Background: Soybean pathogens and pests reduce grain production worldwide. Biotic interaction cause extensive changes in plant gene expression profile and the data produced by functional genomics studies need validation, usually done by quantitative PCR. Nevertheless, this technique relies on accurate normalization which, in turn, depends upon the proper selection of stable reference genes for each experimental condition. To date, only a few studies were performed to validate reference genes in soybean subjected to biotic stress. Here, we report reference genes validation in soybean during root-knot nematode (Meloidogyne incognita) parasitism and velvetbean caterpillar (Anticarsia gemmatalis) attack. Findings: The expression stability of nine classical reference genes (GmCYP2, GmELF1A, GmELF1B, GmACT11, GmTUB, GmTUA5, GmG6PD, GmUBC2 and GmUBC4) was evaluated using twenty-four experimental samples including different organs, developmental stages, roots infected with M. incognita and leaves attacked by A. gemmatalis. Two different algorithms (geNorm and NormFinder) were used to determine expression stability. GmCYP2 and GmUBC4 are the most stable in different organs. Considering the developmental stages, GmELF1A and GmELF1B genes are the most stable. For spatial and temporal gene expression studies, normalization may be performed using GmUBC4, GmUBC2, GmCYP2 and GmACT11 as reference genes. Our data indicate that both GmELF1A and GmTUA5 are the most stable reference genes for data normalization obtained from soybean roots infected with M. incognita, and GmCYP2 and GmELF1A are the most stable in soybean leaves infested with A. gemmatalis. Conclusions: Future expression studies using nematode infection and caterpilar infestation in soybean plant may utilize the reference gene sets reported here.

The root‐knot nematodes are the most devastating worms to worldwide agriculture with Meloidogyne incognita being the most widely distributed and damaging species. This parasitic and ecological success seems surprising given its supposed obligatory clonal reproduction. Clonal reproduction has been suspected based on cytological observations but, so far, never confirmed by population genomics data. As a species, M. incognita is highly polyphagous with thousands of host plants. However, different M. incognita isolates present distinct and overlapping patterns of host compatibilities. Historically, four “host races” had been defined as a function of ranges of compatible and incompatible plants. In this study, we used population genomics to assess whether (a) reproduction is actually clonal in this species, (b) the host races follow an underlying phylogenetic signal or, rather represent multiple independent transitions, and (c) how genome variations associate with other important biological traits such as the affected crops and geographical distribution. We sequenced the genomes of 11 M. incognita isolates across Brazil that covered the four host races in replicates. By aligning the genomic reads of these isolates to the M. incognita reference genome assembly, we identified point variations. Analysis of linkage disequilibrium and 4‐gametes test showed no evidence for recombination, corroborating the clonal reproduction of M. incognita. The few point variations between the isolates showed no significant association with the host races, the geographical origin of the samples, or the crop on which they have been collected. Addition of isolates from other locations around the world confirmed this lack of underlying phylogenetic signal. This suggests multiple gains and losses of parasitic abilities and adaptations to different environments account for the broad host spectrum and wide geographical distribution of M. incognita and thus to its high economic impact. This surprising adaptability without sex poses both evolutionary and agro‐economic challenges.

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in C hile. In B razil, M . ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M . ethiopica isolates from B razil and C hile using AFLP and RAPD markers and to develop a species‐specific SCAR ‐ PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed M eloidogyne species and one isolate of M . ethiopica from K enya were included in the analysis. The results showed a low level of diversity among the M . ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of M eloidogyne sp. showed a high homogeneity and clustered separately from M . ethiopica (100% bootstrap). RAPD screenings of M . ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M . ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.

Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.