Identification of Phytophthora Species Baited and Isolated from Forest Soil and Streams in Northwestern Yunnan Province, China

REVIEW ARTICLE Identiﬁcation of Phytophthora species baited and isolated from forest soil and streams in northwestern Yunnan province, China

By W.-x. Huai1, G. Tian1, E. M. Hansen2, W.-x. Zhao1,5, E. M. Goheen3,N.J.Grunwald€ 4 and C. Cheng1

1Research Institute of Forest Ecology, Environment and Protection, The Key Laboratory of State Forestry Administration on Forest Protection, Chinese Academy of Forestry, Beijing, 100091, China; 2Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR USA; 3USDA Forest Service, Forest Health Protection, Central Point, OR USA; 4USDA Agricultural Research Service, Corvallis, OR USA; 5E-mail: [email protected] (for correspondence)

Summary Phytophthora species were surveyed by collecting soil samples and placing bait leaves in selected streams during June–October in the years 2005, 2006 and 2010 at three sites in oak forests in Diqing Tibetan Autonomous Prefecture of NW Yunnan province, China. Seventy-three isolates of Phytophthora spp. were recovered from 135 baited leaf samples and 81 soil samples. Eight Phytophthora species were identiﬁed by observation of morphological features and ITS1-5.8S-ITS2 rDNA sequence analysis. The eight taxa included two well-known species P. gonapodyides and P. cryptogea, two recently described species P. gregata and P. plurivora, two named but as yet undescribed taxa, P. taxon PgChlamydo and P. taxon Salixsoil, and two previously unrecognized species, Phytophthora sp.1 and P. sp.2. The most numerous species, P. taxon PgChlamydo, and the second most abundant species, P. taxon Salixsoil, were recovered at all three sites. Phytophthora cryptogea was detected only once at site Nixi. Phytophthora gregata and P. sp.2 were isolated from a stream only at site Bitahai, while the other three species were each found at two sites. Phylogenetic analysis revealed that the isolates belonged to three ITS clades, one species including six isolates in clade 2, six species including 66 isolates in clade 6 and one species in clade 8. There was a relatively rich species and genetic diversity of Phytophthora detected in the investigated regions where the forest biotic and abiotic factors aﬀecting the growth and evolution of Phytophthora populations were diverse.

1 Introduction Phytophthora is a genus that is mainly parasitic on various plant hosts. Some species are host specific and some have broad host ranges. It occupies a small but pathologically significant niche in Oomycota currently belonging to the kingdom Chro- mista (Dick 1995a,b). In 1983, only 43 species of Phytophthora were recognized. Later, about 60 species were described by Erwin and Ribeiro 1996; and by 2008, the number of species was approaching 100 (Gallegly and Hong 2008). The genus Phytophthora has complex life cycles with several spore stages. Chlamydospores and oospores are two kinds of resting spores that Phytophthora produces for long-term survival. Oospores are the outcome of sexual reproduction, produced by mating of a female organ called oogonium, which is fertilized by a male organ called antheridium. When environ- mental conditions become suitable, the resting spores germinate by forming sporangia, which release motile, biflagellate zoospores into the soil water.(Erwin and Ribeiro 1996) Because of the morphological variation encountered in this genus, it is often difficult to identify a given isolate to species merely based on morphological and physiological traits. Advances in molecular techniques, such as DNA sequence data analysis from the internal transcribed spacer region and 5.8S gene of the rDNA operon, the mitochondrial cox1 and cox2, ras-related protein, elicitin and b-tubulin genes have permitted a more rational study of genetic distance and phylogenetic relationships within the genus Phytophthora and enhanced the efficiency and accuracy of identification of species as well as clonal lineages within species (Cooke et al. 2000; Forster et al. 2000; Elliott et al. 2009; Grunwald€ et al. 2009). Over the past decade, a number of new Phytophthora species have been described from forests around the world. Some of these new species, such as P. ramorum (Grunwald€ et al. 2008), cause dramatic tree and woody ornamental diseases. Other times, these new Phytophthora species have been found during broader surveys of soil or streams in forest stands that are not part of direct observations of forest declines (Balci et al. 2007). In China, extensive studies about morphological and molecular detection and identification of Phytophthora species as well as population genetics have been focused on pathogens of agricultural crops or ornamental plants such as soybean (P. sojae), potato and tomato (P. infestans), pepper (P. capsici), etc., and some flowers (Zheng 1997; Li et al. 1999; Wang et al. 2000, 2004; Yang et al. 2008, 2011; Guo et al. 2010). So far, there have been only a few reports investigating Phytoph- thora diseases of woody plants. Some species including P. cinnamomi, P. citricola, P. heveae, P. drechsleri, P. citrophthora and P. palmivora were recorded in association with woody plant and forest diseases (Zheng 1997; Zeng et al.2009). Of these, P. cinnamomi is of particular significance due to its broad host range and worldwide distribution, although it lacks the information on economic impacts in China. It had been identified as the cause of death of black locust, cedar and camellia, etc., (Ho et al. 1984; Shen et al. 1990; Zhou et al. 1993). Moreover, some early identified species may need to be re-examined or revised with the advent of a new taxonomic era highlighting the combination of morphological and molecular classification. So far, there have not been any reports on the comprehensive investigation of Phytophthora species in the forest of the north-west region of Yunnan province, China. Therefore, the objectives of this study were to isolate and

Received: 5.4.2012; accepted: 4.11.2012; editor: L. Belbahri

http://wileyonlinelibrary.com/ 88 W.-x. Huai, G. Tian, E. M. Hansen et al. identify Phytophthora species by baiting techniques from streams and forest soil among or alongside oak forests and to use a combination of morphological and molecular tools to evaluate the genetic diversity of Phytophthora populations. Phytoph- thora ramorum, cause of sudden oak death disease in Europe and North America, was a particular target of the search.

2 Materials and methods

2.1 Study sites The study was carried out in Diqing Tibetan Autonomous Prefecture of NW Yunnan province, mainly in the geographic area of Zhongdian (Shangrila). There are evergreen sclerophyllous Quercus forests in this region distributed from 900 to 4800 m asl, chieﬂy between 2400 and 3600 m. Quercus aquifolioides Rehd. et Wils., Q. pannosa Hand.-Mazz., and Q. longispica (Hand.-Mazz) A. Camus were the most important species in the Quercus forests, accompanied by Rhododen- dron rubiginosum Franch., R. vernicosum Franch., R. oreotrephes W. W. Sm., R. decorum Franch., Larix potaninii Batalin, Picea likiangensis (Franch.) Pritz., P. armandii Franch., Pinus densata Mast., Populus szechuanica Schneid., Sorbus rehderiana Koehne and S. rufo pilosa Schneid. Based on climatological data for the cities of Zhongdian (Shangrila) (27°42′N, 99°42′E), average monthly temperatures range between À1.5°C in January and 15°C in July. The mean annual precipitation is 520.5 mm for Zhongdian. Most of it occurs during the monsoon months of June–October. Three decline sites and streams in Quercus forests were surveyed in this area (Fig. 1). Site Nixi (N) (27°58′N, 99°35′E) was about 500 m downstream of a stream in forest located in Nixi Township north of Zhongdian. Site Bitahai (B) (27°44′N, 99°58′E) was near Yakou Inspec- tion Station in the south of the Bitahai Nature Reserve. The third site, Hutiaoxia (H) (27°12′N, 100°02′E) was chosen at the edge of forest that was close to the Hutiaoxia Forest Farm.

2.2 Sampling procedure and isolation methods Surveys were conducted from June to October in the years 2005, 2006 and 2010 both by collecting soil samples at the base of declining trees and placing baiting leaves in selected streams. Nine soil samples were collected from each declining site per year. Unripe apples (variety Golden Delicious) were used as baits for isolating Phytophthora species from soil (Jeffers and Martin 1986). After 4–6 days of incubation at 24°C, the browning parts of apple flesh around holes were plated onto a selective medium (CARP+: corn meal agar [CMA] with 25 ppm hymexazol [99%], 20 ppm Delvocid [50% natamycin salt], 200 ppm ampicillin sodium salt, 10 ppm rifamycin SV sodium salt and 30 ppm Benlate [benomyl 50WP]) and incubated at 20°C in the dark. Three pieces of Rhododendron (site 2) or Quercus (site N and H) leaves were placed in each nylon mesh bag and were floated in relatively slow-moving water in streams as baits. The leaf baits were removed after 7–10 days when lesions developed. Fifteen bags of baited leaf samples were collected from each stream per sampling year. Pieces about 2-mm2 were cut from the margin of the brown spots and plated in CARP+. Colonies of suspected Phytophthora and Pythium species growing from plated baits were transferred to CARP (CARP+ without Benlate and hymexazol) to confirm purity and then to CMA for characterization and storage.

2.3 Identiﬁcation of Phytophthora

2.3.1 Morphological studies Pure cultures of Phytophthora isolates were cultivated on V8-juice agar amended with 20 ppm b-sitosterol (V8S) and on carrot agar (CA) plates and were incubated in the dark at 20°C. After 5–10 days, the isolates were examined for cultural

Fig. 1. The Map of Diqing Tibetan Autonomous Region in the northwestern part of Yunnan province, showing locations of the sampling and baiting sites. Phytophthora species from soil and streams of China 89 and morphological characteristics (Waterhouse 1963; Erwin and Ribeiro 1996; Gallegly and Hong 2008; Jung and Burgess 2009). Heterothallic isolates were crossed with reference cultures of known mating types of P. cinnamomi and P. cambivora from the laboratory of Everett Hansen, Oregon State University. Sporangia were examined on plugs cut from the margin of colonies grown on V8S plates about 4–7 days and suspended in distilled water in daylight for about 24 h.

2.3.2 DNA extraction, amplification and sequencing The isolates for DNA extraction were grown on potato dextrose agar (PDA) at 20°C for 10 days, and the mycelium was harvested by scraping from the agar surface with a sterile blade and placed in a sterile mortar with 800 llofpreheated(60°C) 29 CTAB extraction buffer (2% (w/v) CTAB, 100 mM Tris–HCl, 1.4 M NaCl, 20 mM EDTA, pH 8.0) and ca 0.3 g sterilized quartz sand (Sigma-Aldrich, St. Louis, MO, USA). Harvested mycelium was ground using a pestle for 3–8 min, and genomic DNA was extracted using the modified CTAB protocol (Huai et al. 2003). Primers ITS6 (5′-GAA GGT GAA GTC GTA ACA AGG-3′) (Cooke et al. 2000) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC- 3′) (White et al. 1990) were used to amplify both ITS1 and ITS2 regions including the 5.8S rDNA. The mitochondrial gene cox1 was amplified with primers FM84 and FM83 (Martin and Tooley 2003). PCR reaction mixtures (50 ll) contained ca 20-ng template DNA, 0.5 mM of each primer, 200 mM of dNTPs, 2.5 U Taq DNA polymerase (Promega, Madison, WI, USA)., 3 mM MgCl2, 5 ml of 109 PCR buffer, 100 mM bovine serum albumin. Amplifications were carried out with an Eppendorf Master- cycler Gradient Thermalcycler 5331 (Eppendorf AG, Hamburg, Germany) with the following run parameters: one cycle at 95°C for 2 min; 35 cycles of 94°C for 1 min, 58°C for 30 s, 72°C for 2 min; and followed by one extension cycle at 72°C for 10 min. Amplicon purification and sequencing in both directions was performed by the sequencing facility of Sangon Biotech (Shanghai) Co., Ltd. (China) in Beijing (http://www.sangon.com/sangonindex.aspx).

2.3.3 Phylogenetic analysis The sequence data of Phytophthora isolates used in this study were compared with other closely related species including some undescribed taxa obtained from GenBank (http://www.ncbi.nlm.nih.gov/balst/). Sequence data were initially assem- bled using STADEN Package 1.6.0 (MRC, Cambridge, England). The phylogenetic tree was constructed by the neighbour- joining algorithm using MEGA 4.0, and bootstrap analysis was performed with 1000 trials (Tamura et al. 2007).

3 Results

3.1 Study areas, samples and isolation of Phytophthora The three sampling and monitoring sites for Phytophthora, Nixi, Bitahai and Hutiaoxia, have various oak species in common, and differ in height above sea level of 3418, 3643 and 1935 m, respectively. Some symptoms resembling Phytophthora infection such as leaf lesions, dieback, wilt, decline and death were observed in all the three forests. Of 112 Phytophthora or Pythium colonies isolated, the Pythium spp. were readily separated from Phytophthora spp. through selective medium and subculturing (Jeffers and Martin 1986). Consequently, 73 pure isolates of Phytophthora spp. were obtained that qualified for further examination and sequence analysis for this study (Table 1). The colony morphology of all the isolates on CARP+ selective medium coincided with literature descriptions of Phytophthora (Erwin and Ribeiro 1996; Zheng 1997). Moreover, the colony growth temperatures as well as other physiological characters also fell into the category of reported Phytophthora.

3.2 Phytophthora identification based on ITS1-5.8S-ITS2 sequence identity The ITS region (ITS1-5.8S-ITS2) of all isolates was amplified and sequenced. All Phytophthora amplicons displayed the same size products of about 850 bp, while normally Pythium spp amplicons were about 950 bp. Target amplicons were sent for direct sequencing without cloning. The results of the BLASTn query indicated that eight different Phytophthora species were included (Table 2): P. cryptogea Pethybridge and Lafferty, P. gonapodyides (Petersen) Buisman, P. gregata T. Jung, M.J.C. Stukely and T. Burgess, P. plurivora T. Jung and T.I. Burgess, P. taxon PgChlamydo, P. taxon Salixsoil, Phytoph- thora sp.1 and P. sp.2. However, P. ramorum, the causal agent of Sudden Oak Death, was not found in the surveys from 2005 to 2010. When representative sequences of these identified species or taxa were submitted to the Phytophthora Database website to do online identification (www.phytophthoradb.org), the results were in agreement with those of the GenBank BLAST query. Complete sequences of ITS1, 5.8s and ITS2 regions of rDNA for all isolates in the study were edited and deposited in GenBank. The sequence data set ranged from 760 to 820 bp. Sequence identity comparison showed that the six isolates, 191, DQ01-11, NQ02h, NX05a, b and c shared 99.61–100% identity in ITS sequences with P. plurivora type culture (FJ665225). Sequences of four isolates, NQ07c, NXQ13-2, DQ13-7 and 7a1 matched 99.39–99.63% identity with reference sequences of P. gonapodyides (AF541889). Three isolates, 23a, BR05c and 34b3 shared 99.63–99.88% identity with P. gregata. Forty-three isolates of P. taxon PgChlamydo were similar in ITS sequence, the identity with P. taxon PgChlamydo (AF541901 and HM004224) ranging from 99.63 to 100% (18 of 43 isolates had 100% identity of ITS sequences), ITS Table 1. identification of 73 isolates from Diqing, Yunnan province based on ITS sequence similarity. 90

Species Isolate no. Waterhouse group ITS clade GenBank accession number The most similar reference and GenBank accession Base identity

Phytophthora plurivora 191 III 2 JQ730711 P. plurivora FJ665225 759/761 DQ01-11 III 2 JQ730712 760/761 NQ02h III 2 JQ730713 761/761 NX05a III 2 JQ730714 757/760 NX05b III 2 JQ730715 757/760 NX05d III 2 JQ730716 757/760 P. gonapodyides NQ07c V/VI 6 JQ730717 P. gonapodyides AF541889 816/819 NXQ13-2 V/VI 6 JQ730718 816/819 DQ13-7 V/VI 6 JQ730719 816/819 7a1 V/VI 6 JQ730720 814/819 P. gregata 23a V/VI 6 JQ730721 P. gregata HQ012942 819/820 BR05c V/VI 6 JQ730722 819/820 34b3 V/VI 6 JQ730723 816/819 P. taxon PgChlamydo 193b V/VI 6 JQ730724 P. taxon PgChlamydo AF541901 HM004224 816/819 BR03a V/VI 6 JQ730725 817/819

BR03f V/VI 6 JQ730726 817/819 al. et Hansen M. E. Tian, G. Huai, W.-x. BR03h V/VI 6 JQ730727 817/819 BR04a V/VI 6 JQ730728 817/819 BR05e V/VI 6 JQ730729 819/819 BR06a V/VI 6 JQ730730 817/819 BR06b V/VI 6 JQ755183 817/819 BR06c V/VI 6 JQ755184 817/819 BR06d V/VI 6 JQ755185 817/819 BR06e V/VI 6 JQ755186 817/819 BR07a V/VI 6 JQ755187 817/819 BR07e V/VI 6 JQ755188 817/819 BR08a V/VI 6 JQ755189 817/819 BR08b V/VI 6 JQ755190 817/819 BR08h V/VI 6 JQ755191 816/819 BR09b V/VI 6 JQ755192 816/819 BR09c V/VI 6 JQ755193 816/819 BR09d V/VI 6 JQ755194 816/819 NQ02b V/VI 6 JQ755195 816/819 NQ05a V/VI 6 JQ755196 817/819 192 V/VI 6 JQ755197 819/819 NX03a V/VI 6 JQ755198 819/819 193a V/VI 6 JQ755199 816/819 23b V/VI 6 JQ755200 817/819 DQ01-1 V/VI 6 JQ755201 819/819 DQ01-3 V/VI 6 JQ755202 819/819 DQ01-5 V/VI 6 JQ755203 818/819 DQ01-6 V/VI 6 JQ755204 819/819 DQ01-10 V/VI 6 JQ755205 819/819 DQ01-19 V/VI 6 JQ755206 819/819 DQ01-20 V/VI 6 JQ755207 819/819 DQ02-1 V/VI 6 JQ755208 819/819 DQ02-2 V/VI 6 JQ755209 819/819 DQ02-3 V/VI 6 JQ755210 819/819 Table 1. Continued

Species Isolate no. Waterhouse group ITS clade GenBank accession number The most similar reference and GenBank accession Base identity

DQ07-2 V/VI 6 JQ755211 818/819 DQ12-1 V/VI 6 JQ755212 819/819 DQ12-2 V/VI 6 JQ755213 819/819 DQ12-3 V/VI 6 JQ755214 817/819 DQ12-4 V/VI 6 JQ755215 819/819 DQ12-6 V/VI 6 JQ755216 819/819 DQ13-1 V/VI 6 JQ755217 819/819 NXQ12-2 V/VI 6 JQ755218 819/819 P. taxon Salixsoil HQ01a V/VI 6 JQ755219 P. taxon Salixsoil AF541909 816/817 34a1 V/VI 6 JQ755220 817/817 DQ02-9 V/VI 6 JQ755221 814/817 DQ10-16 V/VI 6 JQ755222 814/817 DQ10-21 V/VI 6 JQ755223 814/817 DQ12-10 V/VI 6 JQ755224 816/817 Phytophthora DQ13-6 V/VI 6 JQ755225 815/817 NXQ12-1 V/VI 6 JQ755226 816/817 NXQ13-6 V/VI 6 JQ755227 815/817 P. sp.1 HQ01b V/VI 6 JQ755228 P. taxon Forestsoil AF541908 804/819

7a2 V/VI 6 JQ755229 802/819 China of streams and soil from species DQ09-5 V/VI 6 JQ755230 804/819 DQ10-24 V/VI 6 JQ755231 804/819 DQ10-26 V/VI 6 JQ755232 804/819 P. sp.2 34a2 V/VI 6 JQ755233 P. taxon paludosa HQ012953 805/816 34b2 V/VI 6 JQ755234 805/816 P. cryptogea 34b1 VI 8 JQ755235 P. cryptogea FZJ-1 AY742742 797/798 91 92 W.-x. Huai, G. Tian, E. M. Hansen et al.

Table 2. Distribution of Phytophthora species recovered from soil and streams in Quercus forests, Diqing, Yunnan province.

Positive Species isolations Site Isolated from Year

P. gonapodyides 4 N, B Streams 2005, 2010 P. gregata 3 B Streams 2005 P. plurivora 6 N, B Soil and streams 2005, 2006, 2010 P. sp. PgChlamydo 43 N, B, H Soil and streams 2005, 2006, 2010 P. sp Salixsoil 9 N, B, H Streams 2005, 2010 P. cryptogea 1 N Streams 2005 P. sp.1 5 B, H Streams 2005, 2010 P. sp.2 2 B Streams 2005 sequences of nine isolates shared 99.63–100% identity with P. taxon Salixsoil. Two additional groups of isolates, named P. sp1 and 2 are thought to belong to new species because they were closely related to but differing from the closest taxa Forestsoil and Paludosa in ITS sequence identity of 97.92–98.65%. When aligning the sequences within species or taxa, six polymorphic sites were found among five isolates, three P. gregata isolates and the two closely related P. taxon Raspberry isolates (Table 3). Seven P. plurivora isolates had four polymorphic sites, and five P. gonapodyides isolates had six polymorphic sites (Tables 4 and 5). Double base peaks with similarly strong signal in the ITS sequencing chromatogram were observed in isolates of P. cryptogea, P. plurivora, P. taxon PgChlamydo and P. taxon Salixsoil as a consequence of direct sequencing the ITS amplicon rather than cloning and then sequencing. Double peaks were particularly evident in the ITS sequences of P. taxon Salixsoil and P. taxon PgChlamydo isolates (Tables 6 and 7). The double base peaks were detected in about 68% (23/34) of P. taxon PgChlamydo isolates. Double peaks at nucleotide positions 54, 129, 190 and 503 were evident (Table 7). In all cases, however, isolates exhibited the same nucleotide combination at each polymorphic site.

3.3 Phylogenetic analysis After an initial phylogenetic analysis including all sequenced Phytophthora isolates and known species or undescribed taxa from every ITS clade, six isolates fell in clade 2 and 66 isolates resided in clade 6, while only one isolate belonged to clade 8. Consequently, the identification conclusion of eight species was strongly supported. The ITS clade 2 isolates obtained in this study were phylogenetically close and matched more than 99% with GenBank sequences of P. plurivora and were closely related to P. pini and P. citricola (Fig. 2). Among them, one isolate (NQ02h) was identical to the type isolate of P. plurivora at all bases, while the other two isolates (191 and DQ01-11) differed by one or two bases. The remaining three isolates NX05a, b and d grouped into a distinct cluster from the other three isolates in the phylogenic tree and varied at three bases and one deletion from the reference isolate, suggesting they are different strains of P. plurivora. Six species including two previously unidentified taxa were identified in ITS clade 6 (Fig. 3). Phytophthora taxon PgChlamydo, previously reported but not yet formally described, was the most frequently identified species overall. All 43 new isolates of P. taxon PgChlamydo grouped into one consensual cluster with several reference isolates (AF541900, AF541901, HM004224). They were closely related to P. pinifolia and P. taxon Hungarica, and clustered into one large evolutionary branch with four P. gonapodyides isolates and other reference species, P. megasperma and P. thermophila. Nine isolates were identified as P. taxon Salixsoil, another undescribed taxon in ITS clade 6 with 100% bootstrap support. Among the nine isolates, one exactly matched ITS sequence of the European reference isolate, and others differed at one to three nucleotide positions. Isolates 23a, BR06c and 34b3 corresponded to P. gregata, the recently described species in subclade II of ITS clade 6, based on both morphology and ITS sequence data (Jung et al. 2011). In this study, the ITS analysis revealed two previously unrecognized species in Clade 6. Phytophthora sp.1 was represented by five isolates recovered from streams at sites Bitahai and Hutiaoxia. Phytophthora sp.1 isolates grouped into an

Table 3. Polymorphic nucleotides from aligned sequence data of ITS showing the variation between isolates 23a, BR05c, 34b3, Phytophthora gregata and P. taxon Raspberry.

Position

Sample no. 15 bp 674 bp 676 bp 718 bp 729 bp 743 bp

23a A G G T A T BR05c A G G T A T P. gregata HQ012942 A G G T G T 34b3 – AACGT P. taxon raspberry AF541905 – AACGC Phytophthora species from soil and streams of China 93

Table 4. Polymorphic nucleotides from aligned ITS sequence data showing variation among isolates belonging to Phytophthora plurivora.

Position

Sample no. 64 bp 152 bp 395 bp 714 bp

191 – TCG DQ01-11 T C T G NX05a – TTT NX05b – TTT NX05d – TTT NQ02h T C C G P. plurivora type FJ665225 T C C G

Table 5. Polymorphic nucleotides from aligned ITS sequence data showing the variation between isolates belonging to Phytophthora gonapodyides.

Position

Sample no. 134 bp 327 bp 474 bp 553 bp 729 bp 744 bp

NQ07c T T C C T G NXQ13-2 G T T C T G DQ13-7 G T T C T G 7a1 G A T C A G P. gonapodyides AF541889 G T C T T A

Table 6. Polymorphic nucleotides from aligned ITS sequence data showing the variation among isolates closely related to P. taxon Salixsoil and double peaks reading from direct sequencing chromatograms.

Position

Sample no. 45 bp 139 bp 370 bp 457 bp 502 bp 601 bp 717 bp 796 bp

HQ01a A C C C G A/G C C 34a1 A C C C G G C C DQ02-9 A/C C C C/T G G C/T C DQ10-16 A C/G C/T C/T G/T G C T DQ10-21 A/C C C C/T G G C/T C DQ12-10 A C C C G G C T DQ13-6 A C C C/T G G C/T C NXQ12-1 A C C C G G C C/T NXQ13-6 A C C C/T G G C/T C P. taxon Salixsoil AF541909 A C C C G G C C

independent subclade and were distinct from all described species residing in a terminal clade with high bootstrap support. They had ITS sequence closest to P. taxon Forestsoil but diﬀered from it at 15 or 17 bases. Phytophthora sp.2 had nearest sequence and morphology similarity to the newly reported P. taxon paludosa, from Western Australia (Jung et al. 2011). It was sexually sterile, and had persistent, non-papillate, ovoid sporangia that prolifer- ated internally. It was represented by two isolates (34a2 and 34b2) obtained from stream water at site B and both isolates shared identical ITS sequences and diﬀered from the Australian reference isolate at 11 bases plus three in/del positions. Only one isolate (34b1) fell in ITS Clade 8 (Fig. 4). Its ITS sequence matched over 99% with GenBank sequences of P. cryptogea. It varied at one double peak at position 146 from the Chinese reference isolate FZJ-1. It was phylogenetically close to some strains of P. erythroseptica and P. drechsleri but had less relatedness with P. ramorum and P. lateralis, both associated with severe forest diseases in North America and Europe. Using the Maximum composite Likelihood method in MEGA4, pairwise analysis of the sequences of the Phytophthora isolates and reference sequences from GenBank belonging to clade 2 and 6 was performed, respectively. The results showed that the values for estimates of evolutionary divergence between two sequences among compared isolates in clade 2 ranged from 0 to 0.094, while the values among six P. plurivora isolates were 0–0.004. Pairwise analysis of the sequences of the Phytophthora isolates and reference sequences from GenBank belonging to clade 6 showed that the values for estimates of evolutionary divergence ranged from 0 to 0.087, while the maximal value among all the clade 6 isolates was 0.029. 94 W.-x. Huai, G. Tian, E. M. Hansen et al.

Table 7. Polymorphic nucleotides from aligned ITS sequence data showing the variation among isolates compared with P. taxon PgChlamydo and double peaks reading from direct sequencing chromatograms.

Position

Sample no. 54 bp 129 bp 190 bp 530 bp

193b A/G A/C C/T G BR03a G A/C C/T G BR03f G A/C C/T G BR03h G A/C C/T G BR04a G A/C C/T G BR05e G C C G BR06a G A/C C/T G BR06b G A/C C/T G BR06c G A/C C/T G BR06d G A/C C/T G BR06e G A/C C/T G BR07a G A/C C/T G BR07e G A/C C/T G BR08a G A/C C/T G BR08b G A/C C/T G BR08h A/G A/C C/T G BR09b A/G A/C C/T G BR09c A/G A/C C/T G BR09d A/G A/C C/T G NQ02b A/G A/C C/T G NQ05a G A/C C/T G 192 G C C G NX03a G C C G 193a A/G A/C C/T G 23b G A/C C/T G DQ01-1 G C C G DQ01-3 G C C G DQ01-5 G C C T/G DQ01-6 G C C G DQ01-10 G C C G DQ01-19 G C C G DQ01-20 G C C G DQ02-1 G C C G DQ02-2 G C C G DQ02-3 G C C G DQ07-2 G C C T/G DQ12-1 G C C G DQ12-2 G C C G DQ12-3 G A/C C/T G DQ12-4 G C C G DQ12-6 G C C G DQ13-1 G C C G NXQ12-2 G C C G P. taxon PgChlamydo AF541901 G C C G

3.4 Morphological characterization The Phytophthora isolates first identified by ITS sequence analysis were further confirmed by comparing colony growth patterns, morphological features of sporangia, oogonia, antheridia, chlamydospores and hyphal swellings with known isolates and reference species descriptions in the literature. All the isolates produced sporangia, and some produced oospores in sufficient numbers for positive identification. Six isolates, 191, PQ01-11, NQ02h, NX05a and NX05d with semi-papillate sporangia, were placed in Waterhouse group III as the ‘P. citricola complex’ and now identified as the recently described species, P. plurivora (Jung and Burgess 2009), as confirmed with ITS sequence. These isolates were homothallic, with globose or subglobose oogonia and paragynous antheridia. Sporangia were borne terminally on unbranched sporangiophores or laterally attached or intercalary, non-deciduous, semi-papillate, frequently bi- or tripapillate. The shapes of sporangia ranged from ovoid or lemoniform to obpyriform, ellipsoid or distorted shapes (Fig. 5). Isolates of P. taxon PgChlamydo (Brasier et al. 2003a) were sterile and usually formed sporangia rapidly in water (within 24 h). Sporangia were non-papillate, broadly or elongated obpyriform or ovoid, persistent, on simple or loosely simple sympodial sporangiophores; clumps of large globose to subglobose hyphal swellings were often formed in water (Fig. 6). Phytophthora taxon Salixsoil was originally described from Europe (Brasier et al. 2003a), and it was isolated from all three streams sampled. Sporangia of the isolates were non-papillate, broadly obpyriform or ovoid, and persistent on simple Phytophthora species from soil and streams of China 95

Fig. 2. Phylogenetic tree showing the relationship within Phytophthora Clade 2 based on rDNA ITS sequence.

or loosely simple sympodial sporangiophores (Fig. 7). Clumps of globose to subglobose hyphal swellings were often formed in agar. Sporangia of P. gonapodyides were non-papillate, ovoid to obpyriform, proliferating internally and non-deciduous (Fig. 8). Isolates were self-sterile. Three isolates recovered from two streams had non-papillate sporangia and produced oospores in single-isolate culture and were classiﬁed into Waterhouse group V/VI and identiﬁed as Phytophthora gregata supported by ITS sequence. Sporangia of P. gregata were produced abundantly in water, persistent and usually proliferating internally. The shapes of sporangia ranged from ovoid to elongated ovoid, limoniform or ellipsoid, pyriform or obpyriform. Glo- bose to subglobose oogonia were formed in single culture on V8S, antheridia were amphigynous or paragynous (Fig. 9). 96 W.-x. Huai, G. Tian, E. M. Hansen et al.

Fig. 3. Phylogenetic tree showing the relationship within Phytophthora Clade 6 based on rDNA ITS sequence.

3.5 Distribution of Phytophthora species Phytophthora plurivora was recovered from soil and streams at site Nixi and Bitahai. Of eight Phytophthora species detected in this study, P. taxon PgChlamydo was the most widespread (59.72% of all isolates) being recovered from both soil and streams at the three sites. The second most common species isolated was P. taxon Salixsoil, originally described from Europe. Phytophthora gonapodyides as well as Phytophthora plurivora were isolated from stream water at site N and B. Five Phytophthora species from soil and streams of China 97

Fig. 4. Phylogenetic tree showing the relationship within Phytophthora Clade 8 based on rDNA ITS sequence.

(a) (b)

Fig. 5. Structures of a Phytophthora plurivora isolate formed on V8 agar ﬂooded with distilled water. (a, b) Semi-papillate ovoid sporangia, young growing sporangia and empty, bipapillate sporangia with distorted shapes; (c, d) oogonia with paragynous antheridia. —Scale bar = 20 lm. 98 W.-x. Huai, G. Tian, E. M. Hansen et al.

(a) (b)

Fig. 6. Structures of Phytophthora taxon PgChlamydo isolates formed on V8 agar ﬂooded with distilled water. (a–c) Non-papillate sporangia; (d) chlamydospores and hyphal swellings. —Scale bar = 20 lm.

(a) (b)

Fig. 7. Sporangia of Phytophthora taxon Salixsoil formed on V8 agar ﬂooded with distilled water. (a–d) Broadly obpyriform or ovoid, non- papillate sporangia; (b, c) ovoid empty sporangia with internal nested proliferation. —Scale bar = 20 lm.

P. sp1 isolates from site B and N and one P. cryptogea isolate from site N were recovered from streams only in 2005. The undescribed species P. sp 1 and P. sp 2 were recovered from two sites in 2 years, and one site in 1 year, respectively (Table 2). Phytophthora species from soil and streams of China 99

(a) (b)

Fig. 8. Morphological structures of Phytophthora gonapodyides isolates on V8 agar ﬂooded with distilled water. (a–d) ovoid, non-papillate sporangia with internal proliferation. —Scale bar = 20 lm.

(a) (b)

Fig. 9. Structures of Phytophthora gregata isolates on V8 agar ﬂooded with distilled water. (a, b) Ovoid, non-papillate sporangia; (c, d) oogonia with paragynous or amphigynous antheridia—Scale bar = 20 lm.

4 Discussion This study provided the first survey data and new records of Phytophthora species in oak forests in south-western China. It revealed the presence of eight Phytophthora spp. in soils and streams of three oak forests in northwestern Yunnan province during the monsoon months. The eight taxa included two well-known morphospecies P. gonapodyides and 100 W.-x. Huai, G. Tian, E. M. Hansen et al. P. cryptogea, two recently described species P. gregata and P. plurivora, two recognized but as yet undescribed taxa, P. taxon PgChlamydo and P. taxon Salixsoil, and two previously unrecognized species, Phytophthora sp.1 and P. sp.2. Phytoph- thora ramorum, the serious forest pathogen in Europe and US, was not found. This study begins to give us a picture of diversity and relative abundance of Phytophthora species in a limited area in oak forests of China. Seven species were identified at site B, and five at site N. It is difficult at the moment to determine which factors cause the differences among the three sites, but the bait bias is likely to be one of the reasons as rhododendron leaves were used in site B and oak leaves were used in the other two sites. However, the remarkable number of Phy- tophthora species found in oak forest streams and soils in this region of China is not surprising. Similar results have been obtained in Europe. Jung et al. (1996) and Hansen and Delatour (1999) each distinguished eight species. Vettraino et al. (2002) identified 11 Phytophthora species from oak forest soils in Italy, and Reeser et al. (2011a) found 18 different species of Phytophthora in 113 forest streams in Alaska and Oregon. Similarly, 14 species were recovered from diseased plant materials in agricultural lands and forest soil/water samples on Hainan island of South China (Zeng et al. 2009), among which only one species (P. cryptogea) was isolated also in our study. It is likely that the use of additional isolation methods and repeated samplings in different seasons would increase the percentage of positive isolations and perhaps reveal the presence of more Phytophthora species. Among the species identified in the present study, only two species were identified in all three sites, the most numerous species P. taxon PgChlamydo and the second abundant species P. taxon Salixsoil. P. taxon PgChlamydo which was detected in high frequency (59.72% of all isolates), is also frequently isolated from streams, rivers, irrigation water and soils in western North America, Argentina, Europe and Australia (Brasier et al. 1993; Hansen and Delatour 1999; Greslebin et al. 2005; Burgess et al. 2009; Reeser et al. 2011a,b). Molecular studies have demonstrated that each of these eight species is diverse, suggesting definite existence of different strains within every species. The abundance of Phytophthora species and strains might result from the high diversity of forest vegetation, stands and environment in mountainous areas. Their distinctive and variable nature may make them valuable materials for further study of genetic diversity and genotyping. Various genet- ically distinct Phytophthora species may differ from each other in their ability to colonize diverse genotypes of host plants, to utilize mineral and organic nutrients and in their adaptation to abiotic factors, such as soil pH and drought, or to cause plant diseases (Huai et al. 2003). Extreme selection occurred within the population of genotypes of P. infestans in North Ireland in each year with different genotype groups dominating the infection of different resistant potato cultivars (Young et al. 2009). The population of soilborne Phytophthora spp varied markedly depending on the site condition, pH value and geographic substrates (Jung 2009). The case of the double base overlapping peaks observed in sequencing chromatograms was previously reported by Xu (2009) who studied multicopies of thymidylate kinase (tmk) gene of a phytoplasma in association with Chinese jujube witches’-broom disease, which was thought to be related to two or more ITS sequences that were amplified and sequenced simultaneously. He assumed that there might be several heterogeneous tmk sequence variants within a single isolate due to the multiple copies. So, it is strongly assumed that the heterogenous sequences detected in a single Phytophthora, for instance, P. taxon PgChlamydo, might suggest variants of ITS sequence existing inside one Phytophthora isolate, possibly related to the multicopy of rDNA operon (Xu 2009, www.phytophthoradb.org), instead of simply accounting for it as isolate mixture or sequencing error. This explanation is supported by the study of some Pythium spp. and Phytophthora spp. (Vasseur et al. 2005). Phytophthora taxon Salixsoil was the other as yet not formally described species that was recovered from steam baits at all three sites in our survey. It was originally isolated from Salix roots in the UK and root debris of Alnus in Denmark (Brasier et al. 2003a), and is widely spread in flooded habitats (Reeser et al. 2011a,b). This species (taxon) was suggested to act as a root pathogen of woody plants in alluvial forest ecosystems, and its pathogenicity towards several tree species has been shown in leaf and shoot inoculation assays (Nechwatal and Mendgen 2006; Jung and Nechwatal 2008; Orlikowski et al. 2011). Phytophthora gregata is a newly described species, previously identified as P. taxon Raspberry and P. sp. seven in Wes- tern Australia (Jung et al. 2011). Among the three isolates (23b, BR05c and 34b3) designated as P. gregata in this study, the first two have identical ITS sequence that only varied at one locus from the WA reference isolate (Table 3). While strain 34b3 differed from them at three bases plus one in/del position and was identical with ITS sequences of the European reference isolates of P. taxon Raspberry, it fell in the P. gregata group from cox1 sequence analysis (data not shown). It seems that the addition of our isolates will make it easier to clarify the relationship of P. gregata and P. taxon Raspberry. Phytophthora plurivora is another recently described species, first recognized in Western Australia from isolates previously identified as P. citricola (Jung and Burgess 2009). The six isolates designated as P. plurivora were more variable in ITS sequence than other species identified, but differed from the WA reference in cox1 sequence only at five bases (data not shown), which fell within the range of variation reported in the species description. Elliott et al. (2009) exploited PCR- RFLP markers of the cox1 to identify three lineages of the North American and European populations of P. ramorum. Phy- tophthora plurivora was found associated with decline and dieback on a wide range of hosts causing root and collar rots, bark cankers and dying shoots and necrotic leaves throughout Europe and North America. Phytophthora plurivora has been found in streams in surveys in south-western Oregon (Reeser et al. 2011b), and now, in our study, was also found occa- sionally in stream and soil samples. It is interesting that Clade 6 has become one of the most rapidly expanded clades in the Phytophthora phylogeny with 12 new species or putative new species described in recent literature. Brasier et al. (2003a) discriminate at least 11 taxa in ITS Clade 6, of which two have subsequently been described as P. inundata (Brasier et al. 2003b) and P. rosacearum (Hansen et al. 2009). Additionally, P. pinifolia, a serious foliar pathogen of Pinus radiata in Chile (Duran et al. 2008), Phytophthora species from soil and streams of China 101 P. taxon cranberry, a new Phytophthora species causing root and runner rot of cranberry in New Jersey (Polashock et al. 2005), and P. taxon asparagi (Saude et al. 2008), a still unnamed pathogen of Asparagus officinalis found in Michigan, have also been described. Recently, four new species (P. gibbosa, P. gregata, P. litoralis and P. thermophila) and an informally described taxon (P. taxon paludosa) from Australia, all belonging to clade 6, were described by Jung et al. (2011). The as yet not formally described species are readily distinguished from our two new species candidates, P. sp1 and sp2 by ITS sequences (data not shown). Further work is planned to describe the two new taxa in Clade 6, and to test their pathogenicity. Adding more isolates to both candidate species would help us look into the morphological and molecular uniformity and variability within individual species (Gallegly and Hong 2008). Concerning pathogenicity, it is well known that most Phytophthora spp. can cause different host plant diseases; some species have narrow host ranges (one to two hosts); others have wide host ranges. And all species but a few such as P. gonapodyides can cause damage to plants. Phytophthora gonapodyides is a minor (weak) pathogen on only a few hosts such as alder, oak, apple, pear and rhododendron, etc., causing root rot or fruit rot diseases (Erwin and Ribeiro 1996; Corcobado et al. 2010). From our preliminary pathogenicity tests using detached leaves as described in De Dobbelaere et al. (2010), of four plant species, Ilex purpurea Hassk, Quercus variabilis Blume, Magnolia denudata Desr. and Syringa oblata Lindl., with representative isolates of P. gonapodyides(7a1), P. taxon Salixsoil (HQ01a), P. cryptogea (34b1) and P. sp.2 (34a2) as inoc- ula, apparent lesions were associated with all four isolates, indicating their definite pathogenicity (data not shown). In fact, the baiting method might exploite the selective pathogenicity of a Phytophthora species to infect and cause lesions on living host tissue. As heavy losses are often the result of delays in recognition of Phytophthora as the causal agent of the disease under investigation, and the symptoms of many diseases caused by Phytophthora spp. can often be mistaken for damage from other pathogens or abiotic agents, isolation and identification of pathogens offers the only accurate method of early detection and disease diagnosis. Most Phytophthora species are soilborne pathogens and survive long periods without their hosts. Given access to a susceptible plant in the presence of adequate moisture, they resume their pathogenic phase (Erwin and Ribeiro 1996). DNA sequence analysis commonly using the ITS region of ribosomal DNA is the most accurate method for identification of isolates to a species (www.phytophthoradb.org). Therefore, the isolation of Phytophthora from streams and soil within or adjacent to forest stands, in combination with ITS region analysis and morphological observation should provide a simple and efficient way for investigating Phytophthora pathogens as well as associated diseases. A comprehensive study and new findings on Phytophthora in natural forest in a limited area such as Diqing will provide us with a better foundation for approaches on further survey and diagnosis of forest diseases, especially to the native dominant plant species such as oak and rhododendron as well as establishing the association of Phytophthora population genetic diversity with local forest types and ecological environments. In order to detect Phytophthora pathogens and diagnose the diseases accurately, the specific and sensitive PCR techniques such as nested PCR and real-time PCR should be developed for detect- ing the pathogens from diseased plant tissue (Martin et al. 2008). Some Phytophthora (P. polonica, P. quercina) were described on oak and beech (P. citricola, P. cambivora) in Poland, compared genetic structure of the studied stands (Nowa- kowska et al. 2007; Nowakowska and Oszako 2008). Future research on the temporal and spatial distributions of populations of these eight Phytophthora species, along with study of origin and maintenance of genetic variation may promote an understanding of how populations of these coexisting organisms interact and evolve in the succession of forest ecosystems (Huai et al. 2003). Based on an ecological niche model, a study by Kluza et al. (2007) on the origins of sudden oak death caused by P. ramorum speculated that the geographic origin of the pathogen was Eastern Asia including Eastern China. Fujian and Yunnan provinces were identified as high-priority areas for the search for native P. ramorum populations. The pest risk analysis of P. ramorum in China by Shao (2008) also suggested that Yunnan should be included in the list of the most suitable areas for SOD occurrence and outbreak. However, from the results of this survey and other surveys in south-east China (data not shown), we have not found the SOD pathogen in this region or in other parts of China (Goheen et al. 2006). Although an isolate of P. cryptogea, also in clade 8 with P. ramorum, was identified, it was definitely different from P. ramorum in morphological and molecular traits as well as pathogenicity. It is still imperative to take precautions to pre- vent this pathogen from entering this region (Shao 2008).

Acknowledgements We thank all cooperators from Forest Pest Control and Quarantine Station of Diqing Tibetan Autonomous Prefecture for their help with baiting and sampling. We are grateful to Paul Reeser, Wendy Sutton (Oregon State University, USA) and Thomas L. Kubisiak (USDA Forest Service) for their invaluable technical assistance. Special thank to Ms. Chunli Jiang, Ms. Lina Shao and Dr. Yanxia Yao for assistance with ﬁeld work and laboratory routines. This research was supported by the State Forestry Administration ‘948’ Project (No. 2009-4-35).

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