Download Characterization of Seasonal Diversity of the Spinach (Spinacea

Comparison of the Bacterial Community Naturally Occurring
on Spinach Seeds and Seedlings
Phyllis
1,2
Carder ,
Gabriela
1
Lopez-Velasco ,
Monica
1 Departments
1
Ponder
and Gregory
2
Welbaum
2Horticulture
of Food Science and Technology and
Virginia Polytechnic Institute and State University - Blacksburg, VA
Introduction
Seeds 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.
Results: Community analysis
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
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.
Harvested spinach
Rinsing with sterile water to remove soil
Menorca full-savoy cultivar
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.
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
6.40
Discussion
6.20
6.00
5.80
Incubation period of 10 days
at 25°C
5.60
5.40
DNA isolation
(Zymo soil microbe DNA isolation kit)
5.20
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
5.00
4.80
seed
cotyledon
mature
PCR amplification of 16S rDNA
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.
Using universal primers
341-GC forward and 907r reverse
Conclusions
DGGE enables assessment of microbial populations that are not culturable.
Gel analysis
Microbial diversity increases with plant development as new microbes populate seedlings after germination.