The seed and its microbial community
In a ripe fruit, a seed remains associated with the microorganisms surrounding it. Some occupy the surface of the seed coat, while others occur in internal tissues. In research, the community of these microorganisms and their genetic material is called the seed microbiome. Their distribution matters because the two areas differ in access to water, nutrients and environmental microorganisms. Available surfaces also change at germination, and the radicle begins growing beyond the seed's protective covering. [Mascot-Gómez et al. 2021, p. 2]
Mascot-Gómez, Flores and López-Lozano studied Echinocactus platyacanthus, Ferocactus latispinus, F. pilosus and Stenocereus queretaroensis. Ripe, closed fruits of the first three species came from Guadalcázar, and those of the fourth from Sierra de Álvarez, in San Luis Potosí state. Collection took place during the rainy season in 2018. At least ten plants were included for each species. After removal, the seeds were pooled into a single sample per species, allowing a collective description of their microorganisms. [Mascot-Gómez et al. 2021, p. 2]
Separating the surface from the interior
Surface microorganisms were recovered by slowly mixing seeds in a buffer solution with a small amount of detergent. The resulting liquid contained cells detached from the surface. To investigate the interior, another sample underwent a series of washes, including ethanol and sodium hypochlorite, after which the seeds were ground. The absence of microbial growth on a medium after washed seeds were placed on it, together with scanning microscopy images, was used to check the effectiveness of surface-cell removal. [Mascot-Gómez et al. 2021, pp. 2, 8]
The authors called the surface communities episeminal and the internal communities endoseminal. This is an operational division defined by the sampling method. It shows where material was recovered and facilitates comparisons between groups. Microorganisms can, however, move during plant development, so each sample represents the state at the time of preparation. Reconstructing microbiome origins requires observations of successive stages, from flowers and fruits to the seedling. [Mascot-Gómez et al. 2021, pp. 2, 8]
Two ways of detecting life
In the first approach, microorganisms were grown on culture media. Sixty bacterial isolates were obtained from four species. Culturing allowed their properties to be tested later. In the second approach, DNA was isolated and markers were sequenced, the bacterial 16S rRNA gene and the fungal ITS region. Comparing these sequences with reference databases allowed reads to be assigned to taxonomic groups. The analysis included reagent controls and a standard microbial mixture. [Mascot-Gómez et al. 2021, pp. 3–5]
Culturing describes organisms that grow on the media and under the conditions used. DNA analysis reveals a broader set of sequences, including those of microorganisms that could not be cultured. No fungal isolates were obtained from either of the Ferocactus species studied, although their markers occurred in the samples. The relative proportion of reads in DNA analysis describes the structure of the sequence library studied. Living cell numbers and their current activity need to be determined through separate measurements. [Mascot-Gómez et al. 2021, pp. 3–5]
Four species, different communities
In E. platyacanthus, more than 90% of bacterial reads from both the seed interior and surface were assigned to Stenotrophomonas. In F. latispinus, this genus predominated on the surface, while Kluyvera dominated the interior. In F. pilosus, the interior was associated mainly with Stenotrophomonas and the surface with Paenibacillus. Seeds of S. queretaroensis had a more diverse bacterial community. In the fungal data, differences between the surface and interior were particularly clear. [Mascot-Gómez et al. 2021, pp. 4–6]
Culturable bacteria were tested for production of indole compounds and siderophores, chitin degradation capacity and nitrogenase activity. Siderophores bind iron, while nitrogenase activity indicates the capacity to fix atmospheric nitrogen. Every isolate showed at least one measured trait associated with supporting plants. These tests establish a strain's potential under laboratory conditions. Its effect on a particular seedling depends on whether the bacterium colonises it and maintains the activity studied in its environment. [Mascot-Gómez et al. 2021, pp. 4–6]
What changed after the surface community was removed
The functional part was conducted only in E. platyacanthus. Seeds retaining their natural surface were compared with washed seeds and five treatments involving reinoculation of washed seeds. They were inoculated with a mixture recovered from the surface or with individual isolates of Bacillus, Burkholderia, Enterobacter and Aspergillus. Each treatment comprised five dishes of fifteen seeds. Germination and visible fungal hyphae were observed for thirty days on agar at 25°C. [Mascot-Gómez et al. 2021, pp. 3–4, 6–9]
Seeds retaining their natural surface germinated significantly more often than washed seeds. No reinoculation treatment restored germination to their level. Washing removed both microorganisms and part of the mucilage layer. The authors identified this change in the covering as a possible explanation for the result. Separating the effect of mucilage from that of microorganisms requires an experiment in which each feature is changed independently. [Mascot-Gómez et al. 2021, pp. 3–4, 6–9]
Fungal colonisation and strain dependence
Washed seeds were more often covered with hyphae of a fungus also isolated from their interior and identified as closely related to Aspergillus welwitschiae. Inoculation with Enterobacter, the surface isolate of Aspergillus or the recovered microbial mixture reduced colonisation frequency compared with washed seeds. This effect concerned the presence of hyphae on the surface. Treatments with Bacillus and Burkholderia did not produce a significant reduction in colonisation in this comparison. [Mascot-Gómez et al. 2021, pp. 6–9]
Two isolates assigned to the same fungal genus could therefore participate differently in the same system. The assigned genus name helps establish relatedness, while an experiment with a specific strain determines its effect. Some fungal species-level identifications in this paper remained preliminary, based on similarity in one DNA region. The observed decrease in colonisation allows antagonistic interactions between microorganisms to be considered. The precise inhibitory mechanism was not established. [Mascot-Gómez et al. 2021, pp. 6–9]
From a seed sample to species biology
Pooling seeds from many plants into one sample limits the ability to determine variation between maternal plants. The data describe communities associated with the populations studied, the collection season and material preparation. Establishing a repeatable species microbiome requires independent populations and further seasons. The functional study of E. platyacanthus also shows that the seed covering, microorganisms and germination together form a system whose components need to be separated through measurement. [Mascot-Gómez et al. 2021, pp. 8–9]
For sowing studies, the practical consequence is a precise description of seed preparation. Washing, disinfection and mucilage removal change the starting material, and their effects can be assessed through parallel control trials. The article provides a specific example of this relationship in E. platyacanthus. The microbial composition of F. latispinus and F. pilosus is also presented in these plants' profiles, retaining the separate scope of the results. [Mascot-Gómez et al. 2021, pp. 8–9]
Based on the full text of the paper. Sequence detection, isolate culture and the seed reinoculation experiment are described as separate levels of investigation.
The seed-associated microbiome of four cactus species from Southern Chihuahuan Desert
Mascot-Gómez E., Flores J., López-Lozano N. E., The seed-associated microbiome of four cactus species from Southern Chihuahuan Desert, Journal of Arid Environments, 2021, 190:104531, doi: 10.1016/j.jaridenv.2021.104531
A comparison of bacteria and fungi on the surface and inside the seeds of Echinocactus platyacanthus, Ferocactus latispinus, F. pilosus and Stenocereus queretaroensis. The study combines DNA marker sequencing with isolate cultivation and assays of functional traits. A separate experiment in E. platyacanthus assesses germination and fungal colonisation after seed washing and reinoculation with selected microorganisms.