November 8–9, 2017, Brno, Czech Republic 24 years

HERBICIDE PROTECTION OF MILK ( MARIANUM L. GAERTN.) STANDS

LUCIE VAGNEROVA1, HELENA PLUHACKOVA1, ANTONIN VACULIK2 1Department of Crop Science, Breeding and Medicine Mendel University in Brno Zemedelska 1, 613 00 Brno CZECH REPUBLIC 2Department of Plant Protection and Informatics Agritec Plant Research, s.r.o. Zemedelska 2520/16, 787 01 Sumperk CZECH REPUBLIC [email protected]

Abstract: Milk thistle [Silybum marianum (L.) Gaertn.] is currently a sought-after plant commodity, but the problem of herbicide protection, which is a limiting factor in its cultivation, has not been solved yet. Milk thistle achenes can be used in a variety of ways (as dietary supplements, livestock feeding etc.), but above all they are an important raw material for processing in the pharmaceutical industry. The aim of this work was to evaluate the phytotoxicity and the suitability of selected herbicides in small-scale experiments during the years 2014, 2015 and 2016. The results indicate that the preparations used in the study have sufficient selectivity for the milk thistle . According to the results, the preparations TARGA SUPER 5 EC and GALLANT SUPER can be recommended during the cultivation. Key Words: milk thistle, herbicide, weeds

INTRODUCTION Milk thistle [Silybum marianum (L.) Gaertn.] is an annual cultural crop belonging to the family, originating from the Mediterranean. Since the ancient times it has been known for its positive effect in the treatment and prevention of hepatic tissue diseases (Kroll et al. 2007, Abenavoli et al. 2010, Habán et al. 2010, Calani et al. 2012). In the Czech Republic, the milk thistle takes the first place as for the area of cultivation of all the MAPs (medicinal, aromatic and spice plants) (Přibylová et al. 2014). Milk thistle achenes are the product of particular interest, because they contain the silymarin complex, which is a mixture of flavolignans, including silybin A, B (50–60%), isosilybin A, B (5%), silydianin (10%), silychristin (20%) a flavonoid taxifolin (Stancheva et al. 2008, Abbasi et al. 2010, Çağdaş et al. 2011, AbouZid 2012). The total content of the silymarin complex in achene dry matter varies in the range of 1–3% (Andrzejewska et al. 2011, Katar et al. 2013). According to Nasrabadi et al. (2014) the silymarin complex is contained in the whole plant, but it is only advantageous to isolate it from the achenes. The milk thistle achenes contain also high quality oil (15–30%), its main components being linoleic acid (60%), oleic acid (30%) and saturated palmitic acid (about 9%). The achenes contain also proteins (30%), carbohydrates (mostly arabinose, glucose, xylose and rhamnose), tocopherol (0.038%), sterols (0.063%) and last but not least, flavonoids (quercetin, taxifolin) (Abenavoli et al. 2010). The optimum growth conditions, especially in the phase of flowering and ripening, affect the yield enhancement (Stancheva et al. 2008). From an agronomic point of view, milk thistle is considered to be an undemanding crop adaptable to the different conditions. It can be grown on sandy soils, but also on heavy and clay soils (Vereš and Týr 2012, Afshar et al. 2014). One of the limiting factors during the milk thistle cultivation is the weeds infestation and interference. From the crop rotation point of view, the milk thistle is a suitable precursor for corn in a sustainable farming system (Týr and Vereš 2011, Karkanis et al. 2011). According to Zheljazkova et al. (2006) milk thistle is sensitive to a wide range of herbicides used for other

146 November 8–9, 2017, Brno, Czech Republic 24 years cultural crops. Defining capability is an important agronomic characteristic of milk thistle thanks to the rapid creation of leaf rosette and a large amount of leaf matter. However, at the beginning of the vegetation period, especially in the germination stage and during the extensive growth, the competitiveness of milk thistle to weeds is relatively low (Delchev 2016). Most common weeds found in milk thistle stands are: arvensis L., Agropyron repens Beauv., Cirsium arvense Scop., Convovulus arvensis L., Raphanus raphanistrum L., Sinapis arvensis L., Galium aparine L., Chenopodium album L., Mentha crispa L., Stachys arvensis L. and Atriplex patula L. (Delchev 2016). In Bulgaria, in the Plovdiv region, where Zheljazkov et al. (2006) carried out their research during the years 1995 and 1996, typical weed representatives were Amaranthus retroflexus (L.), Setaria viridis (L.) Beauv., Cynodon dactylon (L.) Pers., as well as Amaranthus blitoides S. Wats., Chenopodium album L., Datura stramonium L., Polygonum convolvulus L., Polygonum aviculare L., Solanum nigrum L., Xanthium strumarium L., Abutilon theophrasti Medik., Digitaria sanguinalis L., from the biennial species also Convolvulus arvensis L., Leonurus cardiata L., Cirsium arvense L. and Sorghum halepense (L.) Pers.

MATERIAL AND METHODS Characterization of growing locality, experimental design The herbicidal experiments were established in the form of randomized blocks, each in 3 replicates on experimental plots of the Agritec Plant Research, Ltd., in the years 2014, 2015 and 2016. All variants and their designations are given in the Table 1. The size of the plots was 12.5 m2 of which 10 m2 were harvested. Application of the herbicide was carried out on June 23rd, 2014; June 4th, 2015 and June 19th, 2016. A small-scale sprayer HEGE 32 was used for the application. In all experimental years were found following annual dicotyledonous weeds in the trials: THLAR (Thlaspi arvense L.), CAPBP (Capsella bursa-pastoris (L.) MED.), VERHE (Veronica hederifolia L.), LAMPU (Lamium purpurea L.), MATMA (Matricaria discoidea DC.), MATCH (Matricaria recutita L.), VIOAR (Viola arvensis MURRAY), CHEAL (Chenopodium album L.), and others. From monocotyledons then: ECHCG (Echinochloa crus-galli (L.) P.B.) a AGRRE (Elytrigia repens (L.) NEVSKI) – persistent weed. In 2014, 2015 also annual weed AVEFA (Avena fatua L.). Evaluation of the phytotoxicity of individual preparations was carried out in three terms (7, 14 and 28 DPA - days after application) according to the Methodology for the determination of the phytotoxicity of preparations [EPPO No. 135/1988]. The small-scale combine harvester SAMPO was used for the harvest. The experimental plots were harvested on October 15th, 2014, August 1st, 2015 and September 7th, 2016. After the harvest, the yield from the area unit was determined. The evaluation of results was performed by the analysis of variance (ANOVA) using the statistical program STATISTICA (data analysis software system), StatSoft, Inc. (2013), version 12. Fisher’s test at a significance level of P=0.05 was chosen for subsequent testing. Table 1 Overview of the herbicide treatment variants in the years 2014, 2015 and 2016 No. Preparation Active compound Amount 1 CONTROL - - 2 STOMP 400 SC pendimethalin 2.5 l 3 BETANAL MAXX PRO desmedipham+ethofumesate+phenmedipham+lenacil 1.5 l 4 REFINE 50 SX thifensulfuron-methyl 20 g 5 BUTISAN STAR qinmerac+metazachlor 1.5 l 6 TARGA SUPER 5 EC quizalofop-P-ethyl 2.0 l 7 GALLANT SUPER haloxyfop-R methylester 1.0 l 8 GLEAN 75 WG chlorsulfuron 10 g 9 STARANE 250 EC fluroxypyr 0.5 l

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RESULTS AND DISCUSSION In all experimental years, the phytotoxicity of the herbicide preparations used for milk thistle was evaluated in three terms. The phytotoxicity evaluation in the year 2014 7 DPA (BBCH 16–19) STOMP 400; BETANAL MAXX PRO; REFINE 50 SX - Phytotoxicity > 5%. Slight growth delay compared to control, mild necrosis of the leaves that had already been developed when applying the herbicide. BUTISAN STAR - Phytotoxicity 5–10%. Growth delay, mild necrosis of the leaves that had already been developed when applying the herbicide. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous and perennial weeds. GLEAN 75 WG, STARANE 250 EC - Phytotoxicity 20–30%. Compared to untreated control, chlorotic plants were observed, retardation and slower growth. Necrotic stains on leaves. Death of plants, especially the weaker ones. 14 DPA (BBCH 19–32) STOMP 400 SC; BETANAL MAXX PRO; REFINE 50 SX - Phytotoxicity 0%. Phytotoxicity has subsided. BUTISAN STAR - Phytotoxicity 5%. Growth inhibition, with no noticeable necrosis or color changes on leaves. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous and perennial weeds. GLEAN 75 WG; STARANE 250 EC - Phytotoxicity 20 %. Necrotic stains on newly growing leaves are no longer observed. Growth retardation of milk thistle plants, i.e. the delay of the onset of the individual growth phases compared to the untreated control. Death of plants observed, especially the weaker ones. 28 DPA (BBCH 37–61) STOMP 400 SC; BETANAL MAXX PRO; REFINE 50 SX; BUTISAN STAR - Phytotoxicity 0%. Phytotoxicity has subsided. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous and perennial weeds. GLEAN 75 WG - Phytotoxicity 15%. Necrotic stains on newly growing leaves are no longer observed. Growth retardation of milk thistle plants, i.e. the delay of the onset of the individual growth phases compared to the untreated control. STARANE 250 EC - Phytotoxicity 10%. Necrotic stains on newly growing leaves are no longer observed. Growth retardation of milk thistle plants, i.e. the delay of the onset of the individual growth phases compared to the untreated control. The phytotoxicity evaluation in the year 2015 7 DPA (BBCH 15–18) STOMP 400 SC; BETANAL MAXX PRO; REFINE 50 SX - Phytotoxicity to 5%. Slightly delayed growth, slight necrosis of leaves that were already developed at the time of application. BUTISAN STAR - Phytotoxicity 5%. Delayed growth, slight necrosis of leaves that were already developed at the time of application. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous weeds. GLEAN 75 WG - Phytotoxicity 10%. Compared with the untreated variant, chlorotic plants were observed, as well as retardation and slower growth. Necrotic stains on leaves. Minimal mortality, especially of weaker plants.

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STARANE 250 EC - Phytotoxicity 5%. Delayed growth, slight necrosis of leaves that were already developed at the time of application. 14 DPA (BBCH 18–31) STOMP 400 SC; BETANAL MAXX PRO; REFINE 50 SX - Phytotoxicity 0%. Phytotoxicity has subsided. BUTISAN STAR – Phytotoxicity 3% - Growth inhibition, with no apparent necrosis or color changes. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous and perennial weeds. GLEAN 75 WG - Phytotoxicity 5%. Necrotic stains on newly growing leaves are no longer observed. Growth retardation of milk thistle plants, i.e. the delay of the onset of the individual growth phases compared to the untreated control. Death of plants observed, especially the weaker ones. STARANE 250 EC - Phytotoxicity 3%. Growth inhibition, with no apparent necrosis or color changes. 28 DPA (BBCH 37–59) STOMP 400 SC; BETANAL MAXX PRO; REFINE 50 SX; GLEAN 75 WG; STARANE 250 EC; BUTISAN STAR - Phytotoxicity 0%. Phytotoxicity has subsided. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous and perennial weeds. The phytotoxicity evaluation in the year 2016 7 DPA (BBCH 16–31) STOMP 400 SC; BETANAL MAXX PRO; REFINE 50 SX - Phytotoxicity > 5%. Slightly delayed growth, slight necrosis of leaves that were already developed at the time of application. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous and perennial weeds. GLEAN 75 WG - Phytotoxicity 7%. Compared with the untreated variant, chlorotic plants were observed, a well as retardation and slower growth. Necrotic stains on leaves. Minimal mortality, especially of weaker plants. STARANE 250 EC - Phytotoxicity 5%. Delayed growth, slight necrosis of leaves that were already developed at the time of application. 14 DPA (BBCH 31–35) STOMP 400 SC; BETANAL MAXX PRO; REFINE 50 SX - Phytotoxicity 0%. Phytotoxicity has subsided. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous and perennial weeds. GLEAN 75 WG - Phytotoxicity 3%. Necrotic stains on newly growing leaves are no longer observed. Growth retardation of milk thistle plants, i.e. the delay of the onset of the individual growth phases compared to the untreated control. STARANE 250 EC - Phytotoxicity 3%. Growth inhibition, with no apparent necrosis or color changes. 28 DPA (BBCH 39–67) STOMP 400 SC; BETANAL MAXX PRO; REFINE 50 SX; - Phytotoxicity 0%. Phytotoxicity has subsided. TARGA SUPER 5 EC; GALLANT SUPER - Phytotoxicity 0%. Herbicidal effect only on monocotyledonous and perennial weeds. GLEAN 75 WG; STARANE 250 EC - Phytotoxicity 0%. Phytotoxicity has subsided.

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Table 2 Average yield of milk thistle achenes in g/m in the years 2014, 2015 and 2016 Herbicide 2014 2015 2016 CONTROL 108.3 cd 143.9 a 154.3 a STOMP 400 SC 114.5 cd 177.9 c 168.5 a BETANAL MAXX PRO 133.4 e 156.9 b 163.5 a REFINE 50 SX 105.5 bc 169.5 c 171.0 a BUTISAN STAR 114.3 cd 149.5 ab 161.8 a TARGA SUPER 5 EC 118.7 cd 153.9 ab 166.0 a GALLANT SUPER 121.7 de 151.6 ab 156.3 a GLEAN 75 WG 68.4 a 168.9 c 164.0 a STARANE 250 EC 93.7 b 157.8 b 161.8 a

From the table 2 is evident, that in 2014 the highest yield of the achenes was in the variant treated with herbicide BETANAL MAXX PRO, the lowest yield was in the variant treated with herbicide GLEAN 75 WG, where yield was one half lower compared to the highest yield achieved. In 2015 year the highest yield was get in the variant treated with herbicide STOMP 400 SC, the lowest yield was in the control variant. In 2016 statistically significant differences between the yields of the achenes were not found.

CONCLUSION The monitoring of phytotoxicity of the herbicide treatment in three experimental years shows, that some included herbicides proved high selectivity against the plants of the milk thistle and in years 2014 and 2015 also the positive effect of their application on the achenes yield. In 2014, these were TARGA SUPER 5 EC, STOMP 400, BETANAL MAXX PRO, but also GALLANT SUPER and BUTISAN STAR. In 2015, these were mainly preparations: TARGA SUPER 5 EC, GALLANT SUPER, REFINE 50 SX a STOMP 400. Similar results were achieved in 2016. The application of these herbicides had primarily effect on monocotyledonous and perennial weeds. From the point of view of phytotoxicity the application of these herbicides was the best during the experimental years: TARGA SUPER 5 EC a STOMP 400, and next already registered herbicide REFINE 50 SX. There was no phytotoxicity for products TARGA SUPER 5 EC and GALLANT SUPER in the first term. Phytotoxicity up to 5% was monitored for STOMP 400 and REFINE 50 SX. The use of herbicides also corresponds to the yields of the achenes. In 2014, the highest yields were obtained after the application of BETANAL MAXX PRO and GALLANT SUPER. In 2015, the highest yield was obtained after the application of STOMP 400 SC, GLEAN 75 WG and REFINE 50 SX. Comparable yields were harvested also in 2016. However, as already stated in previous papers, the yields are affected by the course of weather in the given growing year, especially.

ACKNOWLEDGEMENTS The research was financially supported by the Grant IGA TP 1/2016: New trend in the cultivation and use of milk thistle (Silybum marianum L.) in agriculture.

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