Local Network Properties of Soil and Rhizosphere Microbial Communities in Potato Plantations Treated with a Biological Product Are Important Predictors of Crop Yield.

Understanding the effectiveness and potential mechanism of action of agricultural biological products under different soil profiles and crops will allow more precise product recommendations based on local conditions and will ultimately result in increased crop yield. This study aimed to use bulk soil and rhizosphere microbial composition and structure to evaluate the potential effect of a Bacillus amyloliquefaciens inoculant (strain QST713) on potatoes and to explore its relationship with crop yield. We implemented next-generation sequencing (NGS) and bioinformatics approaches to assess the bacterial and fungal biodiversity in 185 soil samples, distributed over four different time points-from planting to harvest-from three different geographical locations in the United States. In addition to location and sampling time (which includes the difference between bulk soil and rhizosphere) as the main variables defining the microbiome composition, the microbial inoculant applied as a treatment also had a small but significant effect in fungal communities and a marginally significant effect in bacterial communities. However, treatment preserved the native communities without causing a detectable long-lasting effect on the alpha- and beta-diversity patterns after harvest. Using information about the application of the microbial inoculant and considering microbiome composition and structure data, we were able to train a Random Forest model to estimate if a bulk soil or rhizosphere sample came from a low- or high-yield block with relatively high accuracy (84.6%), concluding that the structure of fungal communities gives us more information as an estimator of potato yield than the structure of bacterial communities. IMPORTANCE Our results reinforce the notion that each cultivar on each location recruits a unique microbial community and that these communities are modulated by the vegetative growth stage of the plant. Moreover, inoculation of a Bacillus amyloliquefaciens strain QST713-based product on potatoes also changed the abundance of specific taxonomic groups and the structure of local networks in those locations where the product caused an increase in the yield. The data obtained, from in-field assays, allowed training a predictive model to estimate the yield of a certain block, identifying microbiome variables-especially those related to microbial community structure-even with a higher predictive power than the geographical location of the block (that is, the principal determinant of microbial beta-diversity). The methods described here can be replicated to fit new models in any other crop and to evaluate the effect of any agricultural input in the composition and structure of the soil microbiome.

Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions.

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.

Segregation and mapping in the root-knot nematode Meloidogyne hapla of quantitatively inherited traits affecting parasitism.

The root-knot nematode Meloidogyne hapla can reproduce on a wide range of crop species but there is variability in host range and pathogenicity both within and between isolates. The inbred strain VW9 causes galling but does not reproduce on Solanum bulbocastanum clone SB22 whereas strain VW8 causes little galling and reproduces poorly on this host. Comparison of reproduction on SB22 of nematode F2 lines generated from hybrids between strains VW8 and VW9 revealed that, whereas over half the lines produced no progeny, some lines reproduced to higher levels than did either parental strain. Using a genetic map previously generated using the same set of F2 lines, three quantitative trait loci (QTLs) were identified and positioned on linkage groups. A combination of two QTL alleles from one parent and one from the other was highly represented in F2 lines that exhibited higher reproduction than either parental strain but was absent from lines that failed to reproduce on SB22. This result suggests that sexual hybridization and assortment of opposing alleles leads to segregation of individuals with improved reproductive ability on a particular host. The genome sequence and integrated genetic and physical linkage map of M. hapla provide resources for identification of genes responsible for the identified QTL.

A sequence-anchored linkage map of the plant-parasitic nematode Meloidogyne hapla reveals exceptionally high genome-wide recombination.

Root-knot nematodes (Meloidogyne spp.) cause major yield losses to many of the world's crops, but efforts to understand how these pests recognize and interact with their hosts have been hampered by a lack of genetic resources. Starting with progeny of a cross between inbred strains (VW8 and VW9) of Meloidogyne hapla that differed in host range and behavioral traits, we exploited the novel, facultative meiotic parthenogenic reproductive mode of this species to produce a genetic linkage map. Molecular markers were derived from SNPs identified between the sequenced and annotated VW9 genome and de novo sequence of VW8. Genotypes were assessed in 183 F2 lines. The colinearity of the genetic and physical maps supported the veracity of both. Analysis of local crossover intervals revealed that the average recombination rate is exceptionally high compared with that in other metazoans. In addition, F2 lines are largely homozygous for markers flanking crossover points, and thus resemble recombinant inbred lines. We suggest that the unusually high recombination rate may be an adaptation to generate within-population genetic diversity in this organism. This work presents the most comprehensive linkage map of a parasitic nematode to date and, together with genomic and transcript sequence resources, empowers M. hapla as a tractable model. Alongside the molecular map, these progeny lines can be used for analyses of genome organization and the inheritance of phenotypic traits that have key functions in modulating parasitism, behavior, and survival and for the eventual identification of the responsible genes.

Root-knot nematodes exhibit strain-specific clumping behavior that is inherited as a simple genetic trait.

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.

Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism.

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 free-living 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.

Meiotic parthenogenesis in a root-knot nematode results in rapid genomic homozygosity.

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.