Systems of organic farming in spring vetch II: Biological response of Aeolothrips intermedius Bagnall and Coccinella septempunctata L

SUMMARY The effects of four systems of organic farming of spring vetsch on Aeolothrips intermedius Bagnall (Thysanoptera: Aeolothripidae) and Coccinella septempunctata L. ( Coleoptera: Coccinellidae ) population density and the toxicity of several products on predatory insects were studied. The variants were: Control (without using any biological products); combined treatment with Polyversum (biological foliar fertilizer) and Biofa (biological plant growth regulator); treatment with NeemAzal T/S (biological insecticide, a.i. azadirachtin) and treatment with a combination of NeemAzal with Polyversum and Biofa. Variant V was a conventional farming system in which a combination of Nurelle D (synthetic insecticide), Masterblend (foliar fertilizer) and Flordimex 420 (growth regulator) was used as a standard treatment. In the organic farming system that included treatment of plants with the biological insecticide NeemAzal (azadirachtin), the reduction in A. intermedius abundance was 20.7% when it was applied alone and 24.6 % in combination with the organic products Polyversum and Biofa. NeemAzal achieved a lower reduction in the counts of predatory ladybirds C. septempunctata, from 14.9% (alone) to 21.9% (combination). The biological insecticide, applied alone or in combination, was mostly harmless and rarely harmful to A. intermedius . NeemAzal manifested harmlessness to C. septempunctata as its toxic action did not exceed 25%. An analysis of variance regarding product toxicity to A. intermedius and C. septempunctata demonstrated that the type of treatment (the application of insecticides alone or in combination) had the strongest effect on product toxicity. Тhe use of neem-based insecticides can be a substantial contribution towards preservation of biodiversity in ecosystems.


INTROdUCTION
Pest control in modern agro-ecosystems is largely achieved by using pesticides, but relying on them globally has led to evolution of pesticide resistance in many crop pests (Whalon et al., 2008). Current trends in organic agriculture have therefore recognized a need for looking for alternative methods of pest control (Atanasov et al., 2005). Such organic systems, which aim for sustainability, while preserving or increasing biological diversity, and minimization of all forms of contamination, make it critical to search for alternative techniques of control, selective and not harmful, especially not to populations of beneficial insects, such as natural enemies and pollinators (Efrom et al., 2012). Knowing the effects that biological insecticides, applied alone or in combination with other biological products, have on harmful or beneficial insects, and the use of selective products are important for the sustainability of organic systems.
NeemAzal (azadirachtin) has been the subject of a number of studies which reported its broad spectrum of action and high efficacy against pests in the orders Lepidoptera, Hemiptera, Thysanoptera and mites (Isman, 1993;Singh et al, 1999;Pavela, 2009;Andreev et al., 2012). A survey of current literature reveals that few data are available regarding the impact of neem (Hoelmer et al., 1990) оn beneficial insects other than direct contact toxicity studies under field conditions. Because aphids and phytophagous thrips make а food source for а large number of predatory and parasitic insects, the effect of neem оn non-target organisms is particularly important in the management of pest populations. Some authors have found that azadirachtin should be combined with plant oils in order to be more effective against some pest (Höhn et al., 1996;Bessin, 2008).
However, information about this product, whether used alone or in combination with other biological products, in terms of its impact on beneficial insects in the field is almost entirely missing or insufficient.
Therefore, this study aimed to evaluate the effects of some methods of organic farming that included biological products on Aeolothrips intermedius and Coccinella septempunctata density, and products toxicity to these species in a field crop of spring vetch.

MATERIAL ANd METHOdS
The experimental design, trial variants and product characteristics were shown in the first part of this study (Nikolova & Georgieva, 2015).
Harmful and beneficial insects were counted immediately after treatment at the buddingbeginning of flowering stage (one day after treatment) and the counting continued until the aboveground biomass has dried (between mid-May and the end of July -approximately 75 days). Over the period, population density was recorded by sweepings with the entomological net once a week. The data in Table  2 regarding insect abundance in the studied variants were averaged for the indicated 75-day period. The efficacy of insecticides and their combinations against Acyrthosiphon pisum Harris (Hemiptera, Aphididae), Empoasca pteridis Dahlb. (Hemiptera, Cicadellidae) and Thrips tabaci L. (Thysanoptera, Thripidae) was estimated at the budding and flowering stage (see part I). Assessments were made 1, 3, 7 and 12 days after treatment. The efficacy of the insecticides was calculated using Abbott's formula (1925) and the entomological net was used for sweepings.

RESULTS ANd dISCUSSION
Beneficial entomofauna living on organically grown spring vetch was represented by species belonging to the orders Thysanoptera, Coleoptera, Hymenoptera and Hemiptera: suborder Heteroptera. The predator species Aeolothrips intermedius Bagnall (Thysanoptera: Thripidae) and Coccinella septempunctata L. (Coleoptera: Coccinellidae) were present in high numbers in the stands, which allowed the monitoring of their reaction to organic products under field conditions. The family Coccinellidae was represented by another 4 species: Propylea quatuordecimpunctata L., Coccinula quatuordecimpustulata L., Adonia variegata Goeze and Scymnus (Neopulus) quadrimaculatus Herb but their numbers were too low to be recorded and processed in this study.
The average number of predatory thrips differed depending on the system of production (Table 1).
Under organic production, the products Biofa and Polyversum had insignificant impact on population density of A. intermedius and density values insignificantly exceeded that of the control by 10.0% over the years. Probably the products slightly stimulated the density of that species. Treatment with the biological insecticide NeemAzal resulted in a lower number of species -by 20.7% on average for its use alone and 24.6% for the combination with foliar fertilizer and growth regulator, which are biological products. Despite the changeable population density of A. intermedius over the years, its numbers varied over a narrow range both when NeemAzal was used alone and in combination, and the difference was insignificant. Significant differences were found between the untreated control and the combined use of organic products in 2014 and on average for the entire 2012-2014 period, too. Concerning Coccinella septempunctata, the impact of NееmАzal on predatory density followed a similar trend and was relatively less pronounced with the reduction ranging from 14.9 (NeemAzal used alone) to 21.9% (NeemAzal in combination). The differences between these variants and the control were insignificant. Unlike NeemAzal, the synthetic products had a strong negative impact on C. septempunctata abundance and the reduction was 49.4%.
According to several authors, the number of predators оn plants sprayed with neem mау have bееn lower аs а result of reduced aphid populations, and the number of adult coccinellids was highly correlated with aphid density (Wright & Laing, 1980;Frazer, 1988;Nijveldt, 1988;Lowery, 1992). Considering the decrease in aphid numbers and counts of predators in the variants, treatment with NeemAzal mау have overestimated the damaging effect of azadirachtin. However, although an increase in host density is usually followed bу increase in the counts of predators and parasitoids, the relationship is often variable and difficult to determine (Stary, 1970;Coderre, 1988). Therefore, toxicity constitutes the most accurate measure of product impact оn natural enemies.
The data showing direct impact of the insecticides on beneficial species over the years showed that the combination in the synthetic product Nurelle D had the highest toxic effect. Mortality was significantly the highest on the first day after treatment, and it ranged from 86.0 to 100.0% in A. intermedius and from 72.0 to 85.4% in C. septempunctata (P <0.05) (Figure 1). Despite a decreasing toxicity of the combination seven and twelve days after application, Nurelle again demonstrated the highest mortality rates against predatory thrips and ladybugs.
The biological product NeemAzal exhibited significantly less impact as its toxicity on the first day ranged from 11.5 to 20.0% (A. intermedius) and from 6.7 to 12.3% (C. septempunctata). NeemAzal combined with Biofa and Polyversum had higher mortality values but the differences versus its use alone were statistically insignificant in each year (P <0.05). Azadirachtin, applied alone and in combination, increased its activity, reaching maximum values on the 7 th day after treatment. An exception was observed in A. intermedius in 2012 when the highest toxic effect was found on the third day after treatment. Differences between the variants, including NeemAzal, were minimal and insignificant over the reporting years and days for both predators (P <0.05). Compared with the synthetic insecticide, their toxicity was three to four-fold lower. It should be noted that predatory thrips were more sensitive than predatory ladybirds.
According to categorization by the International Organization of Biological Control (IOBC), NeemAzal, used alone or in combination, was harmless (non-toxic) in 75% of the cases, and slightly harmful in only 25% [NeemAzal, applied alone -on the 3 rd day (2012) and 7 th day (2014); NeemAzal, applied in combination: on the 3 rd day (2012), 7 th day (2013), 3 rd and 7 th days (2014)] regarding A. intermedius. It is necessary to note that the biological insecticides were classified as harmless to the predatory ladybird C. septempunctata as their toxic action was not more than 25%. Our results support those of Kaethner (1991). He found that neem was not toxic to adults of C. septempunctata. I -NeemAzal; II -NeemAzal+Biofa+Polyversum; III -NurelleD+Flordimex 420+ Masterblend Means in each row marked by the same letter are not significantly different (P > 0.05) on reporting days; *evaluation categories (% mortality or reduction in beneficial capacity) were: 1 = harmless (< 25%); 2 = slightly harmful (25 to 50%); 3 = moderately harmful (51 to 75%); and 4 = harmful (>75%)  Similar results of low impact of azadirachtin on beneficial ladybirds were also reported by Schmutterer (1997). Azadirachtin may cause a certain level of mortality of larval instars, especially under laboratory conditions, but no or only slight side-effects are, as a rule, observed under semi-field or field conditions (Schmutterer, 1997).
On the other hand, Nurelle D was harmful (in 41.7% of the cases) and moderately harmful (in 41.7%) to A. intermedius. Regarding C. septempunctata, Nurelle D was mainly moderately toxic (in 58.3% of the cases) and less highly toxic (in 25.0%) according to IOBC categorization.
Based on the results presented in this study, it is possible to conclude that NeemAzal, applied alone or in combination with the organic products Biofa and Polyversum, overall showed no significant harmful effects on the two predatory species, although it manifested a slightly harmful effect on A. intermedius. In other words, the use of neem-based insecticides can substantially contribute towards preservation of biodiversity in ecosystems in spite of the fact that they are not completely safe to beneficial insects.
An analysis of variance regarding product toxicity to A. intermedius and C. septempunctata demonstrated that the factor B -type of treatment (application of insecticides alone or in combination) had the strongest effect on product toxicity -76.2 and 85.4% of the total variance in the variants for A. intermedius and C. septempunctata, respectively ( Table 2). The influence of the years (factor A) and reporting days after treatment (factor C) was considerably weaker than factor B, while the impact of year was statistically significant (4.0%) regarding A. intermedius. The highest significant interaction was between the type of treatment and the reporting days after treatment -BxC -8.0% in predatory thrips and 5.7% in ladybugs. The strength of the interaction between AxC was considerably lower and a significant effect was found for A. intermedius -2.7%.
The biological insecticide NeemAzal used in combination with the growth regulators Biofa and Polyversum in organic production provided the best protection of plants against a complex of sucking pests and higher productivity. These biological products were also found to be harmless to C. septempunctata and A. intermedius. The possibility of managing sucking pests in vetch is thus maximized, and farmers are becoming aware of its benefits as an eco-friendly botanical pesticide that causes no residue problems in vetch.

CONCLUSIONS
Under conditions of organic farming, the tested biological insecticide caused a low reduction in the number of predatory ladybirds Coccinella septempunctata, from 14.9 (used alone) to 21.9% (in combination), and had a less pronounced influence than the conventional system.
NeemAzal applied alone and in combination was mostly harmless and rarely slightly harmful to A. intermedius. The biological insecticide was harmless to C. septempunctata, as its toxic action did not exceed 25%.
The analysis of variance for the species A. intermedius and C. septempunctata showed that factor B -type of treatment (application of the insecticide alone or in combination) had the strongest effect on product toxicity.