REVIEW ARTICLE Identiﬁcation of Phytophthora …grunwaldlab-dev.cgrb.oregonstate.edu/sites/default/files/efp12015.pdfREVIEW ARTICLE Identiﬁcation of Phytophthora species baited

REVIEW ARTICLE

Identification of Phytophthora species baited and isolated from forest soil and streamsin northwestern Yunnan province, China

By W.-x. Huai1, G. Tian1, E. M. Hansen2, W.-x. Zhao1,5, E. M. Goheen3, N. J. Gr€unwald4 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 years2005, 2006 and 2010 at three sites in oak forests in Diqing Tibetan Autonomous Prefecture of NW Yunnan province, China. Seventy-threeisolates of Phytophthora spp. were recovered from 135 baited leaf samples and 81 soil samples. Eight Phytophthora species were identifiedby observation of morphological features and ITS1-5.8S-ITS2 rDNA sequence analysis. The eight taxa included two well-known speciesP. 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 numerousspecies, P. taxon PgChlamydo, and the second most abundant species, P. taxon Salixsoil, were recovered at all three sites. Phytophthoracryptogea was detected only once at site Nixi. Phytophthora gregata and P. sp.2 were isolated from a stream only at site Bitahai, while theother three species were each found at two sites. Phylogenetic analysis revealed that the isolates belonged to three ITS clades, one speciesincluding six isolates in clade 2, six species including 66 isolates in clade 6 and one species in clade 8. There was a relatively rich speciesand genetic diversity of Phytophthora detected in the investigated regions where the forest biotic and abiotic factors affecting the growthand 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 broadhost 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 byErwin 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, pro-duced 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, biflagellatezoospores 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 inmolecular techniques, such as DNA sequence data analysis from the internal transcribed spacer region and 5.8S gene of therDNA operon, the mitochondrial cox1 and cox2, ras-related protein, elicitin and b-tubulin genes have permitted a morerational study of genetic distance and phylogenetic relationships within the genus Phytophthora and enhanced the efficiencyand accuracy of identification of species as well as clonal lineages within species (Cooke et al. 2000; Forster et al. 2000;Elliott et al. 2009; Gr€unwald et al. 2009). Over the past decade, a number of new Phytophthora species have beendescribed from forests around the world. Some of these new species, such as P. ramorum (Gr€unwald et al. 2008), causedramatic tree and woody ornamental diseases. Other times, these new Phytophthora species have been found duringbroader 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; Wanget 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. citrophthoraand P. palmivora were recorded in association with woody plant and forest diseases (Zheng 1997; Zeng et al.2009). Ofthese, P. cinnamomi is of particular significance due to its broad host range and worldwide distribution, although it lacksthe information on economic impacts in China. It had been identified as the cause of death of black locust, cedar andcamellia, etc., (Ho et al. 1984; Shen et al. 1990; Zhou et al. 1993). Moreover, some early identified species may need to bere-examined or revised with the advent of a new taxonomic era highlighting the combination of morphological and molecu-lar classification. So far, there have not been any reports on the comprehensive investigation of Phytophthora species in theforest 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

For. Path. 43 (2013) 87–103 doi: 10.1111/efp.12015© 2013 Blackwell Verlag GmbH

http://wileyonlinelibrary.com/

identify Phytophthora species by baiting techniques from streams and forest soil among or alongside oak forests and to usea 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 geographicarea of Zhongdian (Shangrila). There are evergreen sclerophyllous Quercus forests in this region distributed from 900to 4800 m asl, chiefly between 2400 and 3600 m. Quercus aquifolioides Rehd. et Wils., Q. pannosa Hand.-Mazz., andQ. 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, Picealikiangensis (Franch.) Pritz., P. armandii Franch., Pinus densata Mast., Populus szechuanica Schneid., Sorbus rehderianaKoehne 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 �1.5°C in January and 15°C in July. The mean annual precipitation is520.5 mm for Zhongdian. Most of it occurs during the monsoon months of June–October. Three decline sites and streamsin Quercus forests were surveyed in this area (Fig. 1). Site Nixi (N) (27°58′N, 99°35′E) was about 500 m downstream of astream 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 theedge 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 thebase of declining trees and placing baiting leaves in selected streams. Nine soil samples were collected from each decliningsite 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 wereplated 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 ineach nylon mesh bag and were floated in relatively slow-moving water in streams as baits. The leaf baits were removedafter 7–10 days when lesions developed. Fifteen bags of baited leaf samples were collected from each stream per samplingyear. Pieces about 2-mm2 were cut from the margin of the brown spots and plated in CARP+. Colonies of suspectedPhytophthora and Pythium species growing from plated baits were transferred to CARP (CARP+ without Benlate andhymexazol) 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 oncarrot 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 andbaiting sites.

88 W.-x. Huai, G. Tian, E. M. Hansen et al.

and morphological characteristics (Waterhouse 1963; Erwin and Ribeiro 1996; Gallegly and Hong 2008; Jung and Burgess2009). Heterothallic isolates were crossed with reference cultures of known mating types of P. cinnamomi and P. cambivorafrom 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 har-vested by scraping from the agar surface with a sterile blade and placed in a sterile mortar with 800 ll of preheated (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 sterilizedquartz sand (Sigma-Aldrich, St. Louis, MO, USA). Harvested mycelium was ground using a pestle for 3–8 min, and genomicDNA 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 genecox1 was amplified with primers FM84 and FM83 (Martin and Tooley 2003). PCR reaction mixtures (50 ll) contained ca20-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 at95°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°Cfor 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 includingsome 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 Phytophthorainfection 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 morphologyof all the isolates on CARP+ selective medium coincided with literature descriptions of Phytophthora (Erwin and Ribeiro1996; Zheng 1997). Moreover, the colony growth temperatures as well as other physiological characters also fell into thecategory 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 thesame size products of about 850 bp, while normally Pythium spp amplicons were about 950 bp. Target amplicons weresent for direct sequencing without cloning. The results of the BLASTn query indicated that eight different Phytophthoraspecies 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 from2005 to 2010. When representative sequences of these identified species or taxa were submitted to the PhytophthoraDatabase website to do online identification (www.phytophthoradb.org), the results were in agreement with those of theGenBank 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 ofP. 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

Phytophthora species from soil and streams of China 89

Tab

le1.

identification

of73

isolates

from

Diqing,

Yunn

anprovince

basedon

ITSsequ

ence

simila

rity.

Species

Isolateno.

Waterho

use

grou

pITSclad

eGen

Ban

kaccessionnumbe

rThe

mostsimila

rreference

andGen

Ban

kaccession

Baseiden

tity

Phytoph

thoraplurivora

191

III

2JQ7307

11

P.p

lurivora

FJ665

225

759/7

61

DQ01-11

III

2JQ7307

12

760/7

61

NQ02

hIII

2JQ7307

13

761/7

61

NX05a

III

2JQ7307

14

757/7

60

NX05b

III

2JQ7307

15

757/7

60

NX05d

III

2JQ7307

16

757/7

60

P.g

onap

odyides

NQ07

cV/V

I6

JQ7307

17

P.g

onap

odyidesAF5

41889

816/8

19

NXQ13-2

V/V

I6

JQ7307

18

816/8

19

DQ13-7

V/V

I6

JQ7307

19

816/8

19

7a1

V/V

I6

JQ7307

20

814/8

19

P.g

rega

ta23a

V/V

I6

JQ7307

21

P.g

rega

taHQ01

2942

819/8

20

BR05c

V/V

I6

JQ7307

22

819/8

20

34b

3V/V

I6

JQ7307

23

816/8

19

P.taxon

PgC

hlam

ydo

193

bV/V

I6

JQ7307

24

P.taxon

PgC

hlam

ydoAF5

41901

HM004224

816/8

19

BR03a

V/V

I6

JQ7307

25

817/8

19

BR03f

V/V

I6

JQ7307

26

817/8

19

BR03h

V/V

I6

JQ7307

27

817/8

19

BR04a

V/V

I6

JQ7307

28

817/8

19

BR05e

V/V

I6

JQ7307

29

819/8

19

BR06a

V/V

I6

JQ7307

30

817/8

19

BR06b

V/V

I6

JQ7551

83

817/8

19

BR06c

V/V

I6

JQ7551

84

817/8

19

BR06d

V/V

I6

JQ7551

85

817/8

19

BR06e

V/V

I6

JQ7551

86

817/8

19

BR07a

V/V

I6

JQ7551

87

817/8

19

BR07e

V/V

I6

JQ7551

88

817/8

19

BR08a

V/V

I6

JQ7551

89

817/8

19

BR08b

V/V

I6

JQ7551

90

817/8

19

BR08h

V/V

I6

JQ7551

91

816/8

19

BR09b

V/V

I6

JQ7551

92

816/8

19

BR09c

V/V

I6

JQ7551

93

816/8

19

BR09d

V/V

I6

JQ7551

94

816/8

19

NQ02

bV/V

I6

JQ7551

95

816/8

19

NQ05

aV/V

I6

JQ7551

96

817/8

19

192

V/V

I6

JQ7551

97

819/8

19

NX03a

V/V

I6

JQ7551

98

819/8

19

193

aV/V

I6

JQ7551

99

816/8

19

23b

V/V

I6

JQ7552

00

817/8

19

DQ01-1

V/V

I6

JQ7552

01

819/8

19

DQ01-3

V/V

I6

JQ7552

02

819/8

19

DQ01-5

V/V

I6

JQ7552

03

818/8

19

DQ01-6

V/V

I6

JQ7552

04

819/8

19

DQ01-10

V/V

I6

JQ7552

05

819/8

19

DQ01-19

V/V

I6

JQ7552

06

819/8

19

DQ01-20

V/V

I6

JQ7552

07

819/8

19

DQ02-1

V/V

I6

JQ7552

08

819/8

19

DQ02-2

V/V

I6

JQ7552

09

819/8

19

DQ02-3

V/V

I6

JQ7552

10

819/8

19


Tab

le1.

Con

tinued

Species

Isolateno.

Waterho

usegrou

pITSclad

eGen

Ban

kaccessionnumbe

rThe

mostsimila

rreference

andGen

Ban

kaccession

Baseiden

tity

DQ07-2

V/V

I6

JQ755

211

818

/819

DQ12-1

V/V

I6

JQ755

212

819

/819

DQ12-2

V/V

I6

JQ755

213

819

/819

DQ12-3

V/V

I6

JQ755

214

817

/819

DQ12-4

V/V

I6

JQ755

215

819

/819

DQ12-6

V/V

I6

JQ755

216

819

/819

DQ13-1

V/V

I6

JQ755

217

819

/819

NXQ12-2

V/V

I6

JQ755

218

819

/819

P.taxon

Salix

soil

HQ01a

V/V

I6

JQ755

219

P.taxon

Salix

soilAF5

41909

816

/817

34a1

V/V

I6

JQ755

220

817

/817

DQ02-9

V/V

I6

JQ755

221

814

/817

DQ10-16

V/V

I6

JQ755

222

814

/817

DQ10-21

V/V

I6

JQ755

223

814

/817

DQ12-10

V/V

I6

JQ755

224

816

/817

DQ13-6

V/V

I6

JQ755

225

815

/817

NXQ12-1

V/V

I6

JQ755

226

816

/817

NXQ13-6

V/V

I6

JQ755

227

815

/817

P.sp.1

HQ01b

V/V

I6

JQ755

228

P.taxon

ForestsoilAF5

4190

8804

/819

7a2

V/V

I6

JQ755

229

802

/819

DQ09-5

V/V

I6

JQ755

230

804

/819

DQ10-24

V/V

I6

JQ755

231

804

/819

DQ10-26

V/V

I6

JQ755

232

804

/819

P.sp.2

34a2

V/V

I6

JQ755

233

P.taxon

paludosaHQ012

953

805

/816

34b2

V/V

I6

JQ755

234

805

/816

P.cryptog

ea34b1

VI

8JQ755

235

P.cryptog

eaFZ

J-1AY74

2742

797

/798


sequences of nine isolates shared 99.63–100% identity with P. taxon Salixsoil. Two additional groups of isolates, namedP. sp1 and 2 are thought to belong to new species because they were closely related to but differing from the closest taxaForestsoil 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. greg-

ata isolates and the two closely related P. taxon Raspberry isolates (Table 3). Seven P. plurivora isolates had four polymor-phic 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. cryp-

togea, P. plurivora, P. taxon PgChlamydo and P. taxon Salixsoil as a consequence of direct sequencing the ITS ampliconrather than cloning and then sequencing. Double peaks were particularly evident in the ITS sequences of P. taxon Salixsoiland P. taxon PgChlamydo isolates (Tables 6 and 7). The double base peaks were detected in about 68% (23/34) of P. taxonPgChlamydo isolates. Double peaks at nucleotide positions 54, 129, 190 and 503 were evident (Table 7). In all cases, how-ever, 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 taxafrom every ITS clade, six isolates fell in clade 2 and 66 isolates resided in clade 6, while only one isolate belonged to clade8. 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) wasidentical to the type isolate of P. plurivora at all bases, while the other two isolates (191 and DQ01-11) differed by one ortwo bases. The remaining three isolates NX05a, b and d grouped into a distinct cluster from the other three isolates in thephylogenic tree and varied at three bases and one deletion from the reference isolate, suggesting they are different strainsof 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 43new 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 evolu-tionary 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 sup-

port. Among the nine isolates, one exactly matched ITS sequence of the European reference isolate, and others differed atone to three nucleotide positions. Isolates 23a, BR06c and 34b3 corresponded to P. gregata, the recently described speciesin 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 repre-

sented by five isolates recovered from streams at sites Bitahai and Hutiaoxia. Phytophthora sp.1 isolates grouped into an

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

SpeciesPositiveisolations Site Isolated from Year

P. gonapodyides 4 N, B Streams 2005, 2010P. gregata 3 B Streams 2005P. plurivora 6 N, B Soil and streams 2005, 2006, 2010P. sp. PgChlamydo 43 N, B, H Soil and streams 2005, 2006, 2010P. sp Salixsoil 9 N, B, H Streams 2005, 2010P. cryptogea 1 N Streams 2005P. sp.1 5 B, H Streams 2005, 2010P. sp.2 2 B Streams 2005

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

Sample no.

Position

15 bp 674 bp 676 bp 718 bp 729 bp 743 bp

23a A G G T A TBR05c A G G T A TP. gregata HQ012942 A G G T G T34b3 – A A C G TP. taxon raspberry AF541905 – A A C G 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 isolatesshared 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 phylogeneticallyclose to some strains of P. erythroseptica and P. drechsleri but had less relatedness with P. ramorum and P. lateralis, bothassociated 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 resultsshowed that the values for estimates of evolutionary divergence between two sequences among compared isolates inclade 2 ranged from 0 to 0.094, while the values among six P. plurivora isolates were 0–0.004. Pairwise analysis ofthe sequences of the Phytophthora isolates and reference sequences from GenBank belonging to clade 6 showed thatthe values for estimates of evolutionary divergence ranged from 0 to 0.087, while the maximal value among all theclade 6 isolates was 0.029.

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

Sample no.

Position

64 bp 152 bp 395 bp 714 bp

191 – T C GDQ01-11 T C T GNX05a – T T TNX05b – T T TNX05d – T T TNQ02h T C C GP. plurivora type FJ665225 T C C G

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

Sample no.

Position

134 bp 327 bp 474 bp 553 bp 729 bp 744 bp

NQ07c T T C C T GNXQ13-2 G T T C T GDQ13-7 G T T C T G7a1 G A T C A GP. 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 Salixsoiland double peaks reading from direct sequencing chromatograms.

Sample no.

Position

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 C34a1 A C C C G G C CDQ02-9 A/C C C C/T G G C/T CDQ10-16 A C/G C/T C/T G/T G C TDQ10-21 A/C C C C/T G G C/T CDQ12-10 A C C C G G C TDQ13-6 A C C C/T G G C/T CNXQ12-1 A C C C G G C C/TNXQ13-6 A C C C/T G G C/T CP. taxon Salixsoil AF541909 A C C C G G C C


3.4 Morphological characterization

The Phytophthora isolates first identified by ITS sequence analysis were further confirmed by comparing colony growthpatterns, morphological features of sporangia, oogonia, antheridia, chlamydospores and hyphal swellings with known iso-lates and reference species descriptions in the literature. All the isolates produced sporangia, and some produced oosporesin sufficient numbers for positive identification. Six isolates, 191, PQ01-11, NQ02h, NX05a and NX05d with semi-papillatesporangia, were placed in Waterhouse group III as the ‘P. citricola complex’ and now identified as the recently describedspecies, P. plurivora (Jung and Burgess 2009), as confirmed with ITS sequence. These isolates were homothallic, with glo-bose or subglobose oogonia and paragynous antheridia. Sporangia were borne terminally on unbranched sporangiophoresor laterally attached or intercalary, non-deciduous, semi-papillate, frequently bi- or tripapillate. The shapes of sporangiaranged 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 oflarge 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

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

Sample no.

Position

54 bp 129 bp 190 bp 530 bp

193b A/G A/C C/T GBR03a G A/C C/T GBR03f G A/C C/T GBR03h G A/C C/T GBR04a G A/C C/T GBR05e G C C GBR06a G A/C C/T GBR06b G A/C C/T GBR06c G A/C C/T GBR06d G A/C C/T GBR06e G A/C C/T GBR07a G A/C C/T GBR07e G A/C C/T GBR08a G A/C C/T GBR08b G A/C C/T GBR08h A/G A/C C/T GBR09b A/G A/C C/T GBR09c A/G A/C C/T GBR09d A/G A/C C/T GNQ02b A/G A/C C/T GNQ05a G A/C C/T G192 G C C GNX03a G C C G193a A/G A/C C/T G23b G A/C C/T GDQ01-1 G C C GDQ01-3 G C C GDQ01-5 G C C T/GDQ01-6 G C C GDQ01-10 G C C GDQ01-19 G C C GDQ01-20 G C C GDQ02-1 G C C GDQ02-2 G C C GDQ02-3 G C C GDQ07-2 G C C T/GDQ12-1 G C C GDQ12-2 G C C GDQ12-3 G A/C C/T GDQ12-4 G C C GDQ12-6 G C C GDQ13-1 G C C GNXQ12-2 G C C GP. taxon PgChlamydo AF541901 G C C G


or loosely simple sympodial sporangiophores (Fig. 7). Clumps of globose to subglobose hyphal swellings were often formedin 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 cul-

ture and were classified into Waterhouse group V/VI and identified as Phytophthora gregata supported by ITSsequence. 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).

Fig. 2. Phylogenetic tree showing the relationship within Phytophthora Clade 2 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 detectedin this study, P. taxon PgChlamydo was the most widespread (59.72% of all isolates) being recovered from both soil andstreams at the three sites. The second most common species isolated was P. taxon Salixsoil, originally described fromEurope. Phytophthora gonapodyides as well as Phytophthora plurivora were isolated from stream water at site N and B. Five




(a) (b)

(c) (d)

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.


P. sp1 isolates from site B and N and one P. cryptogea isolate from site N were recovered from streams only in 2005. Theundescribed 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).

(a) (b)

(c) (d)

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

(a) (b)

(c) (d)

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.


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 Yunnanprovince during the monsoon months. The eight taxa included two well-known morphospecies P. gonapodyides and

(a) (b)

(c) (d)

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

(a) (b)

(c) (d)

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.


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 determinewhich factors cause the differences among the three sites, but the bait bias is likely to be one of the reasons as rhododen-dron 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 beenobtained 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 spe-cies of Phytophthora in 113 forest streams in Alaska and Oregon. Similarly, 14 species were recovered from diseased plantmaterials in agricultural lands and forest soil/water samples on Hainan island of South China (Zeng et al. 2009), amongwhich only one species (P. cryptogea) was isolated also in our study. It is likely that the use of additional isolation methodsand repeated samplings in different seasons would increase the percentage of positive isolations and perhaps reveal thepresence 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 detectedin high frequency (59.72% of all isolates), is also frequently isolated from streams, rivers, irrigation water and soils in wes-tern 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 strainsmight result from the high diversity of forest vegetation, stands and environment in mountainous areas. Their distinctiveand 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 causeplant diseases (Huai et al. 2003). Extreme selection occurred within the population of genotypes of P. infestans in NorthIreland in each year with different genotype groups dominating the infection of different resistant potato cultivars (Younget al. 2009). The population of soilborne Phytophthora spp varied markedly depending on the site condition, pH value andgeographic 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 jujubewitches’-broom disease, which was thought to be related to two or more ITS sequences that were amplified and sequencedsimultaneously. He assumed that there might be several heterogeneous tmk sequence variants within a single isolate dueto the multiple copies. So, it is strongly assumed that the heterogenous sequences detected in a single Phytophthora, forinstance, P. taxon PgChlamydo, might suggest variants of ITS sequence existing inside one Phytophthora isolate, possiblyrelated to the multicopy of rDNA operon (Xu 2009, www.phytophthoradb.org), instead of simply accounting for it as isolatemixture 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 suggestedto act as a root pathogen of woody plants in alluvial forest ecosystems, and its pathogenicity towards several tree specieshas been shown in leaf and shoot inoculation assays (Nechwatal and Mendgen 2006; Jung and Nechwatal 2008; Orlikowskiet 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). Whilestrain 34b3 differed from them at three bases plus one in/del position and was identical with ITS sequences of theEuropean reference isolates of P. taxon Raspberry, it fell in the P. gregata group from cox1 sequence analysis (data notshown). It seems that the addition of our isolates will make it easier to clarify the relationship of P. gregata and P. taxonRaspberry. Phytophthora plurivora is another recently described species, first recognized in Western Australia from isolatespreviously identified as P. citricola (Jung and Burgess 2009). The six isolates designated as P. plurivora were more variablein ITS sequence than other species identified, but differed from the WA reference in cox1 sequence only at five bases (datanot 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 beenfound 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 taxain 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 (Dur�an et al. 2008),


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, havealso been described. Recently, four new species (P. gibbosa, P. gregata, P. litoralis and P. thermophila) and an informallydescribed taxon (P. taxon paludosa) from Australia, all belonging to clade 6, were described by Jung et al. (2011). The asyet not formally described species are readily distinguished from our two new species candidates, P. sp1 and sp2 by ITSsequences (data not shown). Further work is planned to describe the two new taxa in Clade 6, and to test their pathogenic-ity. Adding more isolates to both candidate species would help us look into the morphological and molecular uniformityand 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 suchas alder, oak, apple, pear and rhododendron, etc., causing root rot or fruit rot diseases (Erwin and Ribeiro 1996; Corcobadoet al. 2010). From our preliminary pathogenicity tests using detached leaves as described in De Dobbelaere et al. (2010), offour plant species, Ilex purpurea Hassk, Quercus variabilis Blume, Magnolia denudata Desr. and Syringa oblata Lindl., withrepresentative 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 livinghost tissue. As heavy losses are often the result of delays in recognition of Phytophthora as the causal agent of the diseaseunder investigation, and the symptoms of many diseases caused by Phytophthora spp. can often be mistaken for damagefrom other pathogens or abiotic agents, isolation and identification of pathogens offers the only accurate method of earlydetection and disease diagnosis. Most Phytophthora species are soilborne pathogens and survive long periods without theirhosts. Given access to a susceptible plant in the presence of adequate moisture, they resume their pathogenic phase (Erwinand Ribeiro 1996). DNA sequence analysis commonly using the ITS region of ribosomal DNA is the most accurate methodfor identification of isolates to a species (www.phytophthoradb.org). Therefore, the isolation of Phytophthora from streamsand soil within or adjacent to forest stands, in combination with ITS region analysis and morphological observation shouldprovide a simple and efficient way for investigating Phytophthora pathogens as well as associated diseases. A comprehen-sive study and new findings on Phytophthora in natural forest in a limited area such as Diqing will provide us with a betterfoundation for approaches on further survey and diagnosis of forest diseases, especially to the native dominant plant spe-cies such as oak and rhododendron as well as establishing the association of Phytophthora population genetic diversitywith local forest types and ecological environments. In order to detect Phytophthora pathogens and diagnose the diseasesaccurately, 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) weredescribed 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 popula-tions of these eight Phytophthora species, along with study of origin and maintenance of genetic variation may promote anunderstanding 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 andYunnan provinces were identified as high-priority areas for the search for native P. ramorum populations. The pest riskanalysis of P. ramorum in China by Shao (2008) also suggested that Yunnan should be included in the list of the most suit-able areas for SOD occurrence and outbreak. However, from the results of this survey and other surveys in south-eastChina (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 fromP. 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 withbaiting and sampling. We are grateful to Paul Reeser, Wendy Sutton (Oregon State University, USA) and Thomas L. Kubisiak (USDA ForestService) for their invaluable technical assistance. Special thank to Ms. Chunli Jiang, Ms. Lina Shao and Dr. Yanxia Yao for assistance withfield work and laboratory routines. This research was supported by the State Forestry Administration ‘948’ Project (No. 2009-4-35).

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