Phytophthora cinnamomi suppressive soils

B. Keen and T. Vancov*

Wollongbar Primary Industries Institute. Industry & Investment NSW, 1243 Bruxner Highway, Wollongbar, NSW, 2477,

Australia * Corresponding author: Tony Vancov, Wollongbar Primary Industries Institute, 1243 Bruxner Highway, Wollongbar,

NSW 2477, Australia. Tel: +61 2 6626 1359; Fax: +61 2 6628 3264; E -mail: tony.vancov@industry.nsw.gov.au

Phytophthora cinnamomi suppressive soils were first reported in Australia in 1971 and have since been identified in many

other locations. Studies of P. cinnamomi suppressive soils frequently aim to identify specific organisms responsible for the

phenomenon. The collective outcome has been a modest list of bacterial and fungal suspects with proven antagonism

toward P. cinnamomi. However, no single bacterial or fungal isolate within this cohort has been identified as the specific

cause of suppression. A great majority of the work in this field has been limited to culture based studies, the limitations of

which are well known as is the potential for microbiomic methods to permit much more comprehensive examination of

soil microbial communities. To date there are only a few studies that have utilised microbiomic methods to examine the

structural characteristics of microbial communities in P. cinnamomi suppressive soils. In this mini-review we summarise

more than 35 years of P. cinnamomi suppressive soils research and discuss opportunities to apply recent technologies to

further elucidate the microbiological conditions under which P. cinnamomi suppression occurs.

Keywords root rot; Phytophthora cinnamomi; disease suppression; suppressive soil; microbial community

1. Introduction

Phytophthora cinnamomi (Fig. 1) is a soil and water-borne Oomycete first described by Rands in 1922 [1] as the causal

agent of stripe canker in Cinnamomum burmannii (cinnamon tree). Phylogenetic studies indicate that P. cinnamomi

most likely originated from across a wide area within New Guinea-Malaysia-Celebes and has been introduced to all

other parts of the world where it exists [2]. Renowned as one of the most ubiquitous, invasive and destructive plant

pathogens, P. cinnamomi has a host range exceeding 1000 plant species and has spread to every continent except

Antarctica [3]. The pathogen mainly infects the roots of its host causing root rot and without treatment the host usually

dies. To destructive potential of P. cinnamomi is amply demonstrated by the decimation of an estimated 202,500 ha of

the jarrah forests (Eucalyptus marginata) of Western Australia between 1927 and 1986 [4].

Options for managing P. cinnamomi in native plant communities are limited to regulating human traffic, machinery

and livestock in vulnerable areas and undertaking management activities that encourage an understorey of tolerant

and/or resistant plants to reduce P. cinnamomi inoculum [5]. In conventional cropping systems, control is achieved

through an integrated approach, involving cultural practices that minimise conditions conducive to P. cinnamomi,

planting tolerant root stocks [6] and chemical control using metalaxyl or, more commonly, phosphonate derivatives [7].

Some cultural practices (e.g. application of organic mulches) aim to achieve biological control by inducing soil

conditions that suppress P. cinnamomi.

The phenomenon of naturally occurring P. cinnamomi suppressive soil was first reported in an avocado orchard on

the east coast of Australia in the early 1970s [8-10]. Since then, P. cinnamomi suppressive soils have been reported in

several other parts of the world and there has been a concerted effort to elucidate and exploit the mechanisms that result

in suppression. In the following discussion we review this research and highlight opportunities to further elucidate the

mechanisms underlying P. cinnamomi suppressive soils.

a) b) c)

Fig. 1 P. cinnamomi: a) coralloid hyphae (x200); b) chlamydospores (x400); c) Non-papillate sporangia (x400)

100 µm 50 µm 50 µm

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2. What are P. cinnamomi suppressive soils?

The concept of a ‘suppressive soil’ was introduced by Menzies [11], who applied the term to describe the phenomenon

of soils that suppressed Streptomyces potato scab. By the 1970s the terms ‘disease suppressive soil’ and its antonym

‘disease conducive soil’ were widely adopted [12]. The first sound definition for a disease suppressive soil was

proposed by Baker and Cook [13] who defined a disease suppressive soil as a soil in which either the pathogen cannot

establish, becomes established but fails to produce disease, or becomes established and causes disease at first but

diminishes with continued cultivation of the crop. Alabouvette et al. [14] argued that the terms ‘disease suppression’

and ‘disease conducive’ fail to account for the fact that, in every natural soil, expression of disease occurs at various

degrees of incidence and severity. Thus suppression occurs along a continuum from ‘highly suppressive’ to ‘highly

conducive’ rather than simply being suppressive or conducive. A P. cinnamomi suppressive soil can therefore be

defined as a soil in which disease incidence and severity remain low, despite the presence or introduction of P.

cinnamomi, a susceptible host plant and favourable environmental conditions.

There are two modes of biological suppression; ‘specific’ and ‘general’. Specific suppression occurs when there are

exclusive interactions involving one or several specific agents that suppress a soil-borne plant pathogen. In soils that are

generally suppressive, suppression results from the cumulative effects of complex interactions between the pathogen

and a multiplicity of factors [15]. Abiotic processes within the soil may also have a direct or indirect effect on the

suppressive capacity of a soil. For instance, physicochemical factors such as clay minerals, soil pH and soil macro and

micro nutrients have each been implicated in disease suppressive soils [16]. However, where associations between

abiotic factors and suppressive soils have been determined, they are frequently associated with biotic factors [16, 17].

P. cinnamomi suppressive avocado soils have been characterised as being well drained, having a pH between 5.5 and

7.0 and possessing high levels of NH4, NO3, Ca cations, cation exchange capacity and organic matter content [9]. Mixed

results from experimental manipulations indicate that these factors may have an indirect role in P. cinnamomi

suppressive soils by providing conditions that encourage and maintain a soil microbial community antagonistic toward

P. cinnamomi [9, 18, 19]. Certainly, the majority of studies have presented cumulative and compelling evidence that P.

cinnamomi suppression is predominantly biological in origin [9, 20-24]. Confirmation of the involvement of biological

processes in P. cinnamomi suppressive soils has been demonstrated many times by terminating biological activity

through autoclaving, fumigating or γ-irradiating the soil, transferring suppression to a conducive soil and by observing

cell degradation of P. cinnamomi propagules in soil extracts [9, 20-24]. However, the specific biological agents and

processes that result in P. cinnamomi suppressive soils continue to elude researchers in this field.

3. Biological agents implicated in P. cinnamomi suppressive soils

3.1 Meso- and microfauna

Protozoa, especially vampyrellid and testate amoebas, physically perforate fungal walls and chemically digest the

cellular contents [25, 26]. Malajczuk [25] observed perforations typical of vampyrellid amoebas in the hyphae of P.

cinnamomi in soils suppressive to the pathogen. Observations have also been made of vampyrellid amoebas attacking

and lysing hyphae and chlamydospores of P. cinnamomi [26] and of small naked amoebas, belonging to the

Hartmannellidae, lysing and ingesting zoospores [20]. The potential role of mesofauna such as nematodes and mites in

P. cinnamomi suppressive soils has received little attention. This is probably because the results presented so far

indicate that nematodes and mites are often associated with increased disease severity. Mycophagous microfauna are

widespread and non-specific, grazing on a range of soil fungi and fungal propagules. High numbers of these organisms

are common in P. cinnamomi suppressive soil [20] and so microfauna may contribute to general suppression by

reducing P. cinnamomi inoculum. However, their non-specific predatory behaviour limits their potential use for specific

biological control of P. cinnamomi and this probably explains why researchers have mostly ignored microfauna found

in P. cinnamomi suppressive soils.

3.2 Bacteria

Broadbent and Baker [9] found higher populations of bacteria and actinomycetes in suppressive avocado soils than in

all but one conducive soil. In other studies, lysis of P. cinnamomi hyphae in soil has been positively correlated with

increases in microbial numbers, including bacteria [27]. Malajczuk and McComb [28, 29] isolated bacteria and

actinomycetes from suppressive and conducive soil and rhizosphere samples associated with the susceptible Eucalyptus

marginata and the less susceptible Eucalyptus calophylla. They observed the largest percentage of antagonistic bacteria

and actinomycetes in non-rhizosphere suppressive loam soil and in rhizosphere soil from E. marginata seedlings grown

in suppressive loam soil. Others have observed similar trends in avocado and forest soils [30-35].

There are two ways by which soil bacteria may suppress Phytophthora root rot. In some suppressive soils, bacteria

that stimulate the production of zoosporangia may be dormant, or the stimulatory compounds they produce might be

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destroyed by other soil microorganisms. The second process involves direct bacterial antagonism. In the suppressive

avocado soils studied by Broadbent and Baker [9, 10], P. cinnamomi was abundant but produced mostly abortive

sporangia. Bacteria were observed swarming around the sporangia walls and these may have caused the sporangia to

abort. Other studies have shown that P. cinnamomi may produce prolific numbers of zoospores but that root infection is

reduced by microorganisms that attack the zoospores [29]. Antagonistic bacteria may also operate to reduce P.

cinnamomi inoculum in soil by attacking the mycelium, sporangia or possibly the more resistant chlamydospores and

oospores [20]. Several researchers have observed bacteria intensively colonising Phytophthora spp. hyphae [9, 10, 29,

36]. This attraction appears to be a chemotactic response to metabolites, possibly phenylalanine and glucose, exuded

from Phytophthora hyphae [37]. It is also possible that in the process of feeding on exudates the bacteria may produce

metabolites that degrade P. cinnamomi cells. Numerous bacteria and actinomycetes capable of involvement in these

processes have been identified (Table 1).

Table 1. Bacteria implicated in P. cinnamomi suppressive soils.

Citation Context Bacteria Broadbent et al. [8]; Broadbent and

Baker [9, 10]

Suppressive avocado soils – eastern

Australia

Pseudomonas spp.

Pseudomonas putida

Pseudomonas fluorescens Chromobacterium spp.

Bacillus subtilis

Actinomycetes

Malajczuk [28, 29]

Rhizosphere of E. marginata and E. sieberi grown in

suppressive forest soil

Western Australia

Unidentified bacteria Unidentified actinomycetes

Halsall [30, 31]

Eucalyptus spp. forest soils

New South Wales, Australia

Streptomyces spp.

Malajczuk et al. [39] In vitro study of antagonistic Rhizobium isolated from native legumes

Western Australia

15 Rhizobium spp. isolates

Murray [32]

Eucalyptus forest,

antagonism in rhizosphere

Western Australia

Unidentified bacteria

Unidentified actinomycetes

Mass and Kotze [42] Disease free avocado soil South Africa

Several Pseudomonas spp.

Duvenhage et al. [22]

Suppressive avocado soils South Africa

Bacillus azotoformans Bacillus megaterium

Pseudomonas spp.

Stirling et al. [43]

Suppressive avocado soil Queensland,

Australia

9 Unidentified actinomycetes

3 fluorescent Pseudomonas spp. 3 Bacillus spp.

Serratia marcescens

El Tarabily et al. [40]

Banksia grandis rhizosphere Australia Micromonospora carbonacea

Streptomyces violascens

You et al. [33, 34]

Suppressive mulch applied to avocado

trees Western Australia

1600 isolates inhibitory in vitro;

in vivo some Streptomyces, Agromyces, Micromonospora and Actinomadura

isolates were suppressive

Yang et al. [44]

Healthy avocado root tips - bacterial

community 16S rDNA DGGE profiles

California, USA

Bacterial rDNA community profile obtained

from suppressive soil. Pseudomonas sp.

2 uncultured soil bacteria

Uncultured Pseudomonas sp. Polyangium sp.

Cytophaga sp.

Unidentified eubacterium

Yin et al. [45] Suppressive avocado soil. Bacteria

identified by combining substrate utilisation assays with rDNA intergenic

sequences

California, USA

Bacillus mycoides

Renibacterium salmoninarum Streptococcus pneumoniae

Keen [46] Bacterial community DNA study of suppressive avocado soil.

NSW, Australia

Several unidentified bacteria, recognized as five discrete operational taxonomic units

(OTUs) associated with transferred

suppression.

The understoreys of eucalyptus forests in Australia are typically dominated by grasses, proteaceous species (e.g.

Banksia spp.) or legumes (e.g. Acacia spp.), depending on the fire regime. P. cinnamomi tends to be highly active in

eucalyptus forests that have a proteaceous understorey [5, 38, 39]. Acacia spp. dominated understoreys have been

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associated with P. cinnamomi suppressive soils in the jarrah forests of Western Australia [5, 38, 39] and the root nodule

bacteria (i.e. rhizobia) that form symbiotic relationships with these Acacia spp., are capable of reducing zoospore

survival [39]. Actinomycetes are also frequently associated with suppressive forest soils in Australia (Table 1).

Antagonistic Micromonospora carbonacea and Streptomyces violascens have been isolated from the rhizosphere of

Banksia grandis [40]. Other Streptomyces spp. have also been implicated in in vitro antagonism [41] and in suppression

of P. cinnamomi in soil supporting Australian native vegetation [30, 31].

Broadbent et al. [8] screened 3500 bacteria and actinomycetes isolated from various soils for antagonism toward

several soil-borne plant pathogens including P. cinnamomi. Most of the antagonistic isolates were from the genera

Bacillus, Pseudomonas and Streptomyces. In subsequent work, Broadbent and Baker [9, 10] demonstrated that

Pseudomonas putida and Pseudomonas fluorescens, isolated from suppressive avocado soils, caused massive lysis of P.

cinnamomi mycelium in vitro. They also found that Bacillus subtilis var. niger isolated from the same soils was

involved in sporangial breakdown. Bacillus spp. and Pseudomonas spp. have also been implicated as antagonistic

bacteria common in suppressive avocado soils in South Africa [22, 42] and Australia [43]. Of the 164 bacteria

(including several actinomycetes) isolated from suppressive avocado soils by Stirling et al. [43] in Australia, 9% were

antagonistic toward P. cinnamomi in vitro. Antagonistic isolates included Serratia marcescens, three Bacillus, three

fluorescent Pseudomonas and nine unidentified actinomycete isolates. However, when individual isolates or a

combination of isolates were added to non-sterile and sterile soil leachates from the suppressive soil, lysis of P.

cinnamomi mycelium only occurred in leachate from the non-sterile suppressive soil [43]. This indicates that either the

bacterial isolates were not actively antagonistic toward P. cinnamomi in vivo or alternatively, unidentified and possibly

unculturable organisms were responsible for the suppressive capacity of the soil.

P. cinnamomi is a weak saprophytic competitor [20], fairing poorly in surface organic layers where larger numbers of

saprophytes dominate [35]. Some of these saprophytes may even utilise P. cinnamomi as a substrate as indicated by one

study in which populations of bacteria and actinomycetes increased sharply following infestation of a suppressive

organic mulch by P. cinnamomi [33]. In a subsequent study, 1600 actinomycetes isolated from the suppressive mulch

inhibited growth of P. cinnamomi in vitro but only a few isolates from the genera Streptomyces, Agromyces,

Micromonospora and Actinomadura demonstrated any potential for suppressing P. cinnamomi in vivo [34].

3.3 Fungi

The most common forms of fungal antagonism against Phytophthora spp. involve mycoparasitism and/or production of

metabolites that inhibit growth or destroy P. cinnamomi propagules [20]. Ectomycorrhizal fungi may also contribute to

the protection of susceptible plant roots from P. cinnamomi by: (i) forming a mantle that provides a physical barrier to

penetration; (ii) producing antibiotics that inhibit growth and reproduction; (iii) utilising surplus plant exudates that may

act as biochemical signals to P. cinnamomi hyphae and zoospores; (iv) providing habitat for antagonistic rhizosphere

microorganisms; (v) improving plant vigour; and (vi) inducing the plant to produce compounds that protect it from

infection [20, 47]. A number of ectomycorrhizal fungi have been linked with P. cinnamomi suppression in plantation

conifers and in eucalyptus forests [25, 28, 48]. However, there are also several examples of P. cinnamomi suppressive

avocado soils where no association between suppression and mycorrhizal fungi was found [20].

Antagonistic fungal species frequently associated with P. cinnamomi suppression include species in the genera

Penicillium, Trichoderma, Aspergillus and, less frequently, Myrothecium and Epicoccum (Table 2). Each of these

produces metabolites that actively inhibit P. cinnamomi in vitro and presumably in vivo [49, 50]. In particular,

Trichoderma spp., Myrothecium spp. and Trichoderma virens isolated from suppressive soil have shown extreme

antagonism toward P. cinnamomi during the saprophytic stage via antibiosis and mycoparasitism [49, 51]. Generally,

lysis of P. cinnamomi hyphae by mycoparasitic fungi is rapid and involves the parasite coiling around the hyphae and

subsequently forming structures that penetrate the cell wall [20]. There are also a number of fungi that are capable of

parasitising the thick-walled oospores of P. cinnamomi. These include certain members of the hyphomycetes and

chytrids [52]. Examples include Humicola fascoatra, Anguillospora pseudolongissima, Hyphochytrium catenoides [53],

Dactylella spermatophaga [52] and Catenaria anguillulae [54]. The succession of fungi that parasitise P. cinnamomi

propagules is determined by the soil water status, with oomycetes and the chytrids favoured under moist conditions

[20].

A number of antagonistic fungi associated with P. cinnamomi suppressive soils have also been assessed in vivo for

their potential use in biocontrol. Reduced avocado seedling root infection during glasshouse experiments and in field

experiments were achieved by introducing a strain of Myrothecium roridum isolated from healthy avocado roots [51].

Epicoccum purpurascens successfully protected Lupinus albus and Erica vagans from P. cinnamomi infection during

pot experiments [55]. Some success in reducing Phytophthora root rot of Rhododendron spp. and citrus grown under

controlled conditions was achieved with a number of different Penicillium spp. isolates [56, 57]. Organic mulches

inoculated with Trichoderma virens and Trichoderma harzianum reduced avocado seedling root infection and reduced

viability of sporangia [58]. Variable success has been achieved in reducing Phytophthora avocado root rot with

Paecilomyces lilacinus, Aspergillus candidus and Trichoderma hamatum in South Africa [59]. Despite the attention that

Trichoderma spp. have received [9, 42, 49, 58, 60-63], they have rarely been effective as long-term biological control

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agents against P. cinnamomi. The reason for this is presumably owing to their poor performance in wet soils, which are

more favourable to P. cinnamomi [64].

Table 2. Fungi implicated in P. cinnamomi suppressive soils.

Citation Context Fungi Malajczuk [25]

Rhizosphere of

E. marginata and E. sieberi grown in suppressive forest soil

Western Australia

Rhizopus spp.

Mucor spp. Chaetomium spp.

Alternaria spp.

Arthrobotrys spp. Aspergillus spp.

Aureobasidium spp. Cladosporium spp.

Fusarium spp.

Penicillium spp. Pullularia spp.

Trichoderma spp.

Ulocladium spp. Several non-sporing and unidentified

isolates

Malajczuk [28]

E. marginata

E. sieberi

Western Australia

2 Boletus spp.

Russula sp.

Lactarius sp. Ramaria sp.

Murray [32]

Eucalyptus forest

Antagonism in rhizosphere

Western Australia

Various fungi not specified

Gees and Coffey [51]

Suppressive avocado soil

California, USA

Myrothecium roridum

Mass and Kotze [42]

Disease free avocado soil South Africa

Penicillium sp. Trichoderma spp.

Casale [60]

Suppressive avocado soil California, USA

Trichoderma sp. and several unidentified fungal isolates

Finlay and McCracken [55] Biocontrol of P. cinnamomi infection of

Lupinus albus and Erica vegans

Epicoccum purpurascens

Duvenhage and Kohne [59]

Duvenhage and Kotze [61]

Suppressive avocado soil

South Africa

Paecilomyces lilacinus

Aspergillus candidus

Trichoderma hamatum

McLeod et al. [62]

Healthy avocado roots South Africa

Trichoderma harzianum Trichoderma hamatum

Chambers and Scott [49] Suppressive chestnut soil

South Australia

Trichoderma virens

Trichoderma hamatum

Trichoderma koningii

Costa et al. [58, 63]

Suppressive mulch

California, USA

Trichoderma virens

Trichoderma harzianum

Borneman and Hartin [66] Fungal community DNA study of suppressive avocado soil

California, USA

Dominant fungal sequences isolated from soil DNA samples were related to:

Tritirachium

Aspergillus Pleospora

Petriella

Monilinia Exophiala

Downer et al. [35] Suppressive eucalyptus wood chip mulch applied to avocado trees

California, USA

Most frequently isolated genera: Penicillium

Aspergillus

Trichoderma Sporothrix

Basidiomycetes:

Phanerochaete chryssorhiza Ceraceomyces tessulatus

Keen [46] Fungal community DNA study of

suppressive avocado soil.

NSW, Australia

One unidentified Ascomycete fungal

sequence associated with transferred

suppression.

4. Microbial community structure and diversity in P. cinnamomi suppressive soils

Microbial community structure appears to be influential in several incidences of soils that suppress soil-borne plant

pathogens [17, 67] other than P. cinnamomi. For example, the structure of microbial communities involved in the

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suppression of Pythium aphanidermatum in rock wool slabs was investigated by cultivation on selective media and by

denatured gradient gel electrophoresis (DGGE) [68]. Culture-based isolation indicated that suppression of P.

aphanidermatum was correlated with an abundance of culturable actinomycetes and Trichoderma spp. Bacterial

community profiles generated by DGGE showed a significant relationship between the composition of microbial

communities and disease suppressiveness. Dominant bands excised from the DGGE gel and sequenced matched

sequences from several actinomycetes including: Streptomyces, Mycobacterium, Microbacterium, Rhodococcus,

Curtobacterium, and Tsukamurella [68]. In another study survival of Fusarium oxysporum f. sp. lycopersici was

reduced by increased activity of facultative anaerobes following solarisation of wheat-bran amended soils [69]. DGGE

analysis of bacterial community DNA revealed the emergence of dominant bands unique to the amended soils. This

implies that bran-amended solarisation caused a shift in bacterial community structure which resulted in reduced

establishment and survival of the pathogen [69].

As outlined in Section 3, a plethora of micro-organisms have been implicated in P. cinnamomi suppressive soils.

Bacteria commonly implicated include those from the genera Bacillus and Pseudomonas, and from the Actinomycetes.

Those in the fungal domain commonly belong to the genera Pencillium, Trichoderma, Aspergillus, Myrothecium and

Epicoccum. Each of these groups, along with other known and unidentified antagonistic organisms, are probably active

in the soil simultaneously. This combined with an apparent lack of evidence for the universal involvement of any single

organism in P. cinnamomi suppressive soils indicates that rather than suppression resulting from a specific microbial

agent, it may develop as a consequence of microbial communities containing a number of key phylogenetic groups. In

this instance, the principal mode of suppression would therefore lay somewhere between specific and general

suppression, because suppression is dependent on the presence and activity of key taxonomic groups in the soil. Within

the field of P. cinnamomi suppressive soils research only a limited number of studies have pursued this line of inquiry.

Results from these studies indicate that microbial community structure may play a role in P. cinnamomi suppressive

soils. In one study DGGE analysis of bacterial 16s rDNA revealed that the composition and structure of bacterial

communities associated with healthy avocado roots were consistently similar to each other but were significantly

different to bacterial communities associated with infected roots [44]. Sequencing of dominant rDNA bands excised

from the DGGE gels associated Pseudomonas sp., Polyangium sp., Cytophaga sp., an uncultured Pseudomonas sp., two

uncultured soil bacteria and an unidentified eubacterium with healthy avocado roots with several of these bacteria

absent from diseased roots. The composition of bacterial communities on healthy roots was also similar to that of

healthy roots taken from plots that had been continuously bioaugmented with the biocontrol agent P. fluorescens strain

513. In another study [45], carbon substrates known to attract P. cinnamomi zoospores were used to bait soil bacteria

with the capacity to utilise the same substrates as P. cinnamomi. Intergenic rDNA sequences obtained from bacteria

growing on these substrates revealed three bacteria, related to Bacillus mycoides, Renibacterium salmoninarum and

Streptococcus pneumoniae, as being characteristic of a post-epidemic soil that had apparently become suppressive.

These and other bacteria that respond to the same biochemical cues that attract zoospores to plant roots may contribute

to suppression by occupying and competing for the same niche targeted by P. cinnamomi.

Notable differences in the phylogenetic composition of fungal communities in suppressive and conducive soils were

observed during a study in which clonal sequences, obtained from P. cinnamomi suppressive and diseased avocado soil

community DNA extracts, were compared [66]. Among the 62 sequenced clones there were 10 genera, four of which

were exclusive to the suppressive soil. In the suppressive avocado soil the dominant genera were Tritirachium,

Aspergillus, Pleospora, Petriella, Monilinia and Exophiala. A significant disparity between results from the

microbiomic approach and a culture-based study of the same soils occurred with the cultured fungi predominantly

representing Aspergillus, Penicillium, Sporothrix, Phoma, Trichoderma and Fusarium genera [35]. Results from

microbiomic studies that provide evidence supporting a possible relationship between soil microbial community

structure and P. cinnamomi suppression, are complicated by a recent study in which no such relationship was found in

naturally occurring P. cinnamomi suppressive avocado soils [46]. However, Keen [46] later implicated several bacterial

and fungal constituents in transferred suppression resulting from adding suppressive soil to conducive soils.

Microbial diversity is considered to be an important factor in the maintenance of soil health which incorporates the

concept of disease suppression [67]. During two complementary studies involving culture-based and culture-

independent DGGE comparisons between permanent grassland, grassland planted to maize, long-term arable land and

arable land turned into grassland, suppression of Rhizoctonia solani was correlated with increasing levels of microbial

diversity which was in turn correlated with increasing levels of plant diversity [67, 70]. Despite a general acceptance

that soil microbial diversity may play a role in disease suppression [71], only one study has attempted to examine the

role of soil microbial diversity in P. cinnamomi suppressive soils, but no relationship was found [46].

5. Microbial metabolites implicated in P. cinnamomi suppression

A number of in vitro studies provide evidence of microbial metabolites that disrupt Phytophthora growth, reproduction

and pathogenicity. Metabolites produced by Trichoderma spp. stimulate homothallic sexual reproduction in

Phytophthora spp. [72, 73]. This stimulatory effect is associated with volatile or soluble metabolites that also act as

inhibitors to vegetative growth [72]. P. cinnamomi mycelial growth is known to be inhibited by terrecyclic acid A,

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produced by an antagonistic Aspergillus terreus strain [74], and by diacetylphloroglucinol produced by several

Pseudomonas spp. [75]. Indole-3-ethanol from Zygorrhynchus moelleri [76] and flavopins from the mycoparasitic E.

purpurascens [77] are known to inhibit zoospore germination. Other fungal compounds that inhibit P. cinnamomi

include 6-(pent-1-enyl)-alpha-pyrone from Trichoderma viride [78], 6-pentyl-alpha-pyrone from T. koningii [79] and

metabolites from five classes of volatile compounds (alcohols, esters, ketones, acids and lipids) produced by Muscodor

albus [80]. The effects of these metabolites on P. cinnamomi have mostly been determined during in vitro experiments.

Observing the effects of these metabolites within the soil environment is more difficult and, for this reason, there is a

lack of specific evidence confirming their role in P. cinnamomi suppressive soils.

Enzymes frequently implicated in antagonism of phytopathogenic fungi include chitinase and glucanases. This

appears logically sound considering that the cell walls of most fungi are principally composed of chitin and glucans

[14]. Several microbial enzymes have also been shown to inhibit growth and lyse Phytophthora propagules during in

vitro studies. For example, Budi et al. [81] hypothesised that the mechanisms by which a Paenibacillus sp. antagonised

P. parasitica included production of extracellular cellulolytic, proteolytic, chitinolytic and pectinolytic enzymes.

However, when Budi et al. [81] treated P. parasitica cultures with commercial enzyme preparations, only proteases

inhibited mycelial growth. In another study, proteases from several Pseudomonas spp. acted as a growth inhibitor, but

cellulase and collagenase also inhibited mycelial growth [75]. Several other studies have found inhibitory and

degradative effects of cellulases on Phytophthora propagules [40, 50, 77) and cellulases are used to isolate various

Phytophthora components. For example, Phytophthora cactorum oospores are separated from mycelial mats by first

degrading the mycelium with cellulase [82] and protoplasts are isolated from Phytophthora cells by completely

degrading the cell walls using cellulase and laminarinase [83]. The degradative effect of these enzymes on

Phytophthora propagules is not surprising considering that, unlike true fungi, the cell walls of Oomycota are principally

composed of cellulosic β-1,4-glucans and non-cellulosic β-1,3- and β-1,6-glucans. In Phytophthora spp., these polymers

constitute 80 to 90% of the cell wall [84].

High microbial activity and large populations of antagonistic bacteria, actinomycetes and fungi are found in soils

with high organic matter content, which is a characteristic of some P. cinnamomi suppressive soils. Therefore,

competitive processes within the soil microbial community may be involved in P. cinnamomi suppression. Populations

of cellulase and laminarinase producing bacteria such as Bacillus spp., Pseudomonas spp. and Actinomycetes and fungi

from the Aspergillus, Epicoccum, Myrothecium, Pencillium and Trichoderma genera also tend to be common residents

in P. cinnamomi suppressive soils (Section 3). Where organic matter levels are adequate the activity of these and other

cellulase and laminarinase producing microorganisms would lead to an accumulation of these enzymes in the soil.

Considering the effects of these enzymes on Phytophthora propagules during in vitro studies, it would be expected that

high levels of cellulase and laminarinase activity in soil would result in conditions less favourable to P. cinnamomi.

Conversely, in soils with limited availability of organic substrates, populations of cellulytic saprophytes are likely to be

much lower and, therefore, P. cinnamomi degrading cellulase and laminarinase would be reduced. This would be

expected to result in a soil environment that is more conducive to P. cinnamomi.

The strongest evidence for the possible involvement of cellulase and laminarinase in P. cinnamomi suppressive soils

comes from a study in which mulch consisting of eucalyptus trimmings was applied to soils beneath avocado trees and

compared with unmulched trees. P. cinnamomi inoculum and root infection were reduced beneath the mulch which was

associated with increased microbial activity. Reductions in P. cinnamomi inoculum and root infection were also

negatively correlated with soil cellulase and laminarinase activities and the activities of unknown enzymes active

against P. cinnamomi cell walls from within the mulch and at the mulch-soil interface [35]. In a laboratory study,

Downer et al. [50] found that at concentrations >10 units mL-1

cellulase prevented the development of chlamydospores,

sporangia and zoospores. Cellulase also caused severe damage to mycelium at concentrations >25 units mL-1

. At

concentrations up to 25 units mL-1

laminarinase had little effect on chlamydospore, sporangia or zoospore formation

and mycelium but reduced zoospore survival and encystment at concentrations bewtween 10 and 100 units mL-1

.

Germination of chlamydospores was stimulated by both enzymes at concentrations <10 units mL-1

. Downer et al. [50]

also noted that chlamydospores formed in dead root tissues were shielded from the destructive effects of cellulase,

which indicates that P. cinnamomi may survive spikes in enzyme activity within field soils by the same means.

While it could be construed that the findings reported by Downer et al. [35, 50] demonstrate that cellulase and

laminarinase are principal mechanisms involved in the suppression of P. cinnamomi, the results from their study do not

provide sufficient evidence to draw such definitive conclusions. The first study merely demonstrated an association

between higher enzyme activities and reductions in P. cinnamomi inoculum and root infections. In the second study,

cellulase and laminarinase clearly affected several life stages of P. cinnamomi, however, the relationship between

enzyme activities measured in the suppressive soils and the enzyme concentrations used in the laboratory experiments

was not determined. In addition, the Downer et al. [35, 50] hypothesis is contradicted by subsequent studies [24, 46] in

which no correlation between soil cellulase and laminarinase activities and naturally occurring P. cinnamomi

suppressive soils was found.

_______________________________________________________________________________________

6. Opportunities for future research

P. cinnamomi suppressive soils research has been dominated by culture based studies with the cumulative outcome

being the identification of numerous antagonists. Insights gained have typically been applied by attempting to induce

biological control through introducing a single antagonist to the soil. This approach has met with limited success and

where progress has been made, such as in the case of P. fluorescens [44], the introduced population is short lived and

repeated bioaugmentation is required to maintain effectiveness. It is apparent that many investigations commence with

assumptions of suppression being specific before the hypothesis is tested. While there are a few antagonists commonly

isolated from P. cinnamomi suppressive soils, evidence of numerous potential antagonists in these soils indicates that

the principal mode of suppression may be general.

We argue that when investigating a suppressive soil, after confirming biological suppression, the investigator should

attempt to determine whether the mode of suppression is specific or general. The limitations of culture based methods

are well known [85] and this may explain why such an approach has mostly been overlooked in the past. More recently

developed microbiomic methods provide tools for overcoming many of these limitations. Specifically they provide tools

for examining the structure and diversity of microbial communities, identifying the presence of unculturable organisms,

and for indentifying and quantifying functional genes. These methods have been applied successfully during

investigations of soils suppressive to several plant pathogens [86-88] but so far only a few investigators have applied

these methods to P. cinnamomi suppressive soils [44-46, 66].

The most comprehensive strategy for inventorying constituents within a microbial community is to extract whole

community DNA from the soil, amplify the DNA using universal primers, construct and sequence clone libraries and

align the resulting sequences against those deposited in ribosomal databases [17]. Obvious limitations to this approach

are that it can be expensive and laborious and while comparisons between communities can be based on presence and

absence of sequence strands, it provides little scope for quantitative analyses. Community DNA profiling techniques

turn-out quantitative information which permits assessments of abundance and diversity and comparisons of structural

differences between microbial communities. These methods include DGGE [89], terminal-restriction length

polymorphism analysis (T-RFLP) [90, 91] and automated ribosomal intergenic sequence analysis (ARISA) [92] which

is also known as length heterogeneity PCR (LH-PCR) [91]. DGGE has the advantage of permitting DNA bands to be

excised from gels for sequencing but has the disadvantage of having much lower resolution than the other two methods,

which can present problems when examining highly diverse communities [93]. T-RFLP involves a number of enzyme

digestion steps to cut long sequences into shorter lengths to facilitate analysis but the method is prone to errors [91, 94].

ARISA provides a more efficient method by utilising natural length variations straddling specific hyper-variable regions

of DNA. The main disadvantage with T-RFLP and ARISA is that both methods denature the DNA during analysis

meaning that, unlike DGGE, any bands of interest can not be recovered and sequenced to identify their phylogenetic

origin. However, after ARISA analysis the PCR product, consisting of amplified community DNA, remains available

for cloning and sequencing.

In determining whether the mode of P. cinnamomi suppression is specific or general, ARISA could be applied as a

rapid method for screening suppressive soils for the presence or absence of unique constituents, some of which may not

be identifiable by culture based methods. In the process of doing so the data that is generated can also be scrutinised for

relationships between soil microbial community structure and diversity and the occurrence of suppressive soils. The

PCR amplicons can be cloned and where DNA fragments specific to the suppressive soil are identified, clones with

DNA inserts of the same length as those identified during ARISA can be targeted for sequencing. This approach has the

potential to reduce costs, time and labour inputs associated with other approaches.

Where constituents unique to suppressive soils are identified by ARISA, the evidence supports a specific suppression

hypothesis. The researcher then knows that it may prove fruitful to allocate resources to isolate the organism, if

culturable, or experiment with environmental modifications to determine the conditions that encourage and maintain

populations of those constituents associated with suppressive soils. Where the organism is unculturable, ARISA can be

used to monitor the population status of suspected suppressive soil constituents. Conversely, if no specific constituents

are identified by ARISA then a general suppression hypothesis is supported. Where general suppression is suspected,

investigating microbial functions influencing suppression becomes the most logical direction for further research.

Evidence of P. cinnamomi cell degradation in suppressive soil leachates [10, 24, 43] provides support for the

involvement of metabolic products in general suppression. There may be one key metabolite or there may be many, the

sum of which results in suppression. Quantifying some metabolites in soil presents challenges but directly targeting

functional genes [86, 87] can facilitate comparative analyses. Many soil enzymes can be rapidly quantitated on a single

microplate containing multiple fluorescently labelled substrates [95, 96]. Where specific metabolites are suspected of

being involved in suppression then their effects on P. cinnamomi propagules must be demonstrated and the relationship

between concentrations affecting P. cinnamomi and concentrations, or modes of action, within the suppressive soil need

to be clear. Once this has been established work can proceed toward identifying practices that increase the

concentration, or effective action, of suppressive metabolites in soil. Organisms that produce suppressive metabolites

may already be present in the soil and biostimulation would be the aim of such practices. Some soils may also benefit

from receiving a bioaugmental boost with a consortium of microorganisms from relevant functional groups.

_______________________________________________________________________________________

7. Conclusions

P. cinnamomi suppressive soils research has clearly demonstrated that the phenomenon exists and is microbiologically

mediated. However, there is considerably more uncertainty surrounding the identity of the microbial agents and

ecological processes that result in P. cinnamomi suppressive soils. Many studies appear to have commenced with an

assumption that suppression is specific. While it is likely that the principle mode of suppression will vary with each

incidence of P. cinnamomi suppressive soil, each study should commence by attempting to determine whether

suppression is specific or general. We believe that this approach is justified as the outcomes then provide a sound

rationale for allocating resources toward future research efforts. The past dominance of culture based studies has

imposed limitations on our ability to test a specific suppression hypothesis. While not without their limitations,

microbiomic methods currently provide the best tool for examining this question. In the field of P. cinnamomi

suppressive soils research only a few investigators have taken advantage of microbiomic methods.

ARISA appears to provide a suitable tool for rapid screening of P. cinnamomi suppressive soils for the presence of

bacterial or fungal constituents that may be absent or less dominant in conducive soils. The data generated from ARISA

may also facilitate biodiversity and community structure comparisons. Where common constituents in P. cinnamomi

suppressive soils are observed resources can then be directed toward determining the identity of these and defining the

conditions required to stimulate their suppressive activity. If common constituents are not identified then support is

provided for arguing that resources should be directed toward investigating factors contributing to general suppression.

Under a general suppression hypothesis microbial metabolic functions are likely to play a key role.

Studies that have aimed to understand the role of microbial metabolites in regulating P. cinnamomi suppression have

mainly focused on specific microorganisms and their interactions with P. cinnamomi in vitro. In addition to the

involvement of lytic enzymes in mycoparasitism and antagonism through direct contact with P. cinnamomi, their

accumulation in soil may contribute to general suppression and the subsequent development and maintenance of P.

cinnamomi suppressive soils. Relatively few studies have investigated this hypothesis.

In conclusion, P. cinnamomi remains a serious threat to natural and agricultural systems. For now metalaxyl and

phosphosphonate derivatives provide effective chemical management solutions in conventional crops. Their application

is severely limited and barred in native vegetation and organic production systems, respectively. If available, biological

control would not only reduce dependence on chemical control but provide a solution in situations where chemical

application is not practical or prohibited. Continuing to study soils that naturally suppress P. cinnamomi offers the

greatest potential for discovering effective biological control options.

Acknowledgements The support provided by Dr Alison McInnes and Dr Percy Wong and the University of Western Sydney and

Industry and Investment NSW is gratefully acknowledged.

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