Development of a new in vivo protocol through soil inoculation to investigate sugar beet resistance towards Ditylenchus dipsaci penetration

Abstract

Summary The stem nematode, Ditylenchus dipsaci, causes severe damage in sugar beet. To date, nematode inoculation through the leaf axil has been used as the standard method to investigate D. dipsaci interaction with sugar beet under in vivo conditions. To get as close as possible to field conditions, we established a new screening mechanism to perform soil inoculation. The most suitable inoculation time point, inoculum level and positioning on sugar beet, as well as rearing process on carrots, were determined. At a 15:8°C day:night temperature regime, penetration rates of D. dipsaci were at maximum following soil inoculation at plant emergence. Up to 115 nematodes penetrated sugar beet seedlings 22 days post-planting with an inoculum level of 1000 nematodes into the soil at plant emergence. Ditylenchus dipsaci penetration rate was higher in plants with soil inoculation than with inoculation on to the leaf axil. High soil moisture increased nematode migration into seedlings when D. dipsaci inoculation was carried out in four holes 1 cm from the plant base. Rearing the nematodes for 35 days at 20°C on carrot discs resulted in an infective inoculum containing up to 50% eggs. We recommend a soil inoculation of 1000 freshly extracted nematodes per pot at plant emergence. The nematode suspension has to be previously reared for 35 days on carrot discs to obtain active D. dipsaci inoculum. This system will allow for the selection of suitable sugar beet genotypes that suppress nematode penetration, in support of breeding for resistance against D. dipsaci.

Screening of sugar beet pre-breeding populations and breeding lines for resistance to Ditylenchus dipsaci penetration and reproduction

“…The high susceptibility of 'DIT_005', the paternal line of BERETTA KWS, suggests no direct relation between the resistance of one hybrid component towards D. dipsaci development and tolerance of a respective hybrid to the fungal and bacterial infection introduced by D. dipsaci. The nematode inoculation success depends on environmental conditions and the viability of the inoculated nematode population (Storelli et al 2021). This was also demonstrated by the experiments shown here.…”

“…Indeed, it has been observed in the present study that 'DIT_119' emerged earlier compared to the other genotypes. The period of susceptibility of sugar beets to D. dipsaci penetration occurs at the emergence of the plant (Storelli et al 2021). The rapid growth of 'DIT_119' may then reduce this period of susceptibility.…”

“…Fourth-stage juveniles (J4) and adult nematode stages were hand-picked after morphological identification of the tail, median bulb, stylet, and lip shape using an optical microscope at 40x magnification. After suspending the nematodes in an antibiotic solution containing 0.1% streptomycin sulphate (w/v) and 0.1% amphotericin-B (w/v) for 30 min, 50 nematodes were inoculated per surface sterilized (1% NaOCl) carrot disc (2.5 x 5 cm) and incubated for 45 days at 20 °C in the dark (Kühnhold et al 2006;Storelli et al 2021). The nematodes were extracted from the carrot discs, stored in the dark at 6-8 °C, and after 24 hr the sugar beet plants were inoculated.…”

Virulence and pathogenicity of four Ditylenchus dipsaci populations on sugar beet

“…Afterward, they were re-suspended for 30 min in a sterile antibiotic solution containing 0.1% streptomycin sulfate (w/v) and 0.1% amphotericin-B (w/v), and finally washed twice with sterile demineralized water (Kühnhold et al, 2006). The nematodes were then inoculated on the surface sterilized carrot disc callus with 50 nematodes per cylinder and incubated for 45 days in the dark at 20 ± 2°C (Storelli, Keiser, et al, 2021a). Nematodes were extracted from carrot discs for 24 h using Oostenbrink dishes and collected on a 20 μm mesh sieve.…”

“…In Europe, D. dipsaci is an important threat for sugar beet (Beta vulgaris L.) (Dewar & Cook, 2006;Leipertz, 2007). In Germany and Switzerland, some major sugar beet growing regions are particularly affected by the stem and bulb nematode (Leipertz, 2007;Storelli, Keiser, et al, 2021a). Its penetration on sugar beet seedlings leads to the dissolution of the middle lamellae and swelling of the hypocotyl (Duncan & Moens, 2013;Madani et al, 2015).…”