Influence of Pure Mulches on Suppressing Phytophthora Root Rot Pathogens

Soil borne pathogens such as Phytophthora spp. can result in high mortality rates and/or impaired growth during the production of woody ornamental trees and shrubs. The positive benefits of mulching as a means of culturally managing Phytophthora spp. are becoming widely recognised. The influence of a pure mulch, i.e., mulch derived solely from one tree species, on reducing Phytophthora root rot severity has received little study. Results of this investigation show that use of a pure mulch derived from European beech (Fagus sylvatica L.), common hawthorn (Crataegus monogyna JACQ), silver birch (Betula pendula ROTH.), common cherry (Prunus avium L.), evergreen oak (Quercus ilex L.) and English oak (Q. robur L.) reduced root lesion severity caused by P. cactorum and P. criticola by 33–63%. Selection of an appropriate pure mulch is important, however, as reductions in Phytophthora root rot lesion severity varied between mulches.

Phytophthora is a genus of plant-damaging pathogens whose member species are capable of causing enormous economic losses to arable crops worldwide, as well as environmental damage in natural, forest and urban ecosystems (13, 18). Phytophthora pathogens are also widespread and damaging to woody plants that are commonly found in managed town and city landscapes (37). Several species of Phytophthora attack the fine absorbing roots of plants and may invade larger roots and the root collar. These pathogens grow in the cambium and sapwood causing death of the tissue. Loss of water and nutrient absorbing capacity and stored carbohydrate reserves in the root cause a gradual or sometimes-rapid decline of the above ground portion of the plant (13). Physiologically, stem canker-causing Phytophthora species (e.g., P. cinnamomi and P. cactorum) are known to kill phloem tissue leading to plant death through girdling, and also to colonize and block xylem, leading to altered plant water relations (2). Control of Phytophthora species is primarily by the use of synthetic chemical drenches and/or injections around the root collar (37). Increased pathogen insensitivity to chemical plant protection products coupled with public demands to reduce pesticide use, stimulated by greater awareness of environmental and health issues has placed more emphasis on alternative pathogen control strategies (1, 17, 28). Consequently, non-chemical management options are increasingly being sought to preserve valuable infected specimen trees and protect non-infected hosts (37).

Mulching as a means of reducing soil moisture stress, weed suppression and fertilising have been used in arboricultural, agricultural, fruit and ornamental crops production systems for decades (3). Mulching as a means of reducing soil borne diseases such as Phytophthora spp, is now recognised as a potentially useful cultural management option (6, 7, 8, 9, 10). Physically, mulches reduce splashing of rain or irrigation water, which can carry spores of disease causing organisms to the stems or leaves of susceptible host species. Additionally, the populations of beneficial microbes that colonize organic based mulches can reduce soil pathogens either through direct competition for resources or through chemical inhibition. Regardless of the mechanism involved, disease reduction is an important benefit of many mulches (3, 8, 33).

Limited studies exist focusing on the efficacy of mulches derived solely from one tree species, defined as an pure mulch for the purposes of this study, on suppression of Phytophthora pathogens. However, information available indicates the use of a pure mulch can have a powerful influence on transplant success and subsequent survival of trees. The effect of fresh and composted pure organic mulches of Eucalyptus cladocalyx mulch was found to have a positive effect in transplant performance of Platanus racemosa (8) while fresh pine bark was found to affect Quercus robur establishment through weed suppression but not through an effect of mulching itself (15). Pure mulches derived from the common hawthorn (Crataegus monogyna JACQ), and common cherry (Prunus avium L.) increased survival rates of European beech (Fagus sylvatica L.) from 10 to 70% following containerisation and under field conditions enhanced fruit tree crown volume and fruit yield by 53 and 100% compared to non-mulched trees (29).

The purpose of the conducted research was to determine if a range of pure mulches can i) reduce the development and impact of pathogen severity caused by Phytophthora cactorum and P. criticola spp. on containerised horse chestnut (Aesculus hippocastanum), and ii) quantify alterations in host plant vitality with respect to leaf photosynthetic efficiency, leaf chlorophyll content, photosynthetic rates and root carbohydrate concentration.

Field site and experimental trees. Two-year-old, bare-rooted stock of horse chestnut (Aesculus hippocastanum L.) was obtained from a commercial supplier and planted into 20 litre pots containing a general tree compost (loamy texture, with 23% clay, 46% silt, 31% sand, 3.1% organic carbon, pH 6.6) supplemented with the controlled release nitrogen-based N:P:K (29:7:9) fertiliser Bartlett BOOST (The Doggett Corporation, Lebanon, NJ, USA) at a rate of 5.0 g·kg⁻¹ soil. Following potting, trees remained outdoors on a free-draining mypex covered surface at the University of Reading Shinfield Experimental Site, Reading, Berkshire (51°43′ N, 1°08′ W) subject to natural environmental conditions and watered as required. For the following two years from 2007 to 2009 trees were trained to produce a central-leader system to an average height of 1.0 ± 0.15 m (3.3 ± 0.5 ft) with mean trunk diameters of 8.0 ± 1.5 cm (3.2 ± 0.6 in) at 60 cm (24 in) above ground level. A minimal insecticide and fungicide program to prevent horse chestnut leaf miner and Guignardia blotch outbreaks were performed based on the pyrethroid insecticide deltamethrin and triazole fungicide penconazole (product names Bandu and Topas, respectively, Headland Agrochemicals Ltd, Saffron Walden, Essex, UK) and applied every six week during the growing season. All sprays were applied using a hand-held sprayer at 7.5 ml (0.25 fl oz) deltamethrin and 3.5 ml (0.12 fl oz) penconazole per litre (35 fl oz) of water. Trees were sprayed until runoff, generally 0.15 litre (5.3 fl oz) per tree.

Pure mulches. Branches ≤8 cm (3.2 in) of European beech (Fagus sylvatica L.), common hawthorn (Crataegus monogyna JACQ), silver birch (Betula pendula ROTH.), common cherry (Prunus avium L.), evergreen oak (Quercus ilex L.) and English oak (Q. robur L.) were pruned from mature landscape trees and chipped with a commercial brush chipper to produce 4–6 cm (1.6 2.4 in) long chips. Each mulch was used fresh, i.e., immediately after chipping without any form of composting. All mulches were made when trees were fully dormant (February) when, with the exception of evergreen oak, no foliage was present on the tree. All mulches were prepared from trees located at the University of Reading Shinfield Experimental Site, University of Reading, Berkshire (51°43′ N, 1°08′ W). Each pure mulch was applied to each pot at a depth of 10 cm (4 in) on February 13, 2009. Pots were arranged in a randomized block design with 10 trees per mulch treatment. Non-mulched trees acted as controls. Pots were placed outdoors on black polyethylene mat to avoid under growing weeds at 1.5 m (4.9 ft) spacing to reduce competition for light. Pots were re-randomized every 4 weeks. No fertilizer was applied during the experimental period and any weeds were removed manually from pots when observed. Irrigation was applied as required.

Phytophthora inoculum. Pure cultures of Phytophthora cactorum and P. criticola were obtained from CBS-KNAW Fungal Biodiversity Centre, The institute of the Royal Netherlands Academy of Arts and Sciences, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. Plants were inoculated after bud break with bud break occurring ca. April 12, 2009. Inoculum of both pathogens was produced on sterilized rice grains using the protocol of Holmes and Benson (19). In summary 18 ml (0.6 fl oz) of distilled water was added to 25 g (0.9 oz) of long-grain white rice in 250-ml Erlenmeyer flasks; flasks were autoclaved for 30 min and then autoclaved again 24 hr later for 40 min. After sterilizing, three PARPH-V8 agar plugs taken from the margins of actively growing Phytophthora colonies were added to the rice. Flasks were then placed in the dark at 25C (77F) and gently shaken daily to ensure uniform colonization of the rice grains. Inoculum was incubated for 14 days. Twelve colonized rice grains were added to each 20.0 litre pot around the peripheral edge of the root ball to a depth of 5 cm (2 in). Plants were watered immediately following inoculation and then watered daily to maintain conditions conducive for Phytophthora development. In addition to daily watering, plants were flooded once a week for 24 hr to further encourage root rot development. All plants were located outdoors subject to natural climatic weather for 14 weeks (July 28, 2009) before final evaluation of plant health and Phytophthora pathogen severity.

Phytophthora root rot lesion severity. Severity of both Phytophthora pathogens was assessed by recording the percent infection of each root system using a modified pre-transformed scale based on Lyles et al. (25): 0 = 0% no visible symptoms of Phytophthora infection, 1 = 1 to 15% of the root system infected, 2 = 16 to 50% of the root system infected, 3 = 51 to 85% of the root system infected, and 4 = >85% of the root system infected. All plants were harvested and the root system carefully washed. Water soaked, discoloured regions of the root were classified as Phytophthora infected (20).

Tree vitality. Five leaves randomly selected throughout the crown per tree were used for chlorophyll fluorescence and chlorophyll content measurements. Leaves were adapted to darkness for 30 min by attaching light-exclusion clips to the leaf surface, and chlorophyll fluorescence was measured using a HandyPEA portable fluorescence spectrometer (Hansatech Instruments Ltd, King's Lynn, UK). Measurements were recorded up to 1 sec with a data-acquisition rate of 10 μs for the first 2 ms and of 1 ms thereafter. The fluorescence responses were induced by a red (peak at 650 nm) light of 1500 μmol·m⁻²·s⁻¹ photosynthetically active radiation (PAR) intensity provided by an array of six light-emitting diodes. The ratio of variable (Fv = Fm – Fo) to maximal (Fm) fluorescence, i.e., Fv/Fm where Fo = minimal fluorescence, of dark-adapted leaves was used to quantify any effects on leaf tissue. Fv/Fm is considered a quantitative measure of the maximal or potential photochemical efficiency or optimal quantum yield of photosystem II (35). Likewise Fv/Fm values are the most popular index used as a measure of plant vitality and early diagnostic of stress (26).

Leaf chlorophyll content was measured at the mid point of the leaf next to the main leaf vein by using a hand held optical Minolta chlorophyll meter SPAD-502 (Spectrum Technologies, Inc. Plainfield, IL, USA). Calibration was obtained by measurement of absorbance at 663 and 645 nm in a spectrophotometer (PU8800 Pye Unicam, Portsmouth, UK) after extraction with 80% v/v aqueous acetone (regr. eq. y = 5.66 + 0.055x; r² adj = 0.89, P = <0.01) (23).

The light-induced CO₂ fixation (Pn) was measured in pre-darkened (20 min), fully expanded leaves from near the top of the canopy (generally about 4 nodes down from the apex) by using an Infra Red Gas Analyser (LCA-2 ADC BioScientific Ltd Hoddesdon, Herts, UK). The irradiance on the leaves was 700 to 800 (mol·m⁻² photosynthetically active radiation saturating with respect to Pn; the velocity of the airflow was 1 ml·s⁻¹·cm⁻² of leaf area. Calculation of the photosynthetic rates was carried out according to Von Caemmerer and Farquhar (34). Two leaves per tree were selected for measurements.

The carbohydrate (glucose + fructose + sucrose) and starch concentration within root tissue of insecticide and non-insecticide treated trees was quantified using HPLC (36). Fine roots were collected by using a root density corer. Four 20 cm (8 in) deep, 3 cm (1.2 in) diameter cores were taken (North, South, East, West) 10 cm (4 in) from the base of each tree and stored at 4C until processing. Soil was washed from the roots, and horse chestnut roots were separated from debris and by hand.

Effects of mulch application on chlorophyll fluorescence, photosynthetic rates, chlorophyll concentrations and growth were determined by both two and one way analyses of variance (ANOVA) as checks for normality and equal variance distributions were met using an Anderson-Darling test. Differences between treatment means were separated by Tukey's Honestly Significant Difference test (HSD) at the 95% confidence level (P > 0.05) using the ‘GenStat for Windows 13^th edition’ statistics system (VSN International Ltd., Hemel Hempstead, UK).

There was a significant effect of mulch on growth, tree vitality and the Phytophthora root rot lesion severity scale used in this investigation (Tables 1–3). In all cases, however, none of the mulched or non-mulched control trees died following Phytophthora infection. Irrespective of Phytophthora pathogen, leaf area, leaf, shoot, root and total plant dry weight following application of a pure mulch was, in virtually all instances, significantly (P < 0.05) higher than non-mulched Phytophthora infected controls. However, differences in the magnitude of growth induced between pure mulches following Phytophthora infection were recorded (Tables 2 and 3). Based on increased total plant dry weight as a measure of total plant biomass mulch efficacy following inoculation with P. cactorum was in the order hawthorn > cherry > beech > evergreen oak > silver birch > English oak > control. In the case of P. criticola mulch efficacy was in the order hawthorn > beech > silver birch > cherry > evergreen oak > English oak. Following inoculation with P. cactorum, leaf chlorophyll content, chlorophyll fluorescence Fv/Fm ratios, photosynthetic rates and root carbohydrate concentration ranged from 18–40%, 48–73%, 50–87% and 46 92% higher in pure mulched compared to non-mulched controls. In the case of P. criticola leaf chlorophyll content, chlorophyll fluorescence Fv/Fm ratios, photosynthetic rates and root carbohydrate concentration ranged from 34–48%, 40–59%, 21–43% and 29–57% higher in pure mulched respectively compared to non-mulched controls. Application of a pure mulch had a significant influence on Phytophthora severity of both pathogens based on visual root rot lesion severity symptoms. In the case of P. cactorum root lesion severity was reduced by 53% (hawthorn pure mulch), 45% (cherry, silver birch pure mulch), 61% (English oak, beech pure mulch) and 39% (evergreen oak pure mulch) compared to non-mulched controls. In the case of P. criticola, root lesion severity was reduced by 44% (hawthorn pure mulch), 36% (cherry pure mulch), 33% (silver birch pure mulch), 53% (English oak pure mulch), 39% (evergreen oak pure mulch) and 61% (beech pure mulch) respectively compared to non-mulched controls. In all cases these percent reductions were significant from non-mulched controls.

Results of this study recorded a positive influence of pure mulches on growth and vitality of horse chestnut trees and a reduction in root rot lesion severity caused by P. cactorum and P. criticola following artificial inoculation of containerised white flowering horse chestnut trees. In the case of P. cactorum, reductions in root rot lesion severity ranged from 39–61% while in the case of P. criticola reductions in root rot lesion severity ranged from 33 61% following application of a pure mulch compared to non-mulched controls. As Phytophthora pathogens destroy the fine absorbing roots of plants leading to loss of water and nutrient absorbing capacity as well as stored root carbohydrate reserves then reductions in Phytophthora rot lesion severity would account for the significant improvements in growth (leaf area, leaf, shoot, root and total plant dry weight) and tree vitality (leaf chlorophyll content, chlorophyll fluorescence Fv/Fm ratios, photosynthetic rates and root carbohydrate concentration) recorded in this study (2, 13, 37).

Previous studies have shown mulches can provide an integral cultural control method for suppressing disease development of several plant pathogens. Short-term effects include increased soil moisture, soil temperature moderation, improved soil nutrition, aggregation and drainage. Thus, mulches maintain a soil environment for healthy plant growth and induce a soil environment sub-optimal for opportunistic soil-borne pathogens (33). Likewise the physical presence of a mulch can reduce splashing of rain or irrigation water, which can carry spores of disease organisms up to the stems or leaves of susceptible plant species (3). With respect to elucidating the suppressive nature of mulches on Phytophthora pathogens, work by Downer et al. (6, 7) has been key to identifying mulching effects on the incidence of Phytophthora. Cellulose forms part of the component of the primary cell wall of green plants acting as a structural polymer to provide plant rigidity (12). Following the application of a mulch to a soil surface the concomitant microbial and fungal population buildup promotes a reservoir of enzymatic activity such as cellulase (β-1,4-glucanase) and laminarinase (β-1,3-glucanase) to induce mulch decomposition. Cellulose microfibrils in Phytophthora cell walls are susceptible to enzymatic destruction particularly by cellulases present in mulch litter layers that cause cell wall lysis and, by default, a subsequent reduction in Phytophthora pathogen severity (6, 7). In addition, organic mulches also contain a variety of soil microbes that can exert biological control over soil borne pathogens, either through resource competition or antibiosis (production of antibodies) (11).

Growth effects on pure mulched trees recorded in this study may also relate to allelochemicals released as each mulch degraded over time (10, 21, 29). For example allelopathetic testing of water soluble extracts of pure mulches derived from hawthorn, cherry, silver birch, English and evergreen oak positively increased pea seed germination, relative growth rate and photosynthetic efficiency of established seedlings (29). Extracts of box elder have been shown to stimulate the growth of a range of grasses while in recent studies the effect of fresh and composted pure mulch derived from Eucalyptus cladocalyx was found to have a positive effect in transplant performance of Platanus racemosa (9). Furthermore fresh pine bark mulch has been shown to positively affect establishment of English oak (15). Both hawthorn and cherry mulches have been shown to be naturally high in carbohydrates such as sucrose, glucose and sorbital (24, 31). Applications of carbohydrates to transplanted trees have been shown to be effective at stimulating root vigor and in turn alleviating transplant stress and increasing survival rates of newly planted trees such as oak, birch and beech (14, 27).

On a note of caution, however, other researchers have found application of water soluble extracts obtained from beech, pine, eucalyptus and acacia mulches suppressed germination of a range of weed seeds (30). Likewise pure mulches derived from cypress trees have been shown to reduce the growth of hydrangea, spirea and viburnum compared to a range of garden centre bought mulches (16). Cypress trees are noted for their resistance to decay fungi which is associated with the presence of phenolics compounds within woody tissue. Consequently, it was suggested these phenolics would be leached into the soil in turn inhibiting root growth (4). Pure mulches derived from Eucalyptus foliage have been found to contain phytotoxic organic oil and acid residues three months after application that in turn were toxic to germinating seedlings of several plants (9, 22). Further studies of allelochemicals proving toxic to plants have been reported by Terzi (32) and De Feo (5).

In conclusion, results of this study show that application of a pure mulch can provide a reduction in root rot lesion severity caused by P. cactorum and P. criticola and subsequent increase in tree growth and vitality. With pressures to find non-chemical means of pathogen control stimulated by public and government demands to reduce pesticide usage pure mulches potentially have a positive impact for those involved in the care and maintenance of urban, nursery, forestry and orchard trees as well as horticultural crop production systems. Although further research is required into determining the mechanistic basis for pathogen suppression, practically pure mulches require little capital investment and only small adjustments to standard management aftercare procedures.