Comparison of the Bacterial Community Naturally Occurringon Spinach Seeds and Seedlings

Phyllis Carder1,2, Gabriela Lopez-Velasco1, Monica Ponder1 and Gregory Welbaum2 1 Departments of Food Science and Technology and 2Horticulture

Virginia Polytechnic Institute and State University - Blacksburg, VA

Discussion

The microbial community structure of the phyllopshere was most similar between plants at the 2-3 leaf stages of development and the 6-8 leaf stages of development.

A large number of bands representing members of the spinach microbial community on the seed were also present on the phyllosphere of mature plants.

A loss in diversity and richness was seen in cotyledon samples

Differences in microbial populations could be attributed to changes in nutrients available to the bacteria through different stages of plant development and/or exposure to air, soil and water.

Conclusions

DGGE enables assessment of microbial populations that are not culturable.

Microbial diversity increases with plant development as new microbes populate seedlings after germination.

Results: Community analysis

Menorca full-savoy cultivar

Growing conditions:

A semi savoy cultivar of spinach (Menorca, Seedway) were grown in growth chambers in 4 inch pots containing a maximum of 2 plants per pot.  Plants were maintained at 20-15C and 12 h day length.

Cotyledons were harvested 30 days after planting. Three leaf stage plants were harvested 40 days after planting and 6-8 leaf stage plants were harvested 60 days after planting.

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IntroductionSeeds planted for crop production are known to be populated with bacteria, yeast, fungi, and viruses.  Some seed-borne microorganisms are pathogenic, while others are harmless and some actually benefit plants by competing with plant pathogens for space and nutrients.  To date, the role the seed microbiota plays in the establishment of the microbial community on the plant leaves at different stages of plant development is largely unexplored.  The purpose of this study is to examine changes in microbial communities on the phyllosphere of spinach plants from seed to full plant maturity.

As the plant develops, energy sources available for the microbial community will change, therefore impacting the abundance and functions of the microbial members. It is anticipated that members of the seed community will persist throughout development to establish populations on the phyllosphere or rhizosphere. The identities and abundance of these seed-borne microbes which compete against microbes introduced from exposure to the external environment (soil, air, water), are unknown. A better understanding of the successional microbial community can lead to improving plant health by identifying beneficial microbes and Improving the establishment of bio-control strains against human and plant pathogens

Methods

Harvested spinach

Rinsing with sterile water to remove soil

Suspension of 10 g of spinach in 90 mL of peptone water (3g/L and 1% Tween 80), pulsify for 5 minutes to promote

suspension of attached microorganisms

Microbial counts Collection of peptone water

Serially diluted and plated on to minimal media R2A

Incubation period of 10 days at 25°C

DNA isolation

(Zymo soil microbe DNA isolation kit)

PCR amplification of 16S rDNA

Using universal primers

341-GC forward and 907r reverse

Gel analysis

Results: Microbial plate counts

•The culturable populations of bacteria increase in number with the development of the plant. •The largest population was observed on mature leaves.