Inhibition of Phytophthora cinnamomi mycelial growth with stabilised hydrogen peroxide

The efficacy of hydrogen peroxide (H2O2) was evaluated for the inhibition of mycelial growth of Phytophthora cinnamomi in vitro. Phytophthora cinnamomi infects many crops globally causing root, collar and crown rot, resulting in significant economic losses for producers. Two 30% (w/v) H2O2 products, each stabilised with a different concentration of 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP) (3% versus 0.003% w/v) were compared to determine the most efficacious H2O2 concentration as well as potential interactive effects of the stabilising compound. Inhibition of P. cinnamomi growth was evaluated by amending potato dextrose agar media (PDA) with a range of concentrations of the test solutions. The biocidal activity of H2O2 was enhanced by a higher concentration of HEDP. Concentrations from 6.25 mL/L of the H2O2 product with 3% HEDP provided 100% inhibition of mycelial growth in vitro. Neither the product with 0.003% HEDP, nor HEDP stabiliser without H2O2, achieved comparable inhibition. Our results highlight an opportunity to expand the use of stabilised H2O2 products developed for cleaning of drip irrigation emitters to include the control of Phytophthora spp. and potentially other waterborne plant pathogens.


Introduction
Phytophthora cinnamomi Rands is a soil borne oomycete plant pathogen of great economic significance to plant industries worldwide. It can cause root, crown or collar rot in almost 5000 plant species, resulting in enormous losses in horticulture, forestry, agriculture, and in natural ecosystems across the world (Hardham 2005, Jung et al. 2013. Phytophthora cinnamomi is likely to have originated in Papua New Guinea (Dobrowolski et al. 2003). It was first isolated from cinnamon trees in Sumatra in 1922 and is now found worldwide due to movement of contaminated plant materials (Hardham 2005). In Australia, P. cinnamomi infects many economically important crops including avocado, pineapple, macadamia, chestnut and many stone fruit species, including peach (Hardham 2005).
Soil moisture is essential for the production of sporangia and the release and spread of P. cinnamomi zoospores, which means high rainfall and waterlogged conditions greatly exacerbates the disease (Hardham 2005). Conditions favourable to P. cinnamomi reproduction and spread are often found in irrigated cropping and nursery propagation environments. The regular supply of soil moisture through irrigation or movement of contaminated water can encourage pathogen proliferation and spread and result in devastating losses for producers (Hong and Moorman 2005). Hong and Moorman (2005) suggest that contaminated water is the primary source of Phytophthora inoculum in fruit, vegetable and nursery production systems. Hydrogen peroxide (H 2 O 2 ) has been adopted as a disinfectant in irrigation systems, and can aid the control of water borne pathogens (Bosmans et al. 2016;Hong and Moorman 2005;Raudales et al. 2014).
H 2 O 2 is a naturally occurring molecule produced in the environment (Newman 2004) and plant and animal cells. It is also synthesised for a wide range of commercial uses (Linley et al. 2012). Whilst H 2 O 2 is a powerful oxidising agent, it is considered to be environmentally friendly, as it readily breaks down into water and oxygen leaving no toxic residues (Linley et al. 2012). In horticultural applications it 1 3 has potential for implementation into foliar disease control programs, as it has no withholding or re-entry periods following application, and unlike most fungicides, has little toxicity to humans (Copes 2009). One disadvantage, compared to fungicides, is that H 2 O 2 is readily broken down when it reacts with organic matter or catalysts and therefore leaves no residual protection against subsequent pathogen exposure (Copes 2009). However, if H 2 O 2 is stabilised with a chemical such as 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP), pathogen exposure time to H 2 O 2 would be increased. This could potentially provide continuous protection against waterborne, root disease causing pathogens present in irrigation water if applied at a rate which inhibits pathogen survival, growth and spread without being phytotoxic (Copes 2009).
HEDP, also known as etidronic acid, has the molecular formula C 2 H 8 O 7 P 2 . Phosphonate compounds, such as HEDP, are commonly utilised in water treatment systems as corrosion and scale inhibitors due to their ability to sequester metal ions and mineral salts from solution (FAO 2016;Garcia et al. 2001). Addition of HEDP to antimicrobial solutions containing unstable peroxyacids, such as H 2 O 2 , prevents degradation of H 2 O 2 by contaminants and prolongs storage life (EFSA 2014). HEDP is added to antimicrobial solutions as a stabiliser rather than as an antimicrobial agent (FAO 2004). However, the use of HEDP to stabilise H 2 O 2 in drip irrigation systems to reduce emitter clogging may have implications in the control of crop pathogens.
The aims of this research are to establish a minimum inhibitory concentration (MIC) of HEDP stabilised H 2 O 2 for control of P. cinnamomi mycelia in vitro, and to evaluate the potential interactive effects of HEDP and H 2 O 2 .The outcome of this research will provide a basis for further investigation into optimising the use of H 2 O 2 for irrigation emitter cleaning, as well as suppression of Phytophthora spp. and other waterborne pathogens, in order to reduce losses associated with diseases caused by these pathogens.

Products
A series of laboratory trials were carried out to evaluate the inhibition of mycelial growth of Phytophthora cinnamomi in vitro in treatments containing hydrogen peroxide (H 2 O 2 ) stabilised with 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP), or HEDP alone. Potato dextrose agar (PDA) growing medium was amended with a range of concentrations of two HEDP stabilised H 2 O 2 products (referred to as HP Low and HP High ) (Evonik Industries AG, Essen Germany). While both HP Low and HP High contain the same concentration of H 2 O 2 (30% w/v) they contain different concentrations of the stabiliser HEDP with a stated upper limit concentration of 0.003% and 3% (w/v) respectively. For rate calculations and comparisons, these upper limit concentrations of HEDP were assumed. Redox TM Phosphonate HEDP (58 -62% w/v HEDP), without H 2 O 2, was used to evaluate any effect of the stabiliser alone. For calculations and comparisons, it was assumed the Redox TM Phosphonate HEDP concentration was 60% (w/v). This was diluted to 3% prior to addition to agar media, allowing direct comparison with HP High. (Table 1).

Design and procedures
Four laboratory trials using a range of concentrations of the products were conducted. A completely randomised design (CRD) with six independently cultured biological replicates was used in each trial. Culture maintenance and storage procedures were adapted from Drenth and Sendall (2001) and Lawrence et al. (2017). The agar growing medium amendment procedure was adapted from Bekker et al. (2013).

Culture maintenance
Phytophthora cinnamomi cultures were provided by the Queensland Plant Pathology Herbarium, Department of Agriculture and Fisheries, Dutton Park, Queensland 4102 Australia. One isolate (number 66973) was used for all experiments. Cultures were stored as approximately 10 x 5 mm PDA plugs in glass bottles in sterile, reverse osmosis (RO) Millipore TM filtered water in darkness at approximately 22 °C and cultured onto BD Difco™ PDA (39 g/L) when required for trials. All P. cinnamomi cultures were maintained on PDA and incubated at 25 °C in darkness. Independently grown biological replicates were sub -cultured from a single PDA culture onto V8 agar (V8A) containing 20% (v/v) Campbell's V8™ juice, 1% CaCO 3 (w/v) and 1.5% (w/v) technical agar, seven days prior to the start date of each experiment.

Preparation of amended media
Schott bottles each containing 100 mL of PDA (39 g/L) were autoclaved for 15 minutes at 121 °C and allowed to cool to 50 °C in a water bath. Under sterile conditions in a biohazard hood, PDA solution equivalent to the required volume of treatment product to be added was first extracted from the 100 mL of PDA and discarded. The required product to achieve desired final concentration in media was then pipetted into each bottle (Table 2). This method ensured a final solution volume of 100 mL for each treatment. The solution was then gently swirled and inverted to mix for a minimum of 30 seconds before pouring evenly into six, 90 mm polystyrene sterile Petri dishes. Control media was poured directly into the six plates without any removal of solution or the addition of any treatment product. Petri dishes containing media were then left to cool in the biohazard hood for a minimum of 15 minutes prior to inoculation with P. cinnamomi.

Culture and incubation
An approximately 5 mm 2 agar plug was taken from the actively growing edge of the mycelial mat of the randomly assigned biological replicate growing on V8A and placed onto the centre of the PDA media in each Petri dish. All Petri dishes were then wrapped in aluminium foil to prevent exposure to light and drying of the media, and incubated for 5 -7 days in darkness at 25 °C.

Data collection and analysis
Following incubation, two perpendicular measurements of the mycelial mat diameter on each petri dish were taken using digital calipers. These were averaged to obtain a radial growth measurement. All diameter measurements include the V8A culture plug at the centre of growth when visible growth into the surrounding media was present. The inhibition of growth was calculated using the formula from Bekker et al. (2013): where C is the average diameter of the control fungal colony and T is the average diameter of the treatment fungal colony. Statistical analysis was carried out by ANOVA using Genstat ® , 19 th Ed (VSN International). Pearson correlation analysis and linear and exponential rise to maximum regression of data were performed using SigmaPlot ® 14.0 (Systat Software Inc. California).

Measurement of pH and detection of H 2 O 2
The second and third trials conducted involved measurement of the initial and final pH and detection of H 2 O 2 in the amended media. In these trials, 120 mL of PDA media was prepared as described previously. Following addition of treatment products, 20 mL of media was poured into two sterile 50 mL centrifuge tubes (10 mL in each) and allowed to cool for a minimum of 15 minutes before capping and setting aside for initial pH measurement. The remaining 100 mL of media was poured evenly into the six Petri dishes. Control treatments were handled in the same way without the addition of any treatment product. Initial pH measurements were taken within 48 h of media production. For measurement of final pH, media in each Petri dish (including mycelial culture, if present) was cut with a scalpel into small sections, transferred into 50 mL centrifuge tubes and thoroughly cut and mixed with a scalpel to homogenize. This provided two replicates of initial measurements per treatment and six replicates for final measurements. The pH of the media was measured using a calibrated Testo TM 205 hand -held T bar pH meter. The probe was inserted directly into the media at the bottom of the centrifuge tube and washed and dried between each sample. H 2 O 2 was detected by transferring the media from the centrifuge tubes and Petri dishes into 20 mL plastic syringes and forcing it through Millipore TM 0.45 µm nylon mesh syringe filters. Liquid extracted from the media was collected for testing. Quantofix TM peroxide colorimetric test strips were used in a Macherey-Nagel Quantofix Relax reflectance photometer to detect H 2 O 2 with a lower detection limit of 0.5 mg/L.

Results
Mycelial inhibition data from all four trials were collated and presented as Fig. 1 (Fig. 2).
The concentration range of HEDP stabiliser in HP High and HP Low treatments included in Trials 1 -4 was 0.0002 mM -2.11 mM HEDP. HEDP without H 2 O 2 up to 2.11 mM did not significantly inhibit P. cinnamomi growth. Only at very high concentrations, from ca. 5.3 mM, did HEDP inhibit growth. All lower rates either stimulated pathogen growth or had no effect (Fig. 3). Even the highest tested concentrations of HEDP did not result in 100% inhibition (data not shown due to the lack of efficacy of HEDP in isolation).

Amended Media pH
The initial and final pH of the amended media from the second and third trials were measured to determine whether pH was significantly correlated to P. cinnamomi growth in vitro. The correlation between mycelial diameter and initial pH was not significant for either trial (r = 0.79, P = 0.0044 and r = -0.55, P = 0.5407 for Trial 2 and 3 respectively). The correlation between mycelial diameter and final pH was not significant for Trial 2 (r = 0.17, P = 0.0862) but was significant for Trial 3 (r = 0.98, P = 0.2607) (Fig. 4). All final pH values were lower than the corresponding initial pH values except for the Trial 3 HEDP treatments (ca. 1.8 -2.1 mM HEDP), which all showed a similar increase in pH alongside the same level of stimulation of mycelial diameter.

Detection of H 2 O 2 in media
All initial measurements in the second and third trials detected H 2 O 2 except for HP High treatments at 0.001 mL/L (0.00979 mM H 2 O 2 and 0.0002 mM HEDP) and 0.005 mL/L (0.04895 mM H 2 O 2 and 0.0011 mM HEDP) of product. After seven days of incubation, H 2 O 2 was only detected in HP Low at concentrations of 50 mL/L of product (489.5 mM  (Table 3).

Discussion
The results of these trials demonstrate that HEDP stabilised H 2 O 2 significantly inhibits the growth of P. cinnamomi mycelia in vitro. The results suggest HEDP enhanced the biocidal activity of H 2 O 2, as HP High, which contains a higher concentration of HEDP, was significantly more efficacious than HP Low as a biocide against P. cinnamomi. HEDP alone did not inhibit growth at concentrations equivalent to those in the product HP High , and the higher concentrations of H 2 O 2 did not provide comparable levels of inhibition when the concentration of HEDP was reduced. Complete suppression was not achieved with either HP Low or HEDP treatments alone at the concentrations trialled. These results indicate that HP High is a promising product for incorporation into irrigation to supress P. cinnamomi growth and potentially control plant disease.
Although the exact mechanism by which HEDP increased the biocidal effect of H 2 O 2 in this study is not yet fully understood, there have been many studies performed investigating the additive and synergistic effects of chemicals working in combination with H 2 O 2. Steinberg et al. (1999) reported antibacterial synergism between H 2 O 2 and chlorhexidine against Streptococcus spp. The authors suggested that the chlorhexidine interacted with the bacterial cell surface and allowed H 2 O 2 to enter the cell, thereby increasing the antibacterial effect. Zubko and Zubko (2013) demonstrated both additive and synergistic effects of H 2 O 2 and iodine against bacteria and yeast. Many studies on the enhanced antibacterial activity of combining silver and H 2 O 2 exist (Davoudi et al. 2012;Martin et al. 2015;Pedahzur et al. 1997). Martin et al. (2015) demonstrated the biocidal activity of H 2 O 2 was enhanced by the biocidal activity of silver itself, but also by the interaction of silver with the cell membrane of the target organism, making it easier for H 2 O 2 to enter the cell. The authors also suggested that silver increases the stability of H 2 O 2, ensuring it is not degraded as easily, thereby enhancing its effect.
In our study, HEDP is added to stabilise H 2 O 2 in the products tested and may have protected H 2 O 2 from breakdown in the media, resulting in prolonged H 2 O 2 contact with the mycelial cultures and increased biocidal activity. This is supported by the initial and final H 2 O 2 measurements which detected H 2 O 2 in the product with a higher concentration of HEDP, but not in the product with a lower HEDP concentration, despite treatments containing the same initial H 2 O 2 concentration. However, this observation may also be the result of further breakdown of H 2 O 2 by increased mycelial growth in HP Low treatments due to lower biocidal activity. A more detailed analysis of the residual H 2 O 2 in media in the presence and absence of mycelial growth could determine whether these results are specifically due to stabilisation of H 2 O 2 in media by HEDP, or due to more complex interactions between media, mycelium, H 2 O 2 and HEDP. Increased stability of H 2 O 2 is important in applications where contact with organic matter and metal ions will degrade or catalyse the H 2 O 2, impacting the longevity of effect. If HEDP is increasing the biocidal effect of H 2 O 2 in this way, it is a promising formulation for use in irrigation systems as a cleaning product with the added benefit of controlling diseases caused by P. cinnamomi.
H 2 O 2 is also often formulated with peracetic (peroxyacetic) acid for use as an oxidising agent against bacteria and fungi (Brinez et al. 2006). Raudales et al. (2014) provided a comprehensive review of water treatment options that are potentially able to control pathogens. The authors referenced two in vitro industry studies which reported H 2 O 2 concentration as low as 12.3 mg/L combined with 8 mg/L peroxyacetic acid, and as high as 185 mg/L combined with 120 mg/L peroxyacetic acid achieved 100% mortality of Phytophthora spp. (Choppakatla 2009;Steddom andPruett 2012 in Raudales et al. 2014). These contrasting concentrations indicate a need for more detailed MIC studies for stabilised H 2 O 2 products and their ability to control specific pathogens.
The amendment of media with potential biocides is likely to alter the pH of the media, and possibly influence pathogen growth, as shown by the addition of potassium silicate to PDA media (Bekker et al. 2013). In that study, the authors included treatments of pH adjusted PDA in the absence of potassium silicate to investigate the isolated effect of the pH change of each of the potassium Fig. 3 Phytophthora cinnamomi mycelial growth on PDA media (a) Control (b) -(f) HEDP treatments at concentrations equivalent to those of HP High at 0.5, 1, 2, 4 and 8 mL/L respectively silicate concentrations on pathogen inhibition. In our current study, the initial and final pH of the amended media was measured to identify any significant change in pH, and to assess the potential relationships with mycelial growth. The only significant correlation found was between mycelial diameter and pH for the final pH results in one trial, where it was due to an increase in final pH and stimulation of mycelial growth by 3% HEDP treatments. The authors speculate that the pathogen breaks down and removes HEDP and therefore reduces the acidity of the media, as these 3% HEDP treatments had the largest mycelial diameters. There was no such significant increase in growth for the lower rates of HEDP, potentially explaining the lack of pH reduction for those treatments. Also, there was no significant correlation between mycelial diameter and initial pH for the same trial, suggesting pH did not affect growth, but instead, growth affected pH. This indicates pH had no significant effect on the growth of P. cinnamomi and the products tested had a direct biocidal effect.
Even where HEDP treatments did not affect mycelium diameter, the density of the mycelia was visually increasingly sparser with increasing concentrations of HEDP (Fig. 1). This suggests HEDP in isolation had some effect on the mycelial growth, although not necessarily an inhibitory one. The biocidal action of H 2 O 2 was required to reduce colony diameter significantly at those same HEDP concentrations. For greater understanding of the effects of HEDP and H 2 O 2 on mycelial growth in vitro, more functional parameters other than colony diameter, such as reproductive health, and colony density or mass, are suggested.
The current study focussed on mycelial inhibition in vitro. Many publications reported the amendment of growth media with fungicides to inhibit mycelial growth in vitro as a method to investigate the potential for products to be used to control oomycetes in general, and Phytophthora spp. specifically (Bekker et al. 2013;Bittner and Mila 2016;Hu et al. 2007;Hu et al. 2008;Liu et al. 2014;Miao et al. 2016). However, the main method of P. cinnamomi dissemination Fig. 4 Correlation between pH and Phytophthora cinnamomi mycelial diameter on amended PDA (a) Trial 2 initial pH (b) Trial 2 final pH (c) Trial 3 initial pH (d) Trial 3 final pH is via zoospores in moist environments (Hardham 2005). As the products tested in these trials are intended for use in irrigation systems, the next step would be to investigate the concentration of HP High required to inhibit spore survival in irrigation water. Fungal reproductive structures can be more sensitive to biocides than mycelia (Cayanan et al. 2009). However, longer lasting reproductive propagules (e.g., chlamydospores and oospores) and mycelial fragments within organic matter could potentially survive rates required for the control of zoospores. Oospores can be produced in response to an environmental stress (Jung et al. 2013). It is possible that exposure to a biocide could stimulate a stress response in the pathogen and encourage longevity and proliferation. It will be very important to consider how significant these reproductive propagules are in pathogen spread and disease development.
It is also important to consider the effect of H 2 O 2 on plant health. H 2 O 2 applied with nutrient solution in soilless media has been reported to be phytotoxic at ca. 0.24 mM for lettuce (Nedderhoff 2000 in Raudales et al. 2014) and ca. 3.68 mM for cucumber (Vanninen and Koskula 1998). Results from current trials suggest a minimum concentration of ca. 4.9 mM H 2 O 2 is required to inhibit mycelial growth significantly. However, ca. 78.32 mM H 2 O 2 was required for complete control of the pathogen in vitro. This concentration of H 2 O 2 is likely to be phytotoxic to many plant species. However, as the concentration of stabilised H 2 O 2 required to inhibit zoospore survival in irrigation water is likely to be lower than that for mycelial inhibition, the application of HEDP stabilised H 2 O 2 could be kept below phytotoxic thresholds and still efficiently inhibit the pathogen. Disease progression and pathogen mortality need to be investigated in more detail.
Results suggest the higher concentration of HEDP in HP High enhanced the biocidal activity of H 2 O 2 . However, this was not due to biocidal activity of HEDP. It is likely that the higher concentrations of HEDP stabilised the H 2 O 2 in the PDA media more effectively and slowed the breakdown of H 2 O 2, thereby enhancing the biocidal activity of H 2 O 2 . This work provides the basis for further investigation into the use of H 2 O 2 irrigation products for the control of Phytophthora spp. The adoption of HP High as an irrigation cleaning product could have the added benefit of reducing diseases caused by water and soil borne plant pathogens including P. cinnamomi, as used in this research.