Molecular Plant Pathology On-Line [] 1997/0623gayler

The distribution of elicitin-like gene sequences in relation to elicitin protein secretion within the class Oomycetes

K R Gayler, K M Popa, D M Maksel, DL Ebert and B R Grant

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3052, Australia

Corresponding Author:

K R Gayler, Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3052, Australia


The distribution of elicitin-like gene sequences and the capacity of particular species to secrete biologically active elicitins has been determined within and between genera of the Oomycetes. Species from five families were analysed for elicitins equivalent to those secreted by Phytophthora species (Kamoun, S., Young, M., Forster, H., Coffey, M., and Tyler, B. 1994. Applied Environmental Microbiology 60,1593-1598). Biologically active elicitin proteins were detected by bioassay and elicitin-like gene sequences analysed on Southern blotted genomic DNA using fragments of the coding region of elicitin genes from both Phytophthora and Pythium species. Despite the almost ubiquitous distribution of elicitin secretion within the genus Phytophthora, secretion of these proteins by species from outside of this genus was restricted to a relatively few species and all from the genus Pythium. All species which in liquid culture secreted protein active in the specific elicitin bioassay, contained small families of elicitin-like gene sequences and no such sequences were found elsewhere. Distribution of the elicitin-like gene sequences was compared with previously established phylogenetic relationships between the Oomycete families. It was concluded that elicitin gene distribution provides further evidence subdividing Pythium species into two potential genera.


Elicitins are a family of proteins which induce symptoms closely resembling the hypersensitive response in a number of plant species (Goodman and Novacky, 1994). These symptoms include leaf necrosis (Bonnet et al., 1986; Zanetti et al., 1992), accumulation of pathogen related mRNAs and proteins (Bonnet et al., 1986; Keller et al., 1996) and heightened protection against subsequent pathogen attack (Ricci et al., 1989). Elicitins secreted by members of the genus Phytophthora have been particularly well characterised. Although other secreted proteins which can similarly produce a hypersensitive response have been detected in this genus (Farmer and Helgeson, 1987; Baillieul et al., 1995), elicitins are the most abundant. All Phytophthora elicitins are comprised of 98 amino acids in a highly conserved sequence (Huet et al., 1995). Originally observed in culture filtrates of P. cryptogea (Csinos and Hendrix, 1977), similar elicitins have subsequently been described amongst the proteins secreted by 14 species of Phytophthora (Kamoun et al., 1994). With the exception of a small number of highly virulent isolates of P. parasitica Dastura var nicotianae (Bonnet et al., 1994), secretion of proteins belonging to this elicitin family is ubiquitous amongst all species of Phytophthora examined so far (Ricci et al., 1993; Kamoun et al., 1994). Two proteins with amino acid sequences which were highly homologous to those of the elicitins from Phytophthora species and which exhibited elicitin-like activity upon application to tobacco tissues have also been described in the culture filtrates of Pythium vexans (Huet et al., 1995).

Since one of the responses to treatment of plants with elicitins is the acquisition of systemic acquired resistance (SAR) to subsequent pathogen invasion (Ricci et al., 1989; Bonnet et al., 1996), secretion of elicitins would appear to have the potential to modulate the virulence of those organisms which produce them in plant species capable of reacting to elicitin. Although the range of plants which demonstrate the capacity to sense and respond to elicitins is currently particularly narrow and restricted to tobacco and to specific cultivars of radish and turnip species (Kamoun et al., 1993a), the ability to transfer elicitin sensitivity between plant cultivars has recently been achieved by conventional breeding (Keizer et al., 1997). The prospects for transfer of this trait to a broader range of plants by genetic engineering suggests a potential new approach to increase protection of a much wider range of plant species against those pathogens which produce elicitins.

To date, there has been very limited exploration of the distribution of and genetic basis for the production of elicitins by species within the class Oomycetes. Multiple isoforms of elicitins have been shown to be secreted both by several Phytophthora species (Huet et al., 1992; Le Berre et al., 1994) and by the only Pythium species so far studied (Huet et al., 1995). Southern blotting analysis of genomic DNA from a range of isolates from eight different species of Phytophthora has also identified a small multigene family encoding elicitins in all species (Kamoun et al., 1993b; Ricci et al., 1993). The extent to which each member of such multigene families is expressed remains unclear. For example, secreted proteins corresponding to two of the four elicitin genes clustered as open reading frames within a 6 kb fragment of genomic DNA from P. cryptogea, have not yet been described (Le Berre et al., 1994). In this paper we used DNA analysis to determine the distribution of elicitin-like gene sequences within and between genera from the Oomycetes and used an accompanying bioassay of the secreted proteins as a measure of their ability to produce active elicitins. Distribution of the capacity to secrete elicitins is compared with previously established phylogenetic relationships between the Oomycete families.



Strains and species of Oomycete used in the study were the generous gifts of the following:

Phytophthora cryptogea Pethybr. and Laff., isolate P7407, Phytophthora syringae (Kleb) Kleb, isolate P6208 and Phytophthora melonis Katsura, isolate P3239 (Dr M. Coffey, University of California, Riverside, USA). Phytophthora clandestina Taylor and Greenhalgh (Dr D. de Boer, Institute for Horticultural Development, Knoxfield, Australia). Pythium arrhenomanes, isolate T133-1B, Pythium myriotylum, isolate T133-2A, Pythium graminicola, isolate T133-3A, Pythium ostracodes, isolate T122-3A, Pythium vexans de Bary, isolate T122-5A and Pachymetra chaunorhiza (Dr B.J. Croft, Tully Sugar Experiment Station, Tully, Australia). Achlya ambisexualis J. R. Raper, (Institute of Hygiene and Epidemiology, University of Louvain, Belgium). Peronospora parasitica (Fr.) Fr. (Dr E. Minchington, Institute for Horticultural Development, Knoxfield, Australia). Phytophthora erythroseptica Pethybr. was isolated from infected potato tubers by B R Grant.

Oomycete growth and maintenance

Oomycete cultures were grown in either minimal medium (Ribiero et al., 1975) as modified by Fenn and Coffey (1984) or Rye grain medium (Caten and Jinks, 1968), supplemented with agar (1.5% (w/v)), at 26oC. To determine the capacity of particular Oomycetes to secrete elicitins, 50 ml aliquots of minimal medium were inoculated with plugs of agar containing actively growing mycelia, cultures were grown at 26oC for 14 to 16 days and harvested by passing culture medium through glass fibre filter paper (Advantec GA-55) under vacuum. The culture filtrate was concentrated through a YM10 membrane (Amicon) and the protein concentration determined using the bicinchoninic acid assay (Redinbaugh and Turley, 1986).

Peronospora parasitica was collected from infected seedlings of cauliflower, Brassica oleracea.


SDS-PAGE was carried out using the method of Laemmli (1970) under reducing conditions. To avoid leaching of elicitin bands, gels were stained directly with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 45% (v/v) ethanol, 9% (v/v) acetic acid at 45oC without prior fixation. Gels were destained with 7.5% (v/v) acetic acid at 60oC.

Biological assays

Biological activity of elicitin was assayed on two cultivars of Raphanus sativus, Daikon and White Icicle (Arthur Yates and Co.). A single cotyledon from six to seven day old radish seedlings was injected with 20 µl distilled water containing 3 µg, 10 µg or 500 µg of crude protein filtrate/g cotyledon and monitored for necrosis over 72 h. Daikon is elicitin-sensitive; that is, when purified Beta-cryptogein is applied at 3 µg/g cotyledon, necrosis is observed in the injected cotyledon within 16 h. White Icicle, which is not susceptible to equivalent elicitin treatment was used as a negative control (Kamoun et al., 1993a). Cotyledons of both cultivars injected with water formed a second set of controls.

Isolation of Oomycete genomic DNA

Genomic DNA was prepared from frozen mycelia by the method of Mao and Tyler (1991) and used without CsCl gradient fractionation to remove mitochondrial DNA. DNA which had been precipitated with 70% (v/v) iso-propanol was washed with 100% (v/v) ethanol, air dried and suspended in water. All DNA used in Southern blots was also digested with 0.25 µg Ribonuclease A per µg DNA upon completion of restriction endonuclease digestion.

Polymerase Chain Reactions

PCR reactions were carried out in 50 µl of 10 mM Tris-HCl buffer, pH 8.3, containing 1.5 mM MgCl2, 50 mM KCl, 200 µM each of dATP, dCTP, dGTP and dTTP, 2.5 units TAQ polymerase (Boehringer and Mannheim), oligonucleotide primers (Bresatec) at the concentrations listed below and 100 ng of genomic DNA as template DNA. PCR reactions were carried out in a MiniCycler thermocycler (Bresatec) for 35 cycles including annealing for 45 s at the temperatures given below, extension for 1 min at 72oC and melting for 45 s at 95oC.

Oligonucleotide primers

Unique oligonucleotide primers 19 bp to 23 bp in length corresponding to the cDNA sequences for Beta-cryptogein (Panabieres et al., 1995) and actinA from P. infestans (Unkles et al., 1991) were used at concentrations of 0.2 µM to 0.5 µM and an annealing temperature of 58oC to amplify Beta-cryptogein and actinA genomic DNA respectively.

Oligonucleotide primers to amplify DNA encoding the elicitins from P. vexans were designed by back translation from the protein sequences of elicitins Vex1 and Vex2(Huet et al., 1995). The primers from Vex1 had mean Tms of 48.6oC and 49.6oC with degeneracies of 512 and 256 respectively. The oligonucleotide primers designed from Vex2 had a degeneracy of 256 and mean Tms of 45.5oC and 48.6oC. These degenerate primers were used in amplification reactions at concentrations of 2 or 3 µM and at an annealing temperature of 52oC. Assuming no introns, the expected lengths of the products from amplification from genomic DNA of sequences encoding Vex1 and Vex2 were respectively 219 bp and 213 bp.

Cloning and sequencing of PCR products

PCR products were cloned into the pCR2.1 vector using the Original TA Cloning® Kit (Invitrogen) and plasmids containing inserts of the appropriate size subjected to double stranded dye terminator cycle DNA sequencing using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Invitrogen) and either RSP-25 or USP-17 universal primers.

Southern blotting

DNA was digested with 5 units restriction endonuclease/ µg DNA for 2.5 h at 37oC and subjected to electrophoresis through a 1% (w/v) agarose gel containing 0.4 µg/µl ethidium bromide. DNA was loaded at 4 µg/track. Following electrophoresis, DNA was depurinated with 0.25 M HCl for 15 min then denatured with 0.5 M NaOH for 30 min. DNA was transferred to a Hybond N+ membrane (Amersham) in 10x SSC using the Model 785 Vacuum Blotter (BioRad). The membrane was washed in 2x SSC, dried overnight then baked for 2 h at 80oC.


To prepare probes for hybridisation, cloned PCR fragments were isolated from plasmid DNA stocks as fragments obtained by digestion with EcoRI. The DNA fragments were separated by electrophoresis through a 1.2% (w/v) agarose gel and purified using GENO-BIND (Clontech). Probes were labelled with 32P-dATP (Bresatec) by random priming and purified by passage through a NAP5 column (Pharmacia Biotech). Probes were denatured at 95oC for 3 min then immediately placed on ice.

Membranes were pre-hybridised with Rapid-hyb buffer (Amersham) for 30 min at 65oC, the denatured probe added and hybridisation carried out at 65oC for 90 min. Membranes were washed with 2x SSC and 0.1% (w/v) SDS at room temperature for 20 min followed by two washes with 0.1x SSC and 0.1% (w/v) SDS at 65oC for 15 min and autoradiographed using Kodak BioMax MS Film at -70oC from 4 to 7 days. Bound probes were removed with 0.5% (w/v) SDS at 100oC.


Distribution of elicitin-like proteins amongst Oomycete species

Previous studies have shown the almost ubiquitous secretion of elicitins by all species of Phytophthora studied (Kamoun et al., 1994). Members of the genus Phytophthora were compared in this study with several members of the closely related genus Pythium and with several Oomycetes from outside of the family Pythiaceae.

As shown in Figure 1, within the order Peronosporales five species of Phytophthora including the previously unstudied P. clandestina, P. melonis, P. syringae and P. erythroseptica and five species of Pythium were screened as representatives of the family Pythiaceae, Peronospora parasitica from the family Peronosporaceae and Pachymetra chaunorhiza as an example from the family Veruciaceae. Achlya ambisexualis was included as a member from the separate Oomycete order Saprolegniales and one example of an Ascomycete, Saccharomyces cerevisiae, was included to test organisms of a class other than Oomycetes.

Cultures were examined for the presence of secreted elicitin in two ways. Concentrated filtrates were analysed by SDS-PAGE for a protein of apparent Mr 10,000, the size of all known elicitins (Bonnet et al., 1985; Huet and Pernollet, 1989; Huet and Pernollet, 1993; Huet et al., 1992; Huet et al., 1993; Huet et al., 1994; Nespoulous et al., 1992). Concentrated filtrates were also assayed for biological activity on radish.

As shown in Figure 2, proteins with an apparent Mr between 10,000 and 11,000 were detected in the concentrated filtrates from all species of Phytophthora. As shown in Table 1, the same filtrates of each Phytophthora species were active in an elicitin-specific bioassay. In each bioassay the purified Beta-elicitin, cryptogein was included as a control. Cryptogein induced necrosis in seedlings of the elicitin-sensitive radish cultivar, Daikon, within 16h of the injection of 3 µg protein per g of radish tissue, but did not induce necrosis in seedlings of the relatively insensitive radish cultivar, White Icicle even after 72 h. Cryptogein is known from previous studies to induce necrosis in tobacco tissue when applied at similar concentrations (Pernollet et al., 1993). On the basis of SDS-PAGE (Figure 2), it was estimated that elicitin accounted for a maximum of less than 50% of the total protein in the culture filtrates from Phytophthora species. The bioassays were therefore carried out at 10 µg protein/g of radish tissue.

The filtrate of each Phytophthora species induced necrotic responses in the seedlings of the sensitive radish cultivar, Daikon, following application of 10 µg protein/g radish tissue (Table 1). An equivalent assay on seedlings of the cultivar, White Icicle, showed no response to filtrates from any of the Phytophthora species even after 72 h (Table 1). Increasing the concentration of applied filtrates to 500 µg protein/g radish tissue, caused yellowing and necrosis in both the elicitin-sensitive and elicitin-insensitive cultivars of radish. In bioassays carried out at this concentration it was therefore not possible to distinguish specific effects of elicitins from other toxic effects of the filtrates. Assays in which necrosis was induced in Daikon seedlings and not in White Icicle seedlings, were the only bioassays recorded as positive for elicitin activity.

Concentrated aliquots of culture filtrates from a number of species of Pythium were also tested for secreted elicitins. As shown in (Figure 2), protein with an apparent Mr between 10,000 and 11,000 was detected by SDS-PAGE as a major component of the proteins secreted by both Py. vexans and Py. ostracodes. The filtrates of Py. vexans and Py. ostracodes also induced necrosis in the radish bioassays. The culture filtrates from both Py. vexans and Py. ostracodes induced necrosis in 100% of radish seedlings within 72 h of application of the protein concentrates to the sensitive cultivar, Daikon (Table 1). In control assays with White Icicle seedlings, there was no response to the filtrate from Py. vexans and a minimal response was induced by the filtrate from Py. ostracodes (Table 1).

By contrast with these species, other Pythium species and in fact all other species of Oomycetes tested did not secrete elicitin proteins. Culture filtrates from Py. graminicola, Py. myriotylum and Py. arrhenomanes contained no protein of apparent Mr 10,000. Instead, proteins of apparent Mr 14,000, and 17,000 were the major secreted proteins (Figure 2). The filtrates from Py. graminicola, Py. myriotylum and Py. arrhenomanes produced no response in Daikon seedlings when applied at 10 µg protein/g tissue. Py. vexans and Py. ostracodes were therefore the only Pythium species whose culture filtrates not only contained a protein of apparent Mr 10,000 as detected on SDS-PAGE but also elicited necrotic activity on Daikon seedlings at 10 µg protein/g radish tissue.

Concentrated aliquots of culture filtrates from Pachymetra chaunorhiza and Achlya ambisexualis which were also analysed for elicitin-like proteins did not secrete protein of apparent Mr 10,000 (Figure 2) and were inactive in elicitin-specific bioassays even when applied at 500 µg protein/g radish tissue (Data not shown). Neither was any evidence obtained either by SDS-PAGE or by bioassay for the presence of elicitin in the Ascomycete, Saccharomyces cerevisiae. It was concluded that despite the almost ubiquitous distribution of elicitin secretion within the genus Phytophthora, secretion of these proteins by species from outside of this genus was restricted to a relatively few species, all from the genus Pythium.

Detection of elicitin-like genes in genomic DNA from a range of Phytophthora, Pythium and related Oomycetes

In contrast to the rather detailed knowledge of the amino acid sequences of elicitin proteins, somewhat less is known about the genes which encode them. The picture now emerging is that most if not all Phytophthora species including isolates which do not secrete elicitins contain multiple copies of one or more elicitin genes (Kamoun et al., 1993b; Ricci et al., 1993). To determine the extent to which sequences corresponding to these genes could be observed within the genome of other members of the class Oomycetes, genomic DNA digests were prepared from a range of species, and probed using DNA probes designed on the basis of genes coding for elicitins in Phytophthora cryptogea and Pythium vexans. Probes for three specific genes, encoding respectively Beta-cryptogein, an elicitin from Phytophthora cryptogea, Vex2 , an Alpha-elicitin from Pythium vexans, and actinA from Phytophthora melonis were each used to search for related gene sequences in the genomes of the Oomycetes. Primary DNA probes were prepared using PCR to amplify partial DNA sequences from genomic DNA and their authenticity established by cloning and DNA sequencing. Whereas both actinA and cryptogein sequences were amplified on the basis of known DNA sequences (Panabieres et al., 1995; Unkles et al., 1991), in order to obtain DNA probes for an elicitin from the Pythium genus, it was necessary to use PCRs which relied on oligonucleotide primers designed by back translation from the protein sequences from Pythium vexans (Vex1 and Vex2 ; Huet et al., 1995). Despite the primers being highly degenerate, PCR reactions using Py. vexans genomic DNA as template with the Vex1 and Vex2 primers each yielded products approximately 220 bp, a length consistent with that predicted for amplified sequences encoding Vex1 and Vex2 of 219 and 213 bp respectively, assuming an intronless gene. Sequencing of these amplified products after cloning indicated that both Vex1 and Vex2 gene sequences had been separately amplified. The protein encoded by the 219 bp amplicon cloned into plasmid pVX1 showed 97% identity with the amino acid sequence of the elicitin protein Vex1 and the protein encoded by the 213 bp amplicon cloned into plasmid pVX2 showed 100% identity with the amino acid sequence of the elicitin protein Vex1 . Plasmid pCP containing 309 bp cryptogein coding region and pAA containing 705 bp actinA coding region from P. melonis were similarly prepared by PCR from genomic DNA from P. cryptogea and P. melonis respectively and authenticated by DNA sequencing.

The inserts from plasmids, pVX2 and pCP were used as elicitin probes in Southern blot hybridisation analysis. They encoded fragments of Alpha-elicitin Vex2 and Beta-elicitin cryptogein, respectively.

Multiple bands were detected in Phytophthora genomic DNA digested with either EcoRI or with BamHI and probed with either of the elicitin-specific DNA probes. Two to five EcoRI digested fragments were detected with the Beta-elicitin (cryptogein) probe in P. clandestina, P. erythroseptica, P. cryptogea and P. melonis (Figure 3a). Up to nine fragments were detected in BamHI digests. This suggested that in all these Phytophthora species, elicitins were encoded by a small multigene family similar in size to that previously identified by Kamoun et al. (1993b) in isolates of P. parasitica.

Multiple bands were also detected in the DNA of two Pythium species, Py. vexans and Py. ostracodes, when hybridised with the cryptogein probe (Figure 4a). The number of bands detected were fewer than the number detected in digests of Phytophthora containing equivalent amounts of DNA. Four fragments were detected in EcoRI digests of Py. vexans and Py. ostracodes and four to six fragments were detected in BamHI digests. The reproducibility of all hybridisation patterns was demonstrated by repeated Southern blot analysis of several species and the use of multiple preparations to minimise the possibility that multiple bands were detected due to partial digestion of DNA.

The southern blots of DNA digests from both Phytophthora species and Pythium species were also hybridised with the Alpha-elicitin (Vex2 ) probe. As shown in (Figure 3b), the Vex2 probe bound to multiple bands in the Phytophthora DNA digests and in general bound to the same bands hybridised by the cryptogein probe.

The Vex2 and cryptogein probes also hybridised to identical bands in Py. vexans and to all but two of the bands in Py. ostracodes (Figure 4). Neither the cryptogein nor the Vex2 probes hybridised to genomic DNA digests prepared from either Py. arrhenomanes, Py. graminicola or Py. myriotylum. The insert of pAA which encodes actin was used as a control in such Southern blot hybridisation to confirm the presence of genomic DNA on membranes which otherwise failed to bind elicitin probes.

Hybridisation with the actin probe showed that DNA from Py. vexans, Py. ostracodes, Py. arrhenomanes, Py. graminicola and Py. myriotylum were present in equivalent amounts in each of the digests of genomic DNA (Figure 4c). It was therefore concluded that the failure of elicitin probes to hybridise with Py. arrhenomanes, Py. graminicola and Py. myriotylum was due to the absence of homologous elicitin sequences in the genomes of these species. Genomic DNA from the Oomycetes Pachymetra chaunorhiza, Achlya ambisexualis and Peronospora parasitica were also hybridised with the cryptogein probe. Whilst the actin specific control probe detected intense and discrete bands in the DNA samples prepared from each of these species (data not shown), no such discrete bands were detected in any of these species with the elicitin-specific probe (Figure 5). Even the apparent hybridisation to DNA from Peronospora parasitica in Figure 5 was only at the intensity observed for non-specific binding to phage DNA markers.


The results obtained by probing genomic DNA for elicitin sequences gave a very clear result. Only in the DNA from Phytophthora and some Pythium species were there elicitin-like sequences. No trace was observed in species from orders outside the Peronosporales (Achlya ambisexualis; Saprolegniales) and within the Peronosporales only members of the family Pythiaceae gave positive results. There was no indication that these sequences were present in Peronospora parasitica or in Pachymetra chaunorhiza. Given this restricted distribution it is scarcely surprising that there was no trace of these genes in an ascomycete Saccharomyces cerevisiae.

Moreover, within the genus Pythium there was a clear division. Two species, Py. vexans and Py. ostracodes, contained base sequences which hybridised to the elicitin coding regions. All other Pythium species tested did not. The two species, Py. vexans and Py. ostracodes, and only these two species, also secreted proteins which were active in the elicitin-specific bioassay. In both cases, protein of Mr ca 10,000 constituted approximately 40% of these secreted proteins. Although the sample of Pythium spp examined was small, these results show that two categories of species exist within the genus.

We conclude that the presence or absence of elicitin genes is a potentially useful character for studying the phenological relationships within the genus Pythium, particularly given the ease of bioanalysis for the secreted protein. The distinct patterns obtained in DNA digests with both actinA probe and the two elicitin probes suggest that a major re-organisation of the genome has taken place during the acquisition / loss of the elicitin-like genes. Further analysis of the divergences of ribosomal RNA sequences within the Pythium species as undertaken by Briard et al., (1995) may assist in understanding the origin of this re-organisation. Phylogenetic trees based on ribosomal RNA sequences (Briard et al., 1995) and on earlier DNA analyses (Belkhiri and Dick, 1988), place Pythium vexans as far from other Pythium strains as from Phytophthora. If a third independent genus in the Pythiaceae were to exist as suggested by these authors, we conclude on the basis of the distribution of elicitin genes described in this paper that both Py. vexans and Py. ostracodes would belong together in such a genus.


This work was supported by the Australian Research Council Grant no S09947365

Table 1. Elicitin activities of proteins secreted from axenic cultures of Phytophthora and Pythium cultures.


                                   Necrotic Responses
Radish Cultivar                 Daikon        White Icicle

Phytophthora cryptogea           5/5            0/8
Phytophthora syringae            5/5            0/8    
Phytophthora clandestina         4/5            0/8    
Phytophthora erythroseptica      5/5            0/7
Pythium vexans                   5/5            0/8    
Pythium ostracodes               8/8            2/7    
Pythium graminicola              0/5            6/7    
Pythium myriotylum               0/5       
Pythium arrhenomanes             0/8

Culture filtrates were applied to radish seedlings at 10 µg per g of radish tissue. Response is recorded as the proportion of treated cotyledons that exhibited necrotic lesions after 72 h.

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