Trial establishment

Pots of 16 cm diameter were filled with approximately 2 L of soil. The soil was mixed with controlled-release fertiliser in the form of Osmocote (11-4.8-14.9 NPK + trace elements) added to the soil prior to potting at the rate of 7.5 g per pot. Seeds of sunflower (Helianthus annuus L., ‘Dwarf Sunsation’) were germinated in vermiculite. The seedlings were transplanted into pots after emergence of the cotyledons (two weeks old) and were irrigated daily.

Trial design was a randomised complete block design with three factors. The trial had five replicate blocks with each replicate block containing 20 plants, divided into two groups of ten plants each. The first 10 plants (not inoculated) were sub-divided into 2 groups of five plants each for the orchard and the forest soils. The second set of 10 plants were also divided into two groups of five plants each and inoculated with AMF. The plants were then arranged randomly within the blocks and each block placed onto a different bench in the glasshouse. The factors were soil type (forest, orchard), AMF application (plus, minus) and organic supplement type (four types and a control). Details of the organic supplements are provided below and nutrient composition is described in Table 5.2.

 Control: control treatment – no supplements added

 Ferbon® (FF50SB, a lignite-based soil conditioner) at 0.6 g/pot (300 kg/ha), manufactured by: Interstate Energy Group Pty Ltd, Bacchus Marsh Victoria Australia.

 Compost at 1.6 g/pot equivalent to 800 kg/ha, (Foundation aerobic compost, Pure Living Soil Pty Ltd).

 Soluble humate granules (SHG) at 0.044 g/pot (22 kg/ha), (75% water soluble potassium humate with solubility of 85% and particle size 0.5 - 5 mm, Nutri Tech Solutions®).

 Compost plus SHG at the same rates given above.

The organic supplements were added to the pots two weeks after transplanting. The Ferbon and compost were gently mixed into the top centimetre; the SHG were dissolved in 400 ml water, then added to the pots as a soil drench. The AMF product used was MYCORMAX (Arbuscular Vesicle-Mycorrhizae, manufactured by: JH Biotech, INC, Ventura USA; imported and distributed in Australia by Zadco for Quality Gro PTY LTD); this was applied at 4 g/L as per label recommendation, and mixed into the soil surface without disturbing the

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seedling roots.The components of MYCORMAX product are described in Table 5.3. After transplanting, plants were grown for more than 11 weeks (82 days) under natural lighting conditions. Glasshouse temperature was 20±2° C. To control whiteflies, the glasshouse was fumigated by Pestigas (0.4% natural pyrethrums, 2% Piperonyl Butoxide in Carbon) at week 8.

Table 5.2 The compositional formula for organic applications used in the trial.

N (mg/kg) P (mg/kg) K (mg/kg) S (mg/kg) Ca (mg/kg) Mg (mg/kg) Na (mg/kg) Fe (mg/kg) Cl (mg/kg) Compost 15000 7500 6100 2600 13100 9800 1800 18100 3700 Ferbon 13400 1990 4620 17700 14800 2790 2460 9100 NA Mn (mg/kg) Zn (mg/kg) Cu (mg/kg) Co (mg/kg) B (mg/kg) Mo (mg/kg) PH Electrical Cond. Compost 383.3 199.9 65.1 6.7 30.8 4.4 6.5 2000 uS/cm Ferbon 488.0 142.0 91.0 11.8 54.7 9.0 6.2 3255 uS/cm

Ferbon Org. C 37.5 % and Moisture Con. 35.4 %

Table 5.3 The compositional formula for the AMF application used in the trial.

Treatment Constituents

AMF

(MycormaxTM, 2010)

Active Ingredients 2%, Inert Ingredients 98%

Glomus intraradices (Rhizophagous irregularis) 46 spores/gm, Glomus mosseae 19 CFU/gm.

ectomycorrhizal fungi*: Laccaria bicolor 500 CFU/gm, Pisolithus tinctorius 15,300 CFU/gm, Scleroderma cepa 1,760 CFU/gm, Scleroderma geastrum 1,760 CFU/gm and Scleroderma citrinum 1,760 CFU/gm.

*Note that the ectomycorrhizal fungi Pisolithus tinctorius, Scleroderma cepa, Scleroderma geastrum, Scleroderma citrinum

and Laccaria biocolorare would not have colonized sunflower. The asterids class (including the Asteraceae order, which is

where sunflower is classified) are dominated by AM and ericoid mycorrhizal partnerships (Brundrett, 2009).

5.3.3

Assessments

5.3.3.1 Canopy

Leaf chlorophyll content was assessed with a Minolta SPAD-502 meter at 4, 5, 6, 7, 8 and 9 weeks. Four fully expanded mature leaves were selected on each plant at each assessment date. Plant height and number of nodes were assessed at 5, 6, 7, 8 and 9 weeks. Stem diameter was measured at the fourth internode. Diameter of the primary flower head and number of axillary flowers was measured in week 10. Flowering dates were recorded daily from the beginning of flowering until the last plant flowered. After flowering the stem was cut at the soil surface (week 12), and plants weighed to determine fresh weight. Plant stems

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(with leaves and flower heads) were dried at 40° C, until weight was stable and then dry weight was measured.

Dry matter content was defined as: Dry weight = _{fresh weight}dry weight 𝑥 100

5.3.3.2 Nutrient analysis

After drying at 40° C leaves were taken from each plant from node 5 upwards and ground with a coffee grinder to a fine powder prior to bagging. Roots were removed from each pot following removal of the stems and the soil was allowed to air dry. Once dry, soil from each pot was sub-sampled and samples placed into labelled plastic bags. Leaf and soil samples were sent to a commercial laboratory for analysis (Soil & Plant Laboratory, CSBP Limited, Western Australia).

5.3.3.3 Root colonisation of AMF

To estimate mycorrhizal colonisation in sunflower roots at the end of the trial, roots were gently shaken to remove soil, washed and taken to the laboratory. Fine roots branching from coarse roots were selected, washed with tap water and cut into 1–1.5 cm segments. Roots were cleaned by heating for 3-5 minutes in 5% KOH. The roots were then strained and rinsed twice with tap water, and again rinsed in 3.5% HCl. Roots were stained with 5% black Sheaffer ink in lactic acid according to methods described by Khaosaad et al. (2006) and Toussaint et al. (2007) by reheating for 3 minutes. Roots were rinsed once with tap water and placed in water with a few drops of lactic acid to destain.

5.3.3.4 Estimating AMF colonisation

Following the method described by McGonigle et al. (1990), five slides (five roots for each slide) of stained roots were prepared; roots were placed horizontally, mounted in water, and covered by coverslips. Using a crosshair eyepiece 30 root intersects per slide were inspected, scoring for presence/absence of hyphae, arbuscules and vesicles.

Hypha (H), vesicle (V) or arbuscule (A) presence = ₁₅₀X 𝑥100 Where (X) = hypha, vesicle or arbuscule