Endophytic microbiome variation among single plant seeds

Like other plant compartments, the seed harbors a microbiome. Seed microbiome members are the first to colonize a germinating seedling, and they may initiate the trajectory of microbiome assembly for the next plant generation. Therefore, the members of the seed microbiome are important for the dynamics of plant microbiome assembly and the vertical transmission of potentially beneficial symbionts. However, it remains challenging to assess the microbiome at the individual seed level (and, therefore, for the future individual plants) due to low endophytic microbial biomass, seed exudates that can select for particular members, and high plant and plastid contamination of resulting reads. Here, we report a protocol for extracting microbial DNA from an individual seed (common bean, Phaseolus vulgaris L.) with minimal disruption of host tissue, which we expect to be generalizable to other medium- and large-seed plant species. We applied this protocol to determine the 16S rRNA V4 and rRNA ITS2 amplicon composition and examine the variability of individual seeds harvested from replicate common bean plants grown under standard, controlled conditions to maintain health. Using DNA extractions from individual seeds, we compared seed-to-seed, pod-to-pod, and plant-to-plant microbiomes, and found highest microbiome variability at the plant level. This suggests that several seeds from the same plant could be pooled for microbiome assessment, given experimental designs that apply treatments at the parent plant level. This study adds protocols and insights to the growing toolkit of approaches to understand the plant-microbiome engagements that support the health of agricultural and environmental ecosystems.

Endophytic microbiome variation at the level of a single plant seed

Like other plant compartments, the seed harbors a microbiome. The members of the seed microbiome are the first to colonize a germinating seedling, and they initiate the trajectory of microbiome assembly for the next plant generation. Therefore, the members of the seed microbiome are important for the dynamics of plant microbiome assembly and the vertical transmission of potentially beneficial symbionts. However, it remains challenging to assess the microbiome at the individual seed level (and, therefore, for the future individual plant) due to low endophytic microbial biomass, seed exudates that can select for particular members, and high plant and plastid contamination of resulting reads. Here, we report a protocol for extracting metagenomic DNA from an individual seed (common bean, Phaseolus vulgaris L.) with minimal disruption of host tissue, which we expect to be generalizable to other medium-and large-seed plant species. We applied this protocol to quantify the 16S rRNA V4 and ITS2 amplicon composition and variability for individual seeds harvested from replicate common bean plants grown under standard, controlled conditions to maintain health. Using metagenomic DNA extractions from individual seeds, we compared seed-to-seed, pod-to-pod, and plant-to-plant microbiomes, and found highest microbiome variability at the plant level. This suggests that several seeds from the same plant could be pooled for microbiome assessment, given experimental designs that apply treatments at the maternal plant level. This study adds protocols and insights to the growing toolkit of approaches to understand the plant-microbiome engagements that support the health of agricultural and environmental ecosystems.

Distribution of Xanthomonas hortorum pv. carotae Populations in Naturally Infested Carrot Seed Lots

Bacterial blight of carrot (Daucus carota subsp. sativus), caused by the plant-pathogenic bacterium Xanthomonas hortorum pv. carotae, is a common seedborne disease of carrot wherever the crop is grown. Carrot seed lots were evaluated to determine the variability and distribution of populations of X. hortorum pv. carotae among individual carrot seeds. Twenty-four carrot seed lots harvested between 2014 and 2016 were subjected to a bulk seed wash dilution-plate assay to obtain mean X. hortorum pv. carotae levels. Mean infestation levels resulting from the bulk seed wash assays among the 24 seed lots ranged from 1.2 × 107 and 9.6 × 108 CFU/g seed and averaged 3.6 × 108 CFU/g seed. Individual seeds from the same 24 lots were also tested with a scaled-down wash assay of individual seeds. Among the 1,380 seeds that were individually assayed, 475 X. hortorum pv. carotae-positive seeds were detected (34.4%). Rates of X. hortorum pv. carotae detection on individual seed in seed lots ranged from 0% (not detected) to 97.9%, and the mean and median X. hortorum pv. carotae population on an individual seed was 8.3 × 104 and 6.3 × 101 CFU/seed, respectively. Among individual seeds, X. hortorum pv. carotae populations ranged from 2 (the limit of detection of the assay) to 3.6 × 107 CFU/seed. CFU data for 23 of the 24 seed lots were nonnormal and the Log-Logistic (3P) distribution best described populations of X. hortorum pv. carotae recovered from individual carrot seeds. The influence and impact of nonnormal distributions of X. hortorum pv. carotae in commercial carrot seed lots on seed health tests, seedborne transmission, and bacterial blight epidemiology requires further study.

Quantitative Real-Time PCR Analysis of Individual Flue-Cured Tobacco Seeds and Seedlings Reveals Seed Transmission of Tobacco Mosaic Virus

Tobacco mosaic virus (TMV) is an extensively studied RNA virus known to infect tobacco (Nicotiana tabacum) and other solanaceous crops. TMV has been classified as a seedborne virus in tobacco, with infection of developing seedlings thought to occur from contact with the TMV-infected seed coat. The mechanism of TMV transmission through seed was studied in seed of the K 326 cultivar of flue-cured tobacco. Cross pollinations were performed to determine the effect of parental tissue on TMV infection in seed. Dissection of individual tobacco seeds into seed coat, endosperm, and embryo was performed to determine TMV location within a seed, while germination tests and separation of the developing seedling into seed coat, roots, and cotyledons were conducted to estimate the percent transmission of TMV. A reverse-transcriptase quantitative PCR (RT-qPCR) assay was developed and used to determine TMV concentrations in individual seed harvested from pods that formed on plants from TMV-infected and noninfected crosses. The results showed maternal transmission of TMV to tobacco seed and seedlings that developed from infected seed, not paternal transmission. RT-qPCR and endpoint PCR assays were also conducted on the separated seed coat, endosperm, and embryo of individual seed and separated cotyledons, roots, and seed coats of individual seedlings that developed from infected tobacco seed to identify the location of the virus in the seed and the subsequent path the virus takes to infect the developing seedling. RT-qPCR and endpoint PCR assay results showed evidence of TMV infection in the endosperm and embryo, as well as in the developing seedling roots and cotyledons within 10 days of initiating seed germination. To our knowledge, this is the first report of TMV being detected in embryos of tobacco seed, demonstrating that TMV is seedborne and seed-transmitted in flue-cured tobacco.

Manifestation of positive transgressive variability by the quantitative characteristics of productivity in hybrids f2 of common beans

The analysis of hybrid generations F2 by the frequency and degree of positive transgressions was carried out. The frequency of transgressive forms was significantly higher than the degree of positive transgression. By the indicators of individual seed productivity, the highest frequency and degree of transgression was observed in the hybrid combination Prysadybna/Galaxy.

Starting a conservation collection of Sorbus pseudomeinichii

There are three endemic Sorbus species on the Isle of Arran in Scotland: Sorbus arranensis, S. pseudofennica and S. pseudomeinichii. The latter is the most recently discovered and is represented in the wild by a single individual. Seed propagation and vegetative propagation by chip budding were initiated for all three species at the Royal Botanic Garden Edinburgh (RBGE). This has been successful and there are now conservation collections of these species growing at RBGE.