Comparative efficacy

of disinfectants against Stanley E. Bellgard, Elsa P. Paderes and Ross E. Beever

Landcare Research, Private Bag 92170, Auckland 1142, New Zealand.

Phytophthora Taxon Agathis (PTA) Bellgards@LandcareResearch.co.nz

Introduction

Photo Nick Waipara

Phytophthora taxon Agathis (PTA) has been recognized as posing a threat to the health of kauri (Agathis australis) in northern New Zealand (Beever et al 2009). Affected trees
show a collar rot associated with unusual gummosis leading to tree death. While trees showing such symptoms have been reported widely, they are not universally present. In
order to reduce further movement of PTA, Auckland Regional Council (ARC) has instigated various precautionary policies, including the use of footwear hygiene procedures for
park visitors (Fig 1).

Photograph Nick Waipara

This project has investigated the efficacy of various disinfectants against PTA:
1. Trigene II Advance A mixture of halogenated tertiary amines — Active against fungi, bacteria and viruses — Recommended label rate is 2%
2. Phytoclean™ — A quaternary ammonium compound registered in Australia — For the treatment of Phytophthora cinnamomi — Recommended label rate is 10%
Virkon® S — A dipotassium peroxodisulphate product — Registered in New Zealand for emergency infectious disease control (for use in cleaning and disinfecting Industrial, Animal and Agricultural Facilities)
3.
and is efficacious against a range of viruses, bacteria and fungi — Recommended label rate is 1%.
4. Janola® — A household formulation of sodium hypochlorite — Widely used disinfectant and bleach — Its recommended label rate is 5% to disinfect gardening tools and equipment.
Figure 1: A runner passing-over foot mat soaked
5. Citricidal®— A product based upon grapefruit seed and pulp extracts — Recommended as a bactericide and fungicide in both pre- and post-harvest treatment of fruits — Range between 0.005–0.025% with disinfectant, Auckland Regional Park

Methods and Materials

Mycelial inhibition Zoospore inhibition
Actively growing 6.5 mm diameter plugs of PTA were placed on PDA amended with the five disinfectant Zoospore suspensions produced by incubating
treatments. The plates were incubated at 20ºC and colony growth scored at 4 days. To check on viability PTA colonies growing on V8 juice agar in non-
plugs were removed after 8-days, plated to fresh PDA plates and assessed for growth 3–5 days. sterile soils extract, were added to disinfectant
solutions to give the recommended label rates.
Oospore inhibition
The contents were plated to P5ARP selective
Oospore suspensions (containing c. 250 oospores from 56-day old clarified V8 liquid culture) were
medium for Phytophthora species and colony
added to plates containing 0.6% water agar amended with each of the five disinfectant treatments at
forming units (CFU’s) per ml were counted after 3
levels identified as lethal to mycelium After 10 days (at 20°), viability was assessed using tetrazolium
days.
staining (Jiang & Erwin 1990) as follows: pink=dormant; red=activated; black=non-viable.
Inhibiting PTA in soil Figure 2 & 3: Rubber gumboot being pressed into soil “spiked” with
Two methods were used. In the first, 20g soil PTA oospores (1,500 /g) and then sprayed with disinfectant to run-off

samples spiked with 1500 oospores/g were placed

in mesh bags and soaked in the disinfectants at their label rates. The treated soil was assayed using the
37.5m extended bioassay method (Stack & Millar 1985) and the number of CFUs/ml was also determined.
In the second, boot were surface sterilised and pressed into soil spiked with oospores (Fig 2). The boots
37.5m were then cleaned by spraying the boot to run-off using hand-held, commercial pump-packs sprayer
containing the disinfectants at label rates (Fig 3). The treated soil left adhering to the boot was scraped
Characteristic colony of PTA on Phytophthora-selective PTA Oospore preparation Semi-papillate zoosporangium of PTA
P5ARP media growing from a trimmed leaf-bait of off after the spray treatment and bioassayed for PTA using the extended bioassay method.
Himalayan Cedar (Cedrus deodara)

Results
Mycyelial inhibition (Fig 4) Inhibiting PTA in soil (Table 1)
• TriGene and Phytoclean completely suppressed growth of PTA mycelium at all concentrations and no • TriGene and Phytoclean, completely suppressed PTA and all soil fungi/bacteria (Table 1).
mycelium grew from plugs after 8-days exposure. • Virkon and Janola also completely suppressed PTA (Table 1). However, Virkon- and Janola-treated soil
• Virkon (at 0.2 and 0.1% a.i.) completely suppressed growth of PTA and no mycelium grew after 8-days did not suppress all soil
exposure. fungi and bacteria (Table 1). Table 1: Ability of disinfectants to kill PTA in soil. Data represent total number of leaf baits
• Janola (at 0.2, 0.1, 0.05% a.i.) completely suppressed PTA. No mycelium grew after 8-days exposure. Pythium sp. were found in colonised out of 30 and mean number of colonies formed on P5ARP after 3 days (n = 3).

• Citricidal inhibited PTA growth at all concentrations, but mycelial plugs grew out after 8-days the soils treated with Virkon Soil treatment Leaf Baits
Mean CFUs /ml from soil bioassay water

exposure. PTA Pythium spp. Bacteria

and Janola respectively
TriGene (2%) 0 0 0 0
Oospore inhibition (Fig 5) and bacteria were also Phytoclean (10%) 0 0 0 0
• The majority (c. 80%) of the oospores in the unamended control were dormant, about 10% were associated with the Virkon Virkon (1%) 0 0 17.5 ± 24.8 12.5 ± 17.7
activated and the remainder (c. 10%) were non-viable (Fig 5). treated soil. Janola (5%) 8 Pythium spp. 0 45.0 ± 26.1 0

• Virkon and Janola significantly reduced oospore viability, whereas Trigene Phytoclean and Citricidal • Citricidal and the RO water Citricidal
3 PTA
5.0 ± 5.8 47.5 ± 41.0 13.3 ± 14.1
1 P. cinnamomi
had little effect (Fig 5). (control) did not suppress
RO water control 3 PTA 10.0 ± 5.7 44.7 ± 12.0 67.0 ± 23.0
PTA.
Zoospore inhibition
• Trigene (2%), Phytoclean (10%), Virkon (1%) and Janola (5%) all proved lethal to zoospores. Inhibiting PTA in soil on boots
• Spray treatment of spiked soil on boots with TriGene (2%), Phytoclean (10%) and Virkon (1%)
• Many of the zoospores placed in the Citricidal and Control (i.e. RO water) survived.
significantly decreased the number of leaf baits colonised by soil fungi and completely suppressed PTA.
• Janola did not significantly decrease the amount of soil fungi in total, but did suppress PTA after spray
3
Mycelial Inhibition 200
Colour of 200 oospores application.
a a
2.5
b
• Post-spray treatment, PTA was only recovered from boots sprayed with Citricidal and RO water.
Radial growth rate (mm/day)

e
160
Number of oospores

2 % a.i.
C1 0.2% a.i. 120
Dormant
1.5 C2 0.1% a.i.
C3 0.05% a.i. d
Activated Summary of comparative efficacy experiments
C4 0.025% a.i. 80 Non-viable
1 C5 0.0125% a.i. c Activity against Activity against Activity against Ability to kill PTA
Disinfectant Ability to kill PTA in soil
mycelium oospores zoospores in soil on boots
40
0.5
Complete inhibition
TriGene (2%) Complete mortality Little or none Complete kill Yes
0
(incl. Pythium)
0
Tri Q V NaOCl Cit Cont Tri Q V NaOCl Cit Cont
Complete inhibition
Treatment Treatment Phytoclean (10%) Complete mortality Little or none Complete kill Yes
(incl. Pythium)

Figure 4: Growth rates of PTA (mean of five replicates) on agar Figure 5: Oospore viability counts after 10-days incubation in the 5 dis- Virkon (1%) Inhibited radial growth to 1.5 mm/day Some efficacy Complete kill Complete inhibition Yes
amended with 5 disinfectants (Tri = TriGene; Q = Phytoclean; infectants. Bars with the same letter are not significantly different (P = Janola (5%) Inhibited radial growth to 1.15 mm/day Some efficacy Complete kill Complete inhibition Yes
V = Virkon; NaOCl = Janola; Cit = Citricidal; Cont = unamended 0.05).
control) after 4 days (absence of columns equals zero). Citricidal (0.15%) Fungistatic only - did not kill mycelium None None No effect No

Conclusions References
Beever et al. (2009). Kauri Agathis australis under threat from Phytophthora? Proceedings of the 4th
meeting of the IUFRO Working Party S07.02.09. Phytophthora is forests and natural ecosystems, pp. 74–
• TriGene II Advance (2%) is a suitable disinfectant for controlling PTA, killing propagules of PTA, and reducing the infective capacity of soil containing PTA. 85. Ed. by EM Goheen, SJ Frankel. USDA Forest Service. Pacific Southwest Research Station.
Jiang J, Erwin DC 1990. Morphology, plasmolysis and tetrazolium bromide stain as criteria for
determining viability of Phytophthora oospores. Mycologia 87: 107–113.
• Phytoclean is as effective as TriGene. Consideration could be given to registering this product, or similar quaternary ammonium products, for
Stack JP, Millar RL 1985. Relative survival potential of propagules of Phytophthora megasperma f.sp.
phytosanitary applications in New Zealand. medicaginis. Phytopathology 75: 1398–1404.

Acknowledgements
• Virkon and Janola effectively suppress the spread of PTA inoculum contained in soil. However these products have limited application because of
MAF Biosecurity NZ, acting on behalf of the Kauri Dieback Joint Agency Response, for
reports of corrosivity to metal tools and “bleaching” of clothing. funding, and ARC, espcially Dr Nick Waipara, for on-going logistical support. Clémence
Aliaga for technical support and statistical analysis.

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