Takayuki Mizukubo

Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate.

We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0mM at 1, 24, 48, and 72h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5mM or higher concentrations significantly reduced the infection of RKN in plants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection.

Micro-moulded substrates for the analysis of structure-dependent behaviour of nematodes

Microstructures, consisting of networks of channels of rectangular cross sections (50 μm high, 40-200 μm wide), were formed in 5 mm square areas on transparent substrates made of silicon rubber. An experimental set-up using the network sealed with a flat glass plate has the potential to function in a way similar to the pore space in soil, and is therefore useful for studies of nematode migration. The set-up allowed the migratory activity of nematodes in water-filled, porous and transparent microstructures to be observed with a microscope. By means of substrates with two different channel dimensions, the structure-dependent behaviour of second-stage juveniles of Meloidogyne incognita was visually demonstrated. Their behaviour was examined on the basis of the migration patterns obtained by superimposing recorded serial images of individual juveniles. In a micro-channel network with 40 μm high channels of 200 μm wide elements, juveniles showed marked activity in migration, forming consistent zigzag patterns spread over the network area. In contrast, in a micro-channel network with 80 μm high channels of 400 μm wide elements, migration showed thick, sparse patterns, restricted around the area where the juveniles were initially deposited. This comparison showed that M. incognita juveniles in a narrow, fine network tended to migrate actively and, in contrast, those in a wide, coarse network were prevented from migrating by the network configuration.

Tomato culture filtrate stimulates hatching and activity of Meloidogyne incognita juveniles

Hydroponic culture media were tested after growing tomato, okra, cucumber and bean for their effect on hatching and mobility of Meloidogyne incognita second-stage juveniles (J2) in laboratory experiments. Tomato and okra culture media increased the numbers of J2 that hatched as compared to those in water or fresh culture medium. The tomato culture medium increased hatching even in the presence of fosthiazate, an organophosphate nematicide, and at a concentration that inhibited hatching in the absence of tomato culture medium. Neither heat treatment of the tomato culture medium nor change of pH abolished its hatching stimulatory activity. When active J2 were incubated in the tomato culture media, the percentages of nematodes that became quiescent were lower than those of nematodes incubated in water or in fresh culture medium for 3 and 7 days in two trials. Moreover, the sigmoid movement of J2 was faster in the tomato culture medium than in water. Quiescent J2 became active more rapidly in culture media of tomato, cucumber and okra than in water or fresh culture medium. In experiments using soil containing quiescent J2, more J2 were extracted with the Baermann funnel method from soil treated with tomato culture medium than from soil treated with water. The results confirm that nematode stimulants, which may serve as a potential means in nematode control, exist in the plant culture media.

Sclareol induces plant resistance to root-knot nematode partially through ethylene-dependent enhancement of lignin accumulation.

The root-knot nematode (RKN) is one of the most devastating parasitic nematodes of plants. Although some secondary metabolites released by the host plant play roles as defense substances against parasitic nematodes, the mechanism underlying the induction of such defense responses is not fully understood. We found that sclareol, a natural diterpene known as an antimicrobial and defense-related molecule, inhibited RKN penetration of tomato and Arabidopsis roots. Sclareol induced genes related to ethylene (ET) biosynthesis and signaling and phenylpropanoid metabolism in Arabidopsis roots. In roots of ein2-1, an ET-insensitive mutant line, both sclareol-induced inhibition of RKN penetration and sclareol-induced enhancement of lignin accumulation were abolished. A mutant defective in lignin accumulation did not exhibit such inhibition. Sclareol also activated MPK3 and MPK6, Arabidopsis mitogen-activated protein kinases whose activation is required for triggering ET biosynthesis. Sclareol-induced inhibition of RKN penetration was exhibited by mutants of neither MPK3 nor MPK6. Treatment with a biosynthetic precursor of ET was insufficient compared with sclareol treatment to inhibit RKN penetration, suggesting the existence of an ET-independent signaling pathway leading to RKN resistance. These results suggested that sclareol induced resistance to RKN penetration partially through ET-dependent accumulation of lignin in roots.

The role of pore size on the migration of Meloidogyne incognita juveniles under different tillage systems

Migration of Meloidogyne incognita juveniles was investigated in soils collected from fields under different tillage regimes. Over 7 days, juvenile migration speed was greater in tilled soil (bulk density, 0.64 g cm −3 ) than in non-tilled soil (0.86 g cm −3 ). The volume of pores with diam. > 100 μ m was greater in tilled soil than in non-tilled soil. The effect of bulk density on juvenile migration was investigated using columns filled with soils of six bulk densities (range, 0.60-0.85 g cm −3 ). As bulk density decreased, migration increased, and soil with a lower bulk density had greater volume of pores with diam. > 30 μ m. The migration speed and behaviour of nematodes under saturated conditions were investigated using two micromoulded substrates that had pore spaces measuring 40-100 or 60-160 μ m. As pore size increased from 40 to 160 μ m, the migration speed decreased. The migration speed along a straight line ranged from 8.4 to 70.2 μ m s −1 on these substrates. Migration speed was not constant across a pathway composed of different pore spaces, and the speed was affected greatly by the increased detection behaviour required in the broader space. Our experimental findings suggest that considerable nematode migration can occur in soil through pores with diam. 30-100 μ m and > 100 μ m. Therefore, conventional tillage may enhance the migration and reinfestation of crops by M. incognita .

Absence of geotaxis in soil-dwelling nematodes

Nematodes are found in almost all habitats and exhibit behaviours adapted to diverse environments and food sources. Various studies have examined vertical movement of plantand animal-parasitic nematodes above ground in relation to survival and host-finding. The parasitic nematode, Skrjabinoclava, manipulates the behaviour of its intermediate host, Corophium volutator, to contact a potential definitive host by increasing its surface activity during the day (McCurdy et al., 1999). Several gastrointestinal nematode species position themselves to be eaten by prospective vertebrate herbivore hosts by crawling up foliage (Niezen et al., 1998). The plant-parasitic nematodes Aphelenchoides besseyi and Nothanguina phyllobia crawl to the tip of a plant, where they directly enter the plant organs (Robinson et al., 1979; Togashi & Hoshino, 2003). Geotaxis has been suggested to underlie some of these vertical movements, but many questions remain regarding such automatic responses to gravity across variable experimental conditions (Sciacca et al., 2002). Little is known about the geotaxis of nematodes underground, despite their importance in soil ecosystems. Soil has a complicated structure and is composed of particles of various sizes and shapes, so the movement of soil-dwelling nematodes is always constrained in their natural habitats. To understand geotaxis in soil-dwelling nematodes, it is useful to investigate movements under conditions that mimic natural habitats. In addition, technical difficulties are encountered when devising equipment to investigate nematode behaviour because most nematodes are extremely small, and most nematode habitats are opaque. A recent innovation is the use of two-dimensional micro-moulded substrates, which allow for the direct observation of nematode behaviour in a soil-like structure (Otobe et al., 2004). The purpose of this study was to

Behaviour in constrained spaces reflects habitat adaptations of terrestrial nematodes

Terrestrial nematodes occupy a wide variety of habitats, including soils, plants and animals (McSorley, 2003). Habitat has a major influence on physical adaptation and species diversity, and it seems likely that the variety of habitat characteristics also drives behavioural adaptation, resulting in different physical abilities of nematodes. The inherent obstacle to studying the behaviour of nematodes lies in observing them in natural habitats because their movement is usually constrained by soil or host tissues. The development of micro-moulded substrates may solve this problem by allowing direct observation and can be a promising new tool for nematode research. We test the hypothesis that different environmental structures are a factor that causes behavioural adaptation of nematodes by analysing the movement characteristics of nematodes in three different structures. We devised two micro-moulded substrates (approximately 5 × 5 mm) made of flexible polydimethylsiloxane. One had six long straight pathways 60 μm wide, 50 μm deep and 2 mm long (Fig. 1A). We designed the substrate to reproduce long and narrow pathways in plants, such as resin ducts and tracheids. We measured the locomotion speed of individuals for 30 to 60 s. The other was a micropore network (50 μm deep) designed to investigate the moving pattern in soil (Fig. 1B). We counted the number of 200 μm blocks traversed by the nematodes forwards and backwards and calculated the percentage of distance moved backwards. The speed in the micropore network indicates the total distance moved forward and backward per unit of time. In the network we formed a T-shaped

A new method of studying predatory nematodes using a micropore system

Abstract We developed an innovative micropore system that enables us to directly observe the constrained behaviour of predatory nematodes. The results show that the new system provides a suitable environment for modelling the potential effects of these animals as biocontrol agents in their natural habitat.