Influence of Root Exudates and Soil on Attachment of Pasteuria penetrans to Meloidogyne arenaria.

The bacterium Pasteuria penetrans is a parasite of root-knot nematodes (Meloidogyne spp.). Endospores of P. penetrans attach to the cuticle of second-stage juveniles (J2) and subsequently sterilize infected females. When encumbered by large numbers of spores, juveniles are less mobile and their ability to infect roots is reduced. This study looked at different factors that influence spore attachment of P. penetrans to the root-knot nematode Meloidogyne arenaria. Pretreatment of J2 with root exudates of eggplant (Solanum melongena cv. Black beauty) reduced spore attachment compared with pretreatment with phosphate-buffered saline (PBS), suggesting that the nematode surface coat was altered or the spore recognition domains on the nematode surface were blocked. Spore attachment was equally reduced following exposure to root exudates from both host and nonhost plants for M. arenaria, indicating a common signal that affects spore attachment. Although phytohormones have been shown to influence the lipophilicity of the nematode surface coat, auxins and kinetins did not affect spore attachment compared with PBS. Root exudates reduced spore attachment more in sterilized soil than in natural soil. Sterilization may have eliminated microbes that consume root exudates, or altered the chemical components of the soil solution or root exudates. Root exudates caused a greater decrease in spore attachment in loamy sand than in a sandy loam soil. The sandy loam had higher clay content than the loamy sand, which may have resulted in more adsorption of compounds in the root exudates that affect spore attachment. The components of the root exudates could have also been modified by soil type. The results of this study demonstrate that root exudates can decrease the attachment of P. penetrans endospores to root-knot nematodes, indicating that when these nematodes enter the root zone their susceptibility to spore attachment may decrease.

First Report of Meloidogyne haplanaria Infecting Mi-Resistant Tomato Plants in Florida and Its Molecular Diagnosis Based on Mitochondrial Haplotype.

Meloidogyne haplanaria is a species originally found infesting peanut in Texas and, more recently, in Arkansas. In this study, we confirmed the presence of M. haplanaria in Florida based on morphological and molecular characterization. This species was identified from a sample submitted for diagnosis collected from Mi-resistant tomato rootstock grown in Naples, FL. The major diagnostic criteria to distinguish M. haplanaria from other closely related root-knot nematode (RKN) species are based on morphological differences and host range tests, which are time consuming and labor intensive and require living or well-preserved specimens. In our study, we provide an easy diagnostic strategy to distinguish M. haplanaria from other RKN species based on amplification of two mitochondrial DNA regions. These regions span the intergenic spacer and part of the adjacent large subunit ribosomal RNA gene (lrDNA) and sequence polymorphisms in lrDNA revealed by the restriction pattern following digestion with the restriction enzymes HinfI and MnlI. A unique haplotype pattern, which has not been observed in any of the RKN species described thus far, was observed in M. haplanaria. The outcome of molecular analysis of M. haplanaria aligned with morphological measurement and characteristics as well as perineal pattern originally described for M. haplanaria.

Mitochondrial Haplotype-based Identification of Root-knot Nematodes (Meloidogyne spp.) on Cut Foliage Crops in Florida.

Florida accounts for more than 75% of the national cut foliage production. Unfortunately, root-knot nematodes (RKN) (Meloidogyne spp.) are a serious problem on these crops, rendering many farms unproductive. Currently, information on the Meloidogyne spp. occurring on most commonly cultivated cut foliage crops in Florida, and tools for their rapid identification are lacking. The objectives of this study were to (i) identify specific RKN infecting common ornamental cut foliage crops in Florida and (ii) evaluate the feasibility of using the mtDNA haplotype as a molecular diagnostic tool for rapid identification of large samples of RKN. A total of 200 Meloidogyne females were collected from cut foliage plant roots. Meloidogyne spp. were identified by PCR and RFLP of mitochondrial DNA. PCR and RFLP of mitochondrial DNA were effective in discriminating the Meloidogyne spp. present. Meloidogyne incognita is the most dominant RKN on cut foliage crops in Florida and must be a high target for making management decisions. Other Meloidogyne spp. identified include M. javanica, M. hapla, Meloidogyne sp. 1, and Meloidogyne sp. 2. The results for this study demonstrate the usefulness of the mtDNA haplotype-based designation as a valuable molecular tool for identification of Meloidogyne spp.

Comparative Genomics of Apomictic Root-Knot Nematodes: Hybridization, Ploidy, and Dynamic Genome Change.

The root-knot nematodes (genus Meloidogyne) are important plant parasites causing substantial agricultural losses. The Meloidogyne incognita group (MIG) of species, most of which are obligatory apomicts (mitotic parthenogens), are extremely polyphagous and important problems for global agriculture. While understanding the genomic basis for their variable success on different crops could benefit future agriculture, analyses of their genomes are challenging due to complex evolutionary histories that may incorporate hybridization, ploidy changes, and chromosomal fragmentation. Here, we sequence 19 genomes, representing five species of key root-knot nematodes collected from different geographic origins. We show that a hybrid origin that predated speciation within the MIG has resulted in each species possessing two divergent genomic copies. Additionally, the apomictic MIG species are hypotriploids, with a proportion of one genome present in a second copy. The hypotriploid proportion varies among species. The evolutionary history of the MIG genomes is revealed to be very dynamic, with noncrossover recombination both homogenizing the genomic copies, and acting as a mechanism for generating divergence between species. Interestingly, the automictic MIG species M. floridensis differs from the apomict species in that it has become homozygous throughout much of its genome.

RNA interference in Pratylenchus coffeae: knock down of Pc-pat-10 and Pc-unc-87 impedes migration.

Many of the currently available nematicides used in nematode control are hazardous to the user, environment and beneficial non-target organisms. Therefore the need to develop alternative methods for nematode control such as the development of nematode-resistant crops through RNA-mediated interference (RNAi) holds great promise. The Caenorhabditis elegans genes unc-87 and pat-10 are essential components of the body wall muscle and are thus required for nematode movement. The Pratylenchus coffeae orthologs of these two genes, namely Pc-pat-10 and Pc-unc-87 were cloned and used to test RNAi in this migratory nematode. RNAi was performed by soaking P. coffeae in a solution containing dsRNA of either Pc-unc-87 or Pc-pat-10. The levels of both Pc-unc-87 and Pc-pat-10 mRNAs were significantly reduced in a sequence-specific manner in nematodes soaked for 24h. Nematodes incubated in Pc-pat-10 dsRNA appeared straight and rigid while Pc-unc-87 resulted in nematodes that were coiled, in contrast to the regular sinusoidal movement of the control nematodes. While 88.4 ± 3.9% of the control nematodes successfully migrated to the bottom of the sand column in 12h, only 6 ± 1.3% and 7 ± 2.3%, respectively, of the Pc-pat-10 (RNAi) and Pc-unc-87 (RNAi) nematodes successfully migrated to the bottom. However a recovery in movement as well as transcript level was observed in both treatments when the nematodes were incubated in distilled water for 24h following the dsRNA soaking. The recovery rate was slower in Pc-unc-87 when compared to Pc-pat-10. In summary, this study demonstrates the existence of the RNAi phenomenon in P. coffeae and shows that the function of unc-87 and pat-10 genes has been evolutionarily conserved among free-living and plant parasitic nematodes.

Analysis of the transcriptome of the root lesion nematode Pratylenchus coffeae generated by 454 sequencing technology.

To study interactions between plants and plant-parasitic nematodes, several omics studies have nowadays become extremely useful. Since most data available so far is derived from sedentary nematodes, we decided to improve the knowledge on migratory nematodes by studying the transcriptome of the nematode Pratylenchus coffeae through generating expressed sequence tags (ESTs) on a 454 sequencing platform. In this manuscript we present the generation, assembly and annotation of over 325,000 reads from P. coffeae. After assembling these reads, 56,325 contigs and singletons with an average length of 353bp were selected for further analyses. Homology searches revealed that 25% of these sequences had significant matches to the Swiss-prot/trEMBL database and 29% had significant matches in nematode ESTs. Over 10,000 sequences were successfully annotated, corresponding to over 6000 unique Gene Ontology identifiers and 5000 KEGG orthologues. Different approaches led to the identification of different sequences putatively involved in the parasitism process. Several plant cell wall modifying enzymes were identified, including an arabinogalactan galactosidase, so far identified in cyst nematodes only. Additionally, some new putative cell wall modifying enzymes are present belonging to GHF5 and GHF16, although further functional studies are needed to determine the true role of these proteins. Furthermore, a homologue to a chorismate mutase was found, suggesting that this parasitism gene has a wider occurrence in plant-parasitic nematodes than previously assumed. Finally, the dataset was searched for orthologues against the Meloidogyne genomes and genes involved in the RNAi pathway. In conclusion, the generated transcriptome data of P. coffeae will be very useful in the future for several projects: (1) evolutionary studies of specific gene families, such as the plant cell wall modifying enzymes, (2) the identification and functional analysis of candidate effector genes, (3) the development of new control strategies, e.g. by finding new targets for RNAi and (4) the annotation of the upcoming genome sequence.