Phytophthora cinnamomi suppressive soils

Transcription

1 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: ; Fax: ; 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 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. 100 µm 50 µm 50 µm a) b) c) Fig. 1 P. cinnamomi: a) coralloid hyphae (x200); b) chlamydospores (x400); c) Non-papillate sporangia (x400)

2 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 NH 4, NO 3, 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

3 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] Halsall [30, 31] Malajczuk et al. [39] Murray [32] Mass and Kotze [42] Duvenhage et al. [22] Stirling et al. [43] Rhizosphere of E. marginata and E. sieberi grown in suppressive forest soil Eucalyptus spp. forest soils New South Wales, Australia In vitro study of antagonistic Rhizobium isolated from native legumes Eucalyptus forest, antagonism in rhizosphere Disease free avocado soil South Africa Suppressive avocado soils South Africa Suppressive avocado soil Queensland, Australia Unidentified bacteria Unidentified actinomycetes Streptomyces spp. 15 Rhizobium spp. isolates Unidentified bacteria Unidentified actinomycetes Several Pseudomonas spp. Bacillus azotoformans Bacillus megaterium Pseudomonas spp. 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 1600 isolates inhibitory in vitro; in vivo some Streptomyces, Agromyces, Micromonospora and Actinomadura isolates were suppressive Yang et al. [44] Yin et al. [45] Keen [46] Healthy avocado root tips - bacterial community 16S rdna DGGE profiles Suppressive avocado soil. Bacteria identified by combining substrate utilisation assays with rdna intergenic sequences Bacterial community DNA study of suppressive avocado soil. NSW, Australia Bacterial rdna community profile obtained from suppressive soil. Pseudomonas sp. 2 uncultured soil bacteria Uncultured Pseudomonas sp. Polyangium sp. Cytophaga sp. Unidentified eubacterium Bacillus mycoides Renibacterium salmoninarum Streptococcus pneumoniae 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

4 associated with P. cinnamomi suppressive soils in the jarrah forests of [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

5 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 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] Murray [32] Gees and Coffey [51] Mass and Kotze [42] Casale [60] Finlay and McCracken [55] Duvenhage and Kohne [59] Duvenhage and Kotze [61] McLeod et al. [62] Chambers and Scott [49] Costa et al. [58, 63] Borneman and Hartin [66] Downer et al. [35] Keen [46] E. marginata E. sieberi Eucalyptus forest Antagonism in rhizosphere Suppressive avocado soil Disease free avocado soil South Africa Suppressive avocado soil Biocontrol of P. cinnamomi infection of Lupinus albus and Erica vegans Suppressive avocado soil South Africa Healthy avocado roots South Africa Suppressive chestnut soil South Australia Suppressive mulch Fungal community DNA study of suppressive avocado soil Suppressive eucalyptus wood chip mulch applied to avocado trees Fungal community DNA study of suppressive avocado soil. NSW, Australia 2 Boletus spp. Russula sp. Lactarius sp. Ramaria sp. Various fungi not specified Myrothecium roridum Penicillium sp. Trichoderma spp. Trichoderma sp. and several unidentified fungal isolates Epicoccum purpurascens Paecilomyces lilacinus Aspergillus candidus Trichoderma hamatum Trichoderma harzianum Trichoderma hamatum Trichoderma virens Trichoderma hamatum Trichoderma koningii Trichoderma virens Trichoderma harzianum Dominant fungal sequences isolated from soil DNA samples were related to: Tritirachium Aspergillus Pleospora Petriella Monilinia Exophiala Most frequently isolated genera: Penicillium Aspergillus Trichoderma Sporothrix Basidiomycetes: Phanerochaete chryssorhiza Ceraceomyces tessulatus 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

6 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 cultureindependent 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,

7 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.

8 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.

9 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. 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