Pure Camphor and a Thujone-Camphor Mixture as Eco-Friendly Antifeedants against Larvae and Adults of the Colorado Potato Beetle.

The Colorado potato beetle (CPB) is a serious pest of economically important Solanaceae species. The use of essential oil compounds in pest management has been proposed as an alternative to harmful chemical insecticides that disturb human health and ecosystem functioning. We examined the antifeedant activity of three concentrations (0.125%, 0.25% and 0.5%) of pure camphor and a thujone-camphor mixture against 3rd instar larvae and adults. Their efficacy was evaluated according to the degree of leaf damage and avoidance of treated leaves by the CPB. Treatment of potato leaves significantly reduced leaf damage compared to the control. Leaf protection increased at higher concentrations of the examined compounds. Camphor was more effective against larvae and the thujone-camphor mixture was more effective against adults. Additionally, adults moved faster towards the control leaf disc in the two-choice olfactometer assay if an alternative disc was treated with a thujone-camphor mixture, whereas larvae responded similarly to the two potential repellents. However, after contact with the leaf disc treated with the highest compound concentration, the larvae escaped faster from the thujone-camphor mixture than from pure camphor. In conclusion, both examined compounds are promising eco-friendly antifeedants, but their efficacy depends on the developmental stage of the beetle, compound type and applied concentration.

New Insights Into the Activity of Apple Dihydrochalcone Phloretin: Disturbance of Auxin Homeostasis as Physiological Basis of Phloretin Phytotoxic Action.

Apple species are the unique naturally rich source of dihydrochalcones, phenolic compounds with an elusive role in planta, but suggested auto-allelochemical features related to "apple replant disease" (ARD). Our aim was to elucidate the physiological basis of the phytotoxic action of dihydrochalcone phloretin in the model plant Arabidopsis and to promote phloretin as a new prospective eco-friendly phytotoxic compound. Phloretin treatment induced a significant dose-dependent growth retardation and severe morphological abnormalities and agravitropic behavior in Arabidopsis seedlings. Histological examination revealed a reduced starch content in the columella cells and a serious disturbance in root architecture, which resulted in the reduction in length of meristematic and elongation zones. Significantly disturbed auxin metabolome profile in roots with a particularly increased content of IAA accumulated in the lateral parts of the root apex, accompanied by changes in the expression of auxin biosynthetic and transport genes, especially PIN1, PIN3, PIN7, and ABCB1, indicates the role of auxin in physiological basis of phloretin-induced growth retardation. The results reveal a disturbance of auxin homeostasis as the main mechanism of phytotoxic action of phloretin. This mechanism makes phloretin a prospective candidate for an eco-friendly bioherbicide and paves the way for further research of phloretin role in ARD.

Tissue Culture Response of Ornamental and Medicinal Aesculus Species-A Review.

Species of the genus Aesculus are very attractive woody ornamentals. Their organs contain numerous health-promoting phytochemicals. The most valuable of them-aescin-is used in commercial preparations for the treatment of venous insufficiency. The industrial source of aescin is horse chestnut seeds because the zygotic embryos are the main site of its accumulation. Horse chestnut somatic and zygotic embryos contain similar amount of aescin, hence somatic embryos could be exploited as an alternative source of aescin. Somatic embryogenesis, androgenesis and de novo shoot organogenesis were successfully achieved in several Aesculus species, as well as secondary somatic embryogenesis and shoot organogenesis, which enables mass production of embryos and shoots. In addition, an efficient method for cryopreservation of embryogenic tissue was established, assuring constant availability of the plant material. The developed methods are suitable for clonal propagation of elite specimens selected as the best aescin producers, the most attractive ornamentals or plants resistant to pests and diseases. These methods are also useful for molecular breeding purposes. Thus, in this review, the medicinal uses and a comprehensive survey of in vitro propagation methods established for Aesculus species, as well as the feasibility of in vitro production of aescin, are presented and discussed.

Impact of Storage Temperature on Pollen Viability and Germinability of Four Serbian Autochthon Apple Cultivars.

Globalization has drastically reduced the number of autochthon apple cultivars in the Serbian market and most of them have nearly disappeared; however, some of these cultivars, such as Petrovaca, Budimka, Kolacara Pozna, and Kozara, have extraordinary quality, good pomological characteristics, and pest and disease resistance. The present study was conducted to develop a protocol for the storage of pollen for further use in the conservation and breeding of these cultivars. Viability and germination of the mature pollen were tested in vitro, at four storage temperatures (20, 4, -20, and -80°C), right after harvest or 1, 2, 3, 4, 5, and 6 months after storage. Differences in fresh pollen viability and germination between cultivars were statistically significant and ranged from 60 to 88% and 59 to 98%, respectively. Fresh pollen of cv. Budimka showed the highest viability and germination in comparison with other cultivars, especially cv. Kozara. Pollen viability and germination decreased over the storage period, and it was the lowest after 6 months of storage at room temperature in all tested cultivars. Storage at 4°C prolonged the pollen viability and germinability of 1-5 fold, depending on the cultivar and treatment duration; however, the pollen longevity of all cultivars was significantly extended when stored at -20 or -80°C. After 6 months, pollen of cv. Budimka stored at -20 and -80°C showed 14-15 fold higher germination rates in relation to pollen storage at room temperature for the same period. The results of the present study suggest that the pollen of these apple cultivars could be efficiently maintained at -20°C and could be further used for breeding purposes, e.g., for crossings between cultivars that flower at different times of the year.

Repellent activity of Tanacetum parthenium (L.) and Tanacetum vulgare (L.) essential oils against Leptinotarsa decemlineata (Say).

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is one of the most destructive pest species to have developed resistance to most chemical insecticides. We determined the composition and evaluated the potential of Tanacetum parthenium L. and Tanacetum vulgare L. (Asteraceae family) essential oil (EO) application as an alternative eco-friendly control strategy against L. decemlineata. We assessed the antifeedant activity for L. decemlineata larvae and adults by estimating the damage to potato leaves treated with three concentrations of EOs dissolved in ethanol (0.125, 0.25 and 0.5%). Results showed that T. parthenium EO was more effective against larvae, and T. vulgare was more effective against adults. In an olfactometer assay, the time required to choose an untreated leaf disc did not depend on the Tanacetum species, or life stage examined. However, the concentration of EO exhibited a significant effect on the behaviour of both developmental stages. At higher EO concentrations, both third instar larvae and adults require less time to choose an untreated leaf disc. Additionally, T. parthenium EO provoked more rapid movement away from the treated leaf disc than T. vulgare, especially at the highest concentration. Successful modification of L. decemlineata behaviour by the two Tanacetum oils suggests that they possess the potential for use in potato protection.

In vitro cultivation of tansy (Tanacetum vulgare L.): a tool for the production of potent pharmaceutical agents.

In this study, tansy (Tanacetum vulgare L.) in vitro culture was established from seeds collected from natural populations. The multiplication of plantlets was conducted through shoot tips that exhibited potent apical growth and regeneration capacities on basal medium (BM), without the addition of any plant growth regulators (PGRs). PGRs were also omitted for the establishment and cultivation of tansy root cultures. Both abaxial and adaxial leaf surfaces of in vitro micropropagated plantlets were covered with glandular biseriate trichomes. Histochemical staining showed that glandular secretions were rich in lipid and terpene compounds, confirmed by GC-MS analysis of essential oil (EO). In the total EO, similar portions of oxygenated monoterpenes (38.5% m/m) and oxygenated sesquiterpenes (22.6% m/m) were detected. Chemical profiles of methanol extracts of in vitro cultured tansy shoots and roots varied in quantity and quality from those obtained from wild-growingtansy. HPLC analysis indicated that the methanol extracts of in vitro cultured roots were the richest in 3,5-O-dicaffeoylquinic acid (3,5-O-DCQA), in which the concentration was 6 times higher (10.220 mg/g DW) than that in the extract obtained from roots of wild-growing tansy (1.684 mg/g DW). This result is noticeable in the manner of industrial production of biologically active 3,5-O-DCQA that has been shown to have antioxidant, hepatoprotective, antiviral, antimutagenic, and immunomodulatory activity. Biotechnological interventions on secondary metabolite production taking place in trichomes could further enhance the production of some important tansy metabolites and further investigation will be directed toward the elucidation of the pharmaceutical potential of tansy in vitro obtained metabolites, as mixtures or single moieties.

Variability in somatic embryo-forming capacity of spinach.

High variability in somatic embryo (SE)-forming capacity has previously been observed in several spinach cultivars. Such variability frequently accounted for more variation in embryogenic response of the explants than the factor being investigated. Hence, the variability in embryogenic capacity was examined in the present study at both the population and the single-seedling level, using seeds of spinach cultivar Matador obtained from nine European seed companies. Seed population obtained from Slovenia (Sl) was superior to others, with the highest regeneration frequency (100%) and the highest mean SE number (14.4). A total of 82% of these seedlings had 80-100% of regenerating explants, while in populations with intermediate embryogenic capacity approximately 40% of seedlings had 20-60% of regenerating explants. The explants from the majority of seedlings (52-100%) in the least responsive populations were irresponsive. Furthermore, the explants from Sl seedlings regenerated from 10-20 (43.5%) up to > 20 (27.6%) SEs on average, while the explants from the majority of seedlings belonging to other populations regenerated 1-10 SEs. The present study strongly indicates that the variability of plant material must not be overlooked, because choosing more responsive individuals for one treatment and less responsive ones for another may lead to misinterpretation of the data.

Evaluation of tansy essential oil as a potential "green" alternative for gypsy moth control.

The development of "green" alternatives to chemical pesticides could play a crucial role in integrated pest management (IPM). Their use is considered either as a substitution for or in addition to hazardous synthetic products. We analysed the influence of three concentrations of tansy (Tanacetum vulgare L.) essential oil (EO), previously characterised by GC-MS, on the survival and moulting of the 2nd instar and the nutritional indices of the 4th instar gypsy moth (Lymantria dispar L.) larvae. In a residual contact toxicity assessment, the exposure to tansy EO caused low mortality (< 10%) while larval development was significantly slowed down, i.e., the percentage of larvae that moulted into the 3rd instar was reduced. On the other hand, when tansy EO was incorporated into the diet (digestive toxicity assay), high mortality and a lack of moulting after 120 h of eating were recorded for the highest applied concentration of EO. During 48 h of feeding on EO-supplemented food at concentrations of 0.5 and 1% (v/v), the relative growth rate (RGR) of the 4th instar larvae significantly decreased, which can be explained by a significant reduction of the relative consumption rate (RCR) and significantly or marginally significantly lower efficiency of conversion of ingested food into insect biomass (ECI). Although the RCR was also reduced with the lowest applied EO concentration (0.1%), the ECI was not affected which meant the RGR was as high as it was for the control larvae. ECI changes, when two higher EO concentrations were applied, were due to a reduction in the efficiency of conversion of digested food into biomass (ECD), while approximate digestibility was unaffected by the presence of EO in the food. Our results on the significant negative effects of tansy EO on gypsy moth larval survival, development time, and nutritional physiology suggest that it could be considered in future designs for botanical insecticides for gypsy moth control.

Comparative study of antioxidant status in androgenic embryos of Aesculus hippocastanum and Aesculus flava.

In vivo (leaves and seed embryos) and in vitro (androgenic embryos) antioxidant scavenging activity of Aesculus hippocastanum and Aesculus flava medical plants was examined. Here we report antioxidant enzyme activities of superoxide dismutase, catalase, guaiacol peroxidase and glutathione peroxidase, reduced glutathione quantity, flavonoids, soluble protein contents, quantities of malondialdehyde, and (•)OH radical presence in the investigated plant samples. Total antioxidant capacity of all the samples of A. hippocastanum and A. flava was determined using FRAP, DPPH, and NO(•) radical scavenger capacity. The leaves of A. flava collected from the botanical garden exhibited stronger antioxidant activity (higher activities of SOD, and higher quantities of GSH, TSH, TPC, and scavenging abilities of DPPH and NO(•), and higher FRAP values and lowest quantities of (•)OH and MDA) than in vitro obtained cultures. However, the leaves of A. flava showed higher antioxidant activity than the leaves of A. hippocastanum, and therefore they have a stronger tolerance of oxidative stress. Androgenic embryos of both species had low amount of antioxidants due to controlled in vitro environmental conditions (T, photoperiod, humidity, nutritive factors, and pathogen-free). Our results confirmed that we found optimal in vitro conditions for producing androgenic embryos of both Aesculus species. Also, we assume that horse chestnut androgenic embryos can be used as an alternative source for large-scale aescin production.