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Protección Forestal, Centro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP), CC 14, 9200, Esquel, Chubut, Argentina
Everett M. Hansen
Loretta M. Winton
Department of Botany and Plant Pathology, Oregon State University, Cordley Hall 2082, Corvallis, Oregon 97331-2902
Mario Rajchenberg
Protección Forestal, Centro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP), CC 14, 9200, Esquel, Chubut, Argentina
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ABSTRACT |
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A survey of Phytophthora spp. in declining and healthy Austrocedrus chilensis forest was conducted to obtain an overview of the species that inhabit these forests. Seventeen declining and three healthy stands plus 11 associated streams were surveyed. Five Phytophthora species were recovered. P. syringae was the most common species isolated from soil and/or streams at nine declining sites and one healthy site. P. gonapodyides was isolated from streams only, at five declining sites. P. cambivora was isolated from soil and the undescribed taxa ‘P. taxon Pgchlamydo’ and 22 ‘P. taxon Raspberry’ were isolated from streams at one declining site each. The species were identified by ITS rDNA sequences and morphological features. Brief descriptions of each species and a discussion of their possible relationship with "mal del ciprés" are presented.
Key words: forest decline, "mal del ciprés", Oomycota, Pythiaceae, soil pathogens
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Dimitri 1972).
A. chilensis’ longitudinal distribution embraces a steep rainfall gradient from 1500 mm/y on the W to 600 mm/y on the E. It forms pure and mixed stands with Nothofagus spp. and, among the few conifers inhabiting southern Argentina, it has the largest distribution, covering ca 160 000 ha (Dezzotti and Sancholuz 1991). Along with Araucaria araucana, it is the tree species that grows furthest into the ecotone zone with the Patagonia steppe, where it grows in dry forests or in isolated groups on the eastern border of its distribution. A. chilensis is economically valuable because of the high quality of its wood and it has a great tourist and scenic appeal.
Throughout its distribution area, A. chilensis suffers mortality termed "mal del ciprés" or "cypress wither" (Filip and Rosso 1999), caused by unknown factors.
The "mal del ciprés" decline was detected approximately 50 years ago, the first record having been in 1948 in Isla Victoria, Neuquén Province (Varsavsky et al 1975) and in 1953 in Epuyén, Chubut Province (Hranilovich 1988). Interest and concern have increased due to the constant expansion of the affected area. Because it decreases the mortality of the trees, the disease affects tourism, recreation and commercial forestry. It is impossible to carry out appropriate silvicultural management of affected stands because the appearance and evolution of the disease cannot be predicted. Another serious consequence of the disease is the replacement of native forest with exotic introduced species. This is due mainly to the lack of knowledge of its etiology and of any effective control leading the public institutions in charge of forest management to authorize land owners with affected forests to fell the dead trees and replace them with other species.
The main symptom of the disease is progressive withering and subsequent defoliation of the tree, which finally dies while standing. On the rootlets of affected trees, dead tissue can be observed. This becomes more severe during dry summers (Rosso et al 1994) and could be caused by the action of pythiaceous fungi (Pythiaceae, Oomycota) (Havrylenko et al 1989, 1992).
Different studies attempted to determine the etiology of this disease. Rosso et al (1994) studied the spatial distribution of the affected trees, showing that the mortality is scattered in patches; this characteristic could suggest a contagious process caused by a pathogen in the roots. Barroetaveña and Rajchenberg (1996) studied the wood rots associated with the cypress disease but did not find a causal agent, because the decay fungi were found to be opportunist saprophytes that colonize the sapwood once the tree is already in the decay process. Calí (1996) made den-drochronological studies that showed: (i) The relationship between radial growth decrease and disease progression; and (ii) that the phenomenon is initiated many years before external symptoms can be detected. This author found a relationship between the onset of the disease and the occurrence of earthquakes or climatic disturbances (i.e., 22 periods of drought followed by a humid period) in two localities near San Carlos de Bariloche (Río Negro province).
Abiotic factors such as topography, soil and site quality (Rajchenberg and Cwielong 1993, La Manna and Rajchenberg 2004a, b) or the annual rainfall (Havrylenko et al 1989, Calí 1996, Baccalá et al 1998) are the factors that are correlated most strongly with disease incidence. Declining stands usually are associated with poorly drained sites where low slopes, high moisture (at the end of the dry season) and redoximorphic features (close to the soil surface) might act as predisposing factors (La Manna and Rajchenberg 2004a).
The evidence gathered so far indicates that the disease originates in the root system, where the death of the tissue precedes defoliation of the crown (Hennon and Rajchenberg 2000). A decay of the roots is observed, followed in some cases by the development of brown rots in the sapwood. The origin of the disease in the roots, in addition to its association with poorly drained sites, could indicate the presence of pathogenic organisms from the Pythiaceae (Oomycota) affecting the roots.
The presence of Phytophthora spp. and Pythium spp. in association with cypress disease was studied preliminarily by Rajchenberg et al (1998), but the identification of the species needs critical review. A preliminary survey of Phytophthora that inhabit soil and streams of Austrocedrus chilensis forests was conducted. The main objective was to recover Phytophthora isolates from declining and healthy stands of A. chilensis to obtain an overview of the species, both pathogenic and non-pathogenic, that inhabit these forests. The survey revealed the presence of some species common in forests worldwide, and some unexpected ones, rarely reported in forests or rarely recorded before.
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MATERIALS AND METHODS |
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). Surveys mostly were focused in Río Grande Valley because it is one of the areas where decline is most severe, but stands in Los Alerces National Park, Corcovado and Bariloche also were included. Pure Austrocedrus chilensis stands were surveyed in each area.
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) is located in the west of Chubut Province, along the Río Grande. In this area the decline usually showed up as large areas with scattered dead, declining and apparently healthy trees. Ten sites and seven streams/rivers were surveyed in this area including three healthy sites (1, 9 and 10) and one stream associated with site1. Llao Llao-Bariloche (FIG. 1A) in the west of Río Negro Province. Surveyed sites had steep slopes, and the disease showed up as clear patches of dead trees. Three declining stands with no associated stream/river were surveyed in this area. Los Alerces National Park (FIG. 1B) in the west of Chubut Province. In this area the decline had two different appearances; on low slopes the disease had the same scattered appearance as in Río Grande Valley but, in two stands located on steep slopes, the disease appeared in discrete patches of dead trees surrounded by a ring of symptomatic trees. Four declining sites and two streams/rivers were surveyed in this area. Corcovado (FIG. 1D) in the west of Chubut Province. Surveyed sites showed a decline distribution as found in Río Grande Valley. Three declining sites and two streams/rivers were surveyed in this area. Sampling and isolation methods.— – Isolation of Phytophthora was attempted from soil and streams of Austrocedrus chilensis stands in fall and spring 2001 and 2002. Soil was surveyed from 17 declining, three healthy stands and 11 streams located near those stand. Isolation from streams was attempted using pears and apples as bait. One unripe pear and one apple were placed in each nylon mesh bag and anchored along the streams/river courses at approximately 20 m intervals. Ten bags usually were placed in each stream, but up to 18 were used in longer streams and as few as four in shorter ones. Baits were removed after seven to nine days. After surface sterilization with 96% ethanol, isolation was attempted from distinct, rounded brown spots. When no spot was visible, apples and pears were incubated at room temperature (17–20 C) until brown spots appeared but not longer than 10 d. Pieces were cut from the margin of the rots and placed in PARNP (CMA 17 g/L, pimaricin 10 mg/L, ampicillin 200 mg/L, rifampicin 10 mg/L, nystatin 50 mg/L, pentachloro-nitrobenzene (PCNB) 25 mg/L). Isolation from soil samples was attempted by baiting methods. One soil sample per each tree was collected at about 0.5–0.7 m from the bole of declining and healthy A. chilensis trees. The top 5 cm of soil were removed and 30–35 cm of soil were collected in a plastic bag, transported and stored in cool conditions until processing (no longer than 3 d). Soil samples were mixed carefully and a fraction (100–200 g) was placed in a disposable plastic tray and flooded (4 cm deep) with double-distilled water. One unripe pear and one apple were placed in each tray. Baits were removed after 7–10 d; isolation was attempted from distinct, rounded brown spots as for stream baits. When the soil was dry (dry season) after the first isolation, the remaining soil sample was premoistened with double-distilled water and stored for 7–10 d room temperature, then isolation was attempted a second time by the same methods as before. PARNP plates were incubated 22–25 C in the dark. Colonies of suspected Phytophthora and Pythium species were transferred to V8A and incubated 22–25 C in the dark until studied.
Species identification.— –
Morphospecies first were identified by comparison of colony pattern on V8A and PDA. Morphological features (sporangia, oogonia, antheridia, oospores, chlamydospores, hyphal swellings) were studied, described and drawn. Formation of sporangia was stimulated by flooding small V8A disks (0.5 mm diam) from the edge of a young culture (2–3 d old) in nonsterile soil-extract water (SEW) at room temperature. Soil extract was prepared by flooding 50 g of soil from an A. chilensis stand with 1 L of double-distilled water for 2 d and then filtering the solution through filter paper ( Jung et al 1996). Disks were inspected for sporangia after 48 h. Sporangia were described, measured and drawn. Release of zoospores was induced by chilling (4 C) 30 min. Oogonia of homothallic species were obtained in cultures grown in V8A amended with 20 mg/L ß-sitosterol. Oogonia of P. syringae were obtained in cultures grown in V8A amended with 20 mg/L ß-sitosterol and 6 ml/L vegetable oil at 10 C in the dark. Heterothallic species were crossed with tester strains of known mating type.
ITS analysis.— –
Identification of the species also was attempted by comparison of the nine sequences of their ITS rDNA regions generated at Oregon State University with 10 sequences of known species obtained from GenBank. A small agar plug with mycelium was taken from colonies growing on CMA and genomic DNA was extracted as described in Winton and Hansen (2001).
PCR was performed in 50 µl reactions (1x buffer, 200 nM dNTP, 0.4 µM ITS4 and ITS5 primers (White et al 1990), 0.05 U/µl RedTaq DNA polymerase (Sigma, St. Louis, Missouri) and 1 µl template DNA). Reaction conditions were: 60 s at 94 C, 34 cycles of 60 s at 94 C, 60 s at 55 C and 60 s at 72 C, and a final incubation for 7 min at 72 C. After amplification, PCR products were separated on a 1.5% agarose gel to evaluate concentration and quality. PCR products were prepared for DNA sequencing by addition of 0.5 µl EXOSAP-IT (USB, Cleveland, Ohio) and incubation overnight at 17 C followed by 15 min at 80 C. Direct sequencing of PCR products (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit, Applied Biosystems, Foster City, California) was performed with primers ITS2, ITS3, ITS4 and ITS5 (White et al 1990) and run on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Contigs were assembled and edited with the Staden (1996) software package. Those with evidence of multiple ITS-types (i.e., ambiguous base calls) were cloned with the TOPO TA Cloning Kit for Sequencing (Invitrogen Life Technologies, Carlsbad, California). Cells from two colonies of each cloning reaction were suspended in separate PCR mixtures and amplified and sequenced as described above.
Edited sequences were compared to sequences of the known species available at GenBank with the BLASTN search utility (Altschul et al 1997) and aligned to the datasets of Cooke et al (2000, TreeBase accession number M751) and Brasier et al (2003, GenBank accession number AF541887–AF541914) with the multiple alignment program ClustalX (Thompson et al 1997) (FIG. 3). Distance-based phylogenetic analysis was performed with PAUP* 4.0b4 for Windows (Swofford 1999) and employed the Jukes and Cantor correction with neighbor-joining tree-building options. Support for tree stability was obtained from 1000 bootstrap replicates. Trees were drawn with ATV (Zmasek and Eddy 2001).
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RESULTS |
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).
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, Rosso et al 1994). The pH of horizon A (in water) ranged from 5.97 to 7.13 (La Manna and Rajchenberg 2004a, La Manna pers comm). Five morphospecies were identified on the basis of colony pattern and micromorphological features. The identification of species was confirmed by comparison of ITS rDNA sequences.
Phytophthora syringae (Klebahn) Klebahn was the most widespread species. It was isolated from soil at four of 20 sites and from seven of 11 streams sampled. It was commonly present and abundant in highly and slightly human impacted areas. Colony pattern after 2 wk was slightly stellate on V8A medium. On PDA, the colony margin was densely petaloid, composed of small fans of mycelia densely and concentrically arranged and the colony was merulioid (folded with anatomizing ridges) in the central area. Isolates were homothallic, with sexual structures formed in V8A + ß Sitosterol amended with vegetable oil at 8–10 C. Oogonia were smooth, 27–42 µm diam (33.3 ± 2.15). Antheridia were mostly paragynous, 10–16 x 5–9 µm (11.8 ± 2.5 x 8.6 ± 2.9). Oospores were plerotic, 25–35 µm diam (30.53 ± 2.6), and walls were (1-)2–3 µm thick (2.19 ± 0.43). Sporangia readily formed in nonsterile soil-extract water after 48 h, and sometimes were present though scarce, in solid media (V8A, PDA). Sporangia were semipapillate, typically ovoid, but also elongate obpyriform and some with distorted shapes, sometimes with the stalk attached laterally, 25–74 x16–37 µm [mean: 41.6 (± 7) x 27.7 (± 4.5); L/B 1.5 (± 0.2); n = 323]. Mature sporangia were persistent and not proliferating but direct germination of closed sporangia with formation of a new sporangium frequently was observed. Sporangiophores were simple or formed close or lax sympodia. Chlamydospores were absent. Globose, catenulate hyphal swellings formed in liquid media.
Phytophthora cambivora (Petri) Buisman was isolated twice from soil, from only one site. P. cambivora and P. syringae were the only species isolated from soil. The colony morphology after 2 wk was cottony on V8A and PDA, without a distinct pattern. Both strains from Patagonia were heterothallic; sexual organs formed in about 15 d when paired with an A1 strain of P. cinnamomi. Oogonia were mostly smooth, some of them slightly bullate (sinuose walls) (FIG. 2B) and rarely bullate. They were thick walled, hyaline or yellowish, 33–50 µm diam (40.5 ± 3.74). Antheridia were amphigynous, mostly bicellular, 20–38 x 12–21 µm. (27.5 ± 5.06 x 17.6 ± 2.46). Oospores were plerotic, 31–46 µm diam (37.5 ± 3.51), with walls 3–4.5(–7) µm thick (3.86 ± 0.83), hyaline or yellowish. Sporangia were nonpapillate, ovoid to ellipsoid, 40–81 x 27–46 (59.33 ± 7.8 x 38.67 ± 4.23; L/B 1.54 ± 0.14; N = 100), proliferating, and non-deciduous. Sporangiophores were simple or simple sympodial. Chlamydospores were absent. Hyphal swellings usually were absent but once observed after 7–10 d in SEW (formed later than sporangia). These were globose and catenulate or clustered.
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P. taxon Pgchlamydo (Brasier et al 2003) was isolated from a stream at only one site. This area has a high human impact. The colony pattern after 2 wk was densely cottony with subfelty margins on V8A and petaloid on PDA. Isolates were sterile, with sexual structures not formed even when paired with A1 and A2 mating type tester strains. Sporangia were non-papillate, ellipsoid, ovoid, sometimes obpyriform or distorted shapes, 25–87 x 19–49 (56.6 ± 12.5 x 32.9 ± 5.3 L/B 1.71 ± 0.20, n = 277). They were non-deciduous and proliferating. Sporangiophores were simple or loosely simple sympodial. Globose hyphal swellings were formed.
P. taxon Raspberry (Brasier et al 2003) was isolated from a river at only one site, the same location where P. taxon Pgchlamydo was recovered. The colony morphology after 2 wk was cottony on V8A medium, without a distinct pattern and woolly with a dense, granulose aerial mat on PDA. The isolate was homothallic, with sex organs readily produced in solid and liquid media. Oogonia were globose and smooth, 30–43 µm (35.8 ± 2.96) diam. Antheridia were mostly paragynous but also amphigynous, irregularly ellipsoid or spherical, 7–17 x 7–15 µm. Oospores were mostly aplerotic, 27–35 µm (30.99 ± 2.82) diam, hyaline or slightly yellowish, and thick-walled (2.5–)3–5(–6) µm (4 ± 1.14). Sporangia were nonpapillate, ovoid or globose when young, sometimes with a basal swelling, (35–)40 –68 x (25–)30 –46 µm (50.6 ± 5.76 x 36.8 ± 3.54, L/B = 1.37 ± 0.08). They were nondeciduous and proliferating. Sporangiophores were mostly simple, or occasionally loosely simple sympodial. Hyphal swellings were globose, mostly with radiating hyphae, and often catenulate. Chlamydospores were absent but globose young sporangia might be confused with that structure.
ITS rDNA analysis.— –
Complete sequences of ITS1, 5.8S and ITS2 regions of rDNA for two representative strains of each morphospecies (TABLE II) were obtained and deposited in GenBank. Strains AG5, AG44 and AG45 were cloned because direct sequencing of PCR products yielded ambiguous base calls due to multiple ITS types. The alignment generated for phylogenetic analysis has been deposited at TreeBase (http://www.treebase.org/treebase/SN1859PIN19700). Sequences ranged from 745 bp (AG34) to 860 bp (AG45 clone2). Sequences of morphospecies 1 (strains AG5 and 24 AG66) matched 99% with GenBank sequences of P. syringae (FIG. 3). Sequences of 25 morphospecies 2 (strains AG34 and AG52) matched 100% with sequences of P. taxon Pgchlamydo, an undescribed taxon in the Phytophthora gonapodyides-P. megasperma ITS clade 6 (Brasier et al 2003). Sequences of morphospecies 3 (strains AG7, AG8 and AG56) matched 100% with P. gonapodyides sequences. Sequences of morpho-species 4 (strains AG44 and AG45) matched 99–100% with P. cambivora sequences. Morphospecies 5 (strain AG43) was identified as P. taxon Raspberry, another undescribed taxon in the Phytophthora gonapodyides-P. megasperma ITS clade 6 (Brasier et al 2003). Sequence of this species matched 100% with the sequence of strain P1050 from Sweden and 99% with sequences of strains P1049 and P896 from Australia and Tasmania, respectively.
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DISCUSSION |
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; Barzanti et al 2001; Delatour 2003; Fleischmann et al 2002; Jung et al 2000, 2003; Vettranio et al 2001) associated with oak decline areas. Recently, however, Jung et al (2003) demonstrated that many of these isolates previously identified as P. syringae are P. pseudosyringae, a new species morphologically similar to the former. True P. syringae has been isolated from oak and beech forests throughout Europe, but less frequently than P. pseudosyringae ( Jung pers comm). This pathogen has also been reported causing root rot on several tree species including Castanea, Fagus (Day 1938), Pinus (Newhook 1959) and through artificial inoculation, twig blight on Aesculus, Alnus and Quercus (Erwin and Ribeiro 1996).
P. cambivora has been reported causing root rot, collar root and or stem canker on several tree species (Brasier 2000, Erwin and Ribeiro 1996). It is well known as the cause, together with P. cinnamomi, of "ink disease" of Castanea sativa (Vettraino et al 2001) and it has been reported as root and collar pathogen of Fagus sylvatica (Day 1938, Jung and Blaschke 1996, Jung et al 2003). P. cambivora has been detected in soil of declining oak forests in several countries in Europe: France, Germany, Great Britain and Italy (Delatour 2003; Jönsson et al 2003; Jung et al 1996, 2000; Vettraino et al 2002); and its pathogenicity against Quercus robur rootlets has been demonstrated ( Jung et al 1996). P. cambivora also has been reported from noble fir Christmas tree plantations (Castagner et al 1995) and native vegetation (Hansen pers. comm.) in western North America. It also is known from hardwood plantations in Australia and native vegetation in Papua New Guinea (Old and Dudzinski 2000).
P. gonapodyides is apparently a widespread species commonly isolated from streams, rivers, ponds and riverbank soils around the world (Brasier et al 2003). It is also common in wet forest soils, submerged plant debris and in fallen, green, senescent leaves and fruits on soil (Hansen and Delatour 1999). The species seems to play an important role in plant debris degradation (Brasier et al 2003), but it has been reported as a weak pathogen. P. gonapodyides was isolated from stripe canker of Quercus robur but it was weakly pathogenic against rootlets of this species ( Jung et al 1996). It was one of the species associated with root rot and stem cankers of noble fir Christmas trees (Castagner et al 1995) and root rot of conifer seedlings (Hamm et al 1985, Campbell and Hamm 1989 misidentified as P. drechsleri [Brasier et al 1993]). Brasier et al (1993) state that the species might attack fine feeder roots of trees being in balance if the root system is unstressed but, it might cause a rapid decline if the host is stressed by conditions favorable for Phytophthora (i.e., flooding or prolonged wet soil conditions).
The pathogenicity of the two unnamed species, P. taxon Pgchlamydo and P. taxon Raspberry, is not known totally because they have been identified only recently. According to Brasier et al (2003) P. taxon Pgchlamydo seems to have a habitat similar to P. gonapodyides, being frequently isolated from rivers, but is not as common as this species. It also has been isolated from roots and recently was found in the rhizosphere of nursery grown alder plants (Alnus spp.) in Germany ( Jung and Blaschke 2004). P. taxon Raspberry has been isolated from diseased raspberry roots in Sweden and Australia and from high altitude boggy vegetation in Tasmania (Brasier et al 2003).
None of the isolated species showed a clear relationship with "mal del ciprés" incidence. P. syringae and P. cambivora are perhaps the most suspect because they were isolated from soil and are known to be pathogens of other tree species. P. syringae was isolated from most of the declining stands and also from one healthy stand, suggesting it is a widely distributed species not associated exclusively with declining sites. P. cambivora preliminarily is considered to have low incidence because it was isolated from only one declining stand. Pathogenicity tests should be carried out with all isolated species on A. chilensis, including stress of the trees as a factor. Since other studies (La Manna and Rajchenberg 2004a, b) showed a relationship between the disease and poorly drained sites, it is possible that Phytophthora species that are normally weakly aggressive to A. chilensis are being favored by site conditions and stressed trees. The slow progression of the decline process, that starts many years before external symptoms can be detected (Calí 1996), might also be related to the continuous action of weak pathogens affecting fine roots, causing chronic root reduction.
The situation of A. chilensis in Patagonia can be compared to other native forest declines in other areas of the world. Oak decline in Europe and Chamaecyparis nootakensis decline in Alaska are two decline processes that show similarities with "mal del ciprés". Both of them appear as complex diseases where a single cause could not be identified. However, in the case of oak decline, and depending on site conditions, a clear relationship has been demonstrated between the disease and the action of different Phytophthora spp. causing chronic root damage ( Jung et al 2000). The decline of Alaska yellow-cedar (Chamaecyparis nootakensis) is also a long term decline only present in SE Alaska. Unlike "mal del ci-pres", C. nootakensis mortality is present in mostly pristine areas. Its relationship with different biotic and abiotic factors has been investigated. No biotic factors (fungi, insects, nematodes, viruses or phyto-plasmas) has been identified as a primary cause (Hennon et al 1986a; 1990b; Hennon and McWilliams 1999). Alaska cedar decline appears to be an example of naturally induced forest decline related to abiotic factors (Hennon et al 1990a). It began on bog and semibog sites and subsequently spread onto better drained sites. Once some trees die, the adjacent trees lose protection becoming more vulnerable to weather; freeze and desiccation probably being the cause of the spread (Hennon et al 1990a). Other forest declines caused by Phytophthora such as jarrah (Eucalyptus marginata) dieback in Australia, Port-Orford cedar (Chamaecyparis lawsoniana) root diseas and sudden oak death in USA differ from "mal del ciprés" mostly because an aggressive pathogenic Phytophthora species is clearly involved and because of more rapid death of the trees.
In a second stage of this work, more specific baits (i.e., cypress leaves and seedlings) will be used in order to restrict the isolation to pathogenic or specific species. Two previously reported species (P. pseudotsugae and P. cactorum [Rajchenberg et al 1998]) were not recovered in this study. Because those cultures were lost, it is important to continue the survey to confirm or discard their presence and detect other species that could have been overlooked. In addition, a study of the fine root status on declining and healthy trees will be conducted to evaluate the hypothesis of the chronic root reduction as a cause of "mal del ciprés" decline.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: alina{at}ciefap.cyt.edu.ar
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LITERATURE CITED |
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