GLUCOSINOLATES IN SOME BRASSICA SPECIES AS SOURCES OF BIOACTIVE COMPOUNDS AGAINST ROOT KNOT NEMATODES

G. Sarıkamıs 1 , G. Aydınlı 2 and S. Mennan 3 . 1. Ankara University, Faculty of Agriculture, Department of Horticulture, Ankara, Turkey. 2. Ondokuz Mayıs University, Bafra Vocational High School, Samsun, Turkey. 3. Ondokuz Mayıs University, Faculty of Agriculture, Department of Plant Protection, Samsun, Turkey. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Brassica species are sources of bioactive compounds with several biological properties including biocidal activity against various soil borne pathogens and pests such as parasitic nematodes. Isothiocyanates derived from corresponding glucosinolates are major bioactive compounds responsible for this activity. In this study, glucosinolate content of red and white radish (Raphanus sativus L.), oilseed rape (Brassica napus L.), turnip (Brassica rapa L.) and Arugula (Eruca sativa L.) that were previously assessed for their host suitability level of root-knot nematodes (Meloidogyne arenaria and Meloidogyne incognita) were determined to understand the relationship between glucosinolate content and host-suitability level of these crops. The highest glucosinolate content was in radish. Turnip revealed lower levels compared to radish. However, the lowest glucosinolate content was determined in arugula and oilseed rape. Together with previous findings demonstrating host-suitability levels, the effect of glucosinolates on biocidal potential of Brassicaceae plants to fight against root-knot nematodes were evaluated.

…………………………………………………………………………………………………….... Introduction:-
The incorporation of plants containing specific biologically active compounds into soil against soil-borne pests is a natural plant protection approach. The Brassicaceae plants synthesize glucosinolates (GSL) that are sources of bioactive compounds such as isothiocyanates with several biological properties including biocidal activity. They are classified as aliphatics, aromatics or indoles having different properties and functions. Genetic factors determine the glucosinolate profile of plants, therefore the glucosinolates produced by a plant may vary. Glucosinolate content, however, is under the influence of environmental factors as well as stress factors during the growth period as reviewed by Sarıkamış 2009. Although present in the entire plant, the amount of glucosinolates is variable at different plant parts and plant growth stages (Brown et al., 2003).
Glucosinolates are hydrolyzed by endogenous myrosinases to produce an array of compounds including isothiocyanates, nitriles and indoles. Among these compounds isothiocyanates are associated with several biological activities including the suppression of soil borne pests (Lin et al., 2000). Benzyl isothiocyanate derived from the 272 hydrolysis of glucotropaeolin and 2-phenylethyl isothiocyanate has been shown to have high levels of antibacterial activity (Jang et al., 2010) and toxicity to several soil pathogens including nematodes and fungi (Jensen et al., 2010). Root-knot nematodes are plant parasitic nematodes that cause high losses in plants by reducing its quality and quantity. Chemical nematicides including soil fumigants have effectively controlled nematodes (Nyczepir and Thomas 2009). However, in recent years, restrictions on the use of these chemicals due to adverse effect on environment and human health have increased interest in non-chemical alternatives ( . Nematode-suppressive activity of Brassica species has met with variable results and this variability can be explained by some major factors including glucosinolate profile in plant tissues (Zasada et al., 2010). Soil-borne pests and diseases suppression by product of glucosinolate hydrolysis (most commonly isothiocyanates) released from incorporated plant tissues related to identities and concentrations of glucosinolates in plant tissue (Morra and Kirkegaard 2002). Therefore, it is crucial to know glucosinolate content of the plants to be used for biofumigation.
A good biofumigation crop for nematode management should be a poor host for target nematode species (Edwards and Ploeg 2014) and also have high glucosinolate production (Monfort et al., 2007). In our previous study, we investigated 40 genotypes from 15 different Brassica species initially to assess their host suitability level of rootknot nematodes (M. arenaria and M. incognita) and 12 genotypes were found to be as poor host with low potential of nematode multiplication as cover crop for biofumigation (Aydınlı and Mennan 2016). The objective of our study was to determine the glucosinolates of the selected brassica species that were known as poor hosts for M. arenaria and M. incognita, thus to have an idea of the relation between glucosinolates and biofumigation effect of the Brassica plants.

Materials And Method:-
Plant material:-Brassicaeae spp. including red and white radish (Raphanus sativus L.), oilseed rape (Brassica napus L.), turnip (Brassica rapa L.) and Arugula (Eruca sativa L.) previously tested for host suitability to root-knot nematodes (Aydınlı and Mennan, 2016) were used for the analysis of glucosinolates. These species were selected among the poor hosts for M. arenaria and M. incognita according to Aydınlı and Mennan (2016). Egg masses index, gal index and % RS (Relative Susceptibility= total nematode number on tested plant / total nematode number on susceptible control plant x 100) that are used as criteria to evaluate host status of plant are given for each species in Table 1.

Analysis of glucosinolates:-
Extraction of glucosinolates were performed on lyophilized leaf tissue using 70% (v/v) methanol, desulfated using Type H-1 Sulfatase from Helix pomatia (Sigma®) using DEAE Sephadex TM column, collected in vials and analyzed by HPLC (Shimadzu®) at Ankara University, Faculty of Agriculture, Department of Horticulture. Desulfoglucosinolates of each species were analyzed and separated by HPLC-UV detection using Waters Spherisorb 5μM ODS 2, 4.6x250mm analytical cartridge with a gradient program of 99% water and 1% acetonitrile as 1ml/min for 24 min (Sarıkamış et al. 2006) at a wavelength of 229 nm. Sinigrin from horseradish (Sigma®) or glucotropaeolin (Applichem®) which is not synthesized by the plant itself was used as the internal standard at a concentration of 16mM for the quantification of the peaks and given as μmolg -1 dry weight. A correction factor during calculation of each compound is provided by Brown et al., 2003. The peaks were identified using pure standards glucoraphenin (4-methylsulsulfinyl-3-butenyl), gluconapin (3butenyl), progoitrin (2-hydroxy-3-butenyl), glucoerucin (4-methylthiobutyl) purchased from PhytoLab GmbH&Co., sinigrin (2-propenyl glucosinolate) and glucotropaeolin (benzyl glucosinolate). The standards were desulfated prior to use and run in each sequence as external standards together with plant extracts.

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Statistical Analysis:-The analysis for each species was performed as three replicates. The glucosinolate contents were determined as mean ± standard error (SE) of the mean.
Quantification of the results using glucotrapeolin as the internal standard revealed that glucobrassicin content was 0.57±0.03 µmolg -1 DW, 4-hydroxyglucobrassicin content was 0.20±0.02 µmolg -1 DW, progoitrin was 0.34±0.04 µmolg -1 DW and sinigrin was 0.33±0.08 µmolg -1 DW in oilseed rape (Table 2). Overall, total and individual glucosinolate contents were very low in oilseed rape compared to other brassica species used in the present study.

Glucosinolates in Arugula (Eruca sativa L.)
Glucosativin (4-mercaptobutyl glucosinolate) was determined as the major glucosinolate in Arugula (E. sativa cv. Istanbul) (Fig. 4). Quantification of the results using internal standards suggested that glucosativin content was 1.81±0.14 µmolg -1 DW in the Arugula leaf tissue analyzed ( Table 2). This compound is convertes to 4mercaptobutyl isothiocyanate (sativin), a volatile and pungent metabolite probably responsible for the typical flavor of Arugula.

Discussion:-
Aliphatic and indole glucosinolates in red and white radish, turnip, oilseed rape and arugula revealed as poor hosts for M. arenaria and M. incognita (Aydınlı and Mennan 2016) were determined in the study. According to the findings, the highest level of glucosinolates was quantified in radish containing glucoraphenin as the predominant glucosinolate followed by turnip containing glucobrassicanapin, progoitrin and gluconapin as the major 274 glucosinolates. Aragula revealed lower levels compared to radish with glucosativin as the predominating glucosinolate. The lowest glucosinolate content was determined in oilseed rape determined as progoitrin and sinigrin at very low concentrations. Potter et al., (1998) reported that leaf tissues of high glucobrassicanapin and progoitrin containing B. rapa significantly reduced populations of root lesion nematode Pratylenchus neglectus (66%) when amended in soil. Strong nematicidal activity of isothiocyanates derived from sinigrin (2-propenyl isothiocyanate) was reported in vitro on juveniles of H. schachtii after 24 hours at 0.5% concentration (Lazzeri et al., 1993). Lazzeri et al., (2004) demonstrated stronger activity of the isothiocyanate on M. incognita in vitro. Aside from plant-parasitic nematodes, sinigrin isothiocyanate showed also high biocidal activity on other soil-borne pathogens (Mayton et al., 1996) Glucosativin (4-mercaptobutyl), glucoerucin (4-methylthiobutyl) and glucoraphanin (4-methylsulfinylbutyl)  Brassica crops are important for biofumigation as an effective way instead of synthetic chemicals to control soil borne pests and diseases. This is mainly attributed to glucosinolates which are converted into isothiocyanates on hydrolysis by the enzyme myrosinase. Isothiocyanates can reduce the activity of pathogens and pests in the soil (Ntalli and Caboni, 2017). The current study revealed that among different brassica species, radish (red and white cultivars) had the highest glucosinolates. Aliphatic glucosinolates were the major compounds followed by indoles at low levels in radish. Glucosinolates in other brassica species were much lower compared to radish. Both aliphatics and indoles were almost equal in turnip and oilseed rape. Glucosativin (4-mercaptobutyl glucosinolates), the precursor of sativin (4-mercaptobutyl isothiocyanate) was identified at low concentrations in Arugula. Therefore, radishes in this study may have a more biofumigation potential than other brassica plants if utilized as a green manure amendment. In order to increase the success of biofumigation, non-host or poor host species for target nematode species or population should be grown especially when soil temperature is suitable for nematode activity (Stirling and Stirling 2003;Pattison et al., 2006;Avato et al., 2013). Otherwise, nematode population increases in the soil during cultivation before the incorporation of biofumigant plants into the soil. According to the present findings non-host or poor host brassica with high glucosinolate content converting the majority of glucosinolates into isothiocyanates should be preferred to succeed in biofumigation approaches.