected and analysis of variance (ANOVA) was conducted regarding the null hypothesis. Then the significant differences between means (Po0.05) were compared using HSD Tukey’s test. Statistical evaluation of experimental data was performed with Statgraphics 5 Plus software (Manugistics Inc., 2000).
2.5. Herbicidal activity Each of four tested compounds was studied at three concentrations: 15, 1.5 and 0.015 mM using two means of treatment. The first one was maintained in such a manner that herbicide was taken up by plant via glass capillary. Thus, glass capillary containing 0.1 cm3 of studied solution was introduced 0.1 cm deep in the stem of plants 5 cm above the root and was left there until the solution was absorbed. Furthermore, the same amount of glyphosate was used but at a concentration of 150 mM, which is adequate to its field dose per one plant. The four V. faba plants 10 cm in height were grown in an isolator and treated with herbicide at each concentration (or distilled water as control) using capillary application. Each experiment was done in six repetitions. The second application was a standard spraying of the plants with 0.1 cm3 solutions of tested herbicides or pure water (control). Each experiment was carried out in six repetitions. In both experiments all examined plants were reared in a climate chamber at 2071 1C, 65% relative humidity, 1200 lx in a long-day LD 16:8 h photocycle for 9–10 days. During this period the total height of the green part of plants was recorded everyday and their morphological changes were observed. The values of plant heights recorded each day in each experiment were grouped and then the significant differences between means were compared using MRT Duncan’s test (Po0.05). Statistical evaluation of experimental data was performed with Statgraphics 5 Plus software (Manugistics Inc., 2000).
2.6. Combined influence of herbicides and aphids on plant growth V. faba plants of about 10 cm height, treated with herbicides at concentrations and in a manner described above, were infested with two apterous female aphids 24 h after the treatment. In order to study aphid development and their impact on plant, the number of adults and L1–L4 nymphs was counted everyday for a 9–10day period and also the total length of green part of plants and morphological changes were recorded. Each experiment was replicated six times. Statistical evaluation of the results achieved in these experiments was conducted similar to that described in herbicidal activity tests (MRT Duncan’s test), additionally taking into account the total number of aphids, which were recorded as developed on each infested plant.
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2.7. Aphid growth and development on the artificial diet containing herbicides To test the effect of studied herbicides as a factor influencing aphid feeding behaviour and reproduction, the studies on artificial diet were performed. This diet was a modified form of the one originally prescribed for rearing Myzus persicae (Sulzer) by supplementation with 11 amino acids, namely asparagine, asparaginic acid, phenylalanine, glutamine, histidine, isoleucine, leucine, lysine, serine, threonine and valine (Mittler and Koski, 1976). The pH of the diet was adjusted to 7.0 with KOH and it was filter-sterilized using Pyrex No 5 glass filter. The solution was sandwiched between two aseptic Nescofilm (Bando Chemical Ind. Ltd., Kobe, Japan) membranes placed over a plastic ring (30 mm in diameter, 15 mm in height) and fed to aphids. The diets including herbicides were prepared by adding appropriate amounts of these compounds at a final concentration of 1.5 mM. Three, 1-day-old, adult females were transferred from aphid cultures at V. faba to each ring with artificial diet. The treatments were carried out in glass chambers covered with light green paper and experiments were carried out under long-day (LD 16:8) condition and at a temperature of 2171 1C. Six hours after the experiments were established and then for the next 3 days with 24 h intervals, the number of aphids was recorded and their developmental stage was observed. The average number of offspring per aphid was determined, taking into consideration the number of rearing aphids. Each experiment was carried out in four replications. The total numbers of aphids, which were recorded daily in each experiment, were evaluated using the analysis of variance followed by HSD Tukey’s test in order to compare the significant differences between means (Po0.05).
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plants) (Table 2). The activity of two of bisphosphonic acids, namely PMBA and 3MPMBA, examined at higher concentrations strongly depended on the mode of treatment, with the application of herbicide via glass capillary being more effective. If they were applied at 15 mM concentration by means of capillary, it caused the death of the plants at the third day of experiment, whereas spraying the plants with a solution of the same concentration had negligible effect on plant growth. Glyphosate appeared to be much less active under the same conditions. Considering the plants, which were treated via capillary and then infested by aphids, significant decrease of their growth potency was observed in relation to plants treated with herbicide alone. Spraying the plants has quite contradictory results in that the kind of additive suppression mentioned above was not observed. The only exception was 3MPMBA, which was also the most effective herbicide when applied by this mode. The additive suppression of herbicides and aphids was best seen from the fourth to the sixth day of experiment (Fig. 1). Prolongation of experiment up to 10 or 11 days resulted in changes in plant colour, shape or leafs and stems vigour. 3.3. Effects of herbicides on aphid development and fecundity
3. Results 3.1. Olfactometer bioassay The results derived from the choice test carried out in an olfactometer indicated that aphids nearly immediately preferred non-treated over treated plants as the food source. This effect was more pronounced after 60 min of the experiment; however, it has already been observed after 15 min of experiment. The mean number of aphids in the chambers containing control plants was two times higher than that in the chambers containing plants treated with glyphosate or 6MPMBA, and six times higher when PMBA and 3MPMBA were used. The examination of data presented in Table 1 clearly shows that two aminomethylenebisphophonic acids, namely non-substituted PMBA and its 3-methyl derivative, most likely act as weak repellents. 3.2. The effect of herbicides on plant and aphid development
No significant differences were observed in the number of insects per one plant between control and plants sprayed with 0.015 and 1.5 mM herbicides during the 8 days of culturing. The only exception was application of PMBA (Table 2). This compound used at the lowest concentration (0.015 mM) caused the decrease in number of aphids up to about 70% in relation to control. This herbicide also caused the highest reduction of aphid development when applied in higher concentrations (1.5; 15 mM) by the spraying mode. The development of aphids on plants treated via capillary was suppressed even if the herbicides were tested at low concentrations. With the exception of 6MPMBA, the average numbers of aphids varied significantly from the control even at a concentration as low as 0.015 mM. This observation might suggest that the compounds acted as weak systemic insecticide. 3.4. Reaction of aphids towards artificial diet supplemented with herbicides
All herbicidally active substances tested in this work influenced plant development in a dose-dependent manner and their herbicidal effect was dependent on the mode of their application. At the lowest concentration (0.015 mM) none of the tested herbicides affected the growth of broad bean plants despite the mode of treatment (e.g. via glass capillary or by spraying the
The insects, which were settled on the artificial diet supplemented with glyphosate, tended to escape from the membranes or were dead at the second or fourth day of experiment (Fig. 2). Three out of the examined herbicidal phosphonates exhibited weak insecticidal activity if applied in a systemic manner. Very
Table 1 Response of adult apterous Aphis fabae females in two-way diffusion olfactometer to the odour of Vicia faba L. plants treated with tested compounds at the concentration of 1.5 mM in a choice test. Experiment
The number of aphids in olfactometer chambers (mean7S.E.) after 5, 15, 30, 60 min of the test 5
Glyphosate Control N-(2-piridylamino) methylene-bisphosphonic acid (PMBA) Control N-[2-(3-methylpiridylamino)]-methylenebisphosphonic acid (3MPMBA) Control N-[2-(6-methylpiridylamino)]-methylenebisphosphonic acid (6MPMBA) Control
7.870.5 8.270.5 3.970.5 8.870.7 3.170.5 8.570.6 8.470.7 8.670.7
15 b b d c d c bc b
Mean7S.E. followed by different letters are significantly different (ANOVA, Tukey’s test; Po0.05)
6.070.7 9.070.8 3.270.5 10.170.3 3.070.5 11.370.7 8.170.6 10.870.5
30 c b d b d b bc a
5.770.7 10.870.7 3.470.5 10.170.7 2.670.5 11.570.4 7.370.5 10.670.9
60 c a d b d b c a
5.670.8 11.570.7 1.970.4 12.370.3 1.570.3 12.870.5 6.170.6 10.170.6
c a e a e a d a
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Table 2 The effect of tested herbicides on development of plants and aphids as measured at the eighth day of experiment. Compound and its concentration in mM
Treatment via glass capillary The average number of aphidsa observed on infested plants
Treatment by spraying the plants The mean height of The mean height of The average number of infested plants uninfested plants aphidsa observed on (cm) (cm) infested plants
The mean height of The mean height of infested plants uninfested plants (cm) (cm)
Glyphosate 150 15 1.5 0.015
21.572.1 a 61.579.2 de 62.8711.1 de 86.3710.0 ef
10.372.0 bc 22.373.6 de 23.871.8 de 22.873.9 de
20.871.9 d 26.172.9 ef 27.573.1 ef 30.072.2 f
28.572.9 b 73.676.1 e 81.279.1 ef 78.9711.4 ef
22.272.1 de 28.972.7 ef 27.072.2 ef 32.673.9 f
20.873.3 d 30.373.5 ef 27.272.1 ef 31.671.7 f
N-2-piridylamino methylene bisphosphonic acid 15 1.5 0.015
89711.8 ef 96.479.7 f
dp 2.770.5 a 28.772.6 ef
dp 9.071.2 b 26.972.6 ef
45.679.2 c 57.777.7 cd 60.979.0 de
26.674.3 df 2973.2 ef 28.176.1 df
29.171.1 f 31.073.1ef 29.872.0 ef
N-2-(3-methylpiridylamino) methylene bisphosphonic acid 15 1.5 0.015
31.576.8 bc 86.3711.7 ef
dp 13.372.1 bc 29.873.7 ef
dp 20.472.8 d 31.174.2 ef
56.679.1 cd 76.174.6 e 92.178.2 ef
12.571.9 c 24.572.2 de 27.174.4 df
16.472.1 cd 28.873.6 ef 27.471.9 ef
15.572.1 c 24.773.9 de 27.572.9 ef 30.372.4 f
21.573.2 de 30.875.2 ef 28.372.9 ef
72.9710.6 e 87.1714.2 ef 80.577.9 ef 84.5713.1 ef
24.573.7 30.473.3 28.372.5 31.574.6
25.772.1 e 29.171.5 ef 25.974.7 df
N-2-(6-methylpiridylamino) methylene bisphosphonic acid 15 1.5 0.015 Controlb Uninfested controlc
88.879.1 ef 90.179.4 ef 10479.8 fg 117.6711.3 g
32.373.2 f
de ef ef ef
29.873.7 ef
dp—death of the plants caused by herbicides before the third day of experiment; ns—not significant result in comparison to appropriate control; a–g—evaluation of significant differences in Duncan’s test. a The average number of aphids per one adult female. b Control—plants untreated with herbicides but infested by aphids. c Uninfested control—plants untreated and uninfested by aphids.
Fig. 1. The total length of the V. faba stems during the experiment with N-(2-piridylamino) methylene bisphosphonic acid (PMBA), which was applied via glass capillary at the concentration of 1.5 mM.
similar effect was observed on diets supplemented with herbicidal bisphosphonates (Fig. 2), with PMBA and 3MPMBA significantly decreasing the number of insects after the third day of experiment.
4. Discussion The herbicides examined when used in sublethal doses suppressed the growth of broad bean plants only in highest
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Fig. 2. The mean number of aphids on artificial diets contained herbicidal aminophosphonates at the final concentration of 1.5 mM during 4 days. The significant differences between means (Tukey’s test, Po0.05) are indicated with asterisks.
tested concentrations, being more effective when they were applied via glass capillary. Such effect has not been seen, or it has been significantly less pronounced, upon application of herbicide by spraying. This might result from the lack of the barrier, which should be overcome by these active substances if they are applied by spraying and had to penetrate plant tissues from the outside surface of leaves and stems into phloem cells. Thus the treatment via capillaries mimics the situation when glyphosate, PMBA or its derivatives are present inside plant tissues and might influence plant metabolism (Forlani et al., 1997; Lejczak et al., 1996; Osiecka and Janas, 1998). Therefore it was also expected that application via capillary should affect aphid development by suppressing shikimic and phenylpropanoid biosynthesis pathway, resulting in the deficiency of plant-defence substances (Marchiosi et al., 2009). Indeed, this phenomenon was observed evoking additive suppression of herbicides and feeding aphids on the growth of infested plants. Similar results were obtained by Williams and co-authors during the study on the combined effect of Fluroxypyr herbicide and colorado potato beetle herbivory on volunteer potatoes (Williams et al., 2004). On the other hand, the changes in metabolism of brad bean plants treated with aminophosphonate herbicides decreased the olfactory attractiveness of plants for black bean aphids. Olfactometer measurements have indicated that aphids preferred intact V. faba leaves over those treated with sublethal doses of herbicides. A. fabae is a heteroecious insect and it exhibits host plant alternation between a narrow range of primary hosts on which the aphids reproduce sexually in autumn, and wide secondary hosts’ range, which support multiple parthenogenetic generations in the summer (Raymond et al., 2001; Tosh et al., 2001). The primary host plant range of A. fabae in Poland is limited usually to Euonymus europaeus (L.), while the secondary host plant range comprises few species. Among them Beta vulgaris (L.), Chenopodium album (L.) and V. faba are chosen more often than others (Wnuk and Gospodarek, 1999). Since the application of herbicide should affect negatively plant resistance to insect action, a slight but significant decrease in the number of aphids on the plants treated with all the herbicides was observed. The studies on aphids cultured on artificial diet supplemented with herbicides revealed that the same herbicides (glyphosate, PMBA, 3MPMBA), application of which decreased the number of aphids on treated plants,
influence negatively the insect development most likely exhibiting weak insecticidal activity. This is in contradiction to results of studies on development of yellow soybean aphids on soybean plants treated with glyphosate under the field conditions (Burrows et al., 2005); however, the tested soybean variety was a glyphosate-resistant one and in this case metabolic stress evoked by glyphosate is hardly expected. On the other hand, experimental results achieved in this work are in a good agreement with the results obtained upon aphid infestation on weeds and sugar beet plants in glyphosate-tolerant sugar beets crop (Dewar et al., 2000) and with the studies on the effectiveness of one, two or three glyphosate application(s) on V. faba for controlling Orobanche crenata (Forsk) infestation (Naguib et al., 1987). Orobanche infestation alongside with glyphosate spraying caused pronounced decrease in the total amino acid content in the leaves of the host plant. However, the decrease induced by the herbivore was higher than that induced by glyphosate, thus showing more complex relation between these two organisms in the presence of herbicide.
5. Conclusions The results of our studies have shown that treatment of V. faba plants infested by A. fabae aphids with sublethal doses of glyphosate or pyridyl derivatives of aminomethylenebisphosphonic acid caused additive suppression of plant growth. Simultaneously the herbicides decreased also the rate of growth and development of the aphid populations, most probably by exhibiting weak insecticidal activity. Regarding these it can be concluded that the combined effect of herbicide-induced stress and reduced plant fitness by insect herbivory should be considered as a factor enabling reduction of applied herbicide doses.
Acknowledgments This research investigation was conducted in the Faculty of Chemistry, University of Opole, Poland. I thank Prof. Pawe" Kafarski for providing with aminomethylenebisphosphonic acids used in the presented experiments and for the fruitful discussion about their mode of action.
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MacKinnon, D.S., Freedman, B., 1993. Effects of silvicultural use of the herbicide glyphosate on breeding birds of regenerating clearcuts in Nova Scotia, Canada. J. Appl. Ecol. 30, 395–406. Marchiosi, R., Ferrarese, M.L.L., Bonini, E.A., Gomes Fernandes, N., Ferro, A.P., Ferrarese-Filho, O., 2009. Glyphosate-induced metabolic changes in susceptible and glyphosate-resistant soybean (Glycine max L.) roots. Pestic. Biochem. Physiol. 93, 28–33. Medrzycki, P., Montanari, R., Bortolotti, L., Sabatini, A.G., Maini, S., Porrini, C., 2003. Effects of imidacloprid administered in sub-lethal doses on honey bee behaviour. Laboratory tests. Bull. Insectol. 56 (1), 59–62. Mittler, T.E., Koski, P., 1976. Development of meridic and oligidic diets for rearing the aphid Myzus persicae. J. Insect Physiol. 22, 1135–1141. Naguib, N.A., El-Baz, F.K., El-Masry, R., 1987. Effect of glyphosate on the amino acids and mineral contents of Vicia faba and Orobanche crenata and its influence on the host-parasite relationship. Egypt J. Physiol. Sci. 12, 91–102. Nazzi, F., Bartlet, E., Watts, N., Wadhams, L.J., 2001. Factors affecting the response of Ceutorhynchus assimilis Payk. (Col. Curculionidae) males to conspecific odour. J. Appl. Entomol. 125, 433–435. Norris, R.F., Kogan, M., 2005. Ecology of interactions between weeds and arthropods. Annu. Rev. Entomol. 50, 479–503. Osiecka, R., Janas, K.M., 1998. Mitodepressive and clastogenic effects of some aminophosphonates, inhibitors of phenylalaninie ammonia-lyase. I 2-Aminoindan-2-phosphonic acid. Plant Physiol. Biochem. 36 (11), 805–808. Pemsl, D., Waibel, H., Orphal, J., 2004. A methodology to assess the profitability of Bt-cotton: case study results from the state of Karnataka, India. Crop Prot. 23, 1249–1257. Raymond, B., Searle, J.B., Douglas, A.E., 2001. On the process shaping reproductive isolation in aphids of the Aphis fabae (Scop.) complex (Aphididae: Homoptera). Biol. J. Linn. Soc. 74, 205–215. Riemens, M.M., Dueck, T., Kempenaar, C., 2008. Predicting sublethal effects of herbicides on terrestrial non-crop plant species in the field from greenhouse data. Environ. Pollut. 155 (1), 141–149. Manugistics Inc., 2000. Statgraphicss Plus for 32-Bit Operating System. Tosh, C.R., Walters, K.F.A., Douglas, A.E., 2001. On the mechanistic basis of plant affiliation in the black bean aphid (Aphis fabae) species complex. Entomol. Exp. Appl. 99, 121–125. Vet, L.E.M., Van Lenteren, J.C., Heymans, M., Mellis, E., 1983. An airflow olfactometer for measuring olfactory responses of hymenopterous parasitoides and other small insects. Physiol. Entomol. 8, 97–106. Wei, H.-Y., Du, J.-W., 2004. Sublethal effects of larval treatment with deltamethrin on moth sex pheromone communication system of the Asian corn borer, Ostrinia furnacalis. Pest. Biochem. Physiol. 80, 12–20. Wijnands, F.G., 1997. Integrated crop protection and environment exposure to pesticides: methods to reduce use and impact of pesticides in arable farming. Eur. J. Agron. 7, 251–260. Williams II, M.M., Walsh, D.B., Boydston, R.A., 2004. Integrated arthropod herbivory and reduced herbicide use for weed management. Weed Sci. 52, 1018–1025. Wnuk, A., Gospodarek, J., 1999. Occurrence of aphidophagous Syrphidae (Diptera) in colonies of Aphis fabae (Sop.) on its various host plants. Ann. Agric. Sci.—Series E–Plant Prot. 28 (1–2), 7–15. Wright, M.A., Kendall, D.A., Smith, B.D., 1985. Toxicity of paraquat, paraquat+diquat and glyphosate to the cereal aphid Rhopalosiphum padi. In: Tests of Agrochemicals and Cultivars. Association of Applied Biologists, London, pp. 8–9.
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Dual action of phosphonate herbicides in plants affected by herbivore—Model study on black bean aphid Aphis fabae rearing on broad bean Vicia faba plants Jacek Lipok Faculty of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
a r t i c l e in fo
abstract
Article history: Received 15 October 2008 Received in revised form 24 February 2009 Accepted 16 March 2009 Available online 17 April 2009
The interactions between plants, herbicides and herbivore insects were studied as an aspect of possible side effect of the using of phosphonate herbicides. The experimental system was composed of phosphonate herbicides, broad bean Vicia faba (L.) plants and black bean aphid Aphis fabae (Scopoli). Two means of herbicide application, namely standard spraying and direct introduction of the herbicide into stem via glass capillary, were examined. The results obtained for N-2-piridylaminomethylene bisphosphonic acid and its derivatives show 10 times higher inhibition of the plant growth if glass capillary mode was used. When plants were infested by aphids 24 h after the use of herbicide, a significant decrease in plant growth rate was observed in relation to plants treated with herbicides alone. Moreover, the sensitivity of aphids towards glyphosate, N-2-piridylaminomethylene bisphosphonic acid and its 3-methyl derivative introduced to artificial diet indicated that these herbicidal phosphonates possessed also insecticidal activity if applied in a systemic manner. Additionally, olfactometer measurements revealed that aphids preferred intact V. faba leaves over those that had been treated with sublethal doses of herbicides. The results achieved in these experiments indicate that the use of phosphonate herbicides decreases plant resistance and influences the number of aphids accompanied with treated plants. Regarding these facts it can be concluded that the combined effect of herbicide-induced stress and insect herbivory reduced plant fitness and thus should be considered as also a factor enabling the reduction of herbicide doses. & 2009 Elsevier Inc. All rights reserved.
Keywords: Phosphonate herbicides Side effect Aphis fabae Vicia faba Additive action
1. Introduction Chemical treatments of crops are today the most effective way to increase the effectiveness of plant breeding through quantity of harvests. However, both chemical fertilisation and plant protection with pesticides result in the introduction of substantial quantities of agrochemicals into ecosystems. Although the research is focused on the required action of these substances, unexpected side effects on non-target organisms are also of great environmental concern (Croft, 1990; Desneux et al., 2007). Many examples show that the objective of high yields of desirable crops led to increased use of pesticides, which were in the past often applied even prophylactically (Fabre et al., 2003; Pemsl et al., 2004; Bortoluzzi et al., 2007). It is one amongst the important reasons as to why there is a renewed support for studies on cultural and biological control accompanying pesticide use (Wijnands, 1997), especially considering the integrated pest management (IPM), in which even sublethal doses of pesticides
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could affect the insect behaviour (Wei and Du, 2004; Medrzycki et al., 2003) and the attractiveness of crops for herbivorous insects (Norris and Kogan, 2005). Similarly the implementation of glyphosate-ready and gluphosinate-ready crops into agricultural practise promotes the incorporation of this commonly used herbicides to IPM strategies. Considering the non-selective mode of action of phosphonate herbicides and their predicted negative effect on non-crop plants (Riemens et al., 2008), there is a requirement for better understanding of some aspects of the environmental impact of these chemicals and thus the possibility to utilise or alleviate the observed side effects in common practise. Glyphosate (N-phosphonomethyl glycine) is now one of the most widely used broad-spectrum, systemic herbicides for vegetation control. A number of examples of side effects of glyphosate and related compounds have been reported so far. They result from over 30 years of its non-target impact on microorganisms, aquatic invertebrates and algae, terrestrial invertebrates, birds, fishes, mammals and finally on humans (Austin et al., 1991; Eijsackers, 1985; MacKinnon and Freedman, 1993; Folmar et al., 1979; Bronstein and Sullivan, 1992). There is, however, surprisingly little information on the direct or
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indirect impact of N-phosphonomethyl glycine on insects, which feed on treated plants. The field experiments on cereal aphid (Rhopalosiphum padi L.) showed that they were rapidly killed by herbicides containing paraquat or paraquat+diquat (1/4 of the recommended field dose of this herbicide) when sprayed either on or off plants, whereas glyphosate had no effect on this insect (Wright et al., 1985). The same results were obtained by cited authors in laboratory tests carried out on aphids fed on sucrose solutions containing herbicides. In the case of glyphosate and other aminophosphonates, potential disruption of aphid population might be of particular interest because of the mode of action of these compounds (Forlani et al., 1996). Representatives of the animal kingdom lack the shikimic acid pathway, a target pathway for the action of glyphosate, and thus should be unaffected by glyphosate. Similar situation should be achieved in the case of pyridyl derivatives of aminomethylenebisphosphonates, which most probably inhibit the first enzyme of shikimic pathway-3-dioxyarabino heptulosonate 7-P synthase (DAHP synthase) (Kafarski et al., 1997). However, the influence of these substances on the shikimic pathway, which provide the chemicals indispensable for plantdefence system and thus change the interactions with pathogens and herbivore insects (De Moraes et al., 2004), might have great importance in agriculture, especially in IPM strategies. The objective of this paper is to find out whether aminophosphonate herbicides are toxic only for host plants or whether, through decreasing the quality of plants as the nutrient source, tested compounds also affect herbivore insects. This effect might be of special interest considering the influence of the nutritional quality of host plants for herbivore insects’ feeding and behaviour (Behmer, 2009) and the combined effect of herbicide-induced stress and insect herbivory to reduce weed fitness (Williams et al., 2004). Thus, this paper describes the sensitivity of black aphid A. fabae towards tested herbicides as the nutrients and the influence of herbicide-treated plants of broad bean V. faba L. on the host plant choice and population development of black aphid A. fabae. The combined effect of sublethal doses of herbicides and presence of aphids on the growth of broad bean plants was also investigated.
2. Materials and methods 2.1. Broad bean Vicia faba culture Dry seeds of broad (fava) bean V. faba cv. White Windsor, a common crop in Opole region, Southwest part of Poland, were used. Seeds were germinated for 5 days between two 0.5-cm-thick wet cotton layers in plastic pots, with 12 seeds in each pot (20 cm 30 cm, 5 cm height). Obtained seedlings having 2 cm root and 2 cm hypocotyl were transferred separately to glass jars, where they were grown hydroponically on cotton gauze over the surface of tap water at 2071 1C, 65% relative humidity (RH), 1200 lx and 14:10 h light:dark. Plants (about 10 cm height) were infested by aphids as soon as the first pair of leaves had unfolded.
2.2. Aphids culture (breading) The culture of A. fabae was established from a single virginoparous apterous female collected from V. faba plant near Opole in May 2000. Aphids were reared on preflowering V. faba (cv. White Windsor) plants in a growth chamber at 2071 1C, 65% relative humidity, 1200 lx in a long-day LD 16:8 h photocycle.
2.3. Tested herbicides In order to study the real effects of pure compounds, which possessed herbicidal activity, all experiments were arranged using active substances only. Pure glyphosate (N-phosphonomethylglycine) was obtained by the author via laboratory procedure: from commercial Roundups 360 SL (Monsanto, MO, USA) formulation by dissolving in water and maintaining the pH of the solution to 1.5–2.0 with hydrochloric acid. This resulted in crystallisation of the pure
herbicide. Its structure and purity were confirmed using 1H, 13C and 31P NMR spectroscopy. The retention time of this substance in capillary electrophoresis was the same as the retention time of glyphosate standard obtained from Monsanto. The bisphosphonic compounds N-(2-piridylamino)methylene bisphosphonic acid (PMBA), N-[2-(3-methylpiridylamino)]methylene bisphosphonic acid (3MPMBA) and N-[2-(6-methylpiridylamino)]methylene bisphosphonic acid (6MPMBA) were gifts from Prof. P. Kafarski (Wroc"aw University of Technology). The chemicals were diluted in water to reach the final concentrations ranging from 150 to 0.015 mM depending on the experiment.
2.4. Olfactometer bioassay The two-chamber diffusion olfactometer was used to measure responses of mating females to herbicide-treated V. faba plants. The olfactometer consisted of the glass tube (4 cm diameter, 25 cm length) with the airflow window located over the arena in the middle of its length and also two connected glass bulb chambers, ending with glass filters. The airflow inside the olfactometer was established by air circulation based on the diffusion process. The lack of constrained airflow distinguished this olfactometer from standard ones (Vet et al., 1983; Nazzi et al., 2001); however, its simplicity and subtle gradient of volatiles concentration allowed one to examine the responses of delicate, wingless aphids. Odour fields were arranged by adding the plants to the glass bulb chambers. All plants used in the experiments were logged immediately after the groups of 20 virgin adult females were introduced into the olfactometer arena, and then the choice test was started. The volume of 0.1 cm3 of tested herbicides at the concentration of 1.5 mM, which is adequate to a sublethal field dose per plant, was applied to the plant tissues via glass capillary. In each experiment 1 g of plant tissue (stems and leaves) treated with appropriate herbicide was placed into one bulb chamber (experiment), while 1 g of V. faba tissue treated in the same manner with water was placed in the opposite bulb chamber (control). The positions of aphids were recorded every 5 min for 60 min. To protect against positional bias, the olfactometer was shielded with white cards and rotated by 1801 between the replications of experiments. Each experiment was carried out in 15 applications. The numbers of aphids recorded after appropriate time in each experiment – each herbicide treatment, respectively, in experimental and control chambers – were collected and analysis of variance (ANOVA) was conducted regarding the null hypothesis. Then the significant differences between means (Po0.05) were compared using HSD Tukey’s test. Statistical evaluation of experimental data was performed with Statgraphics 5 Plus software (Manugistics Inc., 2000).
2.5. Herbicidal activity Each of four tested compounds was studied at three concentrations: 15, 1.5 and 0.015 mM using two means of treatment. The first one was maintained in such a manner that herbicide was taken up by plant via glass capillary. Thus, glass capillary containing 0.1 cm3 of studied solution was introduced 0.1 cm deep in the stem of plants 5 cm above the root and was left there until the solution was absorbed. Furthermore, the same amount of glyphosate was used but at a concentration of 150 mM, which is adequate to its field dose per one plant. The four V. faba plants 10 cm in height were grown in an isolator and treated with herbicide at each concentration (or distilled water as control) using capillary application. Each experiment was done in six repetitions. The second application was a standard spraying of the plants with 0.1 cm3 solutions of tested herbicides or pure water (control). Each experiment was carried out in six repetitions. In both experiments all examined plants were reared in a climate chamber at 2071 1C, 65% relative humidity, 1200 lx in a long-day LD 16:8 h photocycle for 9–10 days. During this period the total height of the green part of plants was recorded everyday and their morphological changes were observed. The values of plant heights recorded each day in each experiment were grouped and then the significant differences between means were compared using MRT Duncan’s test (Po0.05). Statistical evaluation of experimental data was performed with Statgraphics 5 Plus software (Manugistics Inc., 2000).
2.6. Combined influence of herbicides and aphids on plant growth V. faba plants of about 10 cm height, treated with herbicides at concentrations and in a manner described above, were infested with two apterous female aphids 24 h after the treatment. In order to study aphid development and their impact on plant, the number of adults and L1–L4 nymphs was counted everyday for a 9–10day period and also the total length of green part of plants and morphological changes were recorded. Each experiment was replicated six times. Statistical evaluation of the results achieved in these experiments was conducted similar to that described in herbicidal activity tests (MRT Duncan’s test), additionally taking into account the total number of aphids, which were recorded as developed on each infested plant.
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2.7. Aphid growth and development on the artificial diet containing herbicides To test the effect of studied herbicides as a factor influencing aphid feeding behaviour and reproduction, the studies on artificial diet were performed. This diet was a modified form of the one originally prescribed for rearing Myzus persicae (Sulzer) by supplementation with 11 amino acids, namely asparagine, asparaginic acid, phenylalanine, glutamine, histidine, isoleucine, leucine, lysine, serine, threonine and valine (Mittler and Koski, 1976). The pH of the diet was adjusted to 7.0 with KOH and it was filter-sterilized using Pyrex No 5 glass filter. The solution was sandwiched between two aseptic Nescofilm (Bando Chemical Ind. Ltd., Kobe, Japan) membranes placed over a plastic ring (30 mm in diameter, 15 mm in height) and fed to aphids. The diets including herbicides were prepared by adding appropriate amounts of these compounds at a final concentration of 1.5 mM. Three, 1-day-old, adult females were transferred from aphid cultures at V. faba to each ring with artificial diet. The treatments were carried out in glass chambers covered with light green paper and experiments were carried out under long-day (LD 16:8) condition and at a temperature of 2171 1C. Six hours after the experiments were established and then for the next 3 days with 24 h intervals, the number of aphids was recorded and their developmental stage was observed. The average number of offspring per aphid was determined, taking into consideration the number of rearing aphids. Each experiment was carried out in four replications. The total numbers of aphids, which were recorded daily in each experiment, were evaluated using the analysis of variance followed by HSD Tukey’s test in order to compare the significant differences between means (Po0.05).
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plants) (Table 2). The activity of two of bisphosphonic acids, namely PMBA and 3MPMBA, examined at higher concentrations strongly depended on the mode of treatment, with the application of herbicide via glass capillary being more effective. If they were applied at 15 mM concentration by means of capillary, it caused the death of the plants at the third day of experiment, whereas spraying the plants with a solution of the same concentration had negligible effect on plant growth. Glyphosate appeared to be much less active under the same conditions. Considering the plants, which were treated via capillary and then infested by aphids, significant decrease of their growth potency was observed in relation to plants treated with herbicide alone. Spraying the plants has quite contradictory results in that the kind of additive suppression mentioned above was not observed. The only exception was 3MPMBA, which was also the most effective herbicide when applied by this mode. The additive suppression of herbicides and aphids was best seen from the fourth to the sixth day of experiment (Fig. 1). Prolongation of experiment up to 10 or 11 days resulted in changes in plant colour, shape or leafs and stems vigour. 3.3. Effects of herbicides on aphid development and fecundity
3. Results 3.1. Olfactometer bioassay The results derived from the choice test carried out in an olfactometer indicated that aphids nearly immediately preferred non-treated over treated plants as the food source. This effect was more pronounced after 60 min of the experiment; however, it has already been observed after 15 min of experiment. The mean number of aphids in the chambers containing control plants was two times higher than that in the chambers containing plants treated with glyphosate or 6MPMBA, and six times higher when PMBA and 3MPMBA were used. The examination of data presented in Table 1 clearly shows that two aminomethylenebisphophonic acids, namely non-substituted PMBA and its 3-methyl derivative, most likely act as weak repellents. 3.2. The effect of herbicides on plant and aphid development
No significant differences were observed in the number of insects per one plant between control and plants sprayed with 0.015 and 1.5 mM herbicides during the 8 days of culturing. The only exception was application of PMBA (Table 2). This compound used at the lowest concentration (0.015 mM) caused the decrease in number of aphids up to about 70% in relation to control. This herbicide also caused the highest reduction of aphid development when applied in higher concentrations (1.5; 15 mM) by the spraying mode. The development of aphids on plants treated via capillary was suppressed even if the herbicides were tested at low concentrations. With the exception of 6MPMBA, the average numbers of aphids varied significantly from the control even at a concentration as low as 0.015 mM. This observation might suggest that the compounds acted as weak systemic insecticide. 3.4. Reaction of aphids towards artificial diet supplemented with herbicides
All herbicidally active substances tested in this work influenced plant development in a dose-dependent manner and their herbicidal effect was dependent on the mode of their application. At the lowest concentration (0.015 mM) none of the tested herbicides affected the growth of broad bean plants despite the mode of treatment (e.g. via glass capillary or by spraying the
The insects, which were settled on the artificial diet supplemented with glyphosate, tended to escape from the membranes or were dead at the second or fourth day of experiment (Fig. 2). Three out of the examined herbicidal phosphonates exhibited weak insecticidal activity if applied in a systemic manner. Very
Table 1 Response of adult apterous Aphis fabae females in two-way diffusion olfactometer to the odour of Vicia faba L. plants treated with tested compounds at the concentration of 1.5 mM in a choice test. Experiment
The number of aphids in olfactometer chambers (mean7S.E.) after 5, 15, 30, 60 min of the test 5
Glyphosate Control N-(2-piridylamino) methylene-bisphosphonic acid (PMBA) Control N-[2-(3-methylpiridylamino)]-methylenebisphosphonic acid (3MPMBA) Control N-[2-(6-methylpiridylamino)]-methylenebisphosphonic acid (6MPMBA) Control
7.870.5 8.270.5 3.970.5 8.870.7 3.170.5 8.570.6 8.470.7 8.670.7
15 b b d c d c bc b
Mean7S.E. followed by different letters are significantly different (ANOVA, Tukey’s test; Po0.05)
6.070.7 9.070.8 3.270.5 10.170.3 3.070.5 11.370.7 8.170.6 10.870.5
30 c b d b d b bc a
5.770.7 10.870.7 3.470.5 10.170.7 2.670.5 11.570.4 7.370.5 10.670.9
60 c a d b d b c a
5.670.8 11.570.7 1.970.4 12.370.3 1.570.3 12.870.5 6.170.6 10.170.6
c a e a e a d a
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Table 2 The effect of tested herbicides on development of plants and aphids as measured at the eighth day of experiment. Compound and its concentration in mM
Treatment via glass capillary The average number of aphidsa observed on infested plants
Treatment by spraying the plants The mean height of The mean height of The average number of infested plants uninfested plants aphidsa observed on (cm) (cm) infested plants
The mean height of The mean height of infested plants uninfested plants (cm) (cm)
Glyphosate 150 15 1.5 0.015
21.572.1 a 61.579.2 de 62.8711.1 de 86.3710.0 ef
10.372.0 bc 22.373.6 de 23.871.8 de 22.873.9 de
20.871.9 d 26.172.9 ef 27.573.1 ef 30.072.2 f
28.572.9 b 73.676.1 e 81.279.1 ef 78.9711.4 ef
22.272.1 de 28.972.7 ef 27.072.2 ef 32.673.9 f
20.873.3 d 30.373.5 ef 27.272.1 ef 31.671.7 f
N-2-piridylamino methylene bisphosphonic acid 15 1.5 0.015
89711.8 ef 96.479.7 f
dp 2.770.5 a 28.772.6 ef
dp 9.071.2 b 26.972.6 ef
45.679.2 c 57.777.7 cd 60.979.0 de
26.674.3 df 2973.2 ef 28.176.1 df
29.171.1 f 31.073.1ef 29.872.0 ef
N-2-(3-methylpiridylamino) methylene bisphosphonic acid 15 1.5 0.015
31.576.8 bc 86.3711.7 ef
dp 13.372.1 bc 29.873.7 ef
dp 20.472.8 d 31.174.2 ef
56.679.1 cd 76.174.6 e 92.178.2 ef
12.571.9 c 24.572.2 de 27.174.4 df
16.472.1 cd 28.873.6 ef 27.471.9 ef
15.572.1 c 24.773.9 de 27.572.9 ef 30.372.4 f
21.573.2 de 30.875.2 ef 28.372.9 ef
72.9710.6 e 87.1714.2 ef 80.577.9 ef 84.5713.1 ef
24.573.7 30.473.3 28.372.5 31.574.6
25.772.1 e 29.171.5 ef 25.974.7 df
N-2-(6-methylpiridylamino) methylene bisphosphonic acid 15 1.5 0.015 Controlb Uninfested controlc
88.879.1 ef 90.179.4 ef 10479.8 fg 117.6711.3 g
32.373.2 f
de ef ef ef
29.873.7 ef
dp—death of the plants caused by herbicides before the third day of experiment; ns—not significant result in comparison to appropriate control; a–g—evaluation of significant differences in Duncan’s test. a The average number of aphids per one adult female. b Control—plants untreated with herbicides but infested by aphids. c Uninfested control—plants untreated and uninfested by aphids.
Fig. 1. The total length of the V. faba stems during the experiment with N-(2-piridylamino) methylene bisphosphonic acid (PMBA), which was applied via glass capillary at the concentration of 1.5 mM.
similar effect was observed on diets supplemented with herbicidal bisphosphonates (Fig. 2), with PMBA and 3MPMBA significantly decreasing the number of insects after the third day of experiment.
4. Discussion The herbicides examined when used in sublethal doses suppressed the growth of broad bean plants only in highest
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Fig. 2. The mean number of aphids on artificial diets contained herbicidal aminophosphonates at the final concentration of 1.5 mM during 4 days. The significant differences between means (Tukey’s test, Po0.05) are indicated with asterisks.
tested concentrations, being more effective when they were applied via glass capillary. Such effect has not been seen, or it has been significantly less pronounced, upon application of herbicide by spraying. This might result from the lack of the barrier, which should be overcome by these active substances if they are applied by spraying and had to penetrate plant tissues from the outside surface of leaves and stems into phloem cells. Thus the treatment via capillaries mimics the situation when glyphosate, PMBA or its derivatives are present inside plant tissues and might influence plant metabolism (Forlani et al., 1997; Lejczak et al., 1996; Osiecka and Janas, 1998). Therefore it was also expected that application via capillary should affect aphid development by suppressing shikimic and phenylpropanoid biosynthesis pathway, resulting in the deficiency of plant-defence substances (Marchiosi et al., 2009). Indeed, this phenomenon was observed evoking additive suppression of herbicides and feeding aphids on the growth of infested plants. Similar results were obtained by Williams and co-authors during the study on the combined effect of Fluroxypyr herbicide and colorado potato beetle herbivory on volunteer potatoes (Williams et al., 2004). On the other hand, the changes in metabolism of brad bean plants treated with aminophosphonate herbicides decreased the olfactory attractiveness of plants for black bean aphids. Olfactometer measurements have indicated that aphids preferred intact V. faba leaves over those treated with sublethal doses of herbicides. A. fabae is a heteroecious insect and it exhibits host plant alternation between a narrow range of primary hosts on which the aphids reproduce sexually in autumn, and wide secondary hosts’ range, which support multiple parthenogenetic generations in the summer (Raymond et al., 2001; Tosh et al., 2001). The primary host plant range of A. fabae in Poland is limited usually to Euonymus europaeus (L.), while the secondary host plant range comprises few species. Among them Beta vulgaris (L.), Chenopodium album (L.) and V. faba are chosen more often than others (Wnuk and Gospodarek, 1999). Since the application of herbicide should affect negatively plant resistance to insect action, a slight but significant decrease in the number of aphids on the plants treated with all the herbicides was observed. The studies on aphids cultured on artificial diet supplemented with herbicides revealed that the same herbicides (glyphosate, PMBA, 3MPMBA), application of which decreased the number of aphids on treated plants,
influence negatively the insect development most likely exhibiting weak insecticidal activity. This is in contradiction to results of studies on development of yellow soybean aphids on soybean plants treated with glyphosate under the field conditions (Burrows et al., 2005); however, the tested soybean variety was a glyphosate-resistant one and in this case metabolic stress evoked by glyphosate is hardly expected. On the other hand, experimental results achieved in this work are in a good agreement with the results obtained upon aphid infestation on weeds and sugar beet plants in glyphosate-tolerant sugar beets crop (Dewar et al., 2000) and with the studies on the effectiveness of one, two or three glyphosate application(s) on V. faba for controlling Orobanche crenata (Forsk) infestation (Naguib et al., 1987). Orobanche infestation alongside with glyphosate spraying caused pronounced decrease in the total amino acid content in the leaves of the host plant. However, the decrease induced by the herbivore was higher than that induced by glyphosate, thus showing more complex relation between these two organisms in the presence of herbicide.
5. Conclusions The results of our studies have shown that treatment of V. faba plants infested by A. fabae aphids with sublethal doses of glyphosate or pyridyl derivatives of aminomethylenebisphosphonic acid caused additive suppression of plant growth. Simultaneously the herbicides decreased also the rate of growth and development of the aphid populations, most probably by exhibiting weak insecticidal activity. Regarding these it can be concluded that the combined effect of herbicide-induced stress and reduced plant fitness by insect herbivory should be considered as a factor enabling reduction of applied herbicide doses.
Acknowledgments This research investigation was conducted in the Faculty of Chemistry, University of Opole, Poland. I thank Prof. Pawe" Kafarski for providing with aminomethylenebisphosphonic acids used in the presented experiments and for the fruitful discussion about their mode of action.
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