Australasian Plant Pathology

, Volume 42, Issue 4, pp 385–392

Antimicrobial activity of essential oils and pure oil compounds against soilborne pathogens of vegetables

Authors

    • Biosciences Research Division, Department of Primary Industries
    • La Trobe University
    • Research Institute for the Environment and LivelihoodsCharles Darwin University
    • Research Institute for the Environment and LivelihoodsCasuarina Campus, Charles Darwin University
  • K. M. Plummer
    • La Trobe University
  • I. J. Porter
    • Biosciences Research Division, Department of Primary Industries
    • La Trobe University
  • E. C. Donald
    • Biosciences Research Division, Department of Primary Industries
    • Department of AgricultureFisheries and Forestry
Article

DOI: 10.1007/s13313-013-0216-0

Cite this article as:
McMaster, C.A., Plummer, K.M., Porter, I.J. et al. Australasian Plant Pathol. (2013) 42: 385. doi:10.1007/s13313-013-0216-0

Abstract

The antimicrobial effects of essential oils and pure oil compounds on mycelial growth of soilborne pathogens causing vegetable diseases in Australia was investigated. Fourteen essential oils and four pure oil compounds were evaluated using contact in vitro bioassays to determine optimum concentrations which can inhibit growth or kill pathogen mycelium. Three essential oils (thyme, clove bud and origanum) and all four pure oil compounds showed broad-spectrum and dose-dependant inhibitory and/or biocidal activity against mycelium of key soilborne pathogens. These treatments also showed antimicrobial activity against Trichoderma atroviride, which is a beneficial soil fungus. In pots, thyme, clove bud and origanum oils applied pre-planting as 5 % aqueous emulsions (5.0 ml/150 cm3) controlled Rhizoctonia solani AG2.1 infection on broccoli seedlings. However clove bud and origanum oils were phytotoxic at 10 % in soil. This study showed that essential oils have potential for controlling soilborne diseases by reducing the spread and viability of pathogen mycelium in soil. This information will be useful for the development of new and existing products which contain plant extracts for use to manage soilborne vegetable diseases in the field.

Keywords

Essential oilsRhizoctonia solani AG 2.1Fusarium oxysporumTrichoderma atrovirideBroccoli seedlings

Introduction

Soilborne plant pathogens cause root infection (rots), wilts and damping off diseases which are a major problem in vegetable production worldwide. Intensive production with successive plantings of susceptible vegetable crops often result in the build up of high population densities of soilborne pathogens (Abawi and Widmer 2000). Poor soil conditions such as compaction, inadequate drainage and loss of organic matter, associated with intensive farming practices, also lead to increased severity of root diseases. In Victoria, Australia, Pythium, Fusarium, Rhizoctonia and Sclerotinia species are the most economically damaging soilborne pathogens affecting a wide range of vegetable crops (Porter et al. 2007). These pathogens produce survival structures including oospores, chlamydospores, melanised hyphae and sclerotia that allow them to persist and spread in soil. In vegetable production, these and other pathogens have typically been controlled by application of synthetic soil fumigants, such as metham sodium, and fungicides. However, problems associated with the use of these chemicals, such as enhanced microbial degradation, proliferation of resistant strains of the pathogens, and concerns about public health and environment contamination, have increased the need for alternatives, especially in the context of Integrated Pest Management (IPM).

In Australia, effective control of vegetable diseases caused by soilborne pathogens is currently hindered by the lack of effective pre-plant soil treatments for reducing pathogen inoculum in soil (Donald et al. 2010). There is therefore an urgent need for new cost-effective, IPM-compatible, and environmentally friendly soil treatments to enhance soilborne disease control. A wide range of plant extracts are currently being investigated and promoted worldwide as novel alternative controls against plant pathogens. Essential oils are one such group, and many have demonstrated antimicrobial activity in vitro against various micro-organisms including Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum (Barrera-Necha et al. 2009), Fusarium solani, Colletotrichum lindemuthianum (Zambonelli et al. 1996), Sclerotium cepivorum (Montes-Belmont and Prados-Ligero 2006), Sclerotinia sclerotiorum (Soylu et al. 2007), S. sclerotiorum, Rhizopus stolonifer, Mucor sp. (Edris and Farrag 2003), Botrytis cinerea (Wilson et al. 1997) and Phytophthora infestans (Soylu et al. 2006).

Essential oils are complex mixtures of up to 60 compounds (Bakkali et al. 2008). These mixtures usually consist of two or three major compounds at fairly high concentrations (20–70 %) plus other compounds present in trace amounts (Bakkali et al. 2008). The antimicrobial activity associated with essential oils is attributed to phenolic and terpenoid compounds such as carvacrol, eugenol, thymol and p-cymene which show high antimicrobial activity when tested in pure form (Zambonelli et al. 1996; Vukovic et al. 2007; Tullio et al. 2007). The effect of the essential oils has been shown to be dose-dependent resulting in either inhibition of pathogen growth or killing (biocidal) (Szczerbanik et al. 2007). A greater understanding of the mode of action including lowest concentrations that kill inoculum will enable development of plant extracts for disease control and a regime for application of commercial formulations containing plant extracts within integrated soilborne disease control programs in vegetable production.

The aim of this study was to determine the effect of plant extracts, including essential oils and pure oil compounds, on mycelial growth of economically important soilborne pathogens isolated from vegetable farms in Victoria, Australia. The research involved a sequential screening process involving in vitro bioassays that evaluated the antimicrobial activity of plant extract treatments, in the contact phase, to identify concentrations that were biocidal to several target pathogens. Bioassays also tested the effect of the treatments on the beneficial soil fungus Trichoderma atroviride. Seedling (broccoli) bioassays further evaluated promising essential oils as pre-plant soil treatments for their ability to reduce damping off caused by R. solani.

Materials and methods

Culturing of pathogens and T. atroviride

Seven soilborne pathogens were isolated from infected crops across farms in Victoria, Australia and later identified by the diagnostic service (Crop Health Services), Department of Primary Industries, Victoria. The pathogens isolated were: Pythium irregulare VPRI 41859 and Pythium sulcatum VPRI 41858 (both isolated from parsley); Pythium aphanidermatum VPRI 32634 (spinach); Fusarium oxysporum VPRI 13039 (capsicum); Pythium sp. aff. dissotocum (green beans); Sclerotinia minor VPRI 41837 (lettuce); and Rhizoctonia solani anastomosis group (AG) 2.1 VPRI 41912 (broccoli).

The pathogens were maintained on Potato Dextrose Agar (PDA) medium (Oxoid®, 39 g per 1 L deionised water), except for Pythium spp. which were maintained on V8 medium (48 mL V8 juice in 1 L deionised water adjusted to pH 6.5, plus 20 g/L technical agar (Oxoid®)). Trichoderma atroviride (formerly described as T. hamatum) was isolated from a dry formulation of the commercial product, Lettuce Mate® (Agrimm Technologies Ltd. NZ). One gram of Lettuce Mate® was mixed in 9 mL sterile distilled water, shaken, and a 0.5 mL aliquot from this solution spread onto PDA. A colony of T. atroviride was subcultured and maintained on PDA. All cultures were stored by placing plugs of mycelium in sterile distilled water in McCartney bottles and kept at room temperature until used.

Plant extracts

Fourteen essential oils and four pure oil compounds were tested for their antimicrobial activity against pathogens using in vitro bioassays. The essential oils were basil, black pepper, cardamom, clove bud, eucalyptus, fennel sweet, geranium, orange sweet, origanum, peppermint, pine, rosemary, tea tree and thyme (Sydney Essential Oils Company). Chemical analysis of the essential oils, determined by Gas Chromatography, was provided by Sydney Essential Oils Company. This analysis showed thymol (49.3 %), carvacrol (39.0 %), eugenol (77.4 %), and geraniol (17.0 %) were the major chemical constituents in thyme, origanum, clove bud and geranium oils, respectively. The pure compounds tested were: thymol (99.5 % purity); carvacrol (>98 % purity); eugenol (99 % purity); and geraniol (98 % purity) (Sigma-Alrich).

In vitro effect of plant extracts on mycelial growth

Bioassay 1: effect of plant extracts in high concentrations

The effect of the plant extract treatments on mycelial growth in direct contact was investigated using a method similar to that described by Barrera-Necha et al. (2009). Aliquots of the fourteen essential oils and four pure compounds were aseptically pipetted into media (40–45 °C) at concentrations of 500, 1,000 and 2,500 ppm prior to pouring into 95 mm diameter plastic Petri dishes (approximately 20 ml PDA/dish). Each plate was then centrally inoculated with an agar plug (5 mm diameter) taken from the actively growing edge of 4–7 day old cultures of P. irregulare, P. aphanidermatum, P. sulcatum, F. oxysporum and R. solani AG 2.1. The inoculated plates in the same treatments were sealed together with plastic wrap before being incubated inside a laboratory laminar flow cabinet at 20–23.5 °C under natural light (9–14 h per day) for 10 days. There were 4 replicate plates per treatment, with control plates wrapped separately.

Colony diameter of mycelium was measured over several days depending on the growth rate of each pathogen, using a digital Vernier caliper (DVC 150). Measurements were taken on control plates until mycelium had grown to the edge of the plates, then this growth was compared to growth measured on treated plates at that time. After this period, plugs from plates where no growth had been observed were transferred to fresh unamended PDA plates to determine whether the treatment effect had been biocidal or inhibitory. This biossay was repeated with seven of the treatments at 500 ppm and three lower concentrations (bioassay 2 and 3) to further evaluate these treatments for their inhibitory and biocidal antimicrobial activity at lower concentrations.

Bioassay 2 and 3: effect of plant extracts in low concentrations

Three essential oils (clove bud, thyme, origanum) and four pure compounds (thymol, eugenol, carvacrol and geraniol) were tested at reduced concentrations. Tween 80 (Sigma®) at 0.1 % v/v was added as an emulsifier to the media along with the treatments. A preliminary test, not reported here, indicated that Tween 80 at 0.1 % did not affect mycelial growth of the test organisms. Each plate with amended media was inoculated as described before with 4–7 day old day cultures of S. minor, Pythium sp. aff. dissotocum, and two isolates tested in bioassay 1 (F. oxysporum, and R. solani AG2.1). T. atroviride was included in the tests to determine the effect of treatments on this beneficial soil fungus. Inoculated plates were incubated under the conditions described previously, with 4 replicate plates per treatment. Colony diameter of mycelium was measured for each treatment and compared to the control as previously described.

Effect of plant extracts on R. solani AG2.1 inoculum in soil

Pot bioassays 1 and 2

Three essential oils (thyme, origanum, clove bud) which demonstrated antimicrobial activity against R. solani in the in vitro bioassays were tested for their ability to reduce R. solani inoculum in soil. These treatments were compared to the soil fumigant Basamid® [BASF; dazomet 98 % methyl isothiocyanates (MITC)]. Inoculum of R. solani was prepared in wheat grain (255 g plus 345 mL dionised water) that had been twice autoclaved (121 °C for 60 min) 48 h apart. The sterilised grain was inoculated with five plugs of mycelium (5 mm diameter) removed from the actively growing edge of 7 day old cultures of R. solani AG2.1 and incubated at 21 °C for 14 days. Inoculated grain was blended with sterile distilled water into a slurry prior to mixing with pasteurised potting mix (vegetable seed raising mix, BioGro™) in a cement mixer at the rate of 25 g inoculum per kg mix. A control treatment consisting of uninoculated wheat grain was included.

Treatments were added to the inoculated mix inside sealable plastic bags at 5 % and 10 % (oils) as aqueous emulsions (5.0 mL/150 cm3 soil) and at 1.1 and 2.2 g/kg (equivalent to 200 and 400 kg/ha, respectively, Basamid®) and incubated for 48 h. After this period, treated soil was transferred to pots (90 mm diameter), with four replicates per treatment. All pots, including a pathogen only treatment, were arranged according to a randomised complete block design on a glasshouse bench at an ambient temperature of 21 °C (±5 °C). In pot bioassay 1, the pots were then watered using fine overhead irrigation sprinklers at 7 days after the broccoli seeds were planted. In pot bioassay 2, watering began before being planted with broccoli seeds. Five broccoli seeds (Brassica oleracea, cv. Marathon) were planted per pot and pots were watered twice daily for 1 min. The number of seedlings per pot was recorded 14 days after planting. The effect of treatments on infection by R. solani was determined indirectly by comparing seedling survival between pots. This experiment was conducted twice.

Statistical analysis

Colony diameter values were analysed separately for each pathogen due to their different growth rates. Treatment effects for in vitro bioassays were analysed using a two way ANOVA with treatment type and concentration as factors using Genstat, 12th edition (Lawes Agricultural Trust, Rothamstead Experimental Station, Harpenden, UK). Treatment effects for the pot trial (number of seedlings per pot) were analysed also using a two way ANOVA with treatment, rate and inoculum as factors. Significant effects identified by an ANOVA at P ≤ 0.05 were further explored using Fisher’s LSD (least significant difference) tests with mean treatment values. For in vitro bioassays, to facilitate comparison of treatment effects on mycelial growth across the many pathogens and treatment concentrations, all mean values were presented in tables as symbols as follows: no effect on mycelial growth (−), inhibitory effect on growth only (+) and inhibitory plus biocidal effect on mycelium (++), all compared to untreated controls.

Results

In vitro effect of plant extracts on mycelial growth

From the fourteen essential oil treatments tested, only thyme, origanum and clove bud resulted in significant reductions in mycelial growth plus biocidal activity against all four pathogens at all concentrations tested in bioassay 1, except for F. oxysporum at 500 ppm (Table 1). The inhibitory and biocidal activity of these three treatments at 500 ppm against mycelium of R. solani and S. minor (clove bud and origanum), and Pythium spp. (thyme) was confirmed in bioassay 2 (Table 2). Concentrations less than 500 ppm were frequently inhibitory but not biocidal (Table 2). Basil, geranium, peppermint, pine and tea tree oils were biocidal to R. solani at the highest concentration of 2,500 ppm (Table 1). Six of the fourteen essential oil treatments resulted in significant reductions in mycelial growth but not biocidal activity (Table 1).
Table 1

In vitro antimicrobial activity of essential oils and pure compounds on mycelial growth of four soilborne pathogens: P. irregulare (Pi); P. aphanidermatum (Pa); P. sulcatum (Ps); F. oxysporum (Fo); and R. solani (Rs) (bioassay 1)

Treatment

Concentration (ppm)

500

1000

2500

500

1000

2500

500

1000

2500

500

1000

2500

500

1000

2500

P. irregulare

P. aphanidermatum

P. sulcatum

F. oxysporum

R. solani

Clove bud oil

++a

++

nt

++

++

nt

++

++

nt

+

++

++

++

++

++

Eugenol

+

++

++

++

++

++

++

++

++

+

+

++

++

++

++

Thyme oil

++

++

nt

++

++

nt

++

++

nt

+

++

++

++

++

++

Thymol

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

Origanum oil

++

++

nt

++

++

nt

++

++

nt

+

++

++

++

++

++

Carvacrol

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

Geranium oil

+

nt

+

+

nt

+

+

nt

+

+

+

+

Geraniol

+

++

++

++

++

++

++

++

++

+

++

++

++

++

++

Peppermint oil

+

+

nt

+

nt

+

+

nt

+

+

+

+

++

Pine oil

+

nt

+

nt

+

+

nt

+

+

+

+

+

++

Basil oil

B

+

nt

+

nt

+

nt

+

+

+

+

++

Tea tree oil

nt

+

nt

+

+

nt

+

+

+

+

++

Fennel sweet oil

nt

nt

nt

+

+

+

+

+

+

Black pepper oil

nt

nt

+

nt

+

+

+

Cardamom oil

nt

nt

nt

+

+

+

Eucalyptus oil

nt

nt

nt

+

+

+

Rosemary oil

nt

nt

nt

nt

nt

nt

nt

nt

nt

+

+

+

+

Orange sweet oil

nt

nt

nt

+

Mycelial growth measured after 6 (Pi, Pa, Ps) or 8 days (Fo, Rs) of incubation on amended media

a− = no effect; + = mycelial growth significantly (P < 0.001) reduced on amended media compared to untreated control;

++ mycelial growth also significantly (P < 0.001) reduced and treatment was biocidal (mycelium did not growth after plugs transferred to unamended PDA media)

Treatment x concentration effect significant (P < 0.001) for all pathogens

Nt treatment concentration not tested

Table 2

In vitro effect of selected essential oils and pure compounds treatments from bioassay 1 tested at lower concentrations on mycelial growth of four soilborne pathogens: Fusarium oxysporum (Fo); Rhizoctonia solani (Rs); P. sp aff. dissotocum (Pd).; Sclerotinia minor (Sm); and the beneficial soil fungus Trichoderma atroviride (Ta) (bioassay 2 and 3)

Treatment

Concentration (ppm)

F. oxysporum

R. solani

P. sp. aff. dissotocum

S. minor

T. atroviride

Clove bud oila

1

+c

+

+

10

+

+

+

+

100

+

+

+

+

+

500

+

++

+

++

+

Eugenolb

1

+

+

10

+

+

100

+

+

+

+

+

500

+

++

+

+

+

Thyme oila

1

+

+

+

+

10

+

+

+

+

+

100

+

+

+

+

+

500

+

+

++

+

+

Thymola

1

+

10

+

+

+

+

+

100

+

+

+

+

+

500

++

++

++

++

++

Origanum oilb

1

+

+

10

+

+

+

100

+

+

+

+

+

Carvacrolb

500

+

++

+

++

+

1

+

+

+

10

+

+

+

100

+

+

+

+

+

500

++

++

++

++

+

Geraniola

1

+

+

10

+

+

+

+

+

100

+

+

+

+

+

500

+

+

++

++

+

aMycelial growth measured after 4 (Fo, Rs), 3 (Pd), or 2 days (Sm, Ta) of incubation on amended media

bcolony diameter measured after 7 (Fo), 3–4 (Rs, Pd, Ta) or 2 days (Sm) of incubation on amended media

c− = no effect; + = mycelial growth significantly (P < 0.001) reduced on amended media compared to untreated control; ++ mycelial growth significantly (P < 0.001) reduced and treatment was biocidal (mycelium did not resume after plugs transferred to unamended PDA media)

Treatment x concentration effect significant (P < 0.001) for all pathogens

All treatments had 0.1 % Tween added as an emulsifier

All four pure compounds tested (thymol, carvacrol, eugenol, and geraniol) were biocidal to mycelia of P. aphanidermatum, P. sulcatum and R. solani at concentrations of 500, 1,000 and 2,500 ppm (Table 1). Carvacrol and thymol were also biocidal to mycelia of P. irregulare and F. oxysporum at the same concentrations, but geraniol was biocidal to these two pathogens only at the two highest concentrations. The biocidal activity of thymol and carvacrol at 500 ppm against mycelium of two of the pathogens tested in bioassay 1 (F. oxysporum, R. solani) and P. sp. aff. dissotocum and S. minor, was confirmed in bioassay 2 (Table 2). These two treatments resulted in significant reductions in mycelial growth for R. solani and P. sp. aff. dissotocum at 100 and 10 ppm compared to untreated controls (Table 2). Only thymol at 500 ppm was biocidal to T. atroviride (Table 2). Eugenol was also inhibitory and biocidal to P. irregulare at 1,000 and 2,500 ppm and F. oxysporum and 2,500 ppm (Table 1). The biocidal activity of eugenol against R. solani and geraniol against Pythium spp. at 500 ppm was confirmed in bioassays 2 and 3 (Table 2).

When comparing the pure compound to the oil containing the same compound, generally there was similar biocidal activity between the two for eugenol (clove bud oil), thymol (thyme oil) and carvacrol (origanum oil) at most of the higher concentrations (bioassay 1) and pathogens tested (Table 1). The exception was the pure component geraniol which was more effective than geranium oil in killing mycelium in all cases (Table 1). This similar efficacy was repeated only for eugenol (clove bud) on R. solani, thymol (thyme oil) on P. sp. aff. dissotocum, and carvacrol (origanum oil) R. solani and S. minor at 500 ppm in bioassay 2 (Table 2).

Effect of plant extracts on R. solani AG 2.1 inoculum in soil

In pot trial 1, the mean number of broccoli seedlings in pots with R. solani inoculum was also significantly lower (0.4 seedlings/pot) than in untreated controls (4.8 seedlings/pot) (Table 4). Basamid® at 2.2 g/kg was the only treatment with statistically similar levels of seedling number per pot compared to untreated controls. For the oil treatments, broccoli emergence was generally lower in pots from trial 1 than in pots from trial 2 due to the phytotoxicity effect of oils resulting from different watering regimes of treated soil before planting. Although seedling numbers were relatively lower in pot trial 1, soil treated with thyme oil at 10 % had significantly more seedlings (2.8 seedlings/pot) than all other oil treatments (0.0–1.6 seedlings/pot) evaluated.

In pot trial 2, treatment of soil with essential oils resulted in significant differences in mean seedling number among oil treatments and rates with significant interaction between treatment and rate for treatment with similar concentrations (Tables 3 and 4). The mean number of broccoli seedlings in pots with R. solani inoculum was significantly lower (0.3 seedlings/pot) than in untreated controls (4.2–5.0 seedlings/pot) (Table 4). There was no significant difference in seedling number between pots with untreated soil and pots with either uninoculated and inoculated soil both treated pre-plant with thyme oil at 5 %, clove bud oil at 5 % and Basamid® at 2.2 g/kg, indicating that these treatments significantly reduced R. solani inoculum which in turn increased seedling survival (Table 4). However, there was a significant difference in the number of seedlings between inoculated (0.0 seedlings/pot) and uninoculated (4.7 seedlings/pot) soil both treated with 10 % thyme oil. This indicates that thyme oil applied at 10 % did not provide protection of broccoli seedlings against R. solani infection. Basamid® at 1.1 g/kg was also ineffective in reducing R. solani infection. Origanum and clove bud oils at 10 % were phytotoxic to seedlings in uninoculated soil. This suggests that the low seedling survival in inoculated soil may have been due to pathogen and phytotoxicity effects at rates of 10 % (Table 4).
Table 3

Analysis of variance for effects of two rates of thyme, origanum, and clove bud on broccoli seedling survival in inoculated soil

Sourcea

df

F value

P > F

Essential oil

2

14.59

<0.001

Rate

1

205.82

<0.001

Oil x rate

2

5.63

0.006

aData were analysed as mean number of Broccoli seedlings per pot

Table 4

Mean number of broccoli seedlings growing in soil inoculated with R. solani AG 2.1 which was treated pre-sowing with thyme, origanum and clove bud essential oils or a soil fumigant (Basamid®)

Treatment

Rate

Inoculum addedb

Soil treatmentb

Mean number seedlings/potc

Trial 1d

Trial 2d

Untreated soil

 

4.8 d

5.0 e

Substrate onlya

4.8 d

4.2 de

Inoculated soil

+

0.4 a

0.3 ab

Thyme

5 %

+

+

0.2 a

4.0 d

5 %

+

nt

4.8 de

10 %

+

+

2.8 c

0.0 a

10 %

+

nt

4.7 de

Origanum

5 %

+

+

1.6 b

3.0 c

5 %

+

nt

4.7 de

10 %

+

+

0.6 ab

0.5 ab

10 %

+

nt

0.0 a

Clove bud

5 %

+

+

0.0 a

4.2 de

5 %

+

nt

4.0 d

10 %

+

+

0.4 a

0.2 ab

10 %

+

nt

0.7 ab

Basamid®

1.1 g/kg

+

+

0.0 a

1.0 b

1.1 g/kg

+

nt

5.0 e

2.2 g/kg

+

+

5.0 d

4.5 de

2.2 g/kg

+

nt

5.0 e

aSterilised wheat grain was used as a substrate to grow R. solani AG 2.1 mycelial inoculum

b+ = inoculum and treatment added to soil before sowing

cNumber of broccoli seedlings in pots, 14 days after sowing

dMean values within columns followed by the same letter are not statistically different at P = 0.05

Discussion

Three of the fourteen essential oil treatments screened (clove bud, thyme and origanum) were found to have excellent in vitro inhibitory and biocidal antimicrobial activity when mixed with media against mycelial inoculum of several soilborne pathogens isolated from vegetable crops. These oils were biocidal to mycelium at concentrations ranging from 1,000 to 2,500 ppm, efficacy became more variable at 500 ppm, and was only inhibitory or ineffective below these concentrations. Previous studies have demonstrated the antimicrobial activity of thyme, clove and origanum oils against other plant pathogenic organisms (Wilson et al. 1997; Montes-Belmont and Prados-Ligero 2006; Tullio et al. 2007; Soylu et al. 2006, 2007; Lee et al. 2007). In this study, the biocidal activity of these oils varied among isolates tested with F. oxysporum being harder to kill than other isolates at 500 ppm. This may be due to differences in the resilience of mycelium between the species tested. Tullio et al. (2007) also reported differences in the susceptibility of pathogens (including F. oxysporum) to thyme, clove and other essential oils. Research into the specific mode of action of these plant extracts may help to develop commercial antimicrobial products.

The three pure compounds (eugenol, thymol, and carvacrol) found in the best three oil treatments (clove bud, thyme, and origanum, respectively) as well as geraniol found in geranium oil, had excellent in vitro inhibitory and biocidal antimicrobial activity in media against mycelial inoculum of the pathogens tested. Their biocidal efficacy was also consistent at concentrations of 1,000 and 2,500 ppm, more variable at 500 ppm, and only inhibitory or ineffective below this level. Chemical analysis showed that the major compounds in clove bud, thyme and origanum oils were the phenolic compounds eugenol (77.4 %), thymol (49.3 %) and carvacrol (39.0 %), respectively, and that geranium oil contained geraniol (17.0 %). Origanum and thyme oils also contained lower concentrations of thymol (13.5 %) and carvacrol (2.4 %), respectively. Previously, studies have shown that the antimicrobial activity of essential oils is due to their major chemical constituents (Muller-Riebau et al. 1995; Dorman and Deans 2000; Letessier et al. 2001). The fungicidal activity of thyme oil (against R. solani and other pathogenic fungi) and clove oil (against filamentous fungi) were attributed to thymol, and carvacrol and eugenol respectively (Zambonelli et al. 1996; Tullio et al. 2007).

Clove bud, thyme and origanum oils also showed indirect biocidal activity against R. solani mycelial inoculum in soil, by increasing broccoli seedling survival when applied pre-planting at concentrations of 5 %. However, their efficacy in soil was variable with little biocidal effect and phytotoxicity observed at the low and high rates tested, respectively, in the first pot trial. The phytotoxic effect was most likely caused by high levels of oil volatiles still present in dry soil at planting compared to soil in pot trial 2, which were watered several times before planting. We hypothesise that irrigation of treated soil before transplanting or sowing will enhance evaporation or leaching of the plant oils from soil and minimise phytotoxic effects. This information could be used to optimise application of existing commercial products formulated with these plant extracts such as Promax™ (3.5 % thyme oil) for control of soilborne diseases without phytotoxic effects. This could be achieved by identifying suitable irrigation and plant-back periods following in-field treatment application.

The majority of in vitro studies have investigated the antimicrobial effects of essential oils in the liquid or contact phase, yet many recent studies have shown that essential oil vapours also have potential for controlling various plant pathogens (Cavanagh 2007; Lee et al. 2007; Szczerbanik et al. 2007; Kumar et al. 2008). Results from other preliminary laboratory in vitro experiments not presented here also indicated that origanum oil, clove bud oil, thyme oil and eugenol have biocidal activity in the volatile (vapour) phase against some of the target soilborne pathogens tested. Further studies are therefore required to investigate the biocidal activity of promising treatments in the volatile phase to determine optimum concentrations that kill pathogen mycelium under variable soil moisture/air conditions to enhance their biocidal activity in soil. These plant extracts also need to be further tested in vitro against other inoculum structures such as melanised sclerotia and hyphae produced by Sclerotinia spp. and R. solani before being tested for their ability to reduce inoculum and disease in the field.

In this study, several plant extracts were inhibitory and/or biocidal at 500 ppm to the beneficial soil organism, T. atroviride. This indicates that biocontrol products used in vegetable production, for example Tenet™ (T. atroviride) used for onion white rot in New Zealand, should be applied many weeks after application of oil or any other synthetic soil treatments, to ensure that biocontrol proliferation and efficacy is not affected (Villalta pers. comm.). The economic feasibility of applying these plant extracts for disease control in vegetable production systems need to be investigated.

Acknowledgments

This work was funded by the Victorian Department of Primary Industries and Horticulture Australia Ltd (HAL). We thank Dr Elizabeth Minchinton, Desmond Auer, Denise Wite, and Crop Health Services DPI Victoria for pathogen isolates, Agrimm Technologies Pty Ltd for the biocontrol product, and Sydney Essential Oils Company for chemical analysis. We also thank Dr Jacqueline Edwards, Dr Oscar Villalta and Damien McMaster for providing comments which improved the manuscript.

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© Australasian Plant Pathology Society Inc. 2013