We have established Meloidogyne hapla as a tractable model plant-parasitic nematode amenable to forward and reverse genetics, and we present a complete genome sequence. At 54 Mbp, M. hapla represents not only the smallest nematode genome yet completed, but also the smallest metazoan, and defines a platform to elucidate mechanisms of parasitism by what is the largest uncontrolled group of plant pathogens worldwide. The M. hapla genome encodes significantly fewer genes than does the freeliving nematode Caenorhabditis elegans (most notably through a reduction of odorant receptors and other gene families), yet it has acquired horizontally from other kingdoms numerous genes suspected to be involved in adaptations to parasitism. In some cases, amplification and tandem duplication have occurred with genes suspected of being acquired horizontally and involved in parasitism of plants. Although M. hapla and C. elegans diverged >500 million years ago, many developmental and biochemical pathways, including those for dauer formation and RNAi, are conserved. Although overall genome organization is not conserved, there are areas of microsynteny that may suggest a primary biological function in nematodes for those genes in these areas. This sequence and map represent a wealth of biological information on both the nature of nematode parasitism of plants and its evolution.compaction ͉ dauer ͉ development ͉ horizontal gene transfer ͉ gene N ematodes are an abundant and species-rich animal phylum.They share a common body plan on which various adaptations have evolved, enabling Nematoda to occupy essentially all ecological niches, including being parasites of many other organisms (1). Parasitism of plants appears to have arisen independently in three of the major 12 nematode clades (2) and results in annual losses to world agriculture estimated to exceed $US100 billion (3, 4). The majority of damage is caused by sedentary endoparasitic forms in the order Tylenchida, which includes the root-knot nematodes (Meloidogyne spp., RKN). RKN have a cosmopolitan distribution and a host range that spans most crops, although individual RKN species exhibit a more restricted host range. Mature female RKN release hundreds of eggs onto the surface of the root that hatch in the soil as second-stage larvae (L2) and typically reinfect the same plant. RKN L2 are similar in function to dauer larvae (5), which were first described as an adaptation to parasitism to overcome adverse environmental conditions and facilitate dispersal (6), but have been best studied in the free-living nematode Caenorhabditis elegans (7). These larvae are developmentally arrested, motile, nonfeeding, nonaging, and long-lived. Like C. elegans dauers, RKN L2 are detergent-resistant (5), use the glyoxylate pathway (8), and exhibit intestinal morphology with sparse luminal microvilli and numerous lipid storage vesicles that permit long-term survival in the soil. RKN L2 penetrate the root and migrate intercellularly into the vascular cylinder. Migration is accompanied by extensive ...

Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade® and VOTiVO®, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan® probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104–105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.

Many isolates of the plant-parasitic nematode Meloidogyne hapla reproduce by facultative meiotic parthenogenesis. Sexual crosses can occur, but, in the absence of males, the diploid state appears to be restored by reuniting sister chromosomes of a single meiosis. We have crossed inbred strains of M. hapla that differ in DNA markers and produced hybrids and F 2 lines. Here we show that heterozygous M. hapla females, upon parthenogenetic reproduction, produce progeny that segregate 1:1 for the presence or absence of dominant DNA markers, as would be expected if sister chromosomes are rejoined, rather than the 3:1 ratio typical of a Mendelian cross. Codominant markers also segregate 1:1 and heterozygotes are present at low frequency (,3%). Segregation patterns and recombinant analysis indicate that a homozygous condition is prevalent for markers flanking recombination events, suggesting that recombination occurs preferentially as four-strand exchanges at similar locations between both pairs of non-sister chromatids. With this mechanism, meiotic parthenogenesis would be expected to result in rapid genomic homozygosity. This type of high negative crossover interference coupled with positive chromatid interference has not been observed in fungal or other animal systems in which it is possible to examine the sister products of a single meiosis and may indicate that meiotic recombination in this nematode has novel features.

Root knot nematodes (RKN; spp.) cause severe losses worldwide to a wide range of crops. Crop rotations with resistant hosts can be used to control losses, but the wide host range of RKN limits this option. In this study, we found that the wheat cultivar Lassik is resistant to several isolates of the RKN species and , including those that can reproduce on tomato with the resistance gene. Comparison of near-isogenic lines of wheat showed that the wheat resistance gene(s) is localized within a segment of the short arm of chromosome 2N from (Zhuk.) Chennav translocated into common wheat ( L.) chromosome arm 2AS and is associated with a highly significant decrease in RKN eggs in the roots. This RKN resistance gene has been assigned the name . While wheat itself is tolerant of RKN infection, a microplot experiment coupled with tomato bioassays showed less RKN root galling in the tomato samples grown in soil from the previous microplots including RKN resistant wheat varieties than in those including a susceptible wheat isogenic line. This result suggests that rotation with resistant wheat cultivars has the potential to be a valuable component of nematode management for crops that are highly susceptible to nematode damage and for which alternative strategies are limited.

Root-knot nematodes are obligate parasites of a wide range of plant species and can feed only on the cytoplasm of living plant cells. In the absence of a suitable plant host, infective juveniles of strain VW9 of the Northern root-knot nematode, Meloidogyne hapla, when dispersed in Pluronic F-127 gel, aggregate into tight, spherical clumps containing thousands of worms. Aggregation or clumping behavior has been observed in diverse genera in the phylum Nematoda spanning free-living species such as Caenorhabditis elegans as well as both plant and animal parasites. Clumping behavior differs between strains of M. hapla and occurs with other species within this genus where strain-specific differences in clumping ability are also apparent. Exposure of M. hapla juveniles to a gradient formed using low levels of cyanide promotes formation of clumps at a preferred cyanide level. Analysis of F2 lines from a cross of M. hapla strains that differ in clump-forming behavior reveals that the behavior segregates as a single, major locus that can be positioned on the genetic map of this nematode. Clumping behavior may be a survival strategy whose importance and function depend on the niche of the nematode strain or species.