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Background: Pasteuria penetrans is a nonculturable bacterium that obligately parasitizes several species of phytopathogenic nematodes. Unknown endogenous female, plant or microbial reproductive factors are indispensable for multiplication and endospore formation of P. penetrans. Objective: To characterize P. penetrans endospores by 16S rRNA gene sequencing in samples from infected adult female Meloidogyne javanica and microbiota of infected and uninfected adult female M. javanica with P. penetrans by high-throughput sequencing of the V4 hypervariable region of the 16S rRNA gene. Methodology: Infected roots were collected from grapevines; fresh infective juvenile nematodes (J2) were extracted for P. penetrans endospore fixation and inoculation into tomato plants. Genomic and metagenomic DNA from infected and uninfected adult females of M. javanica was extracted for sequencing by sequencing the V4 region of the 16S rRNA gene of P. penetrans and its bacterial microbiome. The generated sequences were processed using bioinformatics software for analyses of alpha and beta diversity indices of the bacterial microbiome. Results: An amplicon of 550 base pairs with 98% identity and homology to P. penetrans was obtained. The taxonomic profile revealed the highest bacterial diversity and richness in the microbiota related to infected adult females, with Proteobacteria occurring in both samples between 45 and 83%, followed by Firmicutes, Actinobacteria and Bacteroidetes with 19, 11 and 8% respectively at the phylum level. Likewise, the most abundant genera associated with the native microbiota of the adult nematode were identified as Pseudomonas, Flavobacterium and Chitinophaga at 82.5, 15 and 2%, respectively. In infected females, Paenibacillus, Pasteuria, Pseudomonas and Streptomyces were recorded with 45, 7, 6 and 5% respectively, the most abundant. Implications: The results suggest the existence of bacterial genera in infected M. javanica females involved in the in vivo development of P. penetrans endospores. Conclusions: This would reveal a reduction of Pseudomonas dominance favoring the colonization of different bacteria that cohabit with P. penetrans, being this change in the microbial composition a possible factor that favors the multiplication of P. penetrans endospores within the host.

Background: The main problem in grapevine cultivation is root-attachment nematodes cause serious yield problems in most crops worldwide. Through their different infection mechanisms these nematodes synthesize and secrete a mixture of protein-based effectors that they use to penetrate the root, migrate and develop into giant root feeding cells in host plants. The use of new molecular tools such as MALDI TOF/TOF (Matrix-Assisted Laser Desorption/Ionization - Time-Of-Flight) mass spectrometry and PCR (Polymerase Chain Reaction) technique have allowed us to know these proteins and genes in different microorganisms. Objective: To characterize the root-knot nematode Meloidogyne javanica by sequencing the 18S rRNA gene from infected root samples and the effector proteins of juvenile (J2) and adult (J4) stage of M. javanica by MALDI TOF/TOF shotgun proteomics dual mass spectrometry. Methodology: Infected roots of grapevine crop were collected to extract fresh galls and J2 of M. javanica, then inoculated on tomato plants. J4 of M. javanica were used for genomic DNA extraction and sequencing at the 18S rRNA gene level. The J2 and J4 stages of M. javanica were disinfected with sodium hypochlorite (0.5%) and sterile distilled water for protein extraction and analysis with MALDI-TOF/TOF. Finally, the sequences obtained were processed with ProteinPilot™ and Protein BLAST software for the identification of effector proteins of M. javanica. Results: The nematode M. javanica was molecularly identified by PCR amplification of the 18S rDNA gene M. javanica with an identity percentage of 98% from infected root samples and by MALDI TOF/TOF mass spectrometry, effector protein sequences were identified such as: Beta-1,4-endoglucan and polygalaturonase, identified from J2, and expansin B2, CLAVATA3/ESR, Pectate lyase and Chorismato mutase from J4, involved in the different infection processes. In addition, we were able to identify 49 nematode non-effector proteins in both stages related to conserved biological development. Implications: The results indicate the existence of effector proteins related to root gill formation. Conclusions: This study confirms that dual mass spectrometry methodology provides in a rapid and reproducible way a proteomic profile that the galls nematode synthesizes to infect root cells and that can be used in other types of pathogens.