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Using of Hydrogen Cyanide Against Ditylenchus Dipsaci Nematode Present on Garlic

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Using of Hydrogen Cyanide Against Ditylenchus Dipsaci Nematode Present on Garlic Vol. 62, 2016, No. 4: 184–188 Plant Soil Environ. doi: 10.17221/28/2016-PSE Using of hydrogen cyanide against Ditylenchus dipsaci nematode present on garlic M. Zouhar1, O. Douda2, M. Dlouhý3, J. Lišková3, M. Maňasová1, V. Stejskal2 1Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Prague, Czech Republic 2Division of Crop Protection and Plant Health, Crop Research Institute, Prague, Czech Republic 3Lučební závody Draslovka a.s., Kolín, Czech Republic ABSTRACT The stem and bulb nematode (Ditylenchus dipsaci) is a serious quarantine pest of vegetables spreading worldwide via seed and planting material. Currently, a hot water technique is used as the pre-seed treatment, which is difficult to execute and the risk of seed damage is high. The objectives of this study were to evaluate the (a) penetration of gaseous hydrogen cyanide (HCN) into garlic tissue; (b) HCN phytotoxicity, and (c) nematicide potential of HCN against D. dipsaci. Penetration of HCN into the core of the garlic clove was approximately 30% of the concentra- tion inside the fumigation chamber after 30 h of exposure. Decreased emergency was observed only in the exposure treatment lasting 16 and more hours. Garlic cloves naturally infested by D. dipsaci were treated with HCN at a concentration of 20 g/m3 for 12, 18 and 24 h in a fumigation chamber, and 99% mortality was achieved in all three exposure times. Keywords: Allium sativum; pesticide; onion vegetable; crop protection In onion crops, the stem and bulb nematode protocol nr. PM 3/2(2), which is the fumigation of (Ditylenchus dipsaci) is an important pest (Aftalion seed material infested with D. dipsaci by methyl and Cohn 1990) with quarantine status in many bromide (EPPO 1998). As methyl bromide is no countries. D. dipsaci dispersion to new localities is longer available as a pesticide, the management mainly a consequence of its presence in seed mate- of D. dipsaci on onion vegetables has relied solely rial. This consequence occurs especially with veg- on hot water treatment in recent years (EPPO etables such as onions, where seeds (e.g. of chives 2000). This technique is often modified using a hot or onion) or seed bulbs (mainly the cases of garlic solution of formaldehyde. As the suspicion of the and leek) can be infested. Another crops e.g. hop carcinogenicity of formaldehyde has gained more could be also affected (Lišková and Renčo 2007). attention since the 1980s (e.g. Kerns et al. 1983) Therefore, treatment of the seed plant material is and some studies have also detected surviving of the key importance and is routinely performed nematodes after the hot water-formaldehyde treat- before planting (Hanks and Linfield 1999). ment (Roberts and Greathead 1986), alternative In the previous EPPO protocols (e.g. EPPO solutions are being investigated for the treatment 1974), hydrogen cyanide (HCN) was available of seed materials against D. dipsaci. Promising for fumigation treatment of bulbs, rhizomes and results were obtained from sodium hypochlorite tubers against D. dipsaci. That EPPO protocol was (Roberts and Matthews 1995) and peroxyacetic withdrawn in 1985, and was substituted with the acid (Hanks and Lindfield 1999). However, most Supported by the Ministry of Agriculture of the Czech Republic, Projects No. QI111B065 and QJ1310226. 184 Plant Soil Environ. Vol. 62, 2016, No. 4: 184–188 doi: 10.17221/28/2016-PSE of the investigated methods require dipping of the and South Korea (Stejskal et al. 2014b). In the past, seed material into liquids. This process has some HCN was extensively employed as a quarantine drawbacks, especially the subsequent drying, which treatment of wood against bark beetles in the can last up to 24 additional hours after treatment USA (Quayle 1922) as well as for seed treatment (Roberts and Matthews 1995). Hot water treat- in the Czech Republic (Stejskal 2014b). As previ- ment also introduces a risk of nematodes escaping ously mentioned, from 1975 to 1984, the EPPO from infested bulbs into the dip tank; these free- standard for D. dipsaci control in onions by HCN swimming nematodes are more difficult to kill by was available (EPPO 1974). However, there are in- temperature alone (Hanks and Lindfield 1999). The sufficient data on the efficacy of HCN fumigation number of newly registered active substances for on the mortality of D. dipsaci nematodes in garlic use as conventional nematicides is currently very cloves. Therefore, the objective of our study was to low. Therefore, alternative chemical treatments collect new data on the (a) speed of penetration of against D. dipsaci in seed plant material should HCN gas into garlic tissue; (b) HCN phytotoxicity be investigated. to seed garlic, and (c) biological efficacy of HCN One of the promising chemicals applicable as against D. dipsaci in infested garlic cloves. a nematode killing agent is HCN. It occurs natu- rally in the environment, it features very high penetration properties, and it is highly reactive MATERIAL AND METHODS and therefore easily and quickly degradable. The risks associated with its high toxicity can be de- Penetration of HCN into garlic tissue. The creased by proper cylinder formulation, use in a evaluation of the penetration of HCN into garlic fumigation chamber, and application by profes- cloves was conducted in a hermetic fumigation sionally trained staff (Stejskal et al. 2014a). HCN chamber. Garlic for testing was prepared by hol- is commercially available and is registered in some lowing the single cloves of garlic and attaching a European Union countries as a biocide for mills, rubber septum onto the hollow area (Figure 1). structural wood and transport fumigation treat- Treatments were conducted in a stainless her- ment (Ducom 2012, Aulicky et al. 2014). Outside metic steel fumigation chamber equipped with of the European Union, HCN is routinely used as an air lock, forced ventilation and rubber glove a plant/commodity quarantine fumigant in several manipulators (Figure 2). An image and technical countries, e.g. India, New Zealand (Navarro 2006), details of this chamber are described in a previous Figure 1. Single clove garlic with attached rubber septum Figure 2. Fumigation chamber 185 Vol. 62, 2016, No. 4: 184–188 Plant Soil Environ. doi: 10.17221/28/2016-PSE work (Stejskal et al. 2014a). The injected dosage of RESULTS AND DISCUSSION HCN in the head space of the fumigation chamber was equivalent to 20 g/m3. HCN samples were The concentration of HCN in the headspace of withdrawn from the garlic cloves via the septum the fumigation chamber during the treatments and using a glass syringe and the HCN concentra- inside the garlic tissue is summarised in Figure 3. tions were determined using a gas chromatograph The HCN concentration in the core of the garlic (Shimadzu GC-17A, RT-QPLOT, 30 m, ID 0.53 mm, cloves was approximately 30% of the initial con- GC Software Clarity DataApex, Kyoto, Japan) after centration inside the chamber headspace after 30 h 6, 12, 18, 24 and 30 h of exposure; the concentra- of treatment. Strong and simple relationship of tion of HCN inside chamber was also measured. exposure time and concentration of HCN inside HCN phytotoxicity. A second experiment tar- garlic tissue (correlation coefficient 0.98) as well geted the evaluation of the phytotoxic effect of as in fumigation chamber (correlation coefficient HCN on seed garlic cloves. The design of this 0.90) enabled estimation of equalizing of concentra- experiment was similar to the previous one. Nine tions in both environments at 50 h of hypothetical cultivars of garlic were obtained and treated with treatment. Our tests on phytotoxicity (Table 1) HCN (concentration 20 g/m3) for 10 exposure pe- further showed that HCN did not affect the viabil- riods. Seven cloves were treated in each cultivar. ity of garlic in the short treatments (up to 14 h); After treatment, garlic cloves were planted and however, there was a decrease in germination after numbers of emerging plants were scored 30 days longer HCN exposures. after planting. Untreated control variants of each Overall, there was good efficacy of the HCN cultivar were included in the experiment. treatment on D. dipsaci mortality (Table 2). Even Biological efficacy on D. dipsaci. Garlic cloves the shortest treatment period significantly de- infested with D. dipsaci were obtained from a creased the number of nematodes in the garlic farm in Central Bohemia. The presence and tissue. No significant differences were observed quantification of the species was confirmed us- among the three exposure times. After all three ing a Baermann funnel extraction technique. To tested exposure times, some living nematodes were determine the location of the nematodes within extracted; however, the number of survivors was the cloves, 56 cloves with peels removed were only approximately 1% of the numbers recorded examined and the removed peels were examined in the untreated control. separately. On average, one clove contained The HCN concentration in the core of the garlic 7 422 D. dipsaci specimens, 7 406 in peels and cloves was lower than in the fumigation chamber; 16 in the bare clove. Ten infested cloves were inserted into fabric sacks (mesh size 45 µm) for 25 fumigation. Five replicates were treated for each gas chamber cultivar. The injected dosage of HCN in the head garlic tissue space of the fumigation chamber was equivalent 20 3 to 20 g/m . The temperature inside the chamber ) 3 15 was maintained at 24°C during the trials. The m / g three exposure times tested were 12, 18 and ( 24 h, and HCN samples were also withdrawn 10 from the chamber at those intervals. After 24 h, the HCN was ventilated from the samples, and 5 surviving nematodes were extracted from treated cloves using the Baermann funnel technique and 0 counted under a stereomicroscope.
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