**Phytophthora Infestations in Forest Ecosystems**

Editors

**Bruno Scanu Thomas Jung**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Bruno Scanu University of Sassari Italy

Thomas Jung Mendel University Czech Republic

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Forests* (ISSN 1999-4907) (available at: https://www.mdpi.com/journal/forests/special issues/ Phytophthora).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

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**ISBN 978-3-0365-0800-9 (Hbk) ISBN 978-3-0365-0801-6 (PDF)**

Cover image courtesy of Thomas Jung.

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## **Contents**

## **About the Editors**

**Bruno Scanu** is currently a researcher at instead of with the Department of Agricultural Sciences of the University of Sassari, where he is in charge of teaching forest pathology and mycology as part of the degree course in forest and environmental sciences. His research focuses primarily on forest tree diseases, with particular emphasis on those caused by the oomycete genus *Phytophthora*. His interests include the ecology, biogeography, pathology, taxonomy, and phylogeny of new and unusual *Phytophthora* species; their spread and impacts on natural ecosystems, plantation forestry, horticulture, and plant production nurseries; management and control strategies for *Phytophthora* diseases. Besides being a member of the University of Sassari, he has been a visiting scientist in several foreign research institutes, which enabled him to build a large network that includes most of the leading international *Phytophthora* researchers. He was appointed a member of the Scientific Council of the Phytophthora Research Centre at Mendel University in Brno, and of the Oomycetes Subject Matter Committee of the International Society of Plant Pathology (ISPP). He has authored or co-authored scientific papers in international peer review journals, his current h-index in Scopus is 19, with more than 900 citations, and he currently serves as the Editor of *Forest Pathology* and an Associate Editor of the *Journal of Plant Pathology*.

**Thomas Jung** is currently the head scientific researcher at the Phytophthora Research Centre of Mendel University in Brno, Czech Republic. His research on *Phytophthora*, *Halophytophthora*, and *Nothophytophthora* is focused on factors driving diversity and speciation in different ecosystems and climatic zones; detection and description of new species in Europe and other continents; assessment of their potential host ranges among European tree species; the roles of introduced and endemic species in the declines of forest ecosystems on a global scale; the behavior and role of invasive species in their centers of origin; biogeography, evolutionary history, adaptability, and evolutionary trends; the evolutionary role of interspecific hybridizations; factors triggering the onset of epidemics; pathways and survival mechanisms; the control of diseases and the development of integrated management concepts for natural ecosystems and nurseries; and screening for high-added-value compounds. Thomas Jung cooperates with many international research groups and has conducted research in 17 countries in Europe, Australia, North Africa, South America, the Caribbean, Indonesia, Japan, Taiwan, and Vietnam. He has participated in 24 national and international research projects and trained scientists from more than 50 research groups in *Phytophthora* methods. He has published 65 papers in international peer review journals and his current h-index in Scopus is 32, with 3553 citations. He has been a speaker or keynote speaker at 21 international conferences and 12 meetings of international networks and was appointed in 2012 as co-chair of the IUFRO Working Party *Phytophthora* in Forests and Natural Ecosystems.

## **Preface to "Phytophthora Infestations in Forest Ecosystems"**

The oomycete genus *Phytophthora* represents one of the most notorious groups of tree pathogens in natural and semi-natural forest ecosystems. Since the discovery in the 1960s of the invasive *P. cinnamomi*, threatening some of the world's richest plant communities in Australia, numerous *Phytophthora* diseases have been reported on forest trees worldwide, which were previously unknown to science. The most notable examples include the oak and beech declines triggered by different *Phytophthora* spp. in Europe and North America, the findings of sudden oak death and sudden larch death caused by *P. ramorum* in the Western USA and the U.K., respectively, and the association of *P. austrocedri* with the mal del cipres´ in Argentina and juniper decline in the U.K. All these epidemic events are driven by exotic invasive *Phytophthora* species, introduced through infested nursery plants from their native overseas environments. In recent years, many independent surveys have studied the diversity of *Phytophthora* species and the diseases they are causing across a diverse range of forests and other natural ecosystems. This Special Issue, which includes 10 scientific p apers from different research groups across three continents, provides new insights into the diversity, ecology, epidemiology, pathogenicity, and new detection methods of *Phytophthora* species.

The first a rticle b y J ung e t a l. h ighlights t he r esults o f s urveys p erformed i n n atural and semi-natural forests in temperate, subtropical montane, and tropical regions of Vietnam, reporting extremely diverse natural *Phytophthora* populations and the important discovery of *P. ramorum*. The most interesting outcomes of this study are the new insights into the origin of several invasive *Phytophthora* pathogens, including *P. cinnamomi* and *P. ramorum*. The latter species, together with *P. gonapodyides*, is the subject of the following paper by Aram and Rizzo, which provides an framework through which the survival and function of these two *Phytophthora* species can be studied in aquatic ecosystems. The next four papers focus on the involvement of *Phytophthora* species in Mediterranean oak decline in Southern Europe. In particular, Seddaiu et al. reveal the high diversity of *Phytophthora* species inhabiting declining *Quercus suber* stands in Sardinia (Italy), underling that not only *P. cinnamomi* is involved in these epidemic events, as had been previously thought; this finding is confirmed by Mora Sala et al . who, using next-generation sequencing (NGS), provide a better understanding of *Phytophthora* assemblages in *Quercus ilex* forests in Spain; Ruiz Gomez ´ et al. experimentally demonstrate the existence of different responses of *Q. ilex* against *P. cinnamomi* infection and water stress; and Sanchez-Cuesta ´ et al. evaluate the effect of several soil and plant parameters on the spatial distribution and aggregation of *P. cinnamomi* inoculum in *Q. ilex*. Another survey by Jung et al. in Sicily (Italy) offers new knowledge about the distribution, host associations, and ecology of numerous *Phytophthora* species in protected natural areas. This is followed by a paper that shows a broad range of *Phytophthora* spp. associated with wild apple forest ecosystems in Northwest China (Xu et al.). In the last two papers, Milenkovic´ et al. provide new data about the detection and pathogenicity of *Phytophthora* species from poplar plantations in Serbia, whereas Corcobado et al. demonstrate the involvement of several *Phytophthora* species in beech decline in Austria and their interaction with other biotic and abiotic factors.

Overall, these studies provide an update on the scientific a dvances r elated t o t his important group of forest pathogens. We hope that the information provided in this book is timely and will help scientists, land managers, and policy makers to better manage and prevent *Phytophthora* infestations in natural and semi-natural ecosystems. We would like to thank the authors for sharing their research and the reviewers and editors for their dedication, which were vital to the success of this *Forests* Special Issue.

#### **Bruno Scanu, Thomas Jung**

*Editors*

## **A Survey in Natural Forest Ecosystems of Vietnam Reveals High Diversity of both New and Described** *Phytophthora* **Taxa including** *P. ramorum*

**Thomas Jung 1,2,\*, Bruno Scanu 3, Clive M. Brasier 4, Joan Webber 4, Ivan Milenkovi´c 1, Tamara Corcobado 1, Michal Tomšovský 1, Mat ˇej Pánek 1,5, József Bakonyi 6, Cristiana Maia 7, Aneta Baˇcová 1, Milica Raco 1, Helen Rees 4,8, Ana Pérez-Sierra 4 and Marília Horta Jung 1,2**

Received: 22 December 2019; Accepted: 10 January 2020; Published: 12 January 2020

**Abstract:** In 2016 and 2017, surveys of *Phytophthora* diversity were performed in 25 natural and semi-natural forest stands and 16 rivers in temperate and subtropical montane and tropical lowland regions of Vietnam. Using baiting assays from soil samples and rivers and direct isolations from naturally fallen leaves, 13 described species, five informally designated taxa and 21 previously unknown taxa of *Phytophthora* were isolated from 58 of the 91 soil samples (63.7%) taken from the rhizosphere of 52 of the 64 woody plant species sampled (81.3%) in 20 forest stands (83.7%), and from all rivers: *P. capensis*, *P. citricola* VII, VIII, IX, X and XI, *P.* sp. botryosa-like 2, *P.* sp. meadii-like 1 and 2, *P.* sp. tropicalis-like 2 and *P.* sp. multivesiculata-like 1 from *Phytophthora* major phylogenetic Clade 2; *P. castaneae* and *P. heveae* from Clade 5; *P. chlamydospora*, *P. gregata*, *P.* sp. bitahaiensis-like and *P.* sp. sylvatica-like 1, 2 and 3 from Clade 6; *P. cinnamomi (Pc)*, *P. parvispora*, *P. attenuata*, *P.* sp. attenuata-like 1, 2 and 3 and *P.* ×*heterohybrida* from Clade 7; *P. drechsleri*, *P. pseudocryptogea*, *P. ramorum (Pr)* and *P.* sp. kelmania from Clade 8, *P. macrochlamydospora*, *P.* sp. ×insolita-like, *P.* sp. ×kunnunara-like, *P.* sp. ×virginiana-like s.l. and three new taxa, *P.* sp. quininea-like, *P.* sp. ×Grenada 3-like and *P.* sp. ×Peru 4-like, from Clade 9; and *P.* sp. gallica-like 1 and 2 from Clade 10. The A1 and A2 mating types of both *Pc* and *Pr* co-occurred. The A2 mating type of *Pc* was associated with severe dieback of montane forests in northern Vietnam. Most other *Phytophthora* species, including *Pr*, were not associated with obvious disease symptoms. It is concluded that (1) Vietnam is within the center of origin of most *Phytophthora* taxa found including *Pc* and *Pr*, and (2) *Phytophthora* clades 2, 5, 6, 7, 8, 9, and 10 are native to Indochina.

**Keywords:** biosecurity; breeding systems; hybridization; *Phytophthora cinnamomi*; biogeography; center of origin

#### **1. Introduction**

The number of devastating declines of trees and other woody plants driven by introduced invasive *Phytophthora* species in natural ecosystems in Australia, Europe, and North America has increased exponentially since the 1960s [1–9]. Therefore, numerous surveys in natural and semi-natural ecosystems have been performed in the past two decades to assess *Phytophthora* diversity in these continents and in Africa, Asia, and South America [4,5,10–19]. As a result of these surveys and molecular re-evaluations of culture collections and several species complexes, the number of described species and informally designated taxa of *Phytophthora* has tripled since 1999 [2,18,20–28]. A conservative estimate predicted the existence of 200–600 unknown *Phytophthora* species in natural ecosystems of as yet unsurveyed regions of the world [26]. These are distributed among 12 major phylogenetic clades [23,28,29].

Accumulating circumstantial evidence suggests that Southeast and East Asia might be one center of origin of the genus. This included the common occurrence of both mating types of several heterothallic *Phytophthora* species, the occurrence of many *Phytophthora* diseases on mainly non-native horticultural trees and crops, and the apparent absence of *Phytophthora* diseases in natural ecosystems, despite the presence of species which cause severe forest dieback elsewhere [2,10,12,13,15,16,30–35]. In 2013, a survey in natural forests and streams of Taiwan demonstrated remarkably high diversity including ten described species and 17 previously unknown taxa of which nine were of hybrid origin. The results suggested that most of these taxa including the A1 mating type of *P. cinnamomi* were indigenous to Taiwan, whereas the A2 mating type of *P. cinnamomi* is introduced; that major *Phytophthora* phylogenetic clades 2, 5, 6, 7 and 9 are native to Southeast and Eastern Asia; and that interspecific hybridisation may have a major role in speciation and radiations in diverse natural ecosystems [10,22].

The high *Phytophthora* diversity in Taiwan probably reflects both the high floristic, geological, and climatic diversity of this island and repeated immigration of *Phytophthora* species from mainland Asia via temporary landbridges during glacial periods in the pleistocene followed by periods of separation and speciation during interglacials [10,22,36–39]. Similarly, due to its complex geology, geomorphology, and orographic climates and the repeated immigration of plant species from both northern latitudes and the numerous islands of Sundaland during glacial periods, Indochina is also a biodiversity hotspot, harbouring 20%–25% of the world s plant species [39–41]. With a north–south extension of 1650 km and a west-east extension ranging between 50 and 600 km, Vietnam is located between 8◦30 and 23◦30 northern latitude and 102◦10 and 109◦27 eastern longitude in eastern Indochina along the South China Sea, covering approximately 330,000 km2. In Vietnam, seven climatic regions are distinguished. In simple terms, northern Vietnam has a humid subtropical monsoon climate with cool winters and hot rainy summers in lowland areas and cold misty winters and warm rainy summers in montane regions. Southern Vietnam has a tropical monsoon climate with warm winters and hot summers and a pronounced rainy period between May and October due to the East Asian monsoon. However, regionally, temperature and precipitation patterns can vary considerably due to orographic influences. The geology and geomorphology of Vietnam are also highly complex. Due to this environmental heterogeneity, the flora of Vietnam is remarkably diverse, comprising more than 10,350 species and 2256 genera of vascular plants, of which 10% and 3%, respectively, are endemic [40]. This includes 245 and 211 native species of the Lauraceae and Fagaceae respectively, families known for the high susceptibility of their European and North American members to introduced *Phytophthora* species [5–7,42–44]. Therefore, as in Taiwan, a high diversity of unknown *Phytophthora* species might be expected in Vietnam. Further, due to their co-evolution with Vietnamese tree genera also present in Europe and North America, some of these might pose a threat to forests and natural ecosystems in the latter two continents.

In spring 2016 and 2017, in the frame of a collaborative research project between the Mendel University in Brno, Forest Research and the University of Sassari, a survey of *Phytophthora* diversity was performed in a diverse range of natural forest types and river systems across Vietnam. This paper reports on the results of this *Phytophthora* survey and the association of *Phytophthora* spp. with disease symptoms of forest trees in Vietnam, and discusses the potential threat posed by previously unknown *Phytophthora* spp. to European and North American forests.

#### **2. Material and Methods**

#### *2.1. Sampling and Phytophthora Isolation*

Twenty-five natural forest stands covering a wide range of tree species, climates, and landscapes across Vietnam were selected for sampling (Figures 1 and 2). The forest stands were located in northern Vietnam in Hoàng Liên National Park (NP) (12 stands) and on two neighboring mountains (two stands), in Ba Vì NP (three stands) and in Cuc Phuong NP (five stands), and in southern Vietnam in Bù Gia Mập NP, U Minh Hạ NP and Côn Đảo NP on Côn Đảo island (each one stand). In addition, 16 rivers and streams were sampled in northern Vietnam (Figures 1 and 2c). Soil sampling and isolation methodology were according to [4,10]. In total, 91 rhizosphere soil samples were taken from 142 mature specimens of 64 native tree and shrub species. Three 20 × 30 × 20 cm soil monoliths were taken around each tree, at a distance of 30–150 cm from the stem base and at a soil depth of 10–30 cm. Aliquots of ca. 2 litres of rhizosphere soil together with roots (diameter ≤5 mm) from all monoliths were bulked, and subsamples of ca. 200 mL were used for isolation tests. Isolations from soil samples were carried out at 18–20 ◦C in an airconditioned laboratory at natural light using 3- to 10-day-old leaflets of native tree species, mainly *Lithocarpus bacgangensis*, *L. corneus*, *Quercus glauca*, *Q. chapaensis*, *Q. gilva*, *Castanopsis indica* and *Chamaecyparis hodginsii*, and the introduced *Acacia mangium* as baits floated over flooded soil. Brownish leaflets were examined at ×80 under a light microscope for presence of *Phytophthora* sporangia. Infected leaflets were blotted dry, necrotic lesions cut into small segments and plated onto selective PARPNH agar (V8-juice agar (V8A) amended with 10 μg/mL pimaricin, 200 μg/mL ampicillin, 10 μg/mL rifampicin, 25 μg/mL pentachloronitrobenzene (PCNB), 50 μg/mL nystatin and 50 μg/mL hymexazol).

In forest stand F07, the isolation of *Phytophthora* was also attempted from a bleeding bark lesion on a surface root of a mature *Castanopsis acuminatissima* (Figure 3e). Necrotic bark pieces were transported in distilled water to the lab and blotted dry on filter paper. Then, ca. 2 mm pieces were cut from the lesion margins and plated onto PARPNH agar.

In forest stand F11, freshly fallen leaves of a mature *Rhododendron arboreum* with necrotic lesions were collected from the forest floor close to forest stream R05 ca. 1 m above the waterline. The isolation of *Phytophthora* from these leaves was carried out as described below for leaves collected from rivers.

**Figure 1.** Location of the 25 forest sites (F01–F25; green triangles) and the 16 riparian sites (R01–R16; blue dots) included in the *Phytophthora* survey in Vietnam; blue triangles represent sites included in both the riparian and forest survey. For geographical coordinates and details of sites see Tables 1 and 2.

**Figure 2.** Representative forest stands and streams sampled in Vietnam; (**a**) Hoàng Liên National Park around the Fansipan mountain with diverse montane evergreen cloud forests and montane evergreen broadleaved forests; (**b**) diverse montane evergreen cloud forest F04 in Hoàng Liên National Park dominated by Fagaceae and Lauraceae species; (**c**) Cat Cat River (R10) running through a diverse montane evergreen forest in Hoàng Liên National Park; (**d**) montane *Chamaecyparis hodginsii*—*Quercus* forest on Sau Chua mountain; (**e**) montane *Alnus nepalensis* stand on Xin Chài mountain; (**f**) diverse, suptropical, humid evergreen forest F15 in Ba Vì National Park; (**g**) Cuc Phuong National Park with diverse, tropical, evergreen lowland rainforests growing on limestone; (**h**) diverse, tropical, evergreen lowland rainforest stand F20. For GPS coordinates see Tables 1 and 2; for location of sites see Figure 1.

**Figure 3.** Disease symptoms of mature native trees in natural forest stands in Vietnam associated with presence of *Phytophthora* species in the rhizosphere; (**a**–**f**) montane evergreen cloud forests in Hoàng Liên National Park; (**a**) crown thinning and dieback of *Quercus glauca* in forest stand F03 (2337 m a.s.l.; *P. cinnamomi* A2); (**b**) crown dieback and mortality of *Castanopsis acuminatissima* and *Neolitsea poilanei* in forest stand F05 (2249 m a.s.l.; *P. attenuata*, *P. castaneae*, *P. cinnamomi* A2); (**c**,**d**,**f**) severe crown dieback and mortality of *C. acuminatissima* in a swampy depression of forest stand F06 close to stream R01 (2083 m a.s.l.; *P. castaneae*, *P. cinnamomi* A2, *P. gregata*); (**f**) the white flowers and young leaves in the crowns of *C. acuminatissima* belong to the epiphytic *Rhododendron leptocladus*; (**e**) bark lesion with staining of the underlying cambium caused by *P. cinnamomi* A2 on a surface root of *C. acuminatissima* in forest stand F07; (**g**) mortality of *Dysoxylum juglans* in suptropical humid evergreen forest stand F15 in Ba Vì National Park (1108 m a.s.l.; *P.* sp. attenuata-like 3).

For the isolation of *Phytophthora* spp. from the 16 rivers and streams, an in-situ baiting technique was used [10,11]. Twelve of the 16 riparian baiting sites were located inside or downstream of natural forests (Figure 2c). At each site, 15–20 non-wounded young leaves of the native *C. indica*, *Citrus sinensis*, *L. bacgangensis, Q. glauca*, and, in some cases, *Carpinus* sp., *C. hodginsii*, *Cinnamomum iners*, *Dipterocarpus alatus*, *Prunus* sp., *Q. gilva* and *A. mangium* were placed as baits in a 25 × 30 cm raft, prepared using

fly mesh and styrofoam, and the raft put to float at a place where water flow was calm. The rafts were collected after 2–3 days. In addition, in 2017 freshly fallen leaves of different tree species and flowers of *Rhododendron arboreum* and *R. leptocladus* were collected from forest streams R01, R02, R10 and R11. Baiting leaves and the collected fallen leaves and flowers were washed in distilled water and blotted dry on filter paper. Five to ten pieces (approximately 2 × 2 mm) were cut from the margins of each watersoaked or necrotic lesion of each leaf or flower, blotted on filter paper and plated onto PARPNH agar.

All Petri dishes with plated leaf, flower or bark pieces were incubated at 20 ◦C in the dark and repeatedly examined under the stereo microscope at ×20 for *Phytophthora*-like hyphae after 12–48 h. Pure cultures were obtained by transferring single hyphal tips from the edge of the colonies onto V8A. Stock cultures were maintained on carrot agar (CA) [45] at 10 ◦C in the dark.

#### *2.2. Molecular Identification of Isolates*

For all *Phytophthora* isolates obtained in this study mycelial DNA was extracted from one-week old V8A cultures. Total DNA was extracted using the Phire Plant Direct PCR Kit (Thermo Fisher Scientific Inc., Waltham, MA USA) following the manufacturer's instructions. DNA was stored at −20 ◦C until further use. For all isolates the region spanning the internal transcribed spacer (ITS1-5.8S-ITS2) region of the ribosomal DNA was amplified using primer-pairs ITS1/ITS4 or ITS6/ITS4 [29,46]. For representative isolates of several known and all putative new species the mitochondrial *cox1* gene was amplified with both primer-pairs COXF4N/COXR4N and FM84/FM83 [47,48]. The PCR reaction mixture and the amplification conditions for ITS and *cox1* were according to [29,47,48]. PCR consumables were provided by Thermo Fisher Scientific. PCR products were purified and sequenced by GATC Biotech (Konstanz, Germany) and by Source Bioscience (Nottinham, UK) in both directions with the primers used for PCR amplification.

Sequences were edited using Geneious (Version 11.1.2, Biomatters Ltd., Auckland, New Zealand). Heterozygous sites observed were labelled according to the IUPAC coding system. Consensus sequences were aligned using the CLUSTAL W algorithm. The consensus sequences were subjected to an NCBI BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) and to a blast search in a local database containing sequences of ex-type isolates or key isolates from published studies to identify the closest related sequences. Isolates were assigned to a species when sequence identities were above a 99% cut-off in respect to those of ex-type isolates or key isolates. ITS and *cox1* sequences from representative isolates of all known and all putative new *Phytophthora* species obtained in this study were deposited at GenBank and accession numbers are given in Supplementary Table S1.

#### *2.3. Classical Identification of Isolates*

Colony growth patterns of 7-d-old cultures grown at 20 ◦C in the dark on V8A, malt-extract agar (MEA; Oxoid Ltd., Basingstoke, UK) and PDA [21] and morphological characters of sporangia, oogonia, antheridia, chlamydospores, hyphal swellings, and aggregations were compared with isolates from known species and with species descriptions in the literature.

Sporangia production and microscopic examinations and measurements of morphological structures at ×400 were according to [21,22] using a compound microscope (Zeiss Axioimager.Z2, Carl Zeiss AG, Oberkochen, Germany), a digital camera (Zeiss Axiocam ICc5) and a biometric software (Zeiss ZEN). Self-sterile isolates were paired on both V8A and CA with known A1 and A2 mating type tester strains of *P. cinnamomi*, *P.* ×*cambivora* and *P.* ×*heterohybrida* (isolates with non-papillate sporangia) or *P. botryosa*, *P. colocasiae* and *P. meadii* (isolates with papillate sporangia). All pairings were examined after 4–6 weeks incubation at 20 ◦C in the dark in order to determine whether self-sterile isolates are heterothallic or sterile and to which mating type heterothallic isolates belong [21]. All isolates are preserved in the culture collections maintained at Mendel University and Forest Research.

#### **3. Results**

In total, 943 oomycete isolates, including 652 *Phytophthora* isolates and 291 isolates from other oomycete genera, were obtained from forest stands (Table 1) and river systems (Table 2) in Vietnam. The *Phytophthora* isolates belonged to 13 described species, five informally designated taxa and 21 previously unknown taxa. From the other oomycete genera, 122 isolates were identified to species level. They could be assigned to the recently described *Nothophytophthora vietnamensis* (26 isolates), *Phytopythium vexans* sensu lato (63 isolates from 14 partly highly different haplotypes), four other known species and three novel taxa of *Phytopythium* (16 isolates), two described species and six novel taxa of *Pythium* (17 isolates) and one novel taxon of *Elongisporangium*. The remaining 169 isolates, which were not identified to species level, belonged to *Phytopythium* (161 isolates), *Pythium* (7 isolates) and *Saprolegnia* (1 isolate), respectively. GenBank accession numbers of ITS sequences of representative isolates of all oomycete taxa and of *cox1* sequences of representative isolates of most *Phytophthora* taxa are given in Supplementary Table S1. Detailed descriptions of morphological characteristics, morphometric and temperature-growth data, and multigene phylogenies for all new *Phytophthora* species will be presented in separate publications.

#### *3.1. Phytophthora Diversity in Natural and Semi-Natural Forest Stands*

In 20 forest stands (80%), 20 *Phytophthora* taxa were isolated from 58 of the 91 soil samples (63.7%) taken from the rhizosphere of 52 of the 64 woody plant species sampled (81.3%); from the root lesion of *C. acuminatissima* in stand F07; and from all four freshly fallen *Rhododendron* leaves collected from the ground in stand F11: *P. attenuata*, *P. castaneae*, *P. chlamydospora*, *P. cinnamomi*, *P. gregata*, *P. heveae*, *P. parvispora*, *P. ramorum*, *P. citricola* VII, three new species related to *P. attenuata*, three new species from the '*Phytophthora citricola* complex', three new species related to *P. botryosa* and *P. meadii*, and one new species related to *P. multivesiculata* and another to *P. tropicalis*, respectively (Table 1). From 29 of the 35 *Phytophthora*-negative soil samples, several known and previously unknown *Phytopythium* or *Pythium* spp. were isolated (Table 1). The only forest site from which no oomycete isolates could be obtained was subalpine *Rhododendron* scrub at 2903 m altitude near the Fansipan peak (F01).

*Phytophthora cinnamomi*, Clade 7c, was isolated from 26 of 66 rhizosphere soil samples (39.4%) collected from 27 of the 50 tree and shrub species (54%) in 13 of the 17 mountainous forest stands sampled (76.5%), making it the most widespread and common *Phytophthora* species above 700 m altitude. The A2 mating type of *P. cinnamomi* was present in 11 forest stands with an altitudinal amplitude ranging from 713 to 2337 m above sea level (a.s.l.). In contrast, the A1 mating type was only found in four forest stands located between 1108 and 2636 m a.s.l. (Figure 1; Table 1). Both mating types co-occurred in one stand in Hoàng Liên NP and another in Ba Vì NP. Interestingly, in Hoàng Liên NP, the A1 mating type was present in the upper montane Rhododendron forest F02 at 2636 m a.s.l. and in the lower montane stands F11 and F12 at 1900 m a.s.l., but was not detected in the eight forest stands (F03–F10) sampled between 2337 and 2022 m a.s.l., all highly infested by the A2 mating type. The latter was also isolated from a bark lesion on a surface root of *C. acuminatissima* in stand F07. Two A2 isolates from stand F11 were able to produce oogonia in single culture on V8A (Table 1). Over all stands, the A1:A2 mating type ratio of the 151 *P. cinnamomi* isolates was 30.5:69.5, whereas in the two stands with co-occurrence of both mating types the A1:A2 ratio of the 44 isolates was 59.1:40.9. Among the 39 *P. cinnamomi* isolates for which ITS sequences were produced, 32 isolates belonged to the same haplotype as the ex-type isolate from Sumatra (CBS 142.22; GenBank accession no. KU899160) (Table S1). Six isolates, representing both mating types, from stands F02 and F05 in Hoàng Liên NP and stand F17 in Ba Vì NP were heterozygous at position 767 (K instead of G) (Table 1S) and shared the same haplotype with an isolate from a subtropical *Quercus* forest in Taiwan (TW213; GenBank accession no. KU682570). Another isolate from stand F17 shared the heterozygous position 767 and was also heterozygous at position 89 (Y instead of C) (Supplementary Table S1). The 15 isolates for which the *cox1* gene was sequenced belonged to eight different haplotype which differed over a 712 bp alignment from the ex-type isolate (KU899315) at 1–4 positions.

*Phytophthora parvispora* was exclusively found in stand F15 in Ba Vì NP where it co-occurred with both mating types of its closest relative *P. cinnamomi* (Table 1). Compared to the ex-type of *P. parvispora* (CBS 132772; KC478667), the three isolates had identical *cox1* sequences and differed in ITS by one heterozygous site at position 73 (Y instead of T) (Supplementary Table S1). In mating tests with A1 and A2 tester strains of *P. cinnamomi*, all isolates were sterile.

*Phytophthora attenuata* from Clade 7a and three previously unknown taxa closely related to *P. attenuata* were recovered from five forest stands in Hoàng Liên NP and Ba Vì NP (Table 1). The individual taxa from this '*P. attenuata* complex' differed in their altitudinal amplitude and geographical distribution (Figure 1; Table 1). *Phytophthora attenuata*, *P.* sp. attenuata-like 1 and *P.* sp. attenuata-like 2 were only found in Hoàng Liên NP. Most widespread was *P.* sp. attenuata-like 1 which was isolated from the rhizosphere of five tree species in three stands located between 2249 and 2636 m a.s.l., followed by *P. attenuata* (three tree species in two stands; 1910–2249 m a.s.l.) and *P.* sp. attenuata-like 2 (2 tree species in 1 stand; 1910 m a.s.l.). In contrast, *P.* sp. attenuata-like 3 was exclusively found between 713 and 1108 m altitude in two of the three forest stands sampled in Ba Vì NP where it was associated with six tree species (Table 1). The ITS and *cox1* sequences of *P. attenuata* isolates from Vietnam differed from the ex-type isolate (CBS 141199; GenBank nos. KU517154 and KU517148) and other isolates of *P. attenuata* from Taiwan at 0–1 and 0–5 positions. *Phytophthora* sp. attenuata-like 1, *P.* sp. attenuata-like 2 and *P.* sp. attenuata-like 3 showed differences to *P. attenuata* in ITS at 0–1, 1–2 and 2–3 positions, respectively, and in *cox1* at 6–8, 9–11 and 6–8 positions, respectively. The *cox1* sequences of the three new taxa differed from each other at 8–17 positions. Heterozygous sites were present in the ITS sequences of all isolates of *P.* sp. attenuata-like 2 (R at position 184) and most isolates of *P.* sp. attenuata-like 3 (Y in position 54; K in position 152). The ITS sequence of one isolate of *P.* sp. attenuata-like 1 from stand F05 contained seven heterozygous sites possibly suggesting hybrid origin.

*Phytophthora castaneae* from Clade 5 showed a similar altitudinal (1108–2242 m a.s.l.) and geographical distribution to *P. cinnamomi* (Figure 1; Table 1). It was isolated from the rhizosphere of 13 tree species from the genera *Castanopsis*, *Lithocarpus*, *Neolitsea*, *Meliosma*, *Illicium* and *Rhododendron* in seven stands in Hoàng Liên NP and Ba Vì NP, and *C. hodginsii* in stand F14 on Sau Chua mountain where it was the only *Phytophthora* species recovered (Table 1). The ITS sequences of all isolates from Hoàng Liên NP and several isolates from Ba Vì NP matched the ex-type of *P. castaneae* (ICMP 19434; GenBank no. KP295319). However, several isolates from Ba Vì NP had a unique polymorphism at position 54 (A or R instead of G) while all isolates from Sau Chua mountain were characterised by having a unique polymorphism at position 590 (A instead of G). The *cox1* sequences of 15 isolates from the seven stands constituted six haplotypes which differed from the ex-type isolate (KP295234) by 0–1 bp. Interestingly, all four tested isolates from Sau Chua mountain had a unique polymorphism at position 421 (A instead of G). Five of the six tested *P. castaneae* isolates from stand F15 in Ba Vì NP shared a T at position 369 with *P. heveae* isolates from the same stand and with the *P. heveae* ex-type (CBS296.29; GenBank nos. HQ643238 and KP295326) whereas *P. castaneae* isolates from the other stands and the *P. castaneae* ex-type have a C at this position. Compared to *P. castaneae*, the other Clade 5 species found in this survey, *P. heveae*, had a lower altitudinal amplitude. *Phytophthora heveae* was isolated from the rhizosphere of 10 tree species in the subtropical lower montane stands F15 and F16 in Ba Vì NP and in four tropical lowland rainforest stands in Cuc Phuong NP, Bù Gia Mập NP and Côn Đảo NP (Figure 1; Table 1). Both Clade 5 species only co-occurred in stand F15. The ITS sequences of all *P. heveae* isolates (Table S1) matched the ex-type of *P. heveae*. The *cox1* sequences of all isolates differed from the ex-type (GenBank no. KP295239) at position 536 (T instead of C). Isolates from Cuc Phuong NP and Bù Gia Mập NP had unique polymorphisms at positions 30 (C instead of A) and 390 (A instead of T), respectively. The morphology of all isolates of *P. castaneae* and *P. heveae* was in accordance with the original descriptions [2].

**Table 1.** *Cont.*

*chamaehyphon* also isolated. k

*Phytopythium*

*cucurbitacearum* also isolated. l

*Phytopythium*

 *vexans* also isolated. m

*Phytopythium* sp. Côn Đảo also isolated.

R06 R07 R08 R09 R10 R11

N22 22.230

1308

tributary of Ngòi Duôi

River; Lào Cai

Forest stream 8;

Sau Chua mountain;

F24; broadleaved

*Cunninghamia*

 *lanceolata*

plantations

 mountain forests and

*Chamaecyparis*

 *hodginsii* forest

Baiting raft Fallen leaves/flowers

BIT, CIT VII, CIT IX, MAC,

QUI, SYL3

CIT IX, KEL, RAM A1 l

E103 52.615

N22 19.372

1193

Forest stream 9, Cat Cat

River; Lào Cai

Hoàng Liên NP; montane broadleaved

 forests

Baiting raft Fallen leaves/flowers

CAP, CIT VII, CIT VIII, SYL1,

SYL3 e

CHL, PSC, QUI, SYL 1, SYL3,

RAM A1, VIE f,j,k

E103 49.780

N22 18.597

1013

Muong Hoa River; Lào

Cai

E103 52.426

N22 20.904

1911

Forest stream 5, Gold

river; Lào Cai

Hoàng Liên NP; montane broadleaved

Hoàng Liên NP; subalpine and montane

Rhododendron

broadleaved

 forests, rice fields

 scrub and forests, montane

 forests

 Baiting raft

Baiting raft

 KEL, PSC, ×KUN

 CIT VII, SYL2, SYL3

> E103 46.259

N22 20.902

1912

tributary of the Gold

Hoàng Liên NP; montane broadleaved

 forests

 Baiting raft

×HET A1, RAM A1

> river; Lào Cai

E103 46.261

N22 20.911

1896

Forest stream 6, tributary of forest

Hoàng Liên NP; montane broadleaved

 forests

 Baiting raft

CHL, ×HET A1 h,i

> stream 5; Lào Cai

Forest stream 7,

E103 46.199

BaitingVII=*P.P.macrochlamydospora*, MUV1 = *P.* sp. multivesiculata-like 1, PSC = *P. pseudocryptogea*, QUI = *P.* sp. quininea-like, RAM = *P. ramorum*, SYL1 = *P.* sp. sylvatica-like 1, SYL2 = *P.* sp. sylvatica-like 2, SYL3 = *P.* sp. sylvatica-like 3, ×GRE3 = *P.* sp. ×Grenada 3-like, ×HET = *P.* ×*heterohybrida*, ×INS = *P.* sp. ×insolita-like, ×KUN = *P.* sp. ×kunnunara-like, ×PER4 = *P.* sp. ×Peru 4-like, ×VIR = *P.* sp. ×virginiana-like s.l., VIE = *Nothophytophthora vietnamensis*. c Mating types: A1, A2, A1ho (homothallic and stimulating oogonia formation in A2 tester strains). d*Elongisporangium* sp. Hoàng Liên also isolated. e Unidentified *Pythium* sp. also isolated. f *Phytopythium vexans* aff. also isolated. g *Pythium senticosum* also isolated. h *Pythium* sp. ×ZSF0056-like also isolated. i *Phytopythium* sp. 1 PB-2013 also isolated. j *Phytopythium litorale* also isolated. k *Pythium* sp. CAL\_2011f also isolated. l *Pythium* sp. 1\_MNS-2013 also isolated. m *Pythium* sp. 2\_ROH-2015 also isolated. n *Phytopythium palingenes* also isolated.

a

In total, nine previously unknown *Phytophthora* species from four of the five subclades within Clade 2 were detected in forest stands. From Clade 2a, *P.* sp. meadii-like 1 was isolated from the montane *A. nepalensis* stand F13 at 1717 m a.s.l. on Xin Chài mountain, while *P.* sp. meadii-like 2 was found in the tropical lowland rainforest stand F20 in Cuc Phuong NP (Figure 1; Table 1). The ITS sequences of all isolates of *P.* sp. meadii-like 1 were identical except for one isolate with an extra T in position 11 and an A instead of a T in position 12 (Supplementary Table S1). This new taxon differed in the ITS from *P. meadii* (isolate P75; GenBank no. GU993903) at positions 137 and 632 which were shared with the ex-type isolate of *P. botryosa* (CBS586.69; GenBank no. HQ643151), and from *P. botryosa* at five positions (72, 152, 444, 460, 773) which were identical with *P. meadii*. In addition, all isolates of *P.* sp. meadii-like 1 had a unique deletion at position 146. The ITS sequences of *P.* sp. meadii-like 2 showed intraspecific variability at positions 11, 13, 22. Most isolates differed from *P.* sp. meadii-like 1, *P. meadii* isolate P75 and the ex-type isolate of *P. botryosa* by having four unique heterozygous positions (161, 444, 502, 713). In addition, *P.* sp. meadii-like 2 showed in the ITS the same differences to *P. meadii* and *P. botryosa* as *P.* sp. meadii-like 1. The ITS sequences of both new taxa showed differences to the ex-type isolate of the recently described *P. mekongensis* from southern Vietnam (CBS135136; GenBank no. KC875838) at eight positions (152, 155, 163, 165, 166, 175, 179, 750). A third new taxon from Clade 2a, *P.* sp. botryosa-like 2, was exclusively isolated from the tropical lowland rainforest stand F25 on Côn Đảo island (Figure 1; Table 1). The ITS sequences of all isolates were identical to each other and differed from *P. botryosa* and *P. meadii* at four (72, 137, 161, 460) and five positions (152, 161, 444, 632, 773), respectively. In a 610 bp alignment of *cox1*, *P.* sp. meadii-like 1, *P.* sp. meadii-like 2 and *P.* sp. botryosa-like 2 differed from *P. meadii* (isolate p75; GU945489) at 14, 13 and 12 positions, respectively, and from *P. botryosa* (HQ261256) at 10, 9, and 8 positions, respectively. According to sequence analyses, the closest relatives of *P.* sp. botryosa-like 2 were an isolate obtained in 1930 from *Cocos nucifera* in Sulavesi (CBS235.30) which differed in ITS (HQ643140) by five heterozygous positions and in *cox1* (HQ708214) at five positions, and an isolate of unknown origin which was obtained from a vanilla plant in 1928 (CBS238.28) and showed differences at five positions in both ITS (HQ643139) and *cox1* (HQ708213) of which three were heterozygous in ITS. All isolates of *P.* sp. botryosa-like 2, *P.* sp. meadii-like 1 and *P.* sp. meadii-like 2 produce caducous papillate sporangia with variable shapes and are heterothallic, exclusively belonging to mating type A1. Oospore abortion rates in mating tests with A2 tester strains of *P. meadii* and *P. botryosa* exceeded 95%.

From the montane *A. nepalensis* stand F13 a new *Phytophthora* species from Clade 2b was isolated which differed from the ex-type isolate of *P. tropicalis*(CBS434.91) in ITS (HQ643369) and *cox1* (HQ708417) at 5 and 7 positions, respectively, and is hence informally designated as *P.* sp. tropicalis-like 2. Similar to *P. tropicalis*, all isolates produce thickwalled chlamydospores and papillate sporangia. *Phytophthora* sp. tropicalis-like 2 differs from *P. tropicalis* [49] by producing sporangia which are only partially caducous and have shorter pedicels (24.7 ± 16.8 μm vs. > 50 μm) and shorter length/breath (l/b) ratio (1.8 ± 0.3 vs. 1.8–2.4).

*Phytophthora citricola* VII, informally designated from a mountain forest in Taiwan [10], and another three new taxa from the '*P. citricola* complex' in Clade 2c, informally designated here as *P. citricola* IX, *P. citricola* X and *P. citricola* XI, were isolated from the montane *A. nepalensis* stand F13, the subtropical evergreen forest stand F16 and the tropical lowland rainforest stands F21 and F25, respectively (Figure 1; Table 1). *Phytophthora citricola* VII, IX, X and XI differ from the authentic type of *P. citricola* s.s. (CBS295.29; ITS–FJ560913; *cox1*—KC855432) in the ITS (771 bp alignment) at 3, 3, 12 and 11 positions, and in *cox1* (1231 bp alignment) at 23, 19, 29, and 15 positions, respectively. Like other members of the '*P. citricola* complex', the four new species are homothallic forming smooth-walled oogonia with paragynous antheridia. The sporangia of *P. citricola* VII and IX resemble those produced by other species from Clade 2c in being semipapillate, persistent and with exclusively external proliferation. In contrast, *P. citricola* X and XI produce mainly papillate sporangia with both external and, infrequently, also internal extended and nested proliferation. In addition, *P. citricola* X is distinguished from all known

related species by forming abundant catenulate hyphal swellings in water. *Phytophthora* citricola VII produces a high proportion of zoospores with a ring-like to oval coiling of both flagella ends.

From a swampy depression in the montane evergreen cloud forest F06 in Hoàng Liên NP, a previously unknown *Phytophthora* species from Clade 2e was isolated which is provisionally named as *P.* sp. multivesiculata-like 1. Its ITS and *cox1* sequences differ from the ex-type isolate of *P. multivesiculata* (CBS545.96; HQ643288 and HQ708340) at eight and 38 positions, respectively. The ITS sequences of two yet undescribed species, *Phytophthora* sp. aquatilis (GenBank no. FJ666126) and *Phytophthora* sp. Costa Rica 5 (KC479200), show differences to *P.* sp. multivesiculata-like 1 at 8 and 6 positions, respectively. Like *P. multivesiculata*, *P.* sp. multivesiculata-like 1 is homothallic with aplerotic oospores and produces in water numerous catenulate hyphal swellings and both nonpapillate and semipapillate sporangia with external and internal proliferation. However, it can easily be distinguished from *P. multivesiculata* [50] by forming considerably larger sporangia (on av. 57.2 × 32.8 vs. 45 × 33 μm), larger oogonia (45 vs. 41 μm) with highly variable shapes ranging from globose, excentric or elongated with long tapering bases to comma-shaped, and exclusively amphigynous antheridia.

Also in Hoàng Liên NP, *P. gregata* from Clade 6b was recovered from the rhizosphere of *Meliosma henryi* and *Neolitsea merilliana* in the montane evergreen cloud forest F06 while the other Clade 6b species *P. chlamydospora* and *P. ramorum* from Clade 8c were isolated from fallen leaves of *R. arboreum* collected from the forest ground in the montane, evergreen broadleaved forest F11 (Table 1).

Besides the recently described *Nothophytophthora vietnamensis* [51] which was isolated from the rhizosphere of *C. acuminatissima* and *Acer campbellii* in the montane evergreen cloud forest F04 and *A. nepalensis* in stand F13 on Xin Chài mountain, a range of *Pythium* and *Phytopythium* species including *Py. intermedium*, *Py. senticosum*, *Ph. chamaehyphon*, *Ph. cucurbitacearum*, *Ph.* sp. 1 PB-2013, 14 haplotypes from the *Ph. vexans* complex and two previously unknown taxa, informally designated as *Py.* sp. conidiophorum-like and *Ph.* sp. Côn Đảo, were obtained from 15 forest stands (Table 1 and Supplementary Table S1).

#### *3.2. Phytophthora Diversity in Natural Forest Streams and Rivers*

Using rafts with leaves of *C. indica*, *C. sinensis*, *L. bacgangensis*, *Q. glauca*, and, less frequently, *Carpinus* sp., *C. hodginsii*, *Cinnamomum iners*, *Dipterocarpus alatus*, *Prunus* sp., *Q. gilva* and *A. mangium* as in situ baits in all 16 rivers and streams tested, and freshly fallen leaves of different tree species and flowers of *R. arboreum* and *R. leptocladus* in four forest streams, seven known species (*P. capensis*, *P. chlamydospora*, *P. drechsleri*, *P. macrochlamydospora*, *P. pseudocryptogea*, *P. ramorum*, *P.* ×*heterohybrida*), five informally designated taxa (*P. citricola* VII, *P.* sp. kelmania, *P.* sp. ×insolita-like, *P.* sp. ×kunnunara-like, *P.* sp. ×virginiana-like s.l.) and 12 previously unknown taxa of *Phytophthora* were isolated (Table 2). The latter included *P.* sp. multivesiculata-like 1, two new species from the '*P. citricola* complex', three and one new species related to the Clade 6 taxa *P.* sp. sylvatica and *P.* sp. bitahaiensis, respectively, three new species from Clade 9 and two new species related to *P. gallica* from Clade 10.

The *Phytophthora* communities in the 11 montane streams above 1000 m a.s.l. with a temperate climate were dominated by species belonging to Clades 2, 6, 7, and 8 whereas from the five lowland rivers with subtropical to tropical climate almost exclusively *Phytophthora* species from Clade 9 were obtained (Figure 1; Table 2).

In montane streams, the most widespread species was *P. ramorum* which could be recovered from seven of the eight forest streams above 1890 m altitude in the Fansipan area and in 8–12 km distance to these sites from stream R11 originating from the *C. hodginsii* forest F24 at Sau Chua mountain in 1300 m altitude. Both mating types were obtained with the A1 mating type occurring in eight streams and the A2 in two streams. In the latter (streams R03, R04) both mating types co-occurred (Table 2). In the two streams (R01, R02) sampled in both 2016 and 2017 only mating type A1 was isolated. The 65 *P. ramorum* isolates exhibited five slightly different ITS genotypes. Eight isolates from four streams (R02, R04, R05, R10) were identical to the ex-type isolate from Germany, which belongs to the EU1 lineage (CBS101553; HQ643339). The most common genotype (46 isolates) differed from the ex-type by having a T instead of a Y at position 616, while three isolates from three streams (R01, R02, R10) had a C at this position. Four *P. ramorum* isolates from streams R02 and R03 were distinguished from the ex-type by being heterozygous at position 682 (R instead of G) while in one isolate from stream R03 both heterozygous positions occurred. For 43 isolates, representative for all eight streams, forest site F11 and both mating types, *cox1* was sequenced and compared to representative isolates of the four known *P. ramorum* lineages EU1, EU2, NA1, and NA2. In a 1240 bp long *cox1* alignment all but one of the Vietnamese *P. ramorum* isolates were identical and differed from four representative isolates of the North American NA2 lineage only at position 123. They differed from EU1 by 5 bp (positions 123, 808, 1141, 1156, 1202), EU2 by 5 bp (624, 966, 1035, 1156, 1240) and NA1 by 4 bp (123, 808, 1156, 1202), respectively. Isolate VN88 differed from the other 42 isolates by having a unique polymorphism at position 1228 (C instead of A). The morphological structures of all isolates were congruent with the original description of *P. ramorum* [52].

From Clade 2c, *P. capensis*, *P. citricola* VII, *P. citricola* VIII, and *P. citricola* IX were isolated from two, one, five, and one montane forest streams, respectively (Table 2). From stream R11, *P. capensis*, and *P. citricola* VII and VIII were obtained while in another two streams (R01, R10) two different species from the '*P. citricola* complex' co-occurred. Compared to the ex-type isolate of *P. capensis* (P1819; ITS—GU191232; *cox1—*GU191275) from South Africa, the ITS sequences of the Vietnamese isolates were identical but their *cox1* sequences were separated in a 598 bp alignment by 9 bp (1.5%). *Phytophthora citricola* VIII differed from the authentic type isolate of *P. citricola* s.s. (CBS295.29; ITS—FJ560913; *cox1*—KC855432) and from *P. citricola* VII, IX, X and XI in ITS (771 bp alignment) at 2, 2–3, 4, 12 and 11 positions, and in *cox1* (1231 bp alignment) at 33, 26, 35, 42, and 37 positions. Being homothallic with paragynous antheridia and producing semipapillate sporangia of variable shapes, *P. citricola* VIII morphologically resembles other species from Clade 2c.

*Phytophthora* sp. multivesiculata-like 1 from Clade 2e was present in streams R01 and R02 which originate from a catchment area around forest stand F06 where this new taxon was also found.

From Clade 6b, which contains numerous predominantly aquatic *Phytophthora* species, *P. chlamydospora*, three new species informally designated as *P.* sp. sylvatica-like 1, 2 and 3, and another new species designated as *P.* sp. bitahaiensis-like were recovered from 8 of the 11 mountain streams (Table 2). Most common were *P.* sp. sylvatica-like 2 (six streams), *P. chlamydospora* (5 streams) and *P.* sp. sylvatica-like 3 (four streams). In five streams more than one Clade 6b species were found. In the ITS (844 bp alignment) and *cox1* (861 bp alignment), *P.* sp. sylvatica-like 1 differs from its closest relative *P.* sp. forestsoil-like from Taiwan (KU682574) at 2 and 7 positions, respectively, while *P.* sp. sylvatica-like 2 and 3 show differences to *P.* sp. forestsoil-like in ITS at 12–13 and 11–12 positions, respectively, and in *cox1* at 29 and 27 positions. *Phytophthora* sp. sylvatica-like 1 differs from *P.* sp. sylvatica-like 2 and 3 in ITS by 9–11 and 7–8 bp, respectively, and in *cox1* by 44 and 46 bp, respectively. The latter two species can be distinguished in the ITS and *cox1* by differences at 2–4 and 11 positions, respectively. *Phytophthora* sp. bitahaiensis-like differs in the ITS from *P.* sp. bitahaiensis (isolate BHL1; KT183432) from a forest stream in Yunnan, China, at 4 positions. Unfortunately, for *P.* sp. bitahaiensis no *cox1* sequences are available. Similar to *P.* sp. forestsoil-like and many other aquatic Clade 6 species [10,21,53], *P.* sp. sylvatica-like 1, 2, and 3 and *P.* sp. bitahaiensis-like are sterile and form abundantly nonpapillate sporangia with internal nested and extended proliferation. All ten sequenced isolates from *P. chlamydospora* were identical in both the ITS and *cox1* and differed from the ex-type isolate of *P. chlamydospora* from the UK (P236; AF541900, MH136867) only by being in the ITS heterozygous at position 57 (R instead of G) while being identical to the ex-type in *cox1*.

From five mountain streams above 1890 m altitude, the recently described Clade 7a hybrid species *P.* ×*heterohybrida* was isolated. All 12 isolates differed in the ITS from the ex-type isolate from Taiwan (CBS141207; KU517151) at position 77 (Y instead T). In addition, three isolates from stream R01 were separated from the ex-type by being homozygous at position 428 (T instead of Y) and by having two unique heterozygous positions (656 and 748). In a 876 bp alignment of *cox1,* one isolate from stream R01 differed at three positions from the ex-type isolate (KU517145) and from all other Vietnamese and Taiwanese isolates which were identical. Mating tests with A1 and A2 tester strains of *P.* ×*heterohybrida* from Taiwan showed that all isolates from Vietnam belonged to the A1 mating type. In addition, two isolates from stream R01 produced oogonia abundantly in single culture. The morphology of the ornamented oogonia, mostly two-celled amphigynous antheridia and nonpapillate sporangia of the Vietnamese isolates matched the original description of *P.* ×*heterohybrida* [22].

*Phytophthora pseudocryptogea* and *P.* sp. kelmania from Clade 8a were isolated from each two of the three lower montane streams R09-R11 (Table 2). The three isolates of *P. pseudocryptogea* from streams R09 and R10 differed in ITS (816 bp alignment) and *cox1* (672 bp alignment) from the Australian ex-type isolate (VHS16118; KP288376, KP288342) at three and one positions, respectively. The five isolates of *P.* sp. kelmania from streams R09 and R11 showed differences in ITS (841 bp alignment) and *cox1* (582 bp alignment) to isolate P10614 (HQ261691, HQ261438) from North America at 3, 4, and 5 positions, respectively. The morphology of all Vietnamese isolates of *P. pseudocryptogea* and *P.* sp. kelmania matched the descriptions in literature [54].

From forest stream R02 in 2007 m altitude two new species from Clade 10 were isolated from naturally fallen leaves. *Phytophthora* sp. gallica-like 1 and *P.* sp. gallica-like 2 were distantly related to *P. gallica* differing in a 885 bp alignment of the ITS from the ex-type isolate of the latter (CBS 111474 = GAL1; DQ286726) at 53 and 68 positions, respectively, while being separated from each other by 49 bp. Morphologically, both new species can easily be distinguished from the sterile *P. gallica* [55] by being homothallic forming smooth-walled oogonia with paragynous antheridia. *Phytophthora* sp. gallica-like 1 produces globose chlamydospores like *P. gallica* whereas *P.* sp. gallica-like 2 does not form chlamydospores.

From Clade 9a2, *P. macrochlamydospora* and a new species related to *P. quininea*, informally designated as *P.* sp. quininea-like, co-occurred in montane forest stream R11. Isolate VN1006 of *P. macrochlamydospora* differed in the ITS (816 bp alignment) and in *cox1* (670 bp alignment) from the Australian ex-type isolate (P10263; FJ801351, MH136923) at 2 and 3 positions, respectively. In accordance with the original description of *P. macrochlamydospora* [2], the Vietnamese isolate was sterile and produced large chlamydospores and semipapillate to non-papillate sporangia. The four isolates of *P.* sp. quininea-like were separated in the ITS and *cox1* from the ex-type isolate of *P. quininea* (CBS407.48 = P8488; HQ261660; AY564200 + HQ708386) from Peru by differences of 7 and 27 bp, respectively. Like *P. quininea*, *P.* sp. quininea-like produces non-papillate sporangia with internal and external proliferation, catenulate irregular hyphal swellings and thick-walled large chlamydospores. Interestingly, two of the four isolates were sterile while the other two isolates were homothallic like *P. quininea* [2] but can be distinguished from the latter by forming amphigynous instead of paragynous antheridia. Only one isolate of the Clade 9a1 hybrid taxon *P.* sp. ×kunnunara-like could be obtained from one lower montane stream (R09) in Hoàng Liên NP (Table 2).

In contrast to the montane streams, the *Phytophthora* communities in the five lowland rivers with subtropical to tropical climate were dominated by *Phytophthora* taxa from the high-temperature tolerant Clades 9a1 and 9a3. From Clade 9a1 potential hybrid isolates related to *P. virginiana* were obtained from the Red River, the Black River and two other streams (R13, R16) (Table 2). The potential hybrids differed in the ITS from the ex-type isolate of *P. virginiana* (46A2; KC295544) by having in total 10 heterozygous positions, with 1–8 heterozygous positions per isolate, which are partly not present in the three hybrid taxa *P.* sp. ×virginiana-like 1, 2, and 3 from Taiwan. Therefore, these Vietnamese isolates are informally designated as *P.* sp. ×virginiana-like sensu lato. *Phytophthora* sp. ×kunnunara-like was found in the Red River and the two streams originating from Ba Vì NP (R13, R14) (Table 2). Compared to *P.* sp. kunnunara from Western Australia, the ITS of the Vietnamese isolates had 10 heterozygous positions, with 1–8 heterozygous positions per isolate, which were only partly shared with Taiwanese isolates of *P.* sp. ×kunnunara-like (KU682602, KU682603). Another swarm of potential hybrid isolates was abundantly obtained from four of the five lowland streams. They differed in the ITS from *P.* sp. Peru 4 (KC479209) at 10 positions which were all heterozygous (1–6 per isolate) and are, hence, informally designated as *P.* sp. ×Peru 4-like. Finally, isolates of another new potential

hybrid taxon, *P.* sp. ×Grenada 3-like were recovered from stream R13 which were distinguished in the ITS from *P.* sp. Grenada 3 (KC479208) by having five instead of one heterozygous position. All isolates of *P.* sp. ×Grenada 3-like, *P.* sp. ×kunnunara-like, *P.* sp. ×Peru 4-like and *P.* sp. ×virginiana-like were in culture fast-growing, self-sterile and produced intercalary or laterally globose, club-shaped to irregular hyphal swellings, mostly globose thin-walled chlamydospores and nonpapillate sporangia with internal nested and extended proliferation, typical features of aquatic Clade 9 species [56].

*Phytophthora* sp. ×insolita-like from Clade 9a3 was found in stream R13. Compared to the Taiwanese ex-type isolate of *P. insolita* (IMI288805; AF271222) the Vietnamese isolates showed differences in the ITS at eight positions of which four were heterozygous and mostly shared with Taiwanese isolates of *P.* sp. ×insolita-like (KU682601). Morphologically, all isolates from stream R13 were similar to both *P. insolita* and *P.* sp. ×insolita-like from Taiwan [2,10], producing in single culture smooth-walled oogonia without antheridia, thin-walled chlamydospores and non-papillate sporangia with internal nested and extended proliferation.

The only *Phytophthora* species recovered from a lowland stream (Black River) and not belonging to Clade 9 was *P. drechsleri* from Clade 8a. The isolates differed from the ex-type isolate of *P. drechsleri* (ATCC 46724 = 23J5; AF266798, MH620076) in the ITS at one position (136; Y instead of T) and in a 862 bp alignment of *cox1* at five positions. All three isolates belonged to the A1 mating type.

With 10, eight and six *Phytophthora* species, respectively, the montane forest streams R10, R11 and R02 harboured the highest diversity of *Phytophthora* species while the lowland rivers contained highly diverse assemblies of Clade 9 hybrids with almost all isolates being different from each other in the ITS.

*Nothophytophthora vietnamensis* was isolated from the montane forest streams R01, R02 and R10 in Hoàng Liên NP. In addition, the novel *Elongisporangium* sp. Hoàng Liên, *Phytopythium litorale*, *Ph. vexans* s.l., *Ph.* sp. 1 PB-2013, *Py. senticosum*, *Py.* sp. CAL\_2011f, *Py.* sp. 1\_MNS-2013, the previously unknown hybrid taxon *Py.* sp. ×ZSF0056-like and unidentified *Pythium* spp. were recovered from eight of the eleven mountain streams (Table 2 and Table S1). In four of the five lowland rivers, *Ph. palingenes*, *Ph.* sp. 1 PB-2013, *Py.* sp. 1\_MNS-2013, *Py.* sp. 2\_ROH-2015 and unidentified *Pythium* spp. were found (Table 2 and Table S1).

#### *3.3. Association between Phytophthora Presence in the Rhizosphere and Disease Symptoms*

In the 20 *Phytophthora*-inhabited forests sampled, the majority of the 52 tree species from which *Phytophthora* species were recovered appeared generally healthy (Figure 2a–f). Symptoms indicative of Phytophthora root diseases were almost exclusively found in eight montane forest stands and were mainly restricted to tree species belonging to the Ericaceae, Fagaceae and Lauraceae (Figures 3 and 4).

In Hoàng Liên NP, scattered dieback of *Rhododendron arboreum* trees with presence of *P. cinnamomi* A1 and *P.* sp. attenuata-like 1 in the rhizosphere was observed in the upper montane *Rhododendron* cloud forest at 2636 m altitude. In the montane evergreen cloud forest F03 at 2337 m altitude, groups of mature *Quercus glauca* trees showed severe thinning and dieback of the crowns (Figure 3a) which was associated with presence of *P. cinnamomi* A2 in the rhizosphere. In contrast, in forest stand F12 at 1895 m altitude, infested by *P. cinnamomi* A1, all *Q. glauca* trees appeared healthy. In six of the nine montane evergreen forest stands sampled between 1895 and 2242 m altitude, *C. acuminatissima* and sometimes also *Neolitsea poilanei* and *N. polycarpa* showed severe thinning and dieback of the crowns and mortality (Figure 3b–d,f). Disease incidence was particularly high in the swampy depression sampled in stand F06 (Figure 3c,d,f). In seven and five of the nine stands, *P. cinnamomi* A2 and *P. castaneae*, respectively, were recovered from rhizosphere soil samples while *P. cinnamomi* A1, *P. attenuata*, *P. gregata*, *P.* sp. attenuata-like 1, *P.* sp. attenuata-like 2 and *P.* sp. multivesiculata-like 1 were only infrequently found (Table 1). On a visual examination root samples from three declining trees each of *C. acuminatissima* and *N. polycarpa*, all infested by *P. cinnamomi* A2 and/or *P. castaneae*, exhibited severe losses of lateral and fine roots and open callussing lesions on coarse roots (Figure 4a–d). In stand F07, *P. cinnamomi* A2 was isolated from a bleeding bark lesion on a surface root of a declining *C. acuminatissima* tree (Figure 3e).

**Figure 4.** Symptoms on root systems of declining *Neolitsea polycarpa* trees in the montane evergreen cloud forest F10 in Hoàng Liên National Park associated with presence of *P. cinnamomi* A2 in the surrounding soil; (**a**,**b**) severe losses of lateral roots and fine roots and open callussing lesions on coarse roots (arrows); (**c**,**d**) detailed view of the open callussing lesions on the coarse roots from Figure 4a.

In contrast to the montane forests of Hoàng Liên NP, in the three submontane (700–1100 m a.s.l.) stands sampled in the subtropical, humid evergreen forests of Ba Vì NP all tree species, including several species from the Fagaceae genera *Castanopsis*, *Lithocarpus* and *Quercus* and the Lauraceae genera *Litsea*, *Machilus* and *Phoebe*, were healthy despite the occurrence of both mating types of *P. cinnamomi* and a range of five other *Phytophthora* species, including *P. castaneae*, *P. heveae*, *P. parvispora*, *P. citricola* IX and *P.* sp. attenuata-like 3, in the soil. The only exception was a small patch dieback of *Dysoxylum juglans* with presence of *P.* sp. attenuata-like 3 in the rhizosphere (Figure 3g).

In the five *Phytophthora*-infested tropical lowland rainforest stands in Cuc Phuong (F18, F20, F21), Bù Gia Mập (F23), and Côn Đảo (F25) National Parks, no symptoms suggestive of *Phytophthora* diseases were observed.

#### **4. Discussion**

Vietnam harbours an extremely diverse flora probably due to its heterogeneous geology, geomorphology and climates and its transitional position between the eastern Himalayas, Yunnan, and the Indomalaysian archipelago on the Asian continental shelf [39–41]. The latter enabled repeated immigrations of plant and most likely also fungal and oomycete species during various glacial periods followed by subsequent speciations and species radiations in the interglacials. A similar scenario was proposed earlier for Taiwan [10,36–38]. We have shown here that the floristic and environmental diversity of Vietnam is reflected by the high diversity of oomycete taxa. In this survey of 25 natural forests and 16 rivers 13 described species, five informally designated taxa and 21 previously unknown taxa of *Phytophthora*, together with *N. vietnamensis* and a range of seven described and ten undescribed species of *Elongisporium*, *Pythium* and *Phytopythium* were obtained. Considering the relatively limited

number and diversity of the sampled sites and ecosystem types it may be assumed that the true *Phytophthora* diversity of Vietnam is markedly higher. The finding of 20 *Phytophthora* taxa in 98 soil and four leaf samples from the 25 forest stands and an additional 15 *Phytophthora* taxa in 11 forest streams in Vietnam indicates a much higher diversity of forest *Phytophthora*s exists in Vietnam than occurs in Europe, the eastern US or the western US. In the latter areas, 39, 7, and 21 *Phytophthora* species, respectively, were detected in numerous surveys involving many more samples collected over much larger areas and a wider range of ecosystems [5,14,23,44,57–59].

The remote location of most sampled forest stands and forest streams in Vietnam, absence of introduced crop or tree species in the catchment areas and, apart from *P. cinnamomi* A2 in higher altitudes, the lack of association of *Phytophthora* with obvious disease symptoms suggest that most of the 35 forest *Phytophthora* species obtained are native to Vietnam. In contrast, only nine of the 32 *Phytophthora* species from European forests are considered indigenous [5,23,60]. The forest *Phytophthora* populations in Vietnam and Europe share only five species, *P. chlamydospora*, *P. cinnamomi* A2, *P. pseudocryptogea*, *P. ramorum* and *P.* sp. kelmania, while Vietnamese and North American forests have only *P. chlamydospora*, *P. cinnamomi* A2 and *P. ramorum* in common. Recent surveys in Taiwan, where floristic diversity is comparable to Vietnam, revealed a comparable *Phytophthora* diversity, with ten described and 17 previously unknown species from 30 forest stands and 25 streams [10,13]. Further, the *Phytophthora* communities revealed in Vietnam and Taiwan shared 12 taxa: *P. attenuata*, *P. capensis*, *P. castaneae*, *P. chlamydospora*, *P. cinnamomi* A1 and A2, *P. citricola* VII, *P. heveae*, *P. parvispora*, *P.* ×*heterohybrida*, *P.* sp. ×insolita-like, *P.* sp. ×kunnunara-like and *P.* sp. ×virginiana-like *s.l.*. In three areas in northern Yunnan, a Chinese province adjacent to northern Vietnam, eight *Phytophthora* species were isolated from streams running through sclerophyllous oak forests but only two of them, *P. chlamydospora* and *P. plurivora*, were recovered from forest soil samples [16]. The only *Phytophthora* species common to Vietnam and northern Yunnan were *P. chlamydospora* and *P. gregata*. In montane forests of the tropical island Hainan, located in the South China Sea close to Vietnam, six *Phytophthora* species were found [12] of which three species, *P. castaneae*, *P. cinnamomi* and *P. heveae*, also occurred in Vietnam. The lower *Phytophthora* diversities in the north Yunnan and Hainan surveys compared to Vietnam were most likely due to the smaller number of sites and forest types sampled and the use of different isolation techniques.

In recent years, an impressive diversity of both known and previously unknown *Phytophthora* species has been revealed from stream surveys in several countries, including the eastern and western USA, Chile, Australia, South Africa and Taiwan [10,11,14,17,18,44,61], as discussed previously [10]. By comparison the riparian *Phytophthora* communities identified here in Vietnam are remarkably rich, with seven described species, five informally designated taxa and 12 previously unknown taxa. Several montane streams with small catchments in the forests around the Fansipan harboured an unprecedented diversity of up to ten *Phytophthora* species per stream.

Interestingly, the most common *Phytophthora* species in Vietnamese forest soils, *P. cinnamomi*, *P. castaneae*, *P. heveae* and the four species from the '*P. attenuata* complex', were never isolated from streams running through or originating from infested forests. Overall, the *Phytophthora* communities found in the forest soils (20 taxa) and in the streams (24 taxa) shared only four species, *P. chlamydospora*, *P. citricola* VII and IX and *P.* sp. multivesiculata-like 1. Similar differences between terrestrial and aquatic *Phytophthora* populations were observed in comparable studies in Europe, Chile, Taiwan, South Africa and the USA [10,11,18,44,58]. This is consistent with previous observations that most *Phytophthora* species are adapted either to a soilborne and root-infecting or aerial foliage-infecting lifestyle, or are aquatic saprotrophs that tend to be opportunistic pathogens [6,10,11,21,53,62]. Consequently, when sampling *Phytophthora* diversity in a diverse environment both soils and streams should be analysed using optimal baiting methods for each or metagenomic approaches based on high-throughput pyrosequencing of environmental DNA with *Phytophthora*-specific primers [19,63]. Ideally, because metagenomic analyses can sometimes result in false molecular operational taxonomic units (MOTUs), and because living isolates are needed for taxonomic descriptions and host range testing, baiting, and metagenomic approaches should be carried out in parallel.

Altitude had a strong influence on *Phytophthora* distribution. The '*P. attenuata* complex', *P. castaneae* and *P. cinnamomi* occurred only in soils of submontane and montane forests above 700 m a.s.l. while *P. heveae* and most taxa from Clade 2a were restricted to forests below 1100 m altitude. The altitudinal influence on aquatic *Phytophthora*s was even more pronounced. While the 11 montane streams above 1000 m altitude with a subtropical to temperate climate contained mainly species belonging to Clades 2c, 2e, 6b, 7a, and 8c, the *Phytophthora* communities in the five lowland rivers with subtropical to tropical climate were dominated by species and hybrids from the high-temperature tolerant Clades 9a1 and 9a3 [28]. Clade 9 species and hybrids were also most common in lowland streams in Taiwan and South Africa [10,18].

The results of this survey offer new insights into the origin of several invasive *Phytophthora* pathogens and of clades and subclades of *Phytophthora*. Most notably, the finding of the highly invasive, wide-host range pathogen *P. ramorum* in eight forest streams around the Fansipan and Sau Chua mountains with both A1 and A2 mating types present, together with an apparent absence of overtly visible disease symptoms on potentially susceptible Ericaceae, Fagaceae, or Lauraceae, suceptible genera where *P. ramorum* is damaging and introduced in Europe and North America [6,7,64], suggests an equilibrium between the pathogen and the north Vietnamese vegetation as a consequence of long term endemism and co-evolution. This is supported by variability in the ITS and *cox1* sequences of the Vietnamese isolates and by *cox1* sequence differences between the Vietnamese isolates and the North American NA1 and NA2 and the European EU1 and EU2 lineages. Because of the implications both for the origin of the pathogen and for international biosecurity a detailed comparative phenotypic and molecular analysis of the Vietnamese *P. ramorum* isolates and the known EU1, EU2, NA1, and NA2 lineages [65] is currently ongoing to further characterise the Vietnamese population and its relationship to the known lineages. Since southern Yunnan, northern Laos, and the eastern Himalayas belong to the same biogeographic area as the Fansipan region mountain forests in these regions may also harbour endemic *P. ramorum* populations. Further surveys are needed to confirm this hypothesis.

*Phytophthora cinnamomi* was the most common soilborne *Phytophthora* species above 700 m. The A2 mating type of *P. cinnamomi* was more widespread, occurring in 11 forest stands between 713 and 2337 m, whereas the A1 occurred only in four forest stands located between 1108 and 2636 m a.s.l. In Taiwan and Papua New Guinea also the A1 mating type occurrs at higher altitudes than the A2 indicating higher tolerance to low temperatures [10,66]. However, in both of these locations the altitudinal differences between the mating types are larger than in Vietnam, the A2 being confined to the lowland forests. In each one stand in Hoàng Liên NP and Ba Vì NP both mating types co-occurred. In Hoàng Liên NP the A1 was present in the upper montane *Rhododendron* forest at 2636 m and in two lower montane stands at 1900 m. However, it was not detected in the eight forest stands between 2337 and 2022 m in which not only the A2 type was present but severe dieback of Fagaceae and Lauraceae was observed (notably *C. acuminatissima*, *Q. glauca*, *N. poilanei* and *N. polycarpa*). Pathogenicity trials are required to fulfill Koch' s postulates for these host-pathogen associations and confirm that *P. cinnamomi* A2 is causing the dieback of these native Fagaceae and Lauraceae species. In contrast, no dieback was observed in the three forest stands in Ba Vì NP between 713 and 1100 m, despite the presence of *P. cinnamomi* A2. In the two stands with the co-occurrence of both mating types the A1:A2 ratio of the 44 isolates was 59.1:40.9, whereas the overall mating type ratio of the 151 isolates from 13 *P. cinnamomi* infested stands was 30.5:69.5. Collectively, these results suggest that, as a consequence of current climatic warming, the more thermophilic but frost sensitive A2 mating type may be spreading into higher altitudes in Vietnam. Such a progression of *P. cinnamomi* A2 into higher latitudes and altitudes with climate change was predicted by CLIMEX modelling [67–69]. In the newly A2 invaded high-altitude forests in Vietnam the A2 may be outcompeting and replacing the native co-evolved A1, causing dieback in the susceptible non-coevolved hosts. The widespread distribution of *P. cinnamomi* in northern Vietnam, the co-occurrence of both mating types in several stands, and the absence

of disease symptoms in lower altitudes also indicates that Vietnam lies within the origin of both mating types. *Phytophthora cinnamomi* is the most invasive member of the genus with a host range of almost 5000 woody plant species [2,70,71]. Two genotypes of the A2 mating type have reached a panglobal distribution causing epidemics in numerous natural and managed ecosystems while the A1 mating type has a limited distribution outside of Asia and has never been associated with epidemic disease [2,5,6,9–11,60,66,72–76].

*Phytophthora attenuata*, recently described from montane forests in Taiwan [22], and three closely related but previously unknown species, were found in the submontane and montane forests of northern Vietnam. *Phytophthora attenuata*, *P.* sp. attenuata-like 1 and *P.* sp. attenuata-like 2 were detected in the temperate, montane cloud forests around the Fansipan. However, *P.* sp. attenuata-like 3 was found only in the subtropical, humid submontane evergreen forests in Ba Vì NP. These four closely related species most likely result from sympatric species radiation, suggesting northern Indochina as the center of origin of the '*P. attenuata* complex'. A pathogenicity trial is required to confirm that *P.* sp. attenuata-like 3 is causing the dieback of *Dysoxylum juglans* in Ba Vi National Park. Another Clade 7a species that was first described from Taiwan, *P.* ×*heterohybrida*, was widespread in Vietnamese montane forest streams. This allopolyploid hybrid species has a functional but peculiar sexual system. In Taiwan, all isolates were selfsterile with both mating types being common and one isolate mating with both mating types [22]. In contrast, almost all Vietnamese isolates were self-sterile and belonged to the A1 mating type while one isolate was prolifically homothallic and stimulated oogonia formation in A2 tester strains.

The results indicate that the '*P. citricola* complex' from Clade 2c also underwent a species radiation process in Vietnam. Besides *P. capensis*, originally described from nursery plants in South Africa and also isolated from natural streams in Taiwan [10,77], and *P. citricola* VII, which was previously reported from a montane forest in Taiwan [10], four previously unknown taxa were found in this survey. Most common was *P. citricola* VII which occurred in mountain streams inside and outside of Hoàng Liên NP and in the rhizosphere of a montane *Alnus* forest, whereas the new taxa *P. citricola* VIII to XI had only cryptic distributions. It is noteable that *P. citricola* X and XI were only found in tropical lowland rainforests and that they differ from all other species of the '*P. citricola* complex' by producing mainly papillate instead of semipapillate sporangia. The occurrence of *P. citricola* VII to X within only ca 300 km in northern Vietnam and the co-occurrence of *P. citricola* VII, VIII and IX in individual streams suggest sympatric species radiation from a common ancestor. In contrast, the exclusive finding of *P. citricola* XI and also *P.* sp. botryosa-like 2 from Clade 2a on Côn Lôn island, situated on the Asian shelf 50 km off the southern Vietnamese coast, are more consistent with allopatric island speciation. The invasive wide-host range pathogen *P. plurivora* occurs in undisturbed, healthy, often deciduous temperate mountain forests in Taiwan, Nepal and Yunnan [10,15,16,43]. However, it was not found here in the subtropical and tropical forests of Vietnam. This suggests that *P. plurivora* is native to temperate mountainous regions of South and East Asia.

The detection of five new species and of *P. capensis* from the '*P. citricola* complex' in Clade 2c, three new species from Clade 2a and *P.* sp. tropicalis-like 2 and *P.* sp. multivesiculata-like 1 from Clades 2b and 2e in this survey, the findings of *P. bisheria*, *P. capensis*, *P. plurivora*, *P. citrophthora*, *P. tropicalis* and the three new Clade 2a species *P.* sp. ×botryosa-like, *P.* sp. ×meadii-like and *P.* sp. occultans-like from natural ecosystems in Taiwan [10,13] together with the widespread occurrence of *P. botryosa*, *P. citricola*, *P. colocasiae* and *P. meadii* across Southeast Asia [2,12,33–35] suggest South, Southeast and East Asia as the center of origin of *Phytophthora* major Clade 2.

Interestingly, all known isolates from the new Clade 2a taxa *P.* sp. meadii-like 1 and 2 and *P.* sp. botryosa-like 2 from Vietnam, as well as *P.* sp. ×botryosa-like and *P.* sp. ×meadii-like from Taiwan [10] are of A1 mating type and are characterised by oospore abortion rates exceeding 95% in mating tests with tester strains of *P. botryosa* and *P. meadii*. It appears that in this complex of aerial *Phytophthora* species the A1 is better adapted to and, hence, more common in natural forests than the A2. It is even possible that these self-sterile taxa, like many aquatic Clade 6 species [21,53], lack the A2 mating

type and have abandoned sexual reproduction in favour of exclusive asexual reproduction, spreading via their caducous sporangia from infected to non-infected above-ground tissues. This possibility is supported by the extremely high oospore abortion rates in mating tests with tester strains of *P. botryosa* and *P. meadii*. More field surveys and laboratory tests are needed to verify this hypothesis.

*Phytophthora castaneae* and *P. heveae* from Clade 5 are also considered being native to Taiwan and Hainan [2,10,12,31]. Their widespread occurrence in Vietnamese forests and the lack of association with disease symptoms in the native vegetation indicate that Indochina also lies within the origin of both species.

As previously demonstrated in Australia, Chile, South Africa and Taiwan putative interspecific hybrids, indicated by multiple heterozygous sites in their ITS sequences, are common in watercourses and can also be found in forest soils [10,11,17,18,78,79]. As with predominantly aquatic species and hybrids from Clade 6, all Clade 9 hybrids from Vietnamese streams, with the exception of *P.* sp. ×insolita-like which produces oogonia without antheridia, are sterile and apparently adapted to rapid and continuous asexual proliferation via zoospores. Also, like many Clade 6 taxa, this may reflect adaptation to a mostly saprotrophic lifestyle as decomposers of naturally fallen leaves [21,53]. As with the Clade 6 hybrid *P. thermophila* × *P. amnicola* in the Valdivia River in Chile [11], no putative parents of the Clade 9 hybrids *P.* sp. ×Grenada 3-like, *P.* sp. ×insolita-like, *P.* sp. ×kunnunara-like, *P.* sp. ×Peru 4-like and *P.* sp. ×virginiana-like were detected in the Vietnamese rivers. Possibly the hybridisation events occurred in the Vietnamese streams and the parents were outcompeted by the better adapted hybrids. Alternatively, the hybrids could be introduced from elsewhere. Since the multicopy ITS locus is of limited use for hybrid studies sequencing of appropriate mitochondrial and nuclear genes are needed to confirm the hybrid status and elucidate the parents of the putative hybrid taxa.

Panglobally distributed pathogens from *Phytophthora* Clades 1 (*P. cactorum*, *P. infestans*, *P. nicotianae*) and 4 (*P. palmivora*) commonly cause diseases of horticultural crops and ornamental plants in mainland China, Hainan and Taiwan [12,33,35]. However, in this survey, as in previous surveys in Taiwan, species from Clades 1 and 4 (exception for one isolate of *P. palmivora* in Taiwan) were not detected in natural forests and streams [10,13], indicating that these two clades are not native to Taiwan and Southeast Asia. The same probably applies to Clades 3, 11 and 12 [23,28].

Although the natural hosts of the putatively endemic Vietnamese forest Phytophthoras obtained in this study are still unknown, it is evident that many native Asian forest Phytophthoras have co-evolved with a variety of tree genera also present in Europe and North America, including Fagaceae, Lauraceae, Aceraceae, Oleaceae, and Pinaceae. In this case high susceptibility of many non-coevolved European and North American trees to these Asian *Phytophthora* species is possible, as already well demonstrated for *P. cinnamomi*, *P. plurivora*, *P.* ×*cambivora* and, more recently, for six new Clade 7a species from Taiwan [22]. An extensive host range study with *Phytophthora* species from Asia, South and Central America has been initiated and will be published separately. In one part of this study, the pathogenicity of five Asian species (*P. castaneae*, *P. heveae* and the three new Vietnamese species *P. citricola* X, *P.* sp. multivesiculata-like 1 and *P.* sp. tropicalis-like 2) to *Castanea sativa*, *Quercus suber* and *Quercus robur* has been investigated and all five caused significant rot and loss of fine roots and suberised lateral roots in all three hosts, *C. sativa* being most susceptible [80].

Against this background, the annual importation of over three billion plants-for-planting into Europe [81], the large numbers of previously unknown *Phytophthora* species in natural and horticultural ecosystems being identified in Asia, South and Central America ([10,11,16,22,82], this study) and the occurrence of at least 47 exotic *Phytophthora* species in European nurseries and associated outplantings [60] represents a significant biosecurity risk for forestry, horticulture, and natural ecosystems in Europe and North America.

Many recent epidemics of trees and horticultural crops have been caused by introduced pathogens that were previously unknown to science, probably due to the organisms being co-evolved and benign in their centres of origin [6,10,83]. Although often introduced via the plants-for-planting pathway, none of them has ever been intercepted pre-emptively during routine phytosanitary controls at the ports of entry [60,81,83,84]. Despite overwhelming scientific evidence, current sanitary and phytosanitary (SPS) protocols largely ignore the risks from unknown, benign, co-evolved and unescaped organisms [6,60,83–85]. However, preventing further introductions of potentially harmful invasive Phytophthoras is a key issue for international forest biosecurity. A series of international research projects and organisations (listed in [10]) have come to similar conclusions. The current, outdated and scientifically flawed species-by-species regulation approach based on random visual inspections for symptoms of described pests and pathogens needs to be replaced by a sophisticated pathway regulation approach using pathway risk analyses, risk-based inspection regimes and molecular high-throughput detection tools [6,60,81,83,84,86,87].

To further define areas of *Phytophthora* diversity, including high-risk areas for the origin of potentially harmful pathogens, more *Phytophthora* surveys are needed in natural ecosystems in unsurveyed areas of Asia, Africa, and South and Central America, followed by host range testing of new taxa on naive tree hosts in Europe and elsewhere. Such surveys should also contribute to a better understanding of the global diversity of *Phytophthora*, the ancient biogeographic radiation of the *Phytophthora* species and Clades, and the influence of local environmental and host factors on breeding strategies and adaptation in the genus.

#### **5. Conclusions**

A remarkable diversity of 13 described species, five informally designated taxa and 21 previously unknown taxa of *Phytophthora* were obtained from 25 natural and semi-natural forest stands and 16 rivers in temperate and subtropical montane and tropical lowland regions of Vietnam. It is concluded that Vietnam is within the center of origin of most *Phytophthora* taxa found, including *P. cinnamomi* and *P. ramorum*, and that *Phytophthora* clades 2, 5, 6, 7, 8, 9, and 10 are native to Indochina.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4907/11/1/93/s1, Table S1: GenBank accession numbers of ITS and partial *cox1* sequences generated in this study for representative *Phytophthora*, *Elongisporangium*, *Nothophytophthora*, *Phytopythium* and *Pythium* isolates from Vietnamese forests and rivers and isolates from related *Phytophthora* species used for comparisons.

**Author Contributions:** Conceptualization: T.J., M.H.J., C.M.B.; data curation: T.J., M.H.J., I.M.; formal analysis: T.J., M.H.J., B.S., M.T., M.P., J.B., H.R., A.P.-S., A.B., M.R., C.M.; investigation: T.J., M.H.J., C.M.B., J.W., T.C., B.S.; methodology: T.J., M.H.J., C.M.B.; writing—original draft: T.J.; writing—review and editing: T.J., C.M.B., B.S., T.C., I.M., M.T., J.B., M.H.J., A.P.-S. All authors have read and agree to the published version of the manuscript.

**Funding:** The authors are grateful to the Czech Ministry for Education, Youth and Sports and the European Regional Development Fund for financing the Project Phytophthora Research Centre Reg. No. CZ.02.1.01/0.0/0.0/15\_003/0000453. This work has also received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 635646, POnTE (Pest Organisms Threatening Europe). Travel and subsistence support for CMB was provided by Brasier Consultancy. DNA sequencing in this study was partly supported by the Hungarian Scientific Research Fund (OTKA) grant K101914.

**Acknowledgments:** The Vietnamese Academy of Forest Sciences in Hanoi is gratefully acknowledged for providing lab space and technical support. We thank Henrieta Datkov ˇ á and Josef Janoušek (PRC, Brno, Czech Republic) for much appreciated technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Phytophthora ramorum* **and** *Phytophthora gonapodyides* **Di**ff**erently Colonize and Contribute to the Decomposition of Green and Senesced** *Umbellularia californica* **Leaves in a Simulated Stream Environment**

### **Kamyar Aram \* and David M. Rizzo**

Department of Plant Pathology, University of California, One Shields Drive, Davis, CA 95616, USA<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
Received: 28 March 2019; Accepted: 19 May 2019; Published: 20 May 2019

**Abstract:** Plant pathogenic as well as saprotrophic *Phytophthora* species are now known to inhabit forest streams and other surface waters. How they survive and function in aquatic ecosystems, however, remains largely uninvestigated. *Phytophthora ramorum*, an invasive pathogen in California forests, regularly occurs in forest streams, where it can colonize green leaves shed in the stream but is quickly and largely succeeded by saprotrophically competent clade 6 *Phytophthora* species, such as *Phytophthora gonapodyides*. We investigated, using controlled environment experiments, whether leaf litter quality, based on senescence, affects how *P. ramorum* and *P. gonapodyides* compete in leaf colonization and to what extent each species can contribute to leaf decomposition. We found that both *Phytophthora* species effectively colonized and persisted on green or yellow (senescing) bay leaves, but only *P. gonapodyides* could also colonize and persist on brown (fully senesced and dried) leaves. Both *Phytophthora* species similarly accelerated the decomposition of green leaves and yellow leaves compared with non-inoculated controls, but colonization of brown leaves by *P. gonapodyides* did not affect their decomposition rate.

**Keywords:** leaf decay; oomycetes; invasive species; aquatic fungi; trophic specialization; saprotroph; pathogen; parasite

#### **1. Introduction**

The ecology of *Phytophthora*, a genus of fungal-like oomycetes historically erected and known for plant pathogenic species primarily associated with destructive diseases in agriculture [1], has undergone substantial reconsideration in recent years [2]. The recent emergence of a number of *Phytophthora*-caused plant epidemics in forests and other non-agricultural ecosystems has clearly shown that many members of the genus have potential as invasive species that can threaten natural ecosystems [2–4]. As a consequence of research in non-agricultural environments, a surprising diversity and abundance of *Phytophthora* species have been discovered, many previously undescribed [2,4]. Incidental to this research has been the discovery that many species of *Phytophthora* are abundant in natural surface waters, especially in streams. Many such species are so widespread and regularly encountered that they are now considered resident, if not endemic, and characteristic of such environments [5–22]. Nevertheless, isolates of well-known plant pathogenic species or species complexes are also regularly recovered, often without discernible symptoms or signs of disease on the vegetation [7,9,15,16,21,23–26].

Though the prevalence of *Phytophthora* in surface waters is now well established, the ecology underpinning this phenomenon is largely speculative. Because these organisms are known primarily as causes of often devastating plant diseases, the nature of their presence in these environments and its implications for the persistence and spread of pathogenic species are important considerations for disease prevention and management. There is also a growing interest to understand the role of *Phytophthora*, among other Peronosporales, in decomposition of vegetative matter in aquatic environments [27]. The biology of *Phytophthora*, a genus of well adapted plant pathogens with a necrotrophic phase [1,28], suggests that their ecological role in leaf decomposition should be early colonization and breakdown of relatively fresh, live vegetative tissue. As they colonize leaves newly exposed in streams, they can open the integral tissues for colonization by saprotrophic organisms less able to penetrate the leaf cuticle, in a process analogous to 'conditioning' of leaf litter for palatability to shredder organisms [29,30]. The co-occurrence of both known plant pathogens and primarily stream-associated *Phytophthora* in aquatic environments also raises the question of whether these taxa have similar or divergent modes of life and whether they compete for resources in these environments.

In streams, vegetative litter is the primary source of nutrients for microorganisms [29,31,32], but the quality of vegetative tissues available varies with respect to senescence and degree of decomposition. Coastal forests of northern California largely consist of evergreen trees and shrubs [33] and so green leaves are a regular component of leaf litter introduced into streams, especially in winter and spring when, based on the region's climate, most rainstorms occur. Nevertheless, much vegetative litter is in the form of senesced leaves [34]. California bay (*Umbellularia californica* (Hook. and Arn.) Nutt.) is a common, broadleaf evergreen component of northern California's coastal forests and a frequently occurring tree species in riparian zones [35,36]. It is also a primary source of *P. ramorum* inoculum in California forests affected by sudden oak death, epidemic mortality of certain species in the beech family (Fagaceae) resulting from *P. ramorum* infection of the vascular cambium of the main trunk [37,38]. California bay leaves are highly conducive to sporulation by *P. ramorum* which, despite causing localized necrotic lesions and spots on leaves, nevertheless causes little damage to the tree species itself [39–41]. Additionally, bay leaves are sclerophyllous, as is typical for broadleaf evergreen plants in this Mediterranean climate, and so they decompose slowly [33]. Bay leaves are therefore both very common as leaf litter in northern California forest streams and a highly suitable substrate for *P. ramorum*.

Leaf senescence in California bay increases in the hot and dry summer months, peaking in late summer [34,37]. Thus, though green leaves often enter streams during winter and spring storms, as summer progresses, most of the bay leaves shed into streams are either dropped directly upon senescence from trees or are blown in from accumulated litter on the forest floor, nearby (as described by [29,42]). In general, fully senesced leaves have as much as 75% reduced protein content compared with green leaves, primarily from the dismantling of chloroplasts, and though yellow, senescing leaves still have live cells with active mitochondria, leaves that have turned brown as a result of drying no longer contain biologically active cells [30,43,44]. Therefore, green, senescing and fully senesced bay leaves are substrates that likely vary in their suitability for colonization by *P. ramorum* and stream-resident clade 6 *Phytophthora* species, taxa that commonly occur at high inoculum levels in northern California coastal forest streams [45,46].

We have shown that there is a difference in trophic specialization between the saprotrophically competent, clade 6 *Phytophthora* species, such as *P. gonapodyides*, and *P. ramorum* [45], an aggressive pathogen on many plant species [38,47,48]. In that study, green California bay leaves were rapidly colonized by *P. ramorum* in streams but were succeeded nearly completely within three weeks by clade 6 *Phytophthora* species [45]. It remains uncertain, however, whether *P. ramorum* was displaced by more competent saprotrophs or receded from an inability to persist in tissues that it had colonized as they progressively decomposed. Additionally, as most leaf litter consists of senesced leaves, it is important to know how these differently adapted taxa can compete for and persist on biologically inactive leaf tissue. Finally, though stream resident *Phytophthora* species are assumed to contribute to leaf decay given their regular recovery from streams and frequent association with decomposing vegetation [2,27], experimental evidence for the kind and extent of this contribution is lacking. Moreover, it is unknown

how the introduction of an exotic and plant pathogenic species, like *P. ramorum,* into a stream ecosystem might affect the decomposition of leaf litter by other organisms, such as resident *Phytophthora* species. Therefore we undertook a laboratory study to determine: (1) How well *P. ramorum* and *P. gonapodyides* could use senesced leaves as a substrate in comparison to green, live leaves, (2) whether colonization by and persistence of *P. ramorum* on leaves was affected by competition with *P. gonapodyides*, and (3) how much each of these *Phytophthora* species contribute to the decay of each leaf type.

#### **2. Materials and Methods**

#### *2.1. Experiment Overview*

To test the capacity of *P. ramorum* and *P. gonapodyides* to colonize green and senesced bay leaves, we conducted controlled environment experiments exposing leaves to an inoculum of each species alone and in combination in microcosms designed to simulate an aquatic environment (Supplemental Figure S1). The experiment consisted of a randomized complete block design with treatments representing a complete factorial of bay leaf type (green/live or brown/senesced), stream water addition (autoclaved or not), and *Phytophthora* inoculation (none, *P. ramorum*, *P. gonapodyides*, or combined *P. ramorum* and *P. gonapodyides*). These 16 treatment combinations were replicated in five blocks arranged in three growth chambers (model PGR-15, Conviron Controlled Environment Ltd). The experimental unit was a mesh packet of five leaves which were sampled at intervals over 16 weeks from microcosms. One treatment packet per sampling served for decomposition as percent biomass loss and another for colonization based on isolations on a selective medium. We repeated the experiment once, with leaf types maintained in the same microcosm in the first and in separate microcosms in the second experiment. We conducted a separate experiment with yellow, senescing leaves collected while still attached to trees and with the cuticle intact, with *P. ramorum*-only and combined *P. ramorum*/ *P. gonapodyides* treatments as well as non-inoculated controls, in a completely randomized design with four reps in a single growth chamber.

#### *2.2. Experiment Preparation*

#### 2.2.1. Leaves

We collected leaves from two sites where our previous field experiments were conducted [45]. One was a canyon through which Graham creek runs at Jack London State Park (38◦21- 2" N, 122◦33- 16" W) which consists of redwood forest with California bay as a dominant riparian tree, along with redwood (*Sequoia sempervirens* [Lamb. ex D. Don] Endl.), Douglas fir (*Pseudotsuga menziesii* [Mirb.] Franco), tanoak (*Notholithocarpus densiflorus* [Hook. & Arn.] P.S. Manos, C.H. Cannon, & S.H. Oh), bigleaf maple (*Acer macrophyllum* Pursh), and less frequently, madrone (*Arbutus menziesii* Pursh) [49,50]. The second included canyons around Copeland Creek at Sonoma State University's Fairfield Osborn Preserve (38◦20- 37" N, 122◦35- 41" W) which is characterized by mixed evergreen forest with a prevalence of California bay, white alder (*Alnus rhombifolia* Nutt.), big leaf maple, and occasionally, tanoak, madrone and coast live oak (*Quercus agrifolia* Née) [39,49]. At each site, we collected green, symptom-free bay leaves with a mature cuticle from trees and brown, recently shed bay leaves from beneath trees in the manner of Wood et al. [51]. Brown leaves were collected from both sites in September 2014, allowed to air dry in the laboratory, and then stored in sealed plastic bags at room temperature until used in experiments. Green leaves were collected on 12 December 2014 from the redwood forest site and on 5 August 2015 from the mixed evergreen forest site. Yellow leaves were collected directly from trees at the mixed evergreen forest site on 7 September 2015. Green leaves were stored at 4 ◦C for up to three weeks prior to use in experiments and yellow leaves were likewise stored but deployed in experiments within one week of collection. We collected leaves primarily from riparian areas around the described creeks, though, at the mixed evergreen forest site, we had to seek symptomless leaves to some extent from plateaus above the canyons. Leaves collected from each forest type were used in separate

experiments. Leaf treatments were in the same microcosm for the first experiment (40 containers) using leaves from the redwood forest and separate in the second experiment (80 containers) with leaves from the mixed evergreen forest. Yellow leaves were collected from the mixed evergreen forest site only. We tested a subsample of 50 of each leaf type for both sites—through isolations attempted on a selective medium as described below—to verify that there were no pre-existing *Phytophthora* infections. Brown leaves were soaked in sterile deionized water at 4 ◦C for two days prior to these test isolations. Leaf packets were prepared for each leaf type by packing five leaves into a flat envelope of 1 mm plastic mesh approximately 20 × 20 cm so that the leaf surfaces were in minimal contact with one another and each packet was sealed by folding over the open lip and securing it with two common metal staples.

#### 2.2.2. Microcosms and Water

We assembled microcosms simulating an aquatic decomposition environment similar to the approach described by Medeiros et al. [52] (Supplemental Figure S1). White plastic buckets (2 gal., 21 × 24 cm, dia. × ht., Argee Corp, Santee, CA, USA) were used in the first (leaf types together) and yellow leaf experiments and opaque plastic containers (8 qt., 19.4 × 27.3 cm, dia. × ht., Continental Carlisle, Oklahoma City, OK, USA) in the second experiment (leaf types separate). Each container was aerated through a tube terminating in an aeration stone (3 cm dia., Uxcell®, Hong Kong, China) fed by an air pump (Commercial Air 1, EcoPlus®, 18W, 793 GPH, 12/Cs, Hawthorne Gardening Co, Vancouver, WA, USA) that was turned on for 30 minutes twice daily using an electric timer (Intermatic TIME-ALL®, TN311, Spring Grove, IL, USA). Aeration intensity was moderated with the addition of adjustable valves inserted in the tubing. A dilute nutrient solution was used as the base for the water mixtures in microcosms in order to avoid osmotic stress on spores. This was achieved by adding Hoagland's #2 salts (Caisson Laboratories, Inc., Smithfield, UT, USA) to autoclaved Millipore®filtered water for a final concentration of 0.01× the standard concentration (1.63 g/L). To test for any effect of natural stream microbiota on *Phytophthora* colonization or leaf decomposition, we included an addition of autoclaved or non-sterilized stream water as a treatment factor. The final composition of water in microcosms consisted of 4 L nutrient solution and 2 L stream water in the first experiment, and 4 L nutrient solution and 1 L stream water in the second. We collected water from streams in a bucket, pouring it through several layers of cotton mesh ("cheesecloth") into 4 L plastic bladders that we consolidated into larger plastic containers or used directly to transport water out of the field. Once brought to the laboratory, stream water was stored in plastic containers in a growth chamber at 12 ◦C and 12 h photoperiod (≈ 1800 lux) for 20 and 23 days prior to deployment in the first and second experiments, respectively. After storing the water for seven days, we submerged symptomless California bay leaves collected at each site as baits in each container for two days to confirm that *Phytophthora* zoospores were not present. We tested baits for infection using the isolation technique described below. No *Phytophthora* infections were detected from baits at this point. In the experiment with yellow leaves, we used only 4 L of a nutrient solution without stream water addition.

We measured stream pH, electrical conductivity (EC) and temperature on site at the time of stream water collection and subsequently in each microcosm throughout the experiments with a portable sensor (Combo pH and EC tester, model 98129N, Hanna Instruments, Woodsocket, RI, USA). Stream pH, EC, and water temperature were 8.55, 208 μS/cm, and 13.5 ◦C, respectively, for the redwood forest stream on the 9 December 2014 collection date, and 8.08, 363 μS/cm, and 17.7 ◦C, respectively, for the mixed evergreen forest stream on the 5 Aug 2015 collection date. To approximate natural stream pH in microcosms, we amended the mix of dilute nutrient solution and stream water in each microcosm with potassium carbonate buffer ("pH UP", General Hydroponics, Santa Rosa, CA, USA) at approximately 10 mg/L and adjusted it with KOH and HCl for a target of pH 8.3. The average pH (± SD) measured in microcosms periodically over the course of experiments was 7.99 (±0.27), 8.33 (±0.38) and 8.24 (±0.27) in the first, second and yellow leaf experiments, respectively. The average EC (±SD) was 208 (±63), 141 (±18), and 98 (±15) in the first, second, and yellow leaf experiments, respectively.

In the first experiment, where green and brown leaves were maintained together in treatment microcosms, the water darkened from leaf leachates shortly after experiment initiation. After 52 days, we removed two liters of water from each microcosm using an auto-siphon—sanitized with a 10% bleach solution in between each treatment—and added a fresh sterile nutrient solution to bring the volume back up to six liters. For the second experiment, we leached leaves prior to deployment in the experiment in approximately 300 mL autoclaved Millipore®-filtered water per 10 leaves and the water did not darken to the extent observed in the first experiment.

During all experiments, we periodically topped off the microcosms with autoclaved Millipore®-filtered water to 6, 5 or 4 L in the first, second, and yellow leaf experiments, respectively.

#### 2.2.3. *Phytophthora* Inoculum

*Phytophthora* inoculum consisted of three isolates per species grown for three weeks at 20 ◦C in 10 mL 10% clarified V8 juice liquid culture (V8®original vegetable juice (Campbells Soup Co., Camden, NJ, USA) neutralized with 15 g/L CaCO3, clarified by centrifuging at 7000 RPM for 10 minutes and diluted with deionized water) for the first experiment and in 5 mL of the same liquid culture for the second and yellow leaf experiments. Inoculum was introduced as mycelial mats to each container to initiate experiments with leaf packets already present for 24 h. Each container received six total inoculum doses: Those receiving only *P. ramorum* or *P. gonapodyides* receiving two doses of each isolate and the combined inoculation treatments receiving one dose of each isolate of each *Phytophthora* species. We used the same isolates in both experiments, all collected from the stream at the redwood forest site described above. *Phytophthora ramorum* isolates Pr-1906, Pr-1907, Pr-1908 and *P. gonapodyides* isolates P-1903, P-1904 and P-1905 are maintained in D.M. Rizzo's laboratory. Isolates of both *Phytophthora* species were originally identified by morphology, and the identity of *P. gonapodyides* isolates was confirmed through ITS sequence BLAST matches in GenBank (GenBank accessions: MK908979, MK908980, MK908981).

#### 2.2.4. Experiment Conditions and Sampling

Experiments were maintained with 12 h photoperiod (≈1800 lux) and 18/14 ◦C light/dark temperatures, respectively, to reflect typical average stream temperatures and also to provide a temperature differential that would potentially encourage *Phytophthora* zoospore release. Temperatures were monitored hourly in each block using iButton®loggers (Maxim Integrated, Inc., San Jose, CA, USA) to verify chamber settings. At 4, 8 and 16 weeks after inoculating microcosms, we sampled one leaf packet for evaluating leaf decomposition as biomass loss and another for evaluating *Phytophthora* colonization. For experiments with green and brown leaves, we included an additional sampling for *Phytophthora* colonization at two weeks. Therefore, in the first experiment each microcosm contained seven packets of each leaf type for a total of 14, and, as leaves were maintained in separate microcosms in the second experiment, each contained a total of seven packets containing either green or brown leaves. In the yellow leaf experiment, microcosms contained six packets each.

#### *2.3. Data Collection*

To determine the rate of decomposition measured as leaf biomass loss [53,54], we weighed leaves to the hundredth decimal of a gram with an analytical balance (model EP612C, Ohaus Corporation, Pine Brook, NJ, USA) prior to packing and we labeled the packets with aluminum tree tags secured with a plastic tie for future identification. We estimated the original dry mass of both leaf types from the average dry weight (determined after oven-drying at 55–60 ◦C for 48 h) of a subsample of 50 fresh or air dried leaves. The average percent dry weight (±SD) for green and brown leaves, respectively, was 40.9 (±2.3) and 94.3 (±0.3) for the first experiment and 53.5 (±0.4) and 92.6 (±0.3) for the second experiment. The average percent dry weight for yellow leaves was 55.0 (±0.3). The average estimated weight in grams (±SD) for five green leaves was 0.84 (±0.08) and 1.13 (±0.06), and that for five brown leaves, 0.91 (±0.08) and 0.85 (±0.06) for the first and second experiments, respectively. For five yellow leaves, the

estimated average weight in grams was 0.98 (±0.06). At each sampling, leaves were retrieved from tagged leaf packets, rinsed gently with deionized tap water to remove adhering debris, oven-dried in a paper envelope or an open aluminum foil envelope at 55–60 ◦C for 48 h, and weighed as described above. The fraction of original biomass was calculated for all leaves in a packet by dividing the weight at the time of sampling by the estimated original dry biomass.

To determine the level of *Phytophthora* colonization of leaves, at each sampling we collected a packet for each leaf type from each container to evaluate by culturing on *Phytophthora*-selective PARP-H medium (corn meal agar 1.7% w/v, pimaricin 5 ppm, ampicillin 250 ppm, rifampicin 10 ppm, PCNB (pentachloronitrobenzene) 50 ppm and hymexazol 25 ppm, [1]). Upon retrieval, leaves were submerged and gently rubbed free of biofilm in 1% household bleach solution (≈65 ppm hypochlorite), surface sterilized in fresh bleach solution for three to seven minutes, rinsed with deionized tap water, and then laid out on paper towels and the excess water allowed to evaporate. Finally, leaves were wrapped in a paper towel and stored at 4 ◦C until isolations by culturing could be performed. Isolations were attempted from all leaves belonging to treatment (a single packet) using a 'mosaic' sampling approach whereby the leaf discs are removed from the petiole, midrib and flanking lobes of the leaf at approximately 1 cm distance from one another in order to collect a representative sample from the entire leaf [45,55]. For experiments with green and brown leaves, isolations were initiated immediately after collection, with most samples (75%) processed within 29 days. All isolations were completed by 46 days after collection. Storage period did not alter results when included as a covariate in models for these experiments and was excluded from the final analyses. Isolations from leaves of the yellow leaf experiment were completed within nine days after collection, and all isolations from a single collection week were completed in one day. The presence of *P. ramorum* and *P*. *gonapodyides* was determined by microscopic examination of isolate morphologies directly from the isolation plates after four to five days and checked again periodically for three weeks [45].

To test for active sporulation from colonized leaves in the microcosms, periodically a California bay leaf disc (12 mm dia.) was floated as bait—either naked or in a roughly 35 mm2 mesh envelope—on the surface of the water in each microcosm for three to seven days, after which it was surface sterilized and isolations attempted from it on selective PARP-H medium. We conducted these tests of sporulation four times during the first and yellow leaf experiments, and three times during the second experiment. Additionally, we tested for sporulation periodically for up to eight weeks after all leaves had been removed from microcosms to determine if *Phytophthora* spores could persist in the absence of a substrate.

The first experiment was initiated on 29 December 2014, but we delayed the first collection at two weeks by two additional weeks because zoospores were not detected in the microcosms until two weeks after inoculation, most likely due to excessive aeration of the water during the first week. All subsequent collection dates were shifted forward by two weeks accordingly. Collections are reported according to the originally planned intervals of 2, 4, 8 and 16 weeks, with time zero being two weeks after inoculation. The final collection for the first experiment was on 1 May 2015 (126 days). Subsequent experiments proceeded as expected and the collection week reflects the period elapsed since introducing inoculum. The second experiment was initiated 28 August 2015 and concluded with the last sampling on 18 December 2015 (112 days). The yellow leaf experiment was initiated on 13 September 2015 and the final collection made on 7 January 2016 (116 days).

#### *2.4. Analysis*

Due to the differences in how each experiment was set up, we analyzed results separately for each.

#### 2.4.1. *Phytophthora* Colonization

To evaluate the colonization of leaves by each *Phytophthora* species in each treatment, we recorded the total number of pieces yielding *P. ramorum* or *P. gonapodyides* out of the total number of pieces sampled for each leaf. The average proportion of leaves colonized by either species was calculated for each packet from this ratio. This average leaf fraction colonized per packet was *logit* transformed to normalize variances, with a +0.005 correction applied to values of zero and −0.005 to values of one before transformation [56]. The transformed average proportions colonized were analyzed in linear mixed models (*lme* function) with the *nlme* package [57] in R statistical software, version 3.3.1 [58]. Replication block and microcosm were set as random variables with microcosm nested in a block. Because *Phytophthora* recovery followed a non-linear trend with respect to time, we treated the collection week as a categorical variable. As one *Phytophthora* species occurred almost exclusively in each treatment (see Results below)—*P. ramorum* and *P. gonapodyides* in the treatments where they were inoculated solely and *P. gonapodyides* in the combined inoculations—we simplified the analysis by comparing leaf colonization by the dominant species across treatments. That is, the response variable in the model was the average fraction of leaf discs colonized by *P. ramorum* in *P. ramorum*-only treatments, and by *P. gonapodyides* in *P. gonapodyides*-only and combined *Phytophthora* inoculum treatments. Therefore, the main independent variables for *Phytophthora* leaf colonization analyses were the inoculation treatment—with non-inoculated treatments excluded—and collection week. Leaf type (green or brown) and stream water type (autoclaved or not) were included as independent variables in the model for the experiments where the distinctions applied. The full set of interactions were included in the models for each experiment (see supplemental Tables S1−S4 and S6). We verified adherence to model assumptions by the Shapiro−Wilk and Levene's tests. We obtained *P*-values using the *anova* function in R with the sum of squares set to type III ("marginal"), and least square means comparisons with the *lsmeans* package [59]. Significance for means comparisons was determined with the default Tukey's HSD.

#### 2.4.2. Leaf Decomposition

For leaf decomposition, we estimated a decay constant (*k*) for each treatment combination in each block based on the fraction of estimated original leaf mass remaining at each collection interval [53,54]. For this, we used the exponential decay equation *M*t = *M*0 · *e*−*kt* where *t* is time as the number of incubation days, *M*t is the fraction of leaf mass remaining at each collection interval, and *M*0, fraction at time zero, is set to one [53,54]. Values for *k* were estimated using the *nls* function in R statistical program. The decay constants for each treatment combination were then analyzed in a mixed model using the *lme* function of the *nlme* package with inoculum, leaf and water type as independent variables and block as a random factor. For the yellow leaf experiment, only inoculum was used as an independent variable, and since replications were not blocked, an analysis of variance was performed using the *aov* function in R. For all experiments, we included treatments not inoculated with either *Phytophthora* species in the analysis to evaluate the effect of *Phytophthora* colonization on leaf decay. Two non-inoculated microcosms in the first experiment were contaminated with both *Phytophthora* species, and one non-inoculated microcosm in the second experiment became contaminated with *P. ramorum*, likely from a rare, undetected leaf infection. We excluded the results from these microcosms from the analysis.

#### **3. Results**

#### *3.1. Phytophthora Leaf Colonization*

When *P. ramorum* was inoculated alone, it rapidly colonized most of the green leaf area and persisted at this level throughout the 16 weeks of incubation (Figure 1). It did not effectively colonize brown, senesced leaves, though it could occasionally be recovered from a few pieces of some leaves. In contrast, *P. gonapodyides* colonized most of the area of both green and brown leaves in microcosms where it was inoculated (Figure 1). However, *P. gonapodyides* colonized brown leaves to a significantly lesser degree than green leaves when the leaves were exposed to inoculum in separate microcosms, while there was no difference between the colonization of green and brown leaves when they were maintained in the same microcosm (Figure 1). In combined inoculations of both *Phytophthora* species, *P. ramorum* was unexpectedly suppressed on both leaf types and the recovery of *P. gonapodyides* from

this treatment was identical to that of *P. gonapodyides*-only treatments (Figure 1). Reflecting these results, in both experiments with green and brown leaves, the interaction of inoculation and leaf type was highly significant (*p* < 0.0001, Tables S1 and S2).

**Figure 1.** Proportion of green, brown and yellow leaves (designated by element shape and line type) colonized by *P. ramorum* or *P. gonapodyides* (designated by element and line shade)—determined as the proportion of leaf pieces colonized out of the total number sampled in "mosaic" isolations—for three different inoculum treatments (horizontal panels) at sampling intervals over 16 weeks of incubation in three different experiments (vertical panels). Two experiments included green and brown leaves, the first with both leaf types in the same microcosm and the second with each leaf type in different microcosms. One experiment included yellow leaves only with only *P. ramorum* and combined *Phytophthora* species inoculation treatments. Non-inoculated treatments are not shown, and results are averaged over stream water treatments which did not have a significant effect, except for the experiment with yellow leaves which used only sterile nutrient solution. Bars represent ± standard error, *n* = 10.

Though the difference was not significant in the overall model, in *P. ramorum*-only inoculated treatments where brown leaves were maintained separately from green leaves, *P. ramorum* colonized brown leaves at consistently higher levels in autoclaved water treatments compared with treatments with non-sterilized stream water added (0.094 and 0.030 mean fraction of leaf discs colonized, respectively). This difference was less apparent with green leaves (Supplemental Figure S2). Nevertheless, there were no statistically significant differences between treatments based on stream water additions (Tables S1 and S2), and therefore, results are presented averaged over this factor.

In the experiment with yellow leaves, *P. ramorum*, when inoculated alone, colonized most of the leaf area and persisted at this level throughout the experiment, similar to the result with green leaves in other experiments (Figure 1). In combined *P. ramorum* and *P. gonapodyides* inoculations, *P. ramorum* was once again completely suppressed and *P. gonapodyides* colonized yellow leaves almost completely, at levels similar to its colonization of green leaves in both other experiments (Figure 1). The colonization of yellow leaves by *P. gonapodyides* in combined *Phytophthora* inoculum treatments was significantly higher than that by *P. ramorum* in *P. ramorum*-only inoculated treatments in this experiment, though both species colonized more than 70% of the leaf area. Thus, in the experiment with yellow leaves, only the effect of *Phytophthora* inoculation was significant (*p* = 0.0159, Table S3). In all experiments, both *Phytophthora* species colonized leaves rapidly, in most cases reaching maximum levels by four weeks, and persisted at these levels throughout the 16 weeks experimental duration. A slight increase in the level of colonization by both *Phytophthora* species was apparent in many cases from two to four weeks, though for brown leaves maintained in separate microcosms in the second experiment, levels appeared to actually decline after the second week. This contrast is reflected in the significant interaction of leaf type and collection week for this experiment (*p* = 0.0135, Table S2).

#### *3.2. Sporulation*

The isolation of *P. ramorum* or *P. gonapodyides* from California bay leaf disc baits deployed on the water surface in microcosms indicated the presence of zoospores. The sum of successful bait isolations for each treatment across the five replication blocks in the green and brown leaf experiments, and across four replications in the yellow leaf experiment, are presented in Tables 1–3. The recovery of *P. gonapodyides* from *P. gonapodyides*-only and combined *Phytophthora* inoculation treatments was from nearly 100% of baits throughout the duration of all experiments. The recovery of *P. ramorum* was more erratic, ranging from 40% to 90% of baits during the experiments with green and brown leaves. However, *P. ramorum* recovery from baits in microcosms that included green leaves and sterile rather than non-sterilized water was closer to 100%, excepting the second baiting of the second experiment, when *P. ramorum* was not recovered from most microcosms. *Phytophthora ramorum* was also rarely recovered by baiting from microcosms in the second experiment with only brown leaves, especially when excluding the first baiting, which was done a few days after inoculation. Consistent with this, brown leaves were colonized at very low levels by *P. ramorum*. Nevertheless, at 14 weeks, *P. ramorum* could still be recovered from several of these microcosms (Table 2). *Phytophthora ramorum* was recovered somewhat more frequently from sterile than non-sterile stream water treatment. Such an effect was not apparent for *P. gonapodyides*. Both *Phytophthora* species were recovered at nearly 100% from baits throughout the yellow leaf experiment which used sterile dilute nutrient solution only (Table 3).

Additionally, we baited microcosms for weeks after all leaf packets had been collected to see how long spores may persist in the absence of leaves. *Phytophthora ramorum* could be recovered from a few microcosms up to six weeks after all leaves were removed, but its frequency generally diminished rapidly. In contrast, *P. gonapodyides* could be recovered for up to 12 weeks after all leaves had been removed from microcosms, and was relatively frequent even six weeks after leaves were removed in the second experiment.

treatment (i.e., out of five possible colonization events per sampling, 35 total. Dash indicates not inoculated and not recovered) in the first experiment where greenand brown leaves were maintained together in microcosms with either sterile (st) or non-sterile (nst) stream water added. Grey shading indicates results from after

**Table 1.** Count of *P. ramorum* (*Pr*) and *P. gonapodyides* (*Pg*) recovery from single leaf disc baits deployed for three to seven days in a total of five microcosms

**Table 2.** Count of *P. ramorum* (*Pr*) and *P. gonapodyides* (*Pg*) recovery from single leaf disc baits deployed for three to seven days in a total of five microcosms per treatment (i.e., out of five possible colonization events per sampling, 35 total. Dash indicates not inoculated and not recovered) in the second experiment where green and brown leaves were maintained in separate microcosms with either sterile (st) or non-sterile (nst) stream water added.. Grey shading indicates results from after all leaves had been removed from the microcosms (112 days).

#### *Forests* **2019** , *10*, 434

 per

8

Inoculum

*Pr* *Pr* + *Pg*

41

*Pr*

 *Pg*

 *Pr*

 *Pg*

 *Pr* 3-4-4-4-

04040404

 *Pg*

 *Pr*

 *Pg*

4 0

4

0

4

0

4

 0

 28

1

0

 20

 -

 *Pr*

 *Pg*

 *Pr*

 *Pg*

 *Pr*

 *Pg*

 *Pr*

 *Pg*

 54

 12

 85

 14

 97

 15

 102

 17

 119

 20

 138

 25

 178

, *10*, 434

#### *3.3. Leaf Decomposition*

In the experiment with green and brown leaves maintained in the same microcosm, only green leaves in microcosms with no *Phytophthora* inoculum decomposed at a slower rate than all other treatments (Figure 2). In fact, on average, they did not lose significant biomass throughout the 16 weeks. Leaves in all other treatments, including brown leaves in non-inoculated microcosms, decomposed at similar rates (Figure 2). The interaction of leaf type and *Phytophthora* inoculation was, therefore, a highly significant predictor in the model (*p* < 0.0001, Table S4). Estimated decay constants are listed in Table S5.