Article Phytophthora Species from Xinjiang Wild Apple Forests in China Xiaoxue Xu 1, Wenxia Huai 1, Hamiti 2, Xuechao Zhang 3 and Wenxia Zhao 1,* 1 The Key Laboratory of National Forestry and Grassland Administration on Forest Protection, Research Institute of Forest Ecology Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China; [email protected] (X.X.); [email protected] (W.H.) 2 Forest Bureau of Emin County, Xinjiang Uygur Autonomous Region, Emin 834600, China; [email protected] 3 Institute of Agricultural Sciences of Ili Kazakh Autonomous Prefecture, Yining 835000, China; [email protected] * Correspondence: [email protected]; Tel.: +86-010-6288-8998 Received: 28 August 2019; Accepted: 17 October 2019; Published: 21 October 2019 Abstract: Phytophthora species are well-known destructive forest pathogens, especially in natural ecosystems. The wild apple (Malus sieversii (Ledeb.) Roem.) is the primary ancestor of M. domestica (Borkh.) and important germplasm resource for apple breeding and improvement. During the period from 2016 to 2018, a survey of Phytophthora diversity was performed at four wild apple forest plots (Xin Yuan (XY), Ba Lian (BL), Ku Erdening (KE), and Jin Qikesai (JQ)) on the northern slopes of Tianshan Mountain in Xinjiang, China. Phytophthora species were isolated from baiting leaves from stream, canopy drip, and soil samples and were identified based on morphological observations and the rDNA internal transcribed spacer (ITS) sequence analysis. This is the first comprehensive study from Xinjiang to examine the Phytophthora communities in wild apple forests The 621 resulting Phytophthora isolates were found to reside in 10 different Phytophthora species: eight known species (P. lacustris being the most frequent, followed by P. gonapodyides, P. plurivora, P. gregata, P. chlamydospora, P. inundata, P. virginiana, and P. cactorum) and two previously unrecognized species (P. sp. CYP74 and P. sp. forestsoil-like). The highest species richness of Phytophthora occurred at BL, followed by XY. P. lacustris was the dominant species at BL, XY, and JQ, while P. gonapodyides was the most common at KE. In the present paper, the possible reasons for their distribution, associated implications, and associated diseases are discussed. Keywords: Phytophthora; diversity; wild apple forest; decline 1. Introduction Xinjiang wild apple (Malus sieversii (Ledeb.) Roem.), the wild ancestor of the domesticated apple, is mainly distributed in the Tian Shan mountains in Central Asia, including the Ili River Valley in northwest China’s Xinjiang Uygur Autonomous Region and southeast and east Kyrgyzstan [1–3]. It is the dominant species in the relict wild fruit wood forests of inner Eurasia and is protected as a vulnerable species among the endangered rare germplasm resources of China [4–6]. However, the wild populations of M. sieversii have experienced a dramatic decrease in recent years, and symptoms of the decline can be observed in many wild apple forests. Affected trees show higher canopy loss, branch dieback, bark and cambium necrosis, and growth reduction. Similar to other countries in Central Asia, several abiotic and biotic factors negatively affect the health status of wild apple forests in China [7], including environmental and climate impacts, insect pests, cambium feeders such as Agrilus mali Matsumura, and infection by pathogenic fungi [8–13] (Figure1). Forests 2019, 10, 927; doi:10.3390/f10100927 www.mdpi.com/journal/forests Forests 2019, 10, 927 2 of 11 Forests 2019, 10, x FOR PEER REVIEW 2 of 11 FigureFigure 1. Declining 1. Declining symptoms symptoms of wild of apple wild appleforests forests in Xinjiang. in Xinjiang. TheThe presence presence of ofPhytophthoraPhytophthora speciesspecies in inforests forests and and natural natural ecosystems ecosystems is considered is considered to tobe beanan importantimportant biotic biotic factor factor responsible responsible for for the the decline, decline, dieback, dieback, and and mortality mortality of oftrees trees [14]. [14 Belonging]. Belonging to to thethe class class Oomycetes, Oomycetes, or or“water “water molds”, molds”, in inthe the kingdom kingdom Chromista, Chromista, these these fungus-like fungus-like organisms organisms can can causecause root root rot, rot, bark bark cankers, cankers, and and diseases diseases leading toto thethe declinedecline and and dieback dieback of of a widea wide range range of plantof plantspecies species worldwide worldwide [15]. [15]. Over Over the pastthe past two two decades, decades, numerous numerous surveys surveys have have shown shown that manythat many known knownand previouslyand previously unknown unknownPhytophthora Phytophthoraspecies species are present are inpresent a variety in a of variety natural of and natural semi-natural and semi-naturalforests and forests river systems and river in Europe, systems America, in Euro Australia,pe, America, South Australia, Africa, andSouth more Africa, recently, and Asia more [16 ]. recently,Some ofAsia these [16].Phytophthora Some of thesespecies, Phytophthora including species,P. cactorum including, P. cinnamomi P. cactorum, and,P. P. plurivora cinnamomi,, have and shown P. plurivorastrong, involvementhave shown strong in the declineinvolvement anddieback in the decline of forests, and whiledieback the of exact forests, role while in forest the exact ecosystems role in offorest many ecosystems other species, of suchmany as P.other cryptogea species,, P. chlamydospora such as P. ,cryptogea and P. gonapodyides, P. chlamydospora, is unclear, and [16]. P. In a gonapodyidesrecent study,, is P.unclear plurivora [16].was In founda recent to causestudy,damage P. plurivora to the was fine found roots to and cause stems damage of M.sieversii to the finein the rootsdeclining and stems wild of fruit M. forestssieversii of in Xinjiang the declining Province, wild China fruit [ 17forests]. However, of Xinjiang the findings Province, described China [17]. in that However,report were the basedfindings on adescribed limited number in that of samples.report were Furthermore, based on thea limited distribution number and ecologicalof samples. roles Furthermore,of P. plurivora theand distribution other Phytophthora and ecologicalspecies roles in wild of P. apple plurivora forests and are other still unknown. Phytophthora species in wild appleIn June forests to October are still 2016–2018,unknown. a survey of Phytophthora diversity was performed at four plots in wildIn appleJune to forests October on 2016–2018, the northern a survey slope of of Tianshan Phytophthora Mountain diversity in Xinjiang was performed using baiting at four assays plots fromin wildrhizosphere apple forests soil, streams,on the northern and canopy slope drip. of ThisTianshan study Mountain presents the in results Xinjiang of thisusing survey baiting related assays to the fromPhytophthora rhizospherespecies soil, associatedstreams, and with canopy the decline drip. or This dieback study of Maluspresents sieversii the .results of this survey related to the Phytophthora species associated with the decline or dieback of Malus sieversii. 2. Material and Methods 2. Material and Methods 2.1. Study Area 2.1. StudyThe Area study was carried out in wild apple forests on the northern slopes of Tianshan Mountain in Xinjiang,The study China. was Wecarried chose out Xin in Yuan wild (XY)apple (83 forests◦330 E, on 43.25 the0 northernN), Ba Lian slopes (BL) of (82 Tianshan◦500 E, 43 Mountain◦150 N), Ku in ErdeningXinjiang, China. (KE) (82 We◦51 chose0 E, 43 Xin◦13 0YuanN), and (XY) Jin (83°33 Qikesai′ E, (JQ)43.25 (83′ N),◦25 Ba0 E, Lian 43◦18 (BL)0 N) (82°50 as the′ E, studied 43°15′trap N), Ku plots, Erdeningas these (KE) locations (82°51 are′ E, where43°13′ theN), wildand Jin apple Qikesai trees (JQ) mostly (83°25 live′ E, [18 43°18] (Figure′ N) as2). the In studied these four trap plots, plots, the as declinethese locations of wild appleare where trees the in XYwild is apple the most trees serious. mostly A live total [18] of (Figure 10 stream 2). sitesIn these that four flow plots, though the the declinedeclining of wild wild apple apple trees trees in were XY setis the at eachmost of seriou the 4s. areas; A total 10 canopyof 10 stream dripsites sites under that flow the decliningthough the wild decliningapple trees wild were apple set trees at each were of theset 4at areas. each Fifteenof the 4 soil areas; sites 10 under canopy the decliningdrip sites trees under were the set declining at XY and wildat BL.apple In trees total, were 40 stream set at sites,each 40of forestthe 4 areas. sites, andFifteen 30 soilsoil samplingsites under sites the weredeclining set in trees these were 4 plots set to at investigateXY and at BL. the In presence total, 40 of streamPhytophthora sites, 40in forest the wild sites, apple and forests.30 soil sampling sites were set in these 4 plots to investigate the presence of Phytophthora in the wild apple forests. ForestsForests 20192019,, 1010,, x 927 FOR PEER REVIEW 3 3of of 11 11 Figure 2. The four plots located in Xinyuang, Gongliu county, China. 2.2. Sampling and PhytophthoraFigure 2. The Isolationfour plots located in Xinyuang, Gongliu county, China. This research used stream, canopy drip, and soil sampling baiting [19–22] at the 4 plots from 2016 2.2. Sampling and Phytophthora Isolation to 2018. All isolates of Phytophthora spp. were recovered from sites by baiting with leaves of wax (FraxinusThis research chinensis usedRoxb). stream, The baiting canopy leaves drip, wereand placedsoil