Fourth-stage Juveniles Research Articles

Phototactic behaviour and the role of light in host transmission of Bursaphelenchus xylophilus

Summary The pine wood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD). To understand the light influence on PWN, we investigated its phototactic behaviour. Our data indicated the mixed population of propagative PWN had a positive response to red, orange, yellow, green, blue and white lights, but a weak negative response to violet. For age-synchronised propagative nematodes, however, phototactic behavioural features changed with development. Interestingly, the dispersal fourth-stage juveniles (JIV) showed negative response to all tested lights, which was almost completely the reverse of the propagative fourth-stage juveniles (J4). Further bioassays proved that green, blue and white lights suppressed the host transmission of dispersal JIV from vector beetle to healthy pine branches. Our results revealed that night could be the peak of host transmission. With the results of previous studies, we speculate volatiles from the host tree and light may play pull-and-push roles to accelerate the host transmission of B. xylophilus.

Differential feeding site development and reproductive fitness of Meloidogyne incognita and M. javanica on zucchini, a source of resistance to M. incognita

The development of Meloidogyne incognita and M. javanica on zucchini ‘Amalthee’ was compared to characterise critical events in plant parasitism. Meloidogyne incognita was much less successful parasitising zucchini than M. javanica despite similarities in penetration rates and juvenile development. The increased frequency of undersized individuals, immature females and empty galls evidenced a failure in M. incognita development. Meloidogyne incognita induced larger feeding sites that contained more and larger giant cells than did M. javanica. Malformation of the M. incognita giant cells and abnormal growth of the surrounding tissues was observed at both 11 and 25 days post-inoculation. Critical events in parasitism differentiating the nematode isolates were the transition from fourth-stage juveniles to females, and the reduced fertility of the egg-laying females. Zucchini can be considered a source of resistance to M. incognita because it restricted nematode proliferation by supporting less fertile egg-laying females and producing fewer egg masses and total eggs.

First Report of Cereal Cyst Nematode (Heterodera filipjevi) on Winter Wheat in Xinjiang Uygur Autonomous Region, China

The cereal cyst nematode Heterodera filipjevi is considered to be one of the most important plant parasitic nematodes attacking most cereals and can cause significant crop losses (Sikora 1988). In China, this nematode was first recorded in Henan province in 2009 and a few years later in Anhui province (Peng et al. 2016). During May 2015 to August 2016, a survey for important plant parasitic nematodes on wheat and sugar beet was undertaken in Xinjiang Uygur Autonomous Region of China; 50 soil samples were collected, and the weight of each sample was about 1 kg. The cysts and second-stage juveniles (J2) were extracted from each soil sample using the sieving-decanting method. Morphological and molecular studies (Subbotin et al. 2010) of cysts and J2 confirmed the identification of H. filipjevi in two wheat-field soil samples from Xinyuan county of Yining city. There were no significant above-ground symptoms on wheat, but a few females were found on roots. The cysts were lemon shaped with light to dark brown cyst walls. Subcrystalline layer thick, valval core ambifenestrate, bullae large and numerous, and underbridge observed. The key morphometrics of cysts (n = 10) were length excluding neck (range = 705 to 887 μm, mean = 775 µm, standard deviation = 30.5); width (478 to 694, 571, 15), fenestra length (45.2 to 63.9, 54.7, 2.1), fenestra width (26.9 to 34.8, 28.4, 1.2), length of vulval slit (7.5 to 12.5, 9.4, 0.5), and length of underbridge (71.4 to 84.5, 76.4, 4.8). Measurements of J2s (n = 20) included length of body (range = 481.1 to 581.5 μm, mean = 540 µm, standard deviation = 7.1), stylet (24.5 to 27, 24.7, 0.2), tail (53.4 to 65.3, 61.2, 1.4), and hyaline tail terminus (30.4 to 42.2, 36.5, 0.7). Genomic DNA was isolated from single cysts (n = 5), and the internal transcribed spacer regions were amplified with primers TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) (De Ley et al. 1999). The obtained sequences (GenBank accession nos. KY448472 and KY448473, 1,054 bp) were found to be 99 to 100% identical to those of H. filipjevi from the United States (KP878490.1 and GU079654.1), Turkey (KR704304.1 and KR704308.1), and China (KT314234.1 and GU083595.1). The species-specific primers were used and produced a single band specific for H. filipjevi (Peng et al. 2013). Pathogenicity of the H. filipjevi was confirmed by infection of wheat and examination of the nematode development and reproduction. Wheat (Triticum aestivum ‘Wenmai 19’), was cultured in 300 cm³ pots filled with autoclaved soil in a glasshouse, and 800 eggs were inoculated into the soil near the roots of 4-day-old seedlings. The parasitic J2s, third and fourth-stage juveniles, and adult females were observed in roots stained with acid fuchsin at 7, 14, 28, and 35 days after inoculation (DAI), and an average of 51 cysts per pot were extracted 50 DAI. The new cysts’ morphological and molecular characteristics were identical to the H. filipjevi cysts from the original soil samples. To our knowledge, this is the first report of H. filipjevi in Xinjiang Uygur Autonomous Region, China. If this nematode continues to spread in China, it could become be a new threat to cereal production, so some effective control should be taken to prevent further spread.

Meloidogyne incognita Fatty Acid- and Retinol- Binding Protein (Mi-FAR-1) Affects Nematode Infection of Plant Roots and the Attachment of Pasteuria penetrans Endospores.

Root-knot nematode (RKN) Meloidogyne incognita is an economically important pest of crops. Pasteuria penetrans, is a nematode hyperparasitic bacterium capable of suppressing the reproduction of RKN and thereby useful for its management. Secreted fatty acid and retinol-binding proteins are unique in nematodes and are engaged in nutrient acquisition, development and reproduction; they are also a component of the nematode cuticle and thought to be involved in the interface between hosts and parasites. Attachment of endospores to the cuticle of second stage juveniles of RKN is the primary step of infection and several factors have been identified to facilitate attachment. In this study, the full length of Mi-far-1 (573 bp) was cloned from M. incognita and characterized. Analysis revealed that the Mi-far-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a glycosylation site. Quantitative PCR showed the highest expression in the fourth stage juveniles and in situ hybridization revealed the presence of Mi-far-1 mRNA in the hypodermis below the cuticle. Single copy insertion pattern of Mi-far-1 in M. incognita genome was detected by Southern blotting. Knockdown of Mi-far-1 showed significantly increased attachment of P. penetrans’ endospores on juvenile cuticle surface and also affected host finding, root infection and nematode fecundity.

Morphological and Molecular Characterization of Gracilacus wuae n. sp. (Nematoda: Criconematoidea) Associated with Cow Parsnip (Heracleum maximum) in Ontario, Canada.

Gracilacus wuae n. sp. from soil associated with cow parsnip in Ontario, Canada is described and illustrated. Morphologically, females have a long stylet ranging from 80 to 93 µm long, the lip region not offset from the body contour, without lateral lips but with large and flat submedian lobes, the mouth opening slit-like elongated laterally and surrounded by lateral flaps, the excretory pore is anterior to the knobs of the stylet; males without stylet and the pharynx degenerated. The fourth-stage juveniles lack a stylet, the pharynx degenerated, and can be differentiated into preadult females and males based on the position of the genital primordia. The third-stage juveniles are similar to females but smaller. Phylogenetic studies using the rDNA small subunit 18S, large subunit 28S D2/D3, and internal transcribed spacer (ITS) sequences collectively provide evidence of a grouping with other Gracilacus and some species of Paratylenchus with stylet length of females longer than 41 µm deposited in GenBank.

Re-description of the slug-parasitic nematode Alloionema appendiculatum Schneider, 1859 (Rhabditida: Alloionematidae)

Alloionema appendiculatum is a common juvenile parasite of many terrestrial molluscs. Its third-stage juveniles (dauers) invade the foot muscle of snails and slugs and develop into fourth-stage juveniles, which then leave the host. Later they mature and reproduce in the soil. A population of A. appendiculatum was isolated from infected individuals of the invasive slug Arion vulgaris (= A. lusitanicus), collected in the city of České Budějovice, Czech Republic, and was designated as the AL strain. This nematode is also able to reproduce on pig kidney in laboratory culture. Our isolate was compared primarily with the description published by Mengert (1953) and in some aspects with the original description published by Schneider (1859). Insufficient morphology, morphometrics, phylogeny and an absence of pictorial material encouraged us to re-describe this frequent slug parasite and to add some new information on its life-cycle. The species is characterised by the absence, in adults, of ridges in the lateral fields. At this life stage the stoma is short, narrow and approximately twice as long as it is broad. Fourth-stage female juveniles produce a mucus-like substance from the phasmids. Males possess six pairs of papillae and a single inconspicuous papilla. The nematode has both parasitic and saprophytic life-cycles. Parasitic adults are bigger than the saprophytes and have a thick, digitate tail, whereas the smaller saprophytic generation has a filiform tail. The species is amphimictic, displaying a higher proportion of females, but males are frequent.

Overwintering strategies of a population of anthelmintic-resistant Haemonchus contortus within a sheep flock from the United States Northern Great Plains

Haemonchus contortus infections have been increasingly reported in ungulates from cold climates even though past studies have shown that the free-living juveniles from this species survive poorly under freezing conditions. Overwintering strategies of H. contortus have not been documented in the Unites States Northern Great Plains. A PCR survey identified H. contortus as vastly predominant trichostrongyle species present (in addition to occasional detections of Teladorsagia sp.) in a closed farm flock of sheep from Brookings County, SD. Benzimidazole (BZ) and avermectin (AV) anthelmintics had been used intensely for many years on this flock. During the autumn season, three fecal egg count reduction tests (doramectin, albendazole, and moxidectin) were performed over a 4 year span to assess drug effectiveness within the flock. Significant drug resistance was found in Haemonchus adults with doramectin (69% efficacy), marginal resistance was found with albendazole (90% efficacy) and no resistance was found in moxidectin (100% efficacy). The following spring, pre-lambing and post-lambing fecals were obtained from albendazole and moxidectin treatment years to assess the resistance of the tissue-dwelling fourth-stage juveniles (J4s) at those times. Albendazole treated pre-lambing fecals averaged only 4 EPG and treated post-lambing fecals increased to 454 EPG, indicating that many of the J4s were not killed during the autumn treatment. Moxidectin pre-lambing fecals averaged only 1 EPG, and post-lambing fecals only increased to 6 EPG in the treated moxidectin population and 1422 EPG in the untreated moxidectin population. In addition to evaluating the ability of H. contortus to overwinter as drug resistant tissue-dwelling J4s, this study also evaluated the overwintering ability of pasture-dwelling, free-living third-stage juveniles at this farm. In the summers of 2010 and 2011, naïve tracer lambs were placed on a H. contortus contaminated pasture for 3 weeks to assess J3 winter survival. In 2010, tracer lambs only averaged 7 EPG whereas drylotted control lambs averaged 2 EPG; in 2011, tracer lambs averaged 2 EPG while the control lambs averaged 1 EPG. These results suggest that at this northern plains farm, yearly transmission of H. contortus is predominately through drug-resistant J4s. This is consistent with other cold-climate, overwintering studies involving H. contortus from Europe.

Comparative cellular responses in susceptible and resistant soybean cultivars infected by Meloidogyne incognita

Meloidogyne incognita, a predominant nematode parasite of soybean in South Africa, increasingly threatens production of the crop as it is expanding to maize-producing areas infected by this nematode. The parasitic relationship between M. incognita and soybean were compared on a susceptible and a resistant cultivar in terms of nematode penetration, development, reproduction and fecundity as well as histopathology studies. Second-stage juveniles (J2) of M. incognita were inoculated on roots of a resistant (LS5995) and a susceptible (Prima2000) cultivar in three concurrent but separate glasshouse trials. For pre-infectional studies, root systems of plants were harvested 2, 4, 10, 16 and 20 DAI. Sampling times for the post-infectional experiment were 4, 10, 20 and 30 DAI, whilst those for the histopathology experiment were 2, 4, 10, 20 and 30 DAI. J2 penetrated roots of both cultivars in comparable numbers 2 DAI but vermiform J2 numbers were significantly lower in roots of LS5995 at 4, 16 and 20 DAI. Final (Pf) J2 population density (vermiform plus swollen individuals) in roots of Prima2000 was significantly higher at all sampling times than those in roots of LS5995. Development of M. incognita J2 to third- (J3) and fourth-stage juveniles (J4) was significantly affected by cultivar susceptibility and time, being slower in LS5995. Development of J2 to J3 and J4 or into mature females was also consistently slower in LS5995 for the duration of this experiment. Adult females in roots of Prima2000 produced significantly more (98%) eggs per egg mass and also maintained significantly more egg and J2 numbers (98.5%) per root system 30 DAI than those in roots of LS5995. Histopathological observations showed that J2 penetrated roots of both soybean cultivars and migrated intercellularly to undifferentiated provascular tissue 2 and 4 DAI, with pronounced cellular changes taking place. A hypersensitive reaction was observed 2 DAI in roots of the resistant cultivar. From 10 to 30 DAI giant cell formation in the differentiated vascular tissue in the roots of LS5995 differed substantially from those in roots of Prima2000. Giant cells that developed in roots of the LS5995 were smaller and fewer compared to those in Prima2000. Giant cells in roots of LS5995 also contained empty as well as sub-optimal giant cells with thicker cell walls than those reported for resistant soybean cultivars in earlier studies. The induction, development and maintenance of giant cells in LS5995 proved to be typically retarded.

Arbuscular mycorrhizal fungi affect both penetration and further life stage development of root-knot nematodes in tomato

The root-knot nematode Meloidogyne incognita poses a worldwide threat to agriculture, with an increasing demand for alternative control options since most common nematicides are being withdrawn due to environmental concerns. The biocontrol potential of arbuscular mycorrhizal fungi (AMF) against plant-parasitic nematodes has been demonstrated, but the modes of action remain to be unraveled. In this study, M. incognita penetration of second-stage juveniles at 4, 8 and 12days after inoculation was compared in tomato roots (Solanum lycopersicum cv. Marmande) pre-colonized or not by the AMF Glomus mosseae. Further life stage development of the juveniles was also observed in both control and mycorrhizal roots at 12days, 3weeks and 4weeks after inoculation by means of acid fuchsin staining. Penetration was significantly lower in mycorrhizal roots, with a reduction up to 32%. Significantly lower numbers of third- and fourth-stage juveniles and females accumulated in mycorrhizal roots, at a slower rate than in control roots. The results show for the first time that G. mosseae continuously suppresses root-knot nematodes throughout their entire early infection phase of root penetration and subsequent life stage development.

Low temperature survival of post-eclosion stages of the potato rot nematode <i>Ditylenchus destructor</i> Thorne (Tylenchida: Anguinidae)

AbstractThe potato rot nematode, Ditylenchus destructor, may experience extreme environmental stress caused by freezing temperatures when overwintering in the field. A series of laboratory experiments were conducted in order to clarify overwintering strategies of the nematode. The current study aimed to examine the cold tolerance of this species in various aqueous environments as well as in plant tissues in order to determine its ability to survive 24 h exposure to subzero temperatures. Cold tolerance of adults and fourth-stage juveniles was significantly lower than that of younger juveniles. The lower lethal temperature of adults was –15°C. Although external ice formation affects all life stages, a few second-stage juveniles were able to survive temperatures as low as –30°C. The results expand the current understanding of freezing survival in the potato rot nematode and will lead to better comprehension of its ability to withstand subzero temperature conditions.

Recovery of infective juveniles of the entomopathogenic nematode Steinernema carpocapsae via factors produced by insect cells and symbiotic bacteria

Abstract Entomopathogenic nematodes, Steinernema carpocapsae, show 'recovery' from the dauer form as infective juveniles (IJ) up to fourth-stage juveniles when host invasion occurs. This recovery also occurs within an insect cell line culturing system. Here we addressed the factor(s) that induce recovery. When IJ were exposed to cell medium obtained from the cultivation of Sf9 cell lines derived from armyworms (Spodoptera frugiperda), approximately 50% of IJ recovered after 4 h. By 16 h, 90% of the IJ had undergone recovery. Other insect cell lines such as silkworm (Bombyx mori)-derived BmN cells and fruit fly (Drosophila melanogaster)-derived S2 cells also secreted the recovery inducing factor(s). By contrast, mammalian cells (NIH/3T3 and HeLa) had no effect on nematode recovery. Our data also suggest that symbiotic bacteria are involved in IJ recovery; axenic IJ did not recover in the cell-cultured medium. When symbiotic bacteria isolated from IJ were propagated within the cell-cultured medium, the supernatant gained recovery-inducing activity against axenic IJ. From these results, we conclude that IJ recovery in S. carpocapsae is induced by multiple factor(s) secreted from insect cells and symbiont bacteria.

Ditylenchus destructor and Ditylenchus dipsaci

<i>Ditylenchus destructor</i> and <i>Ditylenchus dipsaci</i>

Effect of rotation crops on hatch, viability and development of Heterodera glycines

AbstractRotation with non-host crops can be an effective method for reducing soybean cyst nematode (Heterodera glycines) populations in soybean cropping systems. Sunn hemp, Illinois bundleflower, oilseed rape, perennial ryegrass, red clover, corn and H. glycines-susceptible soybean were compared for their effects on H. glycines hatch, viability and development in laboratory and glasshouse experiments. In the laboratory experiments, root exudates in soil leachates, extracts from fresh plant tissues, and extracts from residues of sunn hemp, red clover and soybean stimulated hatch of second-stage juveniles (J2) of H. glycines. All crops appeared to contain hatch inhibitors as well. There was no apparent effect of the root exudates and extracts from any of the crops on egg viability in vitro. When the H. glycines J2 were exposed to root exudates and extracts for 72 h, only the extracts of fresh plants and plant residue from sunn hemp, red clover and soybean, and the extract of plant residue from oilseed rape reduced viability of J2. In glasshouse experiments, residues of all crops, except Illinois bundleflower, reduced egg population density, with sunn hemp providing the greatest reduction. Residues of sunn hemp, red clover, and perennial ryegrass added to soil reduced the reproduction factor, suggesting the residues not only reduced egg population density but also reduced nematode infectivity. While all crops allowed penetration by J2, minimal development (to third- or fourth-stage juveniles only) occurred only in sunn hemp, red clover, oilseed rape and Illinois bundleflower, and full development occurred only in soybean. The results suggest that sunn hemp and red clover were the most effective rotation crops for managing H. glycines, and that stimulating hatch of H. glycines J2 was the main mechanism involved in reducing the H. glycines population density.

Comparison of the life cycle of potato cyst nematode (Globodera rostochiensis) pathotype Ro1 on selected potato cultivars

The life cycle of Globodera rostochiensis pathotype Ro1 was studied under experimental conditions on selected potato cultivars (Korela, Albina, Vivaldi, Veronika, Vera, Monalisa, Victoria, Maranca, Desiree) in Slovakia during two growing seasons. Two peaks of second stage juveniles (J2) were found in the soil; the first peak three and four weeks after planting in the first and second year, respectively. The last J2 were found on 23 September. The number of J2 found in the second peak was much higher. First J2 associated with roots were observed 18 days, on middle early and seritonous cultivars 34 days after planting, but fourth stage juveniles (J4) were observed 40 days after planting in both cultivar groups. First adult males were found in soil 43 and 46 days after planting, respectively, and the last males two weeks later. White females filled with eggs were observed on roots 61 days after planting. The cycle from hatching of J2 in the soil to the hatching of J2 from brown cysts required 68 days in the first year and 60 days in the second year.

Parasitism of Subterranean Termites (Isoptera: Rhinotermitidae: Termitidae) by Entomopathogenic Nematodes (Rhabditida: Steinernematidae; Heterorhabditidae)

In laboratory bioassays, Steinernema riobrave Cabanillas, Poinar and Raulston (355 strain), Steinernema carpocapsae (Weiser) (Mexican 33 strain), Steinernemafeltiae (Filipjev) (UK76 strain), and Heterorhabditis bacteriophora Poinar (HP88 strain) were all capable of infecting and killing three termite species, Heterotermes aureus (Snyder), Gnathamitermes perplexus (Banks), and Reticulitermes flavipes (Kollar) in laboratory sand assays. S. riobrave and S. feltiae caused low levels of Reticulitermes virginicus (Banks) mortality under the same conditions. At 22 degrees C, significant mortality (> or = 80%) of worker H. aureus and G. perplexus was caused by S. riobrave, in sand assays, indicating the need for further study. Because of the short assay time (3 d maximum), reproduction of the nematodes in the target host species was not recorded. All nematode species were observed to develop to fourth-stage juveniles, preadult stages, or adults in all termite species with the exception of R. virginicus. Only S. riobrave developed in R. virginicus. Nematode concentration and incubation time had significant effects on the mortality of worker H. aureus. S. riobrave consistently generated the highest infection levels and mortality of H. aureus in sand assays.

Molecular Diagnostics, Taxonomy, and Phylogeny of the Stem Nematode Ditylenchus dipsaci Species Complex Based on the Sequences of the Internal Transcribed Spacer-rDNA

ABSTRACT The stem nematode Ditylenchus dipsaci is of great economic importance worldwide as a parasite of agricultural crops and horticultural plants. The internal transcribed spacer (ITS) of rDNA from 23 populations of the D. dipsaci complex from various host plants were amplified and sequenced. Seven previously studied populations were also included in the study. The phylogenetic analysis of the full ITS and ITS2 sequence alignments using minimum evolution, maximum parsimony, and Bayesian inference under the complex model of DNA evolution revealed trees with two main clades: (i) D. dipsaci sensu stricto with diploid chromosome numbers and comprising most isolates from agricultural, ornamental, and several wild plants, and (ii) Ditylenchus spp. with polyploid chromosome numbers, reproductively isolated from diploid populations, and subdivided into six subclades ("giant race" from Vicia faba, Ditylenchus species parasitizing various Asteraceae, and a Ditylenchus sp. from Plantago maritima). Using the energy minimization approach and comparative sequence analysis, it has been found that the secondary structure of ditylenchid ITS2 is organized in three main domains. The importance of knowledge on the RNA structure for phylogenetic analysis is discussed. Conventional polymerase chain reaction (PCR) and real-time PCR with SYBR green dye I with a species specific primer have been developed for detection and quantification of D. dipsaci sensu stricto Validation tests revealed a rather high correlation between real numbers of fourth-stage juveniles of the stem nematodes in a sample and expected numbers detected by real-time PCR. Problems of accuracy of quantification are discussed.

Survival of Xiphinema index in Vineyard Soil and Retention of Grapevine fanleaf virus Over Extended Time in the Absence of Host Plants

ABSTRACT Grapevine fanleaf virus (GFLV) is transmitted specifically from grapevine to grapevine by the ectoparasitic root-feeding nematode Xiphinema index. Limited information is available on the survival of X. index in vineyard soil and on the retention of GFLV by X. index over extended periods of time. We addressed these two issues by quantifying the numbers of living X. index recovered from soil samples that were collected in three naturally GFLV-infected vineyards in France and subsequently stored at 7 or 20 degrees C in the absence of host plants. Our data indicated a two- to eightfold decrease in X. index numbers but the recovery of 8 to 10 living fourth-stage juveniles (J4) and adults per kilogram of soil after 4 years of storage regardless of temperature. In addition, GFLV was detected readily in all groups of 20 isolated X. index adults and J4 (except for J4 that were kept 4 years at 20 degrees C) by reverse transcription-polymerase chain reaction using total nematode RNAs and a primer set located in conserved regions at the 3' end of viral genomic RNA 2. Our findings on the long-term survival of viruliferous X. index under adverse conditions emphasize the need for new control strategies against GFLV.

The Root-Knot Nematode Meloidogyne fallax on Strawberry in the Netherlands.

The root-knot nematode Meloidogyne fallax Karssen is closely related to M. chitwoodi Golden, O'Bannon, Santo & Finley. Both species have a wide host range and have been designated as quarantine nematodes by several countries. Strawberry (Fragaria ananassa L.) is considered a poor host for M. chitwoodi (2,3), but the host status for M. fallax in unknown. During 2003, a host suitability study was performed with four common strawberry cultivars (Ciflorette, Elsanta, Kimberly, and Mara des bois) on a field naturally infested with M. fallax near Wintelre, the Netherlands. The identity of the nematode was confirmed, prior to and after the test, by using morphological characteristics, isozymes, and polymerase chain reaction (PCR). The field was not infested with M. hapla Chitwood. The host test included 15 replicates per cultivar and started in April with a mean initial M. fallax density of approximately 4,500 second-stage juveniles per 100 ml of soil. The plants used in the field trial were multiplied by cuttings and grown on artificial substrate free from any Meloidogyne species. Upon plant harvesting in October, the number of root galls were counted and cvs. Mara des bois and Ciflorette did not show any typical root-knot symptoms, while cvs. Elsanta and Kimberly expressed only a few small galls. Additionally, high numbers of nematodes were found in the roots using the centrifugal floating method (1). The mean numbers of isolated nematodes for all cultivars (females, 116; males, 80; second-stage juveniles, 6,636; third- and fourth-stage juveniles, 49; and eggs, 920 per 10 g of roots) suggests that the tested strawberry cultivars are good hosts for M. fallax despite the lack of clear root galling or visible plant grow reduction as has been observed in the past for M. javanica on strawberry (4). To our knowledge, this is the first report of strawberry as a host for M. fallax, and it suggests that M. fallax could be spread by strawberry transplant grown in M. fallax infested soil. The Dutch Plant Protection Service is currently discussing appropriate action, such as an additional root-knot nematode test in addition to visual root inspection, before strawberry plants are shipped and cultivated.

A New Method for Loading Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) on Adult Monochamus alternatus (Coleoptera: Cerambycidae)

A new method was developed for loading the pinewood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, on the beetle Monochamus alternatus Hope. Postdiapause beetle larvae were sterilized with 70 and 99.9% aqueous ethanol and placed singly in flasks where B. xylophilus reproduced on the fungus Ophiostoma minus (Hedgcock) H. et P. Sydow that had been grown on autoclaved barley grain and Pinus densifiora Sieb. et Zucc. wood chips. The fungus produced a large nematode population that developed to a high proportion of third-stage dispersal juveniles that molted to the fourth-stage dispersal juveniles. The survival rate was 80%, and the mean nematode load was 10,096. It took a mean of 5 wk to obtain the nematode-infested beetles after the initiation of nematode rearing.

Boarding abilities of Bursaphelenchus mucronatus and B. xylophilus (Nematoda: Aphelenchoididae) on Monochamus alternatus (Coleoptera: Cerambycidae)

Abstract Bursaphelenchus mucronatus is closely related to Bursaphelenchus xylophilus, the causative agent of pine wilt disease. Both nematodes are transmitted between host pine trees as the fourth-stage dispersal juveniles (JIV) by insect vectors. After the invasion of Japan by B. xylophilus, B. mucronatus, native to Japan, appears to have been replaced in the pine forests during the spread of the disease. To help understand this species replacement, the number of JIV carried by an insect vector (the initial nematode load) was compared between the two nematode species by using the beetle, Monochamus alternatus, in the laboratory. The initial load of B. mucronatus was significantly smaller than that of B. xylophilus although the number of third-stage dispersal juveniles (JIII) concentrated at the pupal chambers did not differ. Statistical analysis showed that the proportion of JIII moulting to JIV was the most important among three components explaining the difference in the initial load of B. mucronatus while the number of JIII concentrated at the pupal chamber was the most important for B. xylophilus. The phoretic affinity between the nematode and its vector is discussed in relation to its role in the species replacement.