Evaluation of Ochrobactrum sp.as a potential bioherbicide for

angled onion (Allium triquetrum L.) in laboratory conditions

Presenter: Parsa Tehranchian

Senior Supervisor: Prof. Ann Lawrie

Second Supervisor: Dr. Robin Adair (DPI, Victoria)

School of Applied Sciences

1. General informationAngled onion• Perennial herb

• Propagated by bulbs, bulblets and seeds

• Cluster of flowers on the triangular stem

Life Cycle• Chasmogamous flowers

• Perennates by bulbs

• Grows May-November

• Dies back in summer

Habitat• Wet areas

Origin• Mediterranean region

Distribution• in Australia

Problems of Angled onion• Reduction of biodiversity in native vegetation (DPI 2008)

• Rapid growth in shaded areas (Parsons & Cuthbertson 1992)

• Allelopathic exudates (DPI 2008)

• Alteration of milk and meat taste (Parsons & Cuthbertson 1992)

• High rate of reproduction (Blood 2001)

• Difficult to manage by herbicides or manual removal

• No current selective control method

Area infested by A. triquetrum near Kinglake National Park, Melbourne, VIC.

Area infested by A. triquetrum, Bendigo Creek, VIC.

2. Ochrobactrum- biological control agent?

• The shrunken bulb was surface sterilized

• Bulbs were placed on water agar and incubated at room temperature for a week.

• The bacterium was isolated by plating the rotted bulbs on nutrient agar (NA) and trypticase soy agar (TSA) (selective for aerobic Gram-negative bacteria).

TSANA

Isolation of Ochrobactrum sp. from rotted Angled onion bulbs

Surface sterilized bulb was incubated on water agar at 25 ºC

Bacterial identification•Bacterium isolated and identified as Ochrobactrum aff. anthropi

•Physiological testing

•Classical physiological and diagnostic tests were performed using API20NE strips.

•Sequencing

•The 16S region of the bacterial genome was amplified usinguniversal FD1 and RP2 primers.

•Sequencing was performed using ABI Big Dye protocol.

Api20 NE

Pathogenicity testing

• Plant regeneration by micropropagation– A. triquetrum (Angled onion)– Other cultivated Allium species

• Pathogenicity testing of Ochrobactrumsp.

– 6 plant replicates– Fresh broth LB culture of Ochrobactrum sp.– No. of bacterial cells adjusted to 107/mL saline– A. triquetrum on water agar in test tubes

inoculated with 107 bacteria– Scoring after 30 days

Pathogenicity testing of Ochrobactrum sp. on A. triquetrumResultsControl plants Infected plants

Pathogenicity of Ochrobactrum sp. on A. triquetrum provenances from across Australia

0

1

2

3

4

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7

8

Belai

r Nati

onal

Park

(SA)

Horsn

ell gu

lly (S

A)

Gardine

r's C

reek

(VIC

)W

aterfa

ll Gull

y (SA

)

Mylor (

SA)

Arar

at (V

IC)

Yarra

Ben

d Par

k (VIC

)Ka

ngaro

o Fla

t (VI

C)Dan

deno

ng (V

IC)

Won

thag

gi (V

IC)

Bend

igo C

reek

(VIC

)

Provenances

Path

ogen

icity

and

viru

lenc

e le

vel

UninfectedInfected

Pathogenicity testing of Ochrobactrum sp. on cultivated AlliumResultsInfected LeekControl plants

Pathogenicity of Ochrobactrum sp. on cultivated Allium seedlings in test tubes

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8

River Brown Pickling Onion Red Rossa Chives Spring onion(White Lisbon)

Leek

Cultivated species

Path

ogen

icity

and

viru

lenc

e le

vel

UninfectedInfected

Wax embedded bulbs also were sectioned and Gram-stained to check for the bacterium in infected cells

Histology and Bacterial re-isolation from infected plants

Bacterial re-isolation

Infected cells with bacteria Control treatment

Bacterial cells inside the plants cells

Conclusion

• Ochrobactrum sp. was pathogenic and virulent on A. triquetrum and cultivated Allium species

• Is it a potential biocontrol agent for A. triquetrum?

• Differences in pathogenicity with provenance

• Does it have the same function in vivo?

• Is it highly virulent to cultivated Allium species?

• Can be used along with S. cepivora?

•Tehranchian, P., Lawrie A.C. and Adair, R. (2010). In vitro assessment of Stromatinia cepivora as a potential biological control agent for angled onion (Allium triquetrum) in Victoria, Australia. Papers and Proceedings of the 17th Australasian Weeds Conference, ed. S.M. Zydenbos. (New Zealand Plant Protection Society) ISBN 978-0-86476-239-9.

3. Interaction of Ochrobactrum sp. and S. cepivora on A. triquetrum provenances in Victoria

•Samples of each provenance were inoculated with bacterium, fungus or both in test tubes.

Bacterium:A. triquetrum in water agar in test tubes was inoculated by injection of 1 ml (107

CFU) of bacterial suspension.

Fungus:7-9 plant replicates (uniform size for each provenance)S. cepivora (VPRI12439a) (supplied by VPRI Herbarium - DPI Knoxfield).Sclerotia were harvested from cultures on PDA (potato dextrose agar) platesSix sclerotia (4 x 4 mm) were placed on the collar region of each plant in its individual test-tube 24 hours later.

Infection:Scored at 4 weeks.Sclerotia of S. cepivora did not germinate in tubes with Ochrobactrum sp.

Interaction of Ochrobactrum sp. and S. cepivora on A. triquetrum provenances from Victoria (Scored by 4 weeks)

Results

Interaction of Ochrobactrum sp. and S. cepivora on A. triquetrum provenances from Victoria in test tubes

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Yellow Gum Park Yarra Bend Park Dandenongs Wonthaggi Gardiners CreekProvenances

Path

ogen

icity

and

viru

lenc

e le

vel

UninfectedInfected by the fungusInfected by the bacteriumInteraction (plants dead by the bacterium, no sclerotial germination)

Conclusions• Both biocontrol agents are pathogenic to A.

triquetrum separately in test tube experiments.

• Can we apply both pathogens together as bioherbicides?

• Interaction result:– Does the bacterium inhibit sclerotial germination?– The bacterium is potentially an effective and

suitable biocontrol agent for A. triquetrum in wet areas (where S. cepivorum does not germinate).

– If the bacterium is not pathogenic to Alliumspecies in vivo, is it a potential biocontrol agent for Allium white rot disease?

• Future work– pot trials– Host specificity testing

References• Blood, K. (2001). Environmental Weeds: A Field Guide for SE Australia.

Chjerram & Associates, Melbourne.

• Department of Primary Industries, Impact Assessment - Angled Onion (Allium triquetrum) in Victoria. Revision date 2008 (Access date 9/9/08).

http://www.dpi.vic.gov.au/dpi/vro/vrosite.nsf/pages/impact_angled_onion

• Parsons, W.T. & Cuthbertson, E.G. (1992). Noxious Weeds of Australia. Inkata Press, Melbourne. Sydney.

• Tehranchian, P., Lawrie A.C. and Adair, R. (2010). In vitro assessment of Stromatinia cepivora as a potential biological control agent for angled onion (Allium triquetrum) in Victoria, Australia. Papers and Proceedings of the 17th Australasian Weeds Conference, ed. S.M. Zydenbos. (New Zealand Plant Protection Society) ISBN 978-0-86476-239-9.

Presenter: Parsa Tehranchian 1*

Senior Supervisor: Prof. Ann Lawrie 1

Second Supervisor: Dr. Robin Adair 2

(Department of Primary Industries)