Compatibility of a Trichoderma atroviride biocontrol agent with management practices of Allium crops

Compatibility of a Trichoderma atroviride biocontrol agent with management practices of Allium crops

Crop Protection 33 (2012) 94e100 Contents lists available at SciVerse ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro...

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Crop Protection 33 (2012) 94e100

Contents lists available at SciVerse ScienceDirect

Crop Protection journal homepage: www.elsevier.com/locate/cropro

Compatibility of a Trichoderma atroviride biocontrol agent with management practices of Allium crops Kirstin L. McLean a, *, John S. Hunt b, Alison Stewart a, Denise Wite c, Ian J. Porter c, Oscar Villalta c a

Bio-Protection Research Centre, P O Box 84, Lincoln University, Canterbury 7647, New Zealand Agrimm Technologies Ltd, P O Box 35, Lincoln, Canterbury 7646, New Zealand c Biosciences, Department of Primary Industries, Victoria, Private Bag 15, Ferntree Gully, Delivery Centre, VIC 3156, Australia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 July 2011 Received in revised form 7 November 2011 Accepted 11 November 2011

Trichoderma atroviride Karsten strain C52 populations were measured in agronomic soils in combination with soil organic amendments, a nitrogen fertiliser, fungicides and diallyl disulphide (DADS) (a Sclerotium cepivorum sclerotial germination stimulant). Trichoderma atroviride C52 populations did not proliferate 2 weeks after inoculation in sandy soils (4.7  102 cfu/g soil) compared with silt loam soils (1.1  106 e5.7  107 cfu/g soil), however, the addition of two blended pellet products containing poultry manure and other organic nutrients or humic acid plus organic matter to the sandy soil enabled populations to proliferate well (1.1e1.3  105 cfu/g soil). In vitro, twice field rate applications of urea reduced in vitro Trichoderma atroviride C52 growth (spore germination, germ tube length and mycelial growth), however, T. atroviride C52 populations were less sensitive to field rate applications of urea in field soil. Overall, T. atroviride C52 populations were not adversely affected when exposed to any of the fungicide soil treatments tested (populations ranged from 1.7  105e1.1  108 cfu/g soil). Volatiles of DADS reduced T. atroviride C52 mycelial growth in vitro when DADS was applied to the medium at standard and twice recommended field rates but not at half field rate. No spore germination occurred under any of the DADS in vitro treatments compared with 100% spore germination in the control. However, when DADS was applied to soil 4, 6 and 8 weeks before application of T. atroviride C52, populations were unaffected. Based on these data, an application strategy for the use of a commercial formulation of T. atroviride C52 in an integrated white rot management programme for onions is proposed. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Trichoderma Organic amendments Nitrogen Fungicides Diallyl disulphide Onion white rot

1. Introduction In New Zealand, an isolate of Trichoderma atroviride C52 has been developed as TenetÒ to control the devastating onion white rot disease. Initial developmental research (Harrison and Stewart, 1988; Kay and Stewart, 1994; McLean and Stewart, 2000; McLean et al., 2005) determined that when Trichoderma atroviride was applied at a rate of 106 cfu/g soil in a pellet formulation, this biological treatment provided onion white rot control equivalent to standard fungicides under low and moderate disease pressure (McLean et al., 2005). Other formulations of T. atroviride C52 were unable to sustain T. atroviride C52 populations at or greater than 1  105 cfu/g soil over the onion (Allium cepa) growing season (McLean et al., 2005), resulting in poor disease control. These

* Corresponding author. Bio-Protection Research Centre, P O Box 84, Lincoln University, Canterbury 7647, New Zealand. Tel.: þ64 3 321 8120; fax: þ64 3 325 3864. E-mail addresses: [email protected] (K.L. McLean), d.gale@agrimm. co.nz (J.S. Hunt), [email protected] (A. Stewart), [email protected]. gov.au (O. Villalta). 0261-2194/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2011.11.018

results and those of additional field trials led the authors to postulate that T. atroviride C52 populations needed to be maintained at or greater than 1  105 cfu/g soil for the duration of the growing season to effectively control disease under low to moderate disease pressure. Under high disease pressure or situations where the onset of disease was delayed, the efficacy of TenetÒ was reduced (McLean et al., 2002; Villalta et al., 2004, 2008). In fields infested with Sclerotium cepivorum, an integrated disease management strategy that includes chemical and biological controls is highly desirable to ensure effective control of onion white rot (Clarkson et al., 2006; Villalta et al., 2008). As high population counts of T. atroviride are required for disease control, it is important that the fungus is compatible with standard crop management practices including organic amendments (routinely applied in nutrient poor sandy soils used for spring onion (Allium fistulosum) production), fertilisers, fungicides and diallyl disulphide (DADS). In the past, fungicides including the dicarboximide and triazole groups have provided good control of onion white rot. However, these chemicals have now become less effective through inconsistent control and enhanced microbial degradation (Slade et al., 1992;

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Tyson et al., 1999). Strategic applications of new fungicides, such as FilanÒ (a.i. boscalid, BASF) and FolicurÒ (a.i. tebuconazole, Bayer), which have provided high levels of onion white rot control in both spring and bulb onions in Australia (Villalta et al., 2004, 2008), are being used to enhance levels of disease control by targeting the fungicide treatments around the plant root zone. Little is known about the effect of currently available fungicide treatments applied in the root zone or in furrow on activity of Trichoderma. The compatibility of T. atroviride with FilanÒ and three additional fungicides formulated in a mixture with boscalid or with activity against S. cepivorum were therefore studied in the present trials. Field trials in New Zealand, Australia and California have shown that applications of diallyl disulphide (DADS), a stimulant of germination of S. cepivorum sclerotia, can reduce S. cepivorum inoculum by 90% or more and disease by up to 70e80% with corresponding yield increases (Davis et al., 2007; Tyson et al., 2000; Villalta et al., 2004, 2008). Therefore, the combination of pre-season applications of DADS with at sowing applications of TenetÒ in the furrow with the seed was also evaluated in the present trials to determine best time for DADS application. The aim of this research was to determine the effect of key Allium crop management practices on T. atroviride C52 soil populations in a nutrient poor Australian soil and an organic rich New Zealand soil. A greater understanding of the population dynamics of T. atroviride C52 in a variety of agronomic scenarios will assist in the development of a suitable strategy for the application of TenetÒ within sustainable integrated disease management programmes to be proposed.

95

Victoria, Australia). Two furrows (10 cm long  1 cm depth) were prepared across each pot. Five treatments were assessed in this trial: 1) control e Tenet, a maize chip coated with a nutrient matrix and active T. atroviride C52 spores to give 107 spores/g product (Agrimm Technologies Ltd, New Zealand); 2) Tenet þ fresh composted poultry manure (NPK analysis 3.5% : 1.7% : 1.4% w/w) from a vegetable farm in Victoria, Australia; 3) Tenet þ poultry manure pellets -Dynamic LifterÔ (NPK analysis: 3.2% : 2.6% : 1.3% w/w), comprised of a pellet blend of composted chicken manure, blood and bone, fish meal and seaweed (76% organic matter; Yates, Australia), Tenet þ humic acid pellets e AgroligÔ pellets (86% organic matter; 75% humic acid, Agrichem, Australia, 2011) and 5) Tenet þ humic acid liquid e Supa HumusÔ (12% liquid humic acid, Agrichem, Australia, 2011). The solid amendment treatments were applied to the base of each furrow at 3 g per 10 cm furrow (equivalent to 900 kg/ha). The Supa HumusÔ was applied as a drench (10 ml/10 cm furrow) using a solution of 3 L product/1000 L water. Spring onion seed cv. ‘Paragon’ and TenetÒ (30 kg/ha) were then added to the base of each furrow. The trial was arranged in a randomized complete block design on glasshouse benches with four replicates per treatment. The glasshouse temperature was set at 20  C  5  C. Pots were watered with fine overhead irrigation as required. Soil samples for T. atroviride C52 soil population analysis were collected as described in section 2.2 at 0 (immediately after setting up the pots) and 2 weeks after the pot trial was established. 2.4. Assessment of the compatibility of T. atroviride with fertilisers and fungicides

2. Materials and methods 2.1. Soil chemical analyses An Australian (Clyde sandy loam) and a New Zealand (Wakanui silt loam) soil were assessed for their chemical composition (Table 1). Chemical analyses were performed for the Australian soil by the State Chemical Laboratory (Department of Primary Industries, Victoria, Australia). A chemical analysis of the New Zealand soil was performed by Hill Laboratories (Hamilton, New Zealand). 2.2. T. atroviride C52 colony forming unit assay TenetÒ, a pellet, grain-based formulation containing conidia and mycelium of T. atroviride C52 at a concentration of 106 cfu/g, was used as the T. atroviride C52 inoculation treatment for all pot trials. Trichoderma atroviride C52 soil population counts were made using a colony forming unit (cfu) assay (Whipps et al., 1989). Briefly, a 1 g sub-sample from a bulk sample of soil taken from a pot was serially diluted in sterile distilled water to a concentration of 106 then 0.5 ml aliquots from each dilution were spread on the surface of four Trichoderma selective media (TSM) plates (McLean et al., 2005). Trichoderma atroviride C52 colonies recovered from soil were distinguished from resident Trichoderma isolates using distinct colony morphology characteristics. All soils used in the following pot trials were sampled prior to T. atroviride C52 inoculation to ensure morphological characters were sufficient to identify T. atroviride C52 from other T. atroviride isolates and isolates of other Trichoderma species resident in the soils. 2.3. Effect of soil amendments on T. atroviride C52 populations in nutrient poor soil 2.3.1. Pot trial 1: effect of organic amendments on Australian nutrient poor soil Plastic pots (16 cm  12 cm  12 cm) were filled to within 2 cm of the top with Clyde sandy loam soil (commercial vegetable farm,

2.4.1. Laboratory assay 1: effect of urea on mycelial growth Nitrogen in the form of urea (which has the highest available nitrogen compared with other nitrogenous fertilisers) was prepared in distilled water at half (18.8 kg/ha), standard (37.5 kg/ha) and twice (75 kg/ha) the recommended field rate (onions receive 150 kg/ha/season, which is applied in four applications (D. Faire, Key Industries Ltd, personal communication)). Aliquots (0.5 ml) of each concentration of urea were aseptically transferred using a pipette and spread over the surface of each of four 95 mm diameter Petri dishes filled with 20 ml of potato dextrose agar (PDA). Sterile distilled water was used as a control. After 30 min standing, each plate was centrally inoculated with an agar plug (5 mm diameter) taken from the actively growing edge of a 5 day old T. atroviride C52 colony grown on PDA at 20  C in the dark. The inoculated plates were incubated upside down at 20  C in the dark for 6 days. Radial mycelial growth was measured for each treatment in four directions away from the inoculum plug on days 1, 2, 3 and 4 after inoculation, or until the plate edge was reached. Plates were kept for a further 4 days to determine whether T. atroviride was able to sporulate in the presence of urea. The average radial growth of each mycelial colony was calculated 3 days after inoculation for the different concentrations of urea and the control. This assay was completed twice. 2.4.2. Laboratory assay 2: effect of urea on spore germination A spore suspension was prepared from 10 day old T. atroviride C52 colonies grown on PDA at 20  C in the light. The stock spore suspension was diluted to 1  105 spores/ml using potato dextrose broth (PDB) to provide the nutrients required for spore germination. Aliquots (0.5 ml) of the spore suspension and urea (0.5 ml) at 18.8, 37.5 (field rate) and 75 kg/ha were added to 1.5 ml conical tubes. Sterile distilled water was used as a control. Each spore/urea treatment mix was replicated four times. The tubes were fastened to the side of a rotating arm in an oven and slowly rotated at 20e25  C for 24 h then four samples were taken from each conical tube for analysis. Spore germination, which was considered to have

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K.L. McLean et al. / Crop Protection 33 (2012) 94e100

occurred when the germ tube length equalled the spore diameter (Hartill et al., 1983), was recorded for 50 spores per sample. This assay was completed twice. 2.4.3. Pot trial 2: effect of urea on T. atroviride C52 populations Plastic pots (8 cm  8 cm  6 cm) were filled with Wakanui silt loam soil, Canterbury, New Zealand. TenetÒ (0.05 g/planting hole) equivalent to a field rate application of 25 kg/ha was then added to each of four planting holes in each pot without seed. Seed was not needed to assess the effect of urea on T. atroviride C52 populations. Toothpicks were inserted into the planting hole to mark the inoculation sites to ensure soil samples were collected from the planting hole. Urea was applied as a drench directly to the soil at either 37.5 kg/ha (field rate) or 75 kg/ha using a plastic 1 L trigger bottle with a spray nozzle. There were three replicate pots for each treatment. A control treatment was included where water replaced the urea application. The soil in each pot was covered with a layer of vermiculite to prevent excessive drying of the soil. Each pot was placed in a separate drip tray, which was positioned according to a randomized complete block design on benches in a glasshouse. The glasshouse temperature was set at 20  C  5  C. Pots were manually watered as required. Before the urea was added (week 0), a teaspoon was used to collect a soil sample from one planting hole and the surrounding soil in each pot. Soil samples were taken in this manner again at 2 and 4 weeks after urea application. Trichoderma atroviride C52 populations (cfu/g soil) were determined using the assay described in section 2.2. This pot trial was completed twice. 2.4.4. Pot trial 3: effect of fungicides on T. atroviride C52 populations Plastic pots (8 cm  8 cm  6 cm) were filled with Wakanui silt loam soil, Canterbury, New Zealand. TenetÒ (0.05 g/planting hole) equivalent to a field rate application of 25 kg/ha was then added to each of four planting holes in each pot and toothpicks used to mark the site of TenetÒ inoculation. The fungicide treatments tested were the dicarboximide fungicide SumisclexÒ 25 (a.i. procymidone), currently used as a seed treatment in Canterbury, New Zealand to control onion white rot (Crop Care Holdings, Nelson, New Zealand) and three additional fungicides (FilanÒ, a.i. boscalid; CometÒ, a.i. pyraclostrobin; PristineÒ, a.i. boscalid and pyraclostrobin) (BASF, Auckland, New Zealand). The treatments were applied as a drench at one or two times the recommended field rates which were 3 L/1000 L water/ha for SumisclexÒ 25, 1.2 kg/500 L/ha for FilanÒ, 0.8 L/500 L/ha for CometÒ and 1.6 kg/500 L/ha for PristineÒ. Each fungicide treatment was applied directly to the soil using a plastic 1 L trigger bottle with a spray nozzle in each of three pots. A control treatment was included where water replaced the fungicide. A layer (0.5 cm) of vermiculite was added to the top of each pot to prevent excessive drying of the soil. Each pot was placed in a separate drip tray, which was positioned on a glasshouse bench using a randomized complete block design. The glasshouse temperature was set at 20  C  5  C. Pots were watered as necessary. A teaspoon was used to collect a soil sample from one planting hole and the surrounding soil in each pot immediately after TenetÒ application and fungicide application (0) and again at 2 and 4 weeks after fungicide applications. Soil populations of T. atroviride C52 were assessed as described in section 2.2. 2.5. Effect of diallyl disulphide on T. atroviride populations 2.5.1. Laboratory assay 3: effect of DADS on mycelial growth and spore germination Diallyl disulphide (DADS; Alli-UpÔ, Elliott Technologies Ltd, New Zealand) was diluted in distilled water to concentrations

equivalent to half (3.75 L/600 L water per hectare), standard (7.5 L/600 L water per hectare) and twice (15 L/600 L water per hectare) the recommended field rate. The methods described for determining the effect of urea on T. atroviride C52 mycelial growth (Section 2.4.1) and spore germination (Section 2.4.2) were followed. These assays were completed twice. 2.5.2. Pot trial 4: effect of DADS on T. atroviride C52 populations Four plastic pots (8 cm  8 cm  6 cm) were filled with Wakanui silt loam soil, Canterbury, New Zealand. Diallyl disulphide was diluted in water to give the standard field rate (7.5 L/600 L water per hectare). Field rate DADS was applied to the Wakanui silt loam soil at 4, 6 and 8 weeks prior to TenetÒ application using a plastic 1 L trigger bottle with a spray nozzle. To minimise the vaporisation of DADS from the soil, the soil surface was compressed using a wooden block to mimic the use of a roller after DADS application in the field. TenetÒ at the rate of 30 kg/ha (or 0.1 g/planting hole) was added to each of three pots treated with DADS 4, 6 or 8 weeks earlier. TenetÒ was placed 0.5 cm deep in the soil in holes equidistant from one another in a grid arrangement. Control treatments were included where sterile distilled water replaced DADS in the solution and treatments were at 4, 6 or 8 weeks prior to inoculation with TenetÒ. Each pot was placed in a separate drip tray, which was arranged in a randomised complete block design in a glasshouse for the duration of the trial. The glasshouse temperature was set at 20  C  5  C. Pots were watered as required. A teaspoon was used to collect a soil sample from each pot immediately after the application of the TenetÒ granules (week 0), then again at 2, 4 and 6 weeks after inoculation. Soil populations of T. atroviride C52 were assessed as described in section 2.2. This pot trial was completed twice. 2.6. Statistical analysis Colony counts from the T. atroviride soil population assays were transformed to logarithmic values before analysis for all experiments. Unless otherwise stated below, treatment effects were analysed using a one-way ANOVA with treatment type as a factor using GenStat (Lawes Agricultural Trust, Rothamstead Experimental Station, Harpenden, UK). Treatment effects for Pot trial 3 (section 2.4.4) and Pot trial 4 (section 2.5.2) were analysed using a two way ANOVA with treatment type and assessment time as factors. Significant effects identified by an ANOVA at P  0.05 for each experimental treatment were further explored using a Fisher’s LSD test with mean treatment values. 3. Results 3.1. Chemical composition of Australian and New Zealand agricultural soils Clyde sandy loam soil from Australia had higher levels of pH, magnesium, sulphur, calcium and phosphorus compared with Wakanui silt loam from New Zealand (Table 1). Wakanui silt loam had higher levels of nitrogen and organic matter compared with the Clyde sandy loam soil (Table 1). 3.2. Effect of soil amendments on T. atroviride C52 populations in nutrient poor soil 3.2.1. Pot trial 1: effect of organic amendments in Australian nutrient poor soil Trichoderma atroviride C52 populations ranged from 4.7  102 to 9.7  105 cfu/g soil (Table 2). At week 0, the T. atroviride C52 population was significantly greater (P  0.05) in the TenetÒ control treated with poultry manure pellets and humic acid pellets

K.L. McLean et al. / Crop Protection 33 (2012) 94e100

75 kg/ha urea treatment compared with the control at all three assessment times (Table 3).

Table 1 Chemical composition of the soils used in the pot trials. Soils

pH g/kg

K g/kg

Mg g/kg

S g/kg

Ca g/kg

P g/kg

N g/kg

OMa %w/w

Clyde sandy loam, Australia Wakanui silt loam, New Zealand

7.1

0.4

0.5

0.2

4.8

1.8

1.0

2.3

5.6

0.5

0.1

0.01

0.8

0.02

3.0

4.4

a

97

OM ¼ organic matter.

compared with all other treatments (Table 2). Two weeks after planting, the T. atroviride C52 population was significantly higher with all organic amendment treatments compared with the control. In addition, T. atroviride C52 populations were significantly higher with poultry manure pellets and humic acid pellets compared with the remaining organic amendments (Table 2).

3.3.4. Pot trial 3: effect of fungicides on T. atroviride C52 population dynamics At week 0, T. atroviride populations ranged from 1.6  106 to 4.1  106 cfu/g soil and there was no significant difference (P  0.05) in population numbers between all treatments (Table 4). After 2 weeks, the T. atroviride C52 population was significantly lower (P  0.05) with the application of the 0.8 L/500 L/ha CometÒ, 1.2 kg/ 500 L/ha FilanÒ and 3.2 kg/500 L/ha PristineÒ compared with the control treatment. After 4 weeks, T. atroviride C52 populations were significantly lower with applications of 1.6 L/500 L/ha CometÒ and 3.2 kg/500 L/ha PristineÒ compared with the control treatment. Over time, T. atroviride was able to proliferate significantly in all rates of each fungicide tested except twice field rate concentrations of CometÒ and PristineÒ (Table 4).

3.3. Assessment of the compatibility of T. atroviride C52 with fertilisers and fungicides

3.4. The effect of diallyl disulphide on T. atroviride C52 population dynamics

3.3.1. Laboratory assay 1: effect of urea on mycelial growth After 3 days, mycelial growth of T. atroviride C52 was significantly inhibited (P  0.05) by urea at 75 kg/ha concentration (colony diameter 24.6 mm) compared with the control, 18.8 kg/ha and 37.5 kg/ha (field rate) concentrations (colony diameters of 27.8, 26.7 and 26.9 mm, respectively). Trichoderma atroviride C52 spore production occurred on all replicate plates (4) in the control treatment by day 8. On agar plates amended with 18.8 kg/ha and 37.5 kg/ha urea concentrations, sporulation occurred on two plates and one plate, respectively. Sporulation did not occur on any of the agar plates amended with urea at the 75 kg/ha rate after 8 days.

3.4.1. Laboratory assay 3: effect of DADS on mycelial growth Diallyl disulphide significantly reduced (P  0.05) T. atroviride C52 radial mycelial growth at 7.5 L/ha (24 mm) and 15 L/ha (15 mm) applications of DADS compared with the control (30 mm). There was no significant difference (P  0.05) in mean mycelial growth between the control (30 mm) and the 3.75 L/ha (field rate) (28 mm) DADS application. In addition, the spore development and maturity was delayed by 2 days in Petri dishes amended with all rates of DADS compared with the control treatment.

3.3.2. Laboratory assay 2: effect of urea on spore germination The percentage of T. atroviride C52 spore germination was significantly lower (P  0.05) in the 75 kg/ha concentration of urea (16% germination) compared with all other treatments (100% germination). The mean germ tube length in the control (3.41 mm), 18.8 kg/ha urea rate (3.11 mm) and 37.5 kg/ha (field rate) urea rate (3.07 mm) were not significantly different (P > 0.05) to one another. The mean germ tube length for the 75 kg/ha urea rate (0.25 mm) treatment was significantly less (P  0.05) than all other treatments. 3.3.3. Pot trial 2: effect of urea on T. atroviride C52 population dynamics in Wakanui silt loam soil Trichoderma atroviride C52 populations ranged from 4.3  104 to 7.4  107 cfu/g soil. There was no significant difference (P  0.05) in T. atroviride C52 populations between the control and 37.5 g/ha urea treatments for the duration of the trial (Table 3). The T. atroviride population was significantly lower (P  0.05) in the Table 2 Trichoderma atroviride C52 soil populations over time in nutrient poor Clyde sandy loam soil, Australia treated with TenetÒ (30 kg/ha) and organic amendments. Organic amendment

T. atroviride populations over time (cfu/g soil)a Week 0

None Fresh composted poultry manure Poultry manure pellets Humic acid pellets Humic acid liquid a

6.9  10 b 4.1  103 b 5.2  104 a 9.7  105 a 3.6  103 b

3.4.3. Pot trial 4: effect of DADS on T. atroviride C52 populations Trichoderma atroviride C52 populations ranged from 4.6  104e9.8  106 cfu/g soil (Table 5). There was no significant difference (P > 0.05) in T. atroviride C52 populations between the control treatment and any of the DADS treatments for the duration of the experiment (Table 5). 4. Discussion The potential integration of Trichoderma biocontrol products into Allium crop growing practices necessitates a greater understanding of the dynamics of Trichoderma populations in agricultural soil. This research monitored T. atroviride C52 populations when Table 3 Trichoderma atroviride C52 soil populations in Wakanui silt loam soil, New Zealand after the application of TenetÒ (25 kg/ha) and urea. Treatment

Week 2 3

3.4.2. Laboratory assay 4: effect of DADS on spore germination Diallyl disulphide significantly inhibited (P  0.05) T. atroviride C52 spore germination at all rates tested. After 24 h incubation, none of the spores incubated with DADS solutions at 3.75 L/ha, 7.5 L/ha and 15 L/ha concentrations, germinated compared with 100% spore germination in the control treatment.

2

4.7  10 c 1.3  103 b 1.3  105 a 1.1  105 a 2.2  103 b

Means within columns followed by the same letter are not significantly different according to Fisher’s LSD test (P ¼ 0.05).

Trichoderma atroviride populations over time (cfu/g soil)a Week 0

Control 37.5 kg/ha ureab 75 kg/ha urea

2.7  106 1.6  106 1.1  106

Week 2 a ab b

5.7  107 1.4  105 4.3  104

Week 4 ab b c

7.4  107 2.0  106 1.3  106

a ab b

a Means within columns followed by the same letter are not significantly different according to Fisher’s LSD test (P ¼ 0.05). b Field rate urea application.

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Table 4 Trichoderma atroviride C52 soil populations after the application of TenetÒ (25 kg/ha) and fungicides over time in Wakanui silt loam soil, New Zealand. Treatmenta

Application rate

T. atroviride populations over time (cfu/g soil)a

Fungicide

Week 0

Control CometÒ CometÒ FilanÒ FilanÒ PristineÒ PristineÒ SumisclexÒ 25 SumisclexÒ 25

3.1 1.6 1.8 2.8 4.1 3.0 2.8 2.3 1.9

a b

0.8 L/500 L/hab 1.6 L/500 L/ha 1.2 kg/500 L/hab 2.4 kg/500 L/ha 1.6 kg/500 L/hab 3.2 kg/500 L/ha 3 L/1000 L/hab 6 L/1000 L/ha

        

106 106 106 106 106 106 106 106 106

Week 2 f fgh fg f f f f f f

4.8 1.8 1.0 4.9 4.7 4.7 1.7 5.1 1.8

        

107 106 106 106 106 106 105 107 107

Week 4 a-e fgh c-f f ef ef L a-d c-f

1.4 3.6 6.5 9.2 9.5 1.1 7.8 7.8 8.0

        

108 107 106 107 107 108 106 107 107

a a-e ef ab ab a-d def abc ab

Means within columns and across rows followed by the same letter are not significantly different according to Fisher’s LSD test (P ¼ 0.05). Field rate concentration.

applied as TenetÒ in Australian and New Zealand agricultural soils alone and in combination with various agronomic treatments. Trichoderma atroviride C52 proliferation following inoculation was different between the two soils examined. While T. atroviride C52 populations were not directly compared in the same experiment requiring caution when comparing between soil types, the fungal populations in the Clyde sandy loam soil were substantially less (102e103 cfu/g soil) than those in Wakanui silt loam soil (>106 cfu/g soil). The colonisation of soil by Trichoderma species is influenced by environmental parameters and the chemical composition of the soil (Adamicki, 2006; Ahmad and Baker, 1988; Alexander and Stewart, 1994; Coley-Smith, 1960; Eastburn and Butler, 1991; Lewis and Papavizas, 1984; Utkhede and Rahe, 1980). It is most likely that the factor with the greatest influence is organic matter given that the addition of organic matter to soil has been known to improve soil aeration, water-holding capacity, moisture retention, permeability and serve as a nutritive source for soil microorganisms (Davey, 1996). In the present trials, the organic matter in the sandy soil was 2.3% w/w compared with 4.4% w/w in the silt loam soil. By amending the sandy soil with products containing a blend of organic matter with other nutrients such as pellet formulations of poultry manure or humic acid (AgroligÔ), the T. atroviride C52 population increased significantly to levels greater than 1  105 cfu/g soil, which is the threshold required to bring about disease control (McLean et al., 2005). The fungal proliferation achieved with the pellet formulation of poultry manure was most likely due to the higher levels of humic acids and organic matter in the blend and their slow release and thus availability in the furrow compared with fresh composted poultry manure. The T. atroviride C52 populations that resulted from soil amended with humic acid plus organic matter pellets would have thrived on the readily available food source compared with less decomposed treatments tested. In addition, it is possible that the liquid Supa HumusÔ Table 5 Trichoderma atroviride C52 populations in Wakanui silt loam soil, New Zealand over time after the application of standard rate (7.5 L/600 L water per hectare) diallyl disulphide, 4, 6 and 8 weeks prior to Tenet (30 kg/ha) application. Treatment

T. atroviride populations over time (cfu/g soil)a

4 4 6 6 8 8

week week week week week week

4.6 1.2 1.7 2.2 2.8 1.1

a

There were no significant differences (P > 0.05) among treatment means.

Week 0 Control DADS Control DADS Control DADS

     

104 106 105 106 105 106

Week 2 1.1 7.7 1.7 3.2 1.5 3.1

     

106 105 106 105 106 106

Week 4 3.5 2.3 2.3 3.3 1.2 9.8

     

106 106 106 106 106 106

Week 6 4.7 2.8 3.0 2.6 1.4 6.0

     

106 106 106 106 106 106

formulation which had lower content of humic acids leached in the sandy soil and the nutrients were not available for fungal growth. In Victoria, Australia, spring onions are routinely grown in sandy soils. In these areas, it would be beneficial to apply soil organic amendments with good levels of organic matter and others nutrients to the sandy loam soils to encourage T. atroviride C52 establishment and proliferation. It would be desirable to apply plant based soil amendments such as residues of green manures or formulations of humic acid mixed with organic matter and other nutrient as the application of animal waste to edible crops can be undesirable (Acosta-Martinez and Harmel, 2006; Ribaudo et al., 2003). Following the present research, the commercially available formulation of TenetÒ was modified to contain humic acid (10% w/w; Agromate; 80% organic matter and 70% humic acid, Agrichem, Australia, 2011) to aid establishment and subsequent proliferation of T. atroviride C52 (D. Gale, Agrimm Technologies, personal communication). The chemical analysis of the soil in Australia and New Zealand showed that the level of nitrogen (pH), magnesium, sulphur, calcium and phosphorus was substantially different between soils. Nitrogen has been shown to have an impact on fungal activity (Yamanaka, 1999; Steyaert et al., 2010). Therefore, as nitrogen in the form of urea is used in Allium cropping systems, the effect of urea on T. atroviride C52 growth was examined in greater detail. The laboratory assays and pot trial showed that T. atroviride C52 was sensitive to higher doses of nitrogen. However, T. atroviride C52 populations in soil in the present trials were able to recover over time. The loss of nitrogen from the pot system through processes such as denitrification and leaching most likely enabled T. atroviride C52 populations to recover. In Pot trial 1, the levels of nitrogen in the fresh composted poultry manure were similar to nitrogen levels in the poultry manure pellets however the T. atroviride C52 populations were significantly different. It is likely that the fresh composted poultry manure was high in ammonia nitrogen (e.g. from urine) that was quickly released leading to poor establishment of T. atroviride C52 as ammonia is toxic to fungi (Johansson et al., 2002). The poultry manure pellets contained nitrogen from various organic sources (e.g. manure, fish meal and blood and bone) formulated as a slow release product. Therefore, nitrogen and other nutrients would most likely have been released at low levels that did not affect T. atroviride C52 establishment. It is possible that T. atroviride C52 populations could proliferate in the presence of other forms of nitrogen than urea. Wakelin et al., 1999 reported that nitrogen added as ammonium sulphate (NHþ 4 N) increased the saprophytic growth of Trichoderma koningii whereas nitrate (NO 3 N) suppressed growth. This supports the findings of Danielson and Davey (1973) who reported that Trichoderma spp.

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grow best when supplied with ammonium-nitrate compared with nitrite-nitrate on artificial culture medium. The fungicides, procymidone (SumisclexÒ 25) and boscalid (FilanÒ) alone or formulated in a mixture with pyraclostrobin (PristineÒ) and pyraclostobin (CometÒ) were assessed in this study. Trichoderma atroviride C52 was compatible with all fungicides tested when applied either at the recommended field rate or twice the recommended field rate. Trichoderma atroviride C52 was shown to be tolerant to procymidone in an earlier study (McLean et al., 2001), when T. atroviride C52 was identified as Trichoderma harzianum C52. In addition, a recent study by Clarkson et al. (2006) reported improved onion white rot control with Trichoderma viride and tebuconazole-based onion seed treatments. The increase in the T. atroviride C52 soil populations from 2 weeks onwards in the present study reflects the ability of Trichoderma to thrive in the biological vacuum created in the soil environment post-fungicide application (Papavizas, 1985). These results indicate the potential for commercial Trichoderma biocontrol products to integrate well with some of the key fungicides used in onion disease management programmes. The DADS in vitro assays showed a similar trend to the nitrogen assays in that T. atroviride C52 mycelial growth and spore germination were sensitive to DADS when in direct contact with the volatiles of DADS. However, the results from the pot trial indicated that T. atroviride C52 populations were not affected by the different concentrations of DADS volatiles, even when DADS was applied 4 weeks prior to the biocontrol application. Current recommendations for DADS involve single and/or twin applications of DADS applied, diluted in water, into fallow soil when soil temperatures are most conducive for S. cepivorum germination and onion white rot development. A resting period of up to 6 weeks is then recommended before the soil is worked up for onion planting. Our results demonstrated that TenetÒ can be safely applied at a minimum of 4 weeks after treatment with DADS. The soil pot trials conducted in the present study used TenetÒ rates of 25 and 30 kg/ha. At the time the trials were completed, the rate used was the manufacturer’s recommendation. Comparisons between pot trials of the control treatments where TenetÒ was added to Wakanui silt loam indicate that higher inoculum densities do not necessarily lead to higher T. atroviride C52 populations in the soil. For example, Wakanui silt loam soil amended with 30 kg/ha TenetÒ resulted in populations of 1.2e3.5  106 cfu/g soil (DADS trial) after 4 weeks, whereas trials amended with 25 kg/ha TenetÒ had greater populations of 7.4  107e1.4  108 cfu/g soil (fertiliser trial and fungicide trials, respectively). The interactions between the T. atroviride C52 inoculum and soil moisture and temperature would most likely be responsible for the variation in population dynamics observed between trials, given that the soil nutrients would have been relatively consistent between the control treatments of the individual trials. Agrimm Technologies currently recommends an application rate of 25 kg/ha for TenetÒ (D. Gale, Agrimm Technologies, personal communication). 4.1. Recommendations for an integrated disease management programme Based on the results of the present research, an application strategy for TenetÒ within sustainable IDM programmes for white rot control is proposed. TenetÒ (25 kg/ha) applied in the furrow with the seed should proliferate adequately in Australian and New Zealand soils high in organic matter (4e5%) such as, clay loam, alluvial and silt loam. However, T. atroviride C52 populations in sandy loam soil will benefit from the addition of organic matter in a readily available form for T. atroviride C52 proliferation. Caution should be used with organic compounds with high nitrogen

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(e.g. fresh poultry manure) as T. atroviride C52 growth may be inhibited if applied too close to application with these types of amendments. Trichoderma atroviride C52 is compatible with field rate applications of important fungicides used in onion production systems, such as boscalid and procymidone fungicides. Most post-sowing, late season foliar applications of fungicides should be compatible as they are targeted to the foliage and minimal fungicide is transported from the foliage to the root rhizosphere. Diallyl disulphide is the cornerstone of integrated onion white rot control for the restoration of fields heavily infested with S. cepivorum to Allium production. Thus, a combined approach, where DADS or another germination stimulant such as composted onion waste (Coventry et al., 2005) was used pre-plant to reduce disease p`ressure, an in furrow application of T. atroviride C52 applied at sowing, carefully timed fertiliser applications and supplementary post-sowing fungicide applications when required has great potential as an IDM strategy for onion white rot. Acknowledgements This research was funded by Horticulture Australia Ltd, Department of Primary Industries Victoria and the (NZ) Foundation for Research Science and Technology. The authors also acknowledge the assistance of Dr Sarah Hunger, Manuscripts Scientific Writing Services for editorial services. References Acosta-Martinez, V., Harmel, R.D., 2006. Soil microbial communities and enzyme activities under various poultry litter application rates. J. Environ. Qual. 35, 1309e1318. Adamicki, F., 2006. Effect of climate conditions on the growth, development, quality and storage potential of onions. Veg. Crops Res. Bull. . Res. Inst. Veg. Crops . Skierniewice 64, 163e173. Agrichem, Australia, 2011. www.agrichem.com.au. (accessed 08.11.11.). Ahmad, J.S., Baker, R., 1988. Implications of rhizosphere competence of Trichoderma harzianum. Can. J. Microbiol. 34, 229e234. Alexander, B.J.R., Stewart, A., 1994. Survival of sclerotia of Sclerotinia and Sclerotium spp. in NZ horticultural soil. Soil Biol. Biochem. 26, 1323e1329. Clarkson, J.P., Scruby, A., Mead, A., Wright, C., Smith, B., Whipps, J.M., 2006. Integrated control of Allium white rot with Trichoderma viride, tebuconazole and composted onion waste. Plant Pathol. 55, 375e386. Coley-Smith, J.R., 1960. Studies of the biology of Sclerotium cepivorum Berk. IV. Germination of sclerotia. Ann. Appl. Biol. 48, 8. Coventry, E., Noble, R., Mead, A., Whipps, J.M., 2005. Suppression of Allium white rot (Sclerotium cepivorum) in different soils using vegetable wastes. Eur. J. Plant Pathol. 111, 101e112. Danielson, R.M., Davey, C., 1973. Carbon and nitrogen nutrition of Trichoderma. Soil Biol. Biochem. 5, 505e515. Davey, C.B., 1996. Nursery soil management-organic amendments. In: Landis, T.D., Douth, D.B. (Eds.), National Proceedings, Forest and Conservation Nursery Associations General Technical Report PNW-GTR-389, pp. 6e18. USDA Forest Service PNWRS. Davis, R.M., Romberg, M.K., Nunez, J.J., Smith, R.F., 2007. Efficacy of germination stimulants of sclerotia of Sclerotium cepivorum for management of white rot of garlic. Plant Dis. 91, 204e208. Eastburn, D.M., Butler, E.E., 1991. Effects of soil moisture and temperature on the saprophytic ability of Trichoderma harzianum. Mycologia 83, 257e263. Harrison, Y.A., Stewart, A., 1988. Selection of fungal antagonists for biological control of onion white rot in New Zealand. New Zeal. J. Agr. Res. 16, 249e256. Hartill, W.F.T., Tompkins, G.R., Kleinsman, P.J., 1983. Development in New Zealand of resistance to dicarboximide fungicides in Botrytis cinerea, to acylalinines in Phytophthora infestans, and to guazatine in Penicillium italicum. New Zeal. J. Agr. Res. 26, 261e269. Johansson, S.M., Pratt, J.E., Asiegbu, F.O., 2002. Treatment of Norway spruce and Scots pine stumps with urea against the root and butt rot fungus Heterobasidion annosum e possible modes of action. For. Ecol. Manag. 157, 87e100. Kay, S.J., Stewart, A., 1994. Evaluation of fungal antagonists for control of onion white rot in soil box trials. Plant Pathol. 43, 371e377. Lewis, J.A., Papavizas, G.C., 1984. A new approach to stimulate population proliferation of Trichoderma species and other potential biocontrol fungi introduced into natural soils. Phytopathology 74, 1240e1244. McLean, K.L., Hunt, J., Stewart, A., 2001. Compatibility of the biocontrol agent Trichoderma harzianum (C52) with fungicides. New Zeal. Plant Prot. 54, 84e88.

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McLean, K.L., Stewart, A., 2000. Application strategies for control of onion white rot. New Zeal. J. Crop Hort. Sci. 28, 115e122. McLean, K.L., Swaminathan, J., Frampton, C.M., Hunt, J.S., Ridgway, H.J., Stewart, A., 2005. Effect of formulation on the rhizosphere competence and biocontrol ability of Trichoderma atroviride C52. Plant Pathol. 54, 212e218. McLean, K.L., Swaminathan, J., Hunt, J.S., Stewart, A., McDonald, M.R., 2002. Biological control of onion white rot in New Zealand. In: Proceedings of the Seventh International Workshop on Allium White Rot. in Prep, Coalinga, California, USA. Papavizas, G.C., 1985. Trichoderma and Gliocladium: biology, ecology, and potential for biocontrol. Annu. Rev. Phytopathol 23, 23e54. Ribaudo, M.O., Gollehon, N.R., Agapoff, J., 2003. Land application of manure by animal feeding operations: is more land needed? J. Soil Water Conserv. (Ankeny) 58, 30e38. Slade, E.A., Fullerton, R.A., Stewart, A., Young, H., 1992. Degradation of the dicarboximide fungicides iprodione, vinclozolin and procymidone in Patumahoe clay loam soil, New Zealand. Pestic. Sci. 35, 95e100. Steyaert, J.M., Weld, R.J., Stewart, A., 2010. Isolate-specfic conidiation in Trichoderma in response to different nitrogen sources. Fungal Biol. 114, 179e188. Tyson, J.L., Fullerton, R.A., Elliott, G.S., Reynolds, P.J., 2000. Use of diallyl disulphide for the commercial control of Sclerotium cepivorum. New Zeal. Plant Prot. 53, 393e397.

Tyson, J.L., Fullerton, R.A., Stewart, A., 1999. Changes in the efficacy of fungicidal control of onion white rot. New Zeal. Plant Prot. 52, 171e175. Utkhede, R.S., Rahe, J.E., 1980. Biological control of onion white rot. Soil Biol. Biochem. 12, 101e104. Villalta, O., Porter, I., Wite, D., Stewart, A., Mclean, K.L., Hunt, J., Murdoch, C.C., 2004. Integrated Control Strategy for Onion White Rot Disease in Spring Onions and Other Bunching Allium Crops. State Goverment of Victoria, Department of Primary Industries, Australia, p. 15. Villalta, O., Pung, H., Duff, A.A., Porter, I.J., Stewart, A., McLean, K.L., Wite, D., 2008. Optimising Diallyl Disulphide (DADS) for the Management of White Rot of Onions. Horticulture Australia Ltd. Wakelin, S.A., Sivasithamparam, K., Cole, A.L.J., Skipp, R.A., 1999. Saprophytic growth in soil of a strain of Trichoderma koningii. New Zeal. J. Agr. Res. 42, 337e345. Whipps, J.M., Budge, S.P., Ebben, M.H., 1989. Effect of Coniothyrium minitans and Trichoderma harzianum on Sclerotinia disease of celery and lettuce in the glasshouse at a range of humidities. In: Cavalloro, R., Pelerents, C. (Eds.), Proceedings of the CEC/IOBC Experts Group Meeting 27-29 May. Integrated Pest Management in Protected Vegetable Crops. A. A. Balkema, Rotterdam, pp. 233e243. Yates, Australia 2011.www.yates.com.au. (accessed 08.11.11.). Yamanaka, T., 1999. Utilization of inorganic and organic nitrogen in pure cultures by saprotrophic and ectomycorrhizal fungi producing sporophores on urea-treated forest floor. Mycol. Res. 103, 811e816.