Free nitrogen – it’s yours for the taking

Dr Sofie De Meyer

MALDIID

Center for Rhizobium studies

Murdoch University

How can you maximise Nitrogen fixation?

• Understand the problem 1. How do the roots look like?

2. Which rhizobia are in the nodules?

3. What does the soil look like?

4. Which herbicides are used?

• Solutions: 1. Nodule scoring system

2. Rhizobial ID using MALDIID

3. Re-inoculation

4. Soil analysis

5. Pro-legume management

Check roots

Healthy? Not Healthy?

Nodule scoring

1 - 3

+

Correct inoculant

No inoculant

Soil analysis

• pH • Phosphorus • Molybdenum • Aluminium

Herbicides

NO SU’s

Inoculate: - Drill in - Re-sowing - Top dress

4 - 8

• Change species • Plant disease control

Summary

Correct inoculant

No inoculant

Check roots Nodule scoring +

Healthy? Not Healthy? 1 - 3 4 - 8

• pH • Phosphorus • Molybdenum • Aluminium

NO SU’s

Inoculate: - Drill in - Re-sowing - Top dress

• Change species • Plant disease control

Soil analysis

Herbicides

Source: Ron Yates, DAFWA

Healthy root systems?

Source: Yates, R.J., Abaidoo, R., and Howieson, J. 2016. Field experiments with rhizobia. Pages 145-166 in: Working with rhizobia, J. Howieson and M. Dilworth, eds. Australian Centre for International Agricultural Research, Canberra.

Nodule scoring system

Breaking down Effective Ineffective

Source: Ron Yates, DAFWA

Good looking nodules?

Correct inoculant

No inoculant

Healthy? Not Healthy? 1 - 3 4 - 8

Check roots Nodule scoring +

• pH • Phosphorus • Molybdenum • Aluminium

NO SU’s

Inoculate: - Drill in - Re-sowing - Top dress

• Change species • Plant disease control

Soil analysis

Herbicides

Mass-to-charge ratio

Rel

ativ

e in

ten

sity

(%)

MALDI-TOF spectrum

ID

Nodule … suspension … 3x wash … spotting … spectra acquisition … ID within minutes

400μl H2O

Centrifuge

+ Wash

Result Bacterial fingerprint unique for each rhizobia

Check roots

Healthy? Not Healthy?

Nodule scoring

1 - 3

+

4 - 8

Correct inoculant

No inoculant

Soil analysis

• pH • Phosphorus • Molybdenum • Aluminium

NO SU’s

Inoculate: - Drill in - Re-sowing - Top dress

• Change species • Plant disease control

Herbicides

Soil chemistry is crucial to success!

1. pH • Suppressed rhizobia activity at pH (CaCl)

Soil chemistry is crucial to success!

1. pH

Source: Drew et al. 2012

Effects of aluminium

Figure 1: Healthy root tip (left) compared to a deformed root tip affected by aluminium toxicity (right). Photos: CSIRO

Figure 2: Wheat seedlings grown in soil with a range of aluminium concentrations demonstrate restricted root growth at high aluminium concentrations. Photo: S Carr

https://www.agric.wa.gov.au/soil-acidity/effects-soil-acidity

Soil chemistry is crucial to success!

2. Molybdenum • Required for nitrogen fixation by rhizobia • Nitrogenase enzyme contains Mo • Less available if low pH

N2 + 8H+ + 8e- + 16ATP 2NH3 + H2 + 16ADP + 16Pi

Nitrogen fixation reaction

• pH • Phosphorus • Molybdenum • Aluminium

Inoculate: - Drill in - Re-sowing - Top dress

• Change species • Plant disease control

Check roots

Healthy? Not Healthy?

Nodule scoring

1 - 3

+

Correct inoculant

No inoculant

4 - 8

Soil analysis

Herbicides

NO SU’s

Different types of herbicides

Group Scientific name Trade name Breakdown inhibitor

B – SU’s Triasulfuron Logran High pH, drought

Chlorsulfuron Glean High pH, drought

Metsulfuron-methyl Ally High pH, drought

B – IMI’s Imazethapyr Spinnaker Low pH, drought

Imazamox Raptor Low pH, drought

D Propyzamide Kerb Stubble, drought

I Clopyralid Lontrel Heavy soil, stubble, drought

K Pyroxasulfone Sakura Drought

Understanding residual danger of herbicides to legumes – be aware of plant back

0

20

40

60

80

100

120

140

1/10 1/100 1/1000 1/10000 1/100000 No SU

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Root length (mm)

Root DW (g)

Logran - Triasulfuron Source: Ron Yates, DAFWA

Triasulfuron application on Sub clover (18days of growth)

3g 0.3g 0.03g 0.003g 0.0003g control

1/10 1/100 1/1000 1/10,000 1/100,000 Source: Ron Yates, DAFWA

Butcher, Brookton study – field history Nodule occupancy of commercial inoculant under herbicide pressure • Previous oat crop, no previous usage of commercial inoculant • Trifolium spumosum cv. Bartolo inoculated with WSM1325 • 9 treatments:

• Spinnaker (Group B, Imazethapyr) – February and April • Kerb (Group D, Propyzamide) – February and April • Spinnaker + Kerb – February and April • Spinnaker post emergence • Control

Brookton herbicide study

• Reduced TDW • Reduced PRL • Increased # nodules

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

Control Kerb April Spin April Spin May

Total dry weights (g/plant) June 2014 - Trifolium spumosum cv. Bartolo (Brookton, WA)

Spinnaker post emergence Control Spinnaker April Kerb April

Brookton herbicide study

Residual herbicides influence nodule occupancy and

subsequent plant performance

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Control Kerb April Spin April Spin PE

Nodule ID

ID suboptimal

ID com. Inoc.

100% 80% 65% 50%

Herbicides take home message!

• Herbicides can take up to 18 months to break down depending on rainfall and climate conditions

• Not all herbicides have been tested and their effects might unknown but damaging

• We know that SU’s like Logran and Clopyralid herbicides like Lontral cause significant damage to legumes

• Spinnaker post emergence spray influences rhizobia nodule occupancy

• pH • Phosphorus • Molybdenum • Aluminium

NO SU’s

Check roots

Healthy? Not Healthy?

Nodule scoring

1 - 3

+

Correct inoculant

No inoculant

4 - 8

Soil analysis

Herbicides

Inoculate: - Drill in - Re-sowing - Top dress

• Change species • Plant disease control

Get the right group for the right plant

• Group AL for Strand Medic and Lucerne

• Group AM for Burr Medic and Barrel medic

• Group BS for Biserrula

• Group C for annual clover

• Group F for Vetch and Pea

• Group G/S for Serradella and Lupin

• Group N for Chickpea

Get the right group for the right plant

Source: Ron Yates, DAFWA

Biserrula ALOSCA mixed 10kg/ha with fertiliser No inoculant

Beckom NSW - Biserrula sown dry 2014

Different types of application

• Reliable • Dry and wet soil • Easy storage • More expensive (10 - 15$

/ha) • Less user friendly for

mixtures

Peat inoculation • Cheap (4 - 6$/ha) • Reliable • Store in fridge • Wet soils • Quick usage after

opening

Pre-coated seed • Easy use • Added fungicides • Expensive (20 –

30$/ha) • Variable results with

clover • Short shelf life

Granular inoculant

Rhizobia persistence in soil

Source: Drew et al. 2012

Check roots

Healthy? Not Healthy?

Nodule scoring

1 - 3

+

Correct inoculant

No inoculant

Soil analysis

• pH • Phosphorus • Molybdenum • Aluminium

Herbicides

NO SU’s

Inoculate: - Drill in - Re-sowing - Top dress

4 - 8

• Change species • Plant disease control

Summary

Special thanks to:

Dominik Ziegler Valentin Pflüger Guido Vogel

Ron Yates Angelo Loi

Regina Carr Tom Edwards John Howieson Brad Nutt

Belinda Hackney Janelle Jenkins Jo Powells Clare Edwards

Sofie De Meyer Daniel Kollehn

Chris Poole Floyd Sullivan