isolation of myrosinase producing microbes from brassica cultivated soils
TRANSCRIPT
The role of myrosinase producing microbes in the soil is poorly understood. It is possible that microbial activity in the soil can improve the effects of biofumigation and aid the suppression of
certain pests such as Potato Cyst Nematodes. Recent descriptions of a soil-dwelling Citrobacter strain that produces myrosinase suggest that many other microbial taxa produce myrosinase
but have not yet been characterised (Albaser et al. 2016). Therefore soil is a suitable environment for bioprospecting of myrosinase producing microbes. This study describes the isolation of
microbes able to degrade sinigrin and their association with 4 different Brassica cultivars.
Microbes were isolated on sinigrin-barium plates and minimal sinigrin broth from soil sites previously used to grow Brassicas .
Morphologically distinct colonies were cultured to form pure isolates and examined for their myrosinase activity using a spectrophotometric assay similar to that described by Palmieri et al.
(1982).
Identification using ITS (fungi) and 16S (bacteria) rRNA genes. Sequences were analysed for closest matches using BLAST.
The highest myrosinase producers were assessed for their ability to survive on brassica roots and their effects on germination of these host plants.
Real-time PCR assays were designed for the highest myrosinase producers to track their presence in environmental samples e.g. soil or plant material.
0 10 20 30 40 50 60 70 80 90 100
Turnip
Swede
Turnip rape
Broccoli
% of seedlings +ve for E7 or E10
Recovery of Cryptococcus from roots of Brassica seedlings
Cryptococcus e10
Cryptococcus e7
Plant E7
germination%
E10
germination%
Control
%
Broccoli (cv. Purple sprouting
late)
94.67 88.67 88
Turnip (cv. Massif) 100 100 99
Turnip rape (cv. Clio) 100 100 97
Swede (cv. Marian) 93.33 93.33 90
The highest myrosinase producing microbes were yeasts (Cryptococcus strains E7 and
E10*) that were able to degrade sinigrin both in the presence and absence of glucose,
(Cryptococcus strains E7 and E10). This is the first account of members of this genus
being able to produce myrosinase. Only one other budding yeast species, Geotrichum
candida, has been implicated in the breakdown of glucosinolates. G. candida was
suggested as an agent for improving the nutritional value of rapeseed meal and oil (Staron
1975). Interestingly, other fungi that were observed to produce myrosinase were from
known pathogenic genera such as Fusarium, Verticillium and Phoma.
Alabaser et al., 2016 Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) Beta-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil, Journal of Agricultural and Food Chemistry, DOI: 10.1021/acs.jafc.5b05381
Palmieri et al., 1982 A Steady-State Kinetics Study of Myrosinase with Direct Ultraviolet Spectrophotometric Assay, Analytical Biochemistry, 123: 320-324
Staron, 1975 Obtention des proteines a partir des graines oleagineuses par des methodes microbiologiques, Revue Francais des CORPS GRAS, 22: 579-589
Germination of 4 Brassica cultivars treated with
Cryptococcus E7 and E10 inoculum.
Cryptococcus E7 and E10 morphology on PDA plates
In all 4 cultivars germination was not negatively affected
by the presence of E7 or E10.
E7 and E10 were found to grow in association with roots
and viable cells were recovered from seedling roots by
culturing on PDA plates. Presence of E7 and E10 on
roots was confirmed using real-time PCR assays.
E7 and E10 were able to degrade sinigrin at a range of pHs and temperatures that are
likely to cover most soil and climatic conditions. In the presence of an alternative carbon
source (Glucose = G) sinigrin was still degraded suggesting that myrosinase is constantly
being produced by these microbes, even when environmental conditions are
unfavourable.
Pot trials with E7*, E10* and four other myrosinase
producing microbes have been set up to examine the
ability of these microbes to improve biofumigation. In
particular, suppression of PCN.
Right: Brassica plants treated with myrosinase
producing microbes from pot trials after 2 months,
before retrieval of PCN cysts
Results and Discussion
Methods
Jason C. SumnerA ; Victoria TaylorB ; Jan RogersB ; Paul ColemanC ; Anthony BarkerD ; David KenyonA
SASA, Roddinglaw Rd, Edinburgh, EH12 9FJA; ARCIS Biotechnology LtdB; Greenvale APC; Barworth AgricultureD
Further work
*Patent application number GB1612424.0