Designing and validation of specific primers for the quantitative detection of bacteria in sugarcane inoculant.

Endophytic diazotrophic plant growth-promoting bacteria Herbaspirillum rubrisubalbicans (HCC103), Herbaspirillum seropedicae (HRC54), Paraburkholderia tropica (Ppe8T), Gluconacetobacter diazotrophicus (Pal5T), and Nitrospirillum amazonense (CBAmC) have been used as inoculants for sugarcane. The genome sequences of these strains were used to design a set of specific primers for the real-time PCR (qPCR) assay. Primer specificity was confirmed by conventional PCR using the genomic DNAs of 25 related bacterial species and the five target strains. The qPCR assays were conducted using root and shoot samples from two sugarcane varieties (RB867515 and RB92579). These samples were collected both with and without inoculation, using the target strains specified in this study. The sugarcane plants were grown in a greenhouse, utilizing a substrate composed of sterile sand and vermiculite in a 2:1 ratio, for a duration of 55 days. The primers designed for this study successfully amplified target DNA fragments from each of the bacterial species, enabling their differentiation at the species level. The total bacterial population present in the sugarcane quantified using qPCR was on average 105.2 cells g-1 of fresh tissue. Across both evaluated varieties, it was observed that the population of inoculated bacteria tended to decrease over time and became more concentrated in the sugarcane roots compared to the aerial parts. The qPCR results suggest that both the host and the microbes influence the endophytic population and the bacterial number decreases with plant age.

Association of mycoheterotrophic Gentianaceae with specific Glomus lineages.

Some plant species took an alternative evolutionary pathway in which they lost their photosynthetic capacity to depend exclusively on carbon supplied by arbuscular mycorrhizal fungi (AMF) in an association called mycoheterotrophy. Among them is Voyriella parviflora, a species of the family Gentianaceae, which is found in tropical regions such as the Amazon basin. Here, we assessed the identity of AMF symbionts associated with this species. DNA was isolated from eight Gentianaceae specimens and from litter and surrounding roots of photosynthetic plants. The atp1 gene was amplified by Sanger sequencing to determine the taxonomic affiliation of the mycoheterotrophic plants. A 280 bp region of the 18S rRNA gene of AMF was amplified with primers NS31/AML2 by high-throughput sequencing. The mycoheterotrophic specimens were assigned to V. parviflora with a bootstrap support of 72%. Glomus was the most abundant AMF genus, both in the mycoheterotrophic plants and in the litter and roots of photosynthetic plants. In addition, a few Glomus genotypes were abundantly enriched in the mycoheterotrophic plants, with only a few specimens colonized by Gigaspora, Acaulospora, and Scutellospora in a low proportion. These genotypes formed a cluster within a larger clade, suggesting that V. parviflora shows a preferential association with a narrow Glomus lineage which is not phylogenetically close to a previously identified V. parviflora's associated lineage. Furthermore, detecting fungi from other families suggests that V. parviflora is colonized by other genera, although with low frequency. These findings provide new insights into the association between AMF and mycoheterotrophic species and highlight the importance of considering trap culture-independent approaches in understanding this symbiosis.