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Article

The First Record of a North American Poplar Leaf Rust Fungus, Melampsora medusae, in China

by
Wei Zheng
1,
George Newcombe
2,
Die Hu
3,4,
Zhimin Cao
1,
Zhongdong Yu
1,* and
Zijia Peng
1
1
College of Forestry, Northwest A&F University, Yangling 712100, China
2
College of Natural Resources, University of Idaho, Moscow, ID 83844, USA
3
College Life of Science, Northwest A&F University, Yangling 712100, China
4
College of Agricultural Science, University of Sydney, Sydney, ID 2570, Australia
*
Author to whom correspondence should be addressed.
Forests 2019, 10(2), 182; https://doi.org/10.3390/f10020182
Submission received: 5 January 2019 / Revised: 15 February 2019 / Accepted: 19 February 2019 / Published: 20 February 2019
(This article belongs to the Section Forest Ecophysiology and Biology)
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Abstract

:
A wide range of species and hybrids of black and balsam poplars or cottonwoods (Populus L., sections Aigeiros and Tacamahaca) grow naturally, or have been introduced to grow in plantations in China. Many species of Melampsora can cause poplar leaf rust in China, and their distributions and host specificities are not entirely known. This study was prompted by the new susceptibility of a previously resistant cultivar, cv. ‘Zhonghua hongye’ of Populus deltoides (section Aigeiros), as well as by the need to know more about the broader context of poplar leaf rust in China. Rust surveys from 2015 through 2018 in Shaanxi, Sichuan, Gansu, Henan, Shanxi, Qinghai, Beijing, and Inner Mongolia revealed some samples with urediniospores with the echinulation pattern of M. medusae. The morphological characteristics of urediniospores and teliospores from poplar species of the region were further examined with light and scanning electron microscopy. Phylogenetic analysis based on sequences of the rDNA ITS region (ITS1, 5.8S rRNA gene, and ITS2) and the nuclear large subunit rDNA (D1/D2) was used to further confirm morphology-based identification. Based on combined analyses, five of the fifteen fully characterized samples were identified as Melampsora medusae: one from Shaanxi and four from Sichuan. Two of the five were from Populus deltoides cv. ‘Zhonghua hongye’. Three others were identified on Populus szechuanica, P. simonii, and P. yunnanensis. Additional samples of M. medusae were collected in Shaanxi in 2017 and 2018, and from Henan in 2015 through 2018. Altogether these findings show that this introduced pathogen is widespread and persistent from year to year in China. This is the first report of this North American poplar leaf rust species, Melampsora medusae, in China. It has previously been reported outside North America in Argentina, Europe, Australia, New Zealand, Japan, and Russia.

1. Introduction

Poplar leaf rust is caused by many species of Melampsora, each of which tends to be host-specific within the genus Populus. Leaf rust can be very damaging to black and balsam poplar species in sections Aigeiros and Tacamahaca, respectively [1,2,3,4]. Melampsora species have native ranges, but many have been inadvertently introduced to other parts of the world [5] since the genus was first described in 1843 [6]. Introductions accompanying the spread of hybrid poplar culture also have provided opportunities for previously isolated species of Melampsora to hybridize [7,8,9]; M. medusae has hybridized with both M. larici-populina [9] and with M. occidentalis [8]. Melampsora medusae is indigenous to Eastern North America in the native range of Populus deltoides [10], but it has also been introduced to Russia [11], Argentina [12], Australia [4], South Africa [4], France [13], India [7], Japan [14], Spain [15], and Portugal [16,17].
Like all rust fungi, Melampsora medusae is an obligate biotrophic plant parasite. It has a heteroecious life cycle in its native range with uredinia, telia, and basidia on P. deltoides, and spermogonia and aecia on Larix laricina. In the European Union, it is an introduced, quarantined fungus [18,19,20,21]. The telial hosts of M. medusae include species of section Aigeiros (e.g., P. deltoides, P. nigra, P. × euramericana) and of section Tacamahaca (e.g., P. maximowiczii, P. simonii, P. trichocarpa). The signs of M. medusae on poplars are typical of many leaf rusts: small, yellow uredinia appear within 10 days of infection on the abaxial surfaces of leaves, or on both sides of the leaves in the case of heavy infections [7,22,23,24].
As recently as five years ago, P. deltoides was largely rust-free in China due to its resistance to the native M. larici-populina. Now, however, P. deltoides and its hybrids experience severe rust from early July until November in Southern China. In Northern China, the new rust season for P. deltoides develops somewhat later. In the mountains of Southern China, some species of section Tacamahaca even become rusted in early July. Unfortunately, the formerly promising cultivar of P. deltoides, ‘Zhonghua hongye’ (Figure 1), is also now rust-susceptible. These recent changes prompted the rust surveys of this study to determine their cause. Between 2015 and 2018, surveys were conducted in Shaanxi, Sichuan, Gansu, Henan, Shanxi, Qinghai, Beijing, and Inner Mongolia. We collected poplar leaf rust specimens and fully characterized 15 collections in this study. By full characterization we mean both morphology- and sequence-based identification [2,25,26]. In addition to the 15, some other samples were identified on the basis of morphology alone.

2. Materials and Methods

2.1. Surveys and Specimens

Poplar leaf rust samples were collected during surveys from 2015 through 2018 in Shaanxi, Sichuan, Gansu, Shanxi, Qinghai, Beijing and Inner Mongolia in China. Specimens were dried in plant presses and deposited in State Key Laboratory of Mycology Institute of Microbiology Chinese Academy of Sciences. Sequences were deposited in GenBank with accession numbers as in Table 1.

2.2. Morphological Observations

Morphological characteristics of uredinia and telia were examined under the light microscope (LM) and the scanning electron microscope (SEM). Fifty spores of each sample were randomly selected and examined with a Leica DM4000B. Length, width, and wall thickness both apically and laterally were measured. The measurements obtained from samples were compared with published descriptions of taxa of Melampsora [2,7,8,19]. The surface echinulation of urediniospores was observed with a SEM. For the SEM, samples were coated with platinum-palladium prior to observations with an S-4800 scanning electron microscope (Hitachi, Tokyo, Japan) operated at 10kV, as in Zhao et al [27].

2.3. Germination of Urediospores on the Surface of 2% Agar

Fresh urediniospores were collected to test germination and to observe nuclei. Dry fresh urediniospores were sprayed on the surface of 2% agar water and sealed in the dish with a little water to maintain 100% relative humidity. After 6 h, 10 h, and 16 h incubation at 25 °C in the dark, urediniospores and germination tubes were dyed by DAPI (4′,6-diamidino-2-phenylindole∙2HCl, Sigma Chemical Company, St. Louis, MO, USA) ethanol solution and observed with a Leica DM4000B [28,29].

2.4. Phylogenetic Analysis

DNA extraction of urediniospores followed Virtudazo et al [30]. Polymerase chain reaction (PCR) was performed with the universal primers ITS1F and ITS4 [31,32], LSU primer [33], under the reaction system of 30 μL volume, containing 0.2 μM primer, 1 unit of TaKaRa Taq DNA polymerase, a 2.5 mM commercial deoxynucleoside triphosphate (dNTP) mixture, and 2 mM Mg2+ Taq reaction buffer. PCR reactions were carried out in a GeneAmp PCR TC-96 (Bioer Technology Co. Ltd., Hangzhou, China) under the following conditions: 95 °C for denaturing for 3 min, then 35 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min, and a final step of 72 °C for 10 min. Products were checked in 1% agarose gel electrophoresis under the UV transilluminator. PCR products were purified and sequenced by Sangon Biotech (Shanghai, China). Sequences were aligned in BioEdit version 7.0.9 and searched in GenBank [34]. All sequences were compared and submitted to NCBI to obtain accession numbers. Other homologous sequences from the GenBank database were downloaded for species comparisons. Multiple alignments were performed using ClustalX version 1.8 [35]. Phylogenetic trees were constructed using the software PAUP* v4.0b10 with the Maximum likelihood (ML) method [36]. Clade support was analyzed with 1000 bootstrap replicates.

2.5. Confirmation of M. medusae

As there still is confusion surrounding the identity of the species of Melampsora on P. tremuloides in North America [37], to confirm M. medusae two sets of specific primers were employed [37]. DNA extraction and PCR amplification were carried out as Section 2.4; annealing temperatures of PCR are shown in Table 2.

3. Results

3.1. Surveyed Poplar Leaf Rust in China, 2015–2018

Five of the fifteen fully characterized samples were identified as Melampsora medusae (Table 1): Two of the five were from Populus deltoides (section Aigeiros) cv. ‘Zhonghua hongye’. Three others were identified on three species of section Tacamahaca: Populus szechuanica, P. simonii, and P. yunnanensis. Additional samples of M. medusae were collected in Shaanxi in 2017 and 2018, and from Henan and Sichuan in 2015 through 2018. The only other rust species recorded on sections Aigeiros and Tacamahaca was M. laricic-populina. The sole representative of section Leucoides in the survey was P. wilsonii, and it revealed M. abietis-populi. Samples of section Populus (i.e., P. alba and P. tomentosa) yielded M. magnusiana. The rust of the sole representative of section Turanga, P. euphratica, from Inner Mongolia, was M. pruinosae. Thus, in all, there were five species of Melampsora.
The uredinia of M. medusae were mainly hypophyllous; a few uredinia were epiphyllous with small or more scattered pustules. Uredinia were roundish and golden orange to orange in color. Abundant capitate paraphyses were intermixed with urediniospores in the uredinia (Figure 2a). Paraphyses were 25.21–43.76 μm in length with stalks of 3.95–5.53 μm in width and swollen apices which were roughly spherical and 12.43–17.95 μm in diameter. Echinulate urediniospores were obovate or oval, with rounded apices; they were typically flattened laterally (Figure 2b,c), 20.64–31.45 × 14.39–20.38 μm, with golden yellow cytoplasm. Urediniospore walls were colorless, and germ pores were indistinct. The wall of the equatorial area of the urediniospores was slightly thickened at 1.76–3.62 μm thickness. Urediniospores were echinulate, except for the smooth equatorial area characteristic of M. medusae that commonly extended from one half to three-quarters of the way around the spore (Figure 2b,c). Spines were smaller near the smooth patch (Figure 2d). Telia were mainly hypophyllous. Their initial color was pale amber brown, but that eventually became deep reddish brown or almost black. Telia were raised slightly above the leaf surface, roughly circular to irregular in outline. Teliospores were roughly cylindrical to angular in shape in the cross-section, and the walls were pale reddish-brown. They were 31.69–44.12 (38.30) μm in length × 10.12–14.50 (11.65) μm in width. The morphological characterizations of all five species of Melampsora found in the surveys are summarized in Table 3.

3.2. Germination of Urediniospores on 2% Agar

Urediniospores of M. medusae germinated after 6 h on 2% agar. As Spiers (1994) described [9], the nuclei moved into germ tubes from the spores. After 10 h, there were typically two nuclei in the germ tubes, and the tops of the germ tubes were swollen; after 16 h, some germ tubes fused together. Usually, there were more than three nuclei in the fusion cell. Whereas urediniospores of M. larici-populina contained 4 germ pores and mostly had more than 2 germ tubes developed (Figure 3d,e), tubes were rope-like without swollen appressorium (Figure 3e), branched randomly, and typically found with 2 nuclei (Figure 3f).

3.3. Molecular Phylogenetic Analysis

Phylogenetic analysis of rDNA ITS regions is shown in Figure 4. The Chinese samples belonged to five clades when M. lini (GenBank accessions: L20283 [38]) and M. helioscopiae (GenBank accessions: AF426197 [39]) were used as outgroups (Figure 4). The analysis revealed that five samples (HMAS-247973, HMAS-247969, HMAS-247970, HMAS-247971 and HMAS-247972) belonged to the clade anchored by M. medusae given the following GenBank accessions: AY375273 (representative of M. medusae in France), AF087711 (M. medusae in U.S.A. [8]), GQ479310 (also a French sample of M. medusae [7]). Five specimens (HMAS-247976, HMAS-247977, HMAS-247975, HMAS-247968, HMAS-247974) belonged to the clade with M. larici-populina given the following GenBank accessions: AY429656 (M. larici-populina in Canada [40]), AB116828 (M. larici-populina in China [2]). Three specimens (HMAS-247979, HMAS-247980, HMAS-247981) were grouped in a clade with M. magnusiana given the following GenBank accessions: GQ479849 (representative of M. magnusiana in Germany [7]), KY364898 (M. magnusiana in Italy [41]). M. pruinosae and M. allii-populina were divided into two separate clades anchored by GenBank accessions AB116858 (M. pruinosae in China [2]) and AB116869 (M. allii-populina in China [2]), respectively (Figure 4), M. pruinosae is closely related to M. magnusiana. nrDNA-ITS phylogeny clearly distinguished M. medusae from the other four Melampsora species of poplars that were found in these surveys in China.
Phylogenetic analysis of D1/D2 regions showed almost the same results as nrDNA-ITS, in that the 15 specimens were distributed in five clades when M. epiphylla (GenBank accessions: AY652947 [26]) and M. ribesii-purpureae (GenBank accessions: AY444770 [26]) were used as outgroups (Figure 5).

3.4. Formae Speciales Identification

The two pairs of primer both showed a single-specified PCR DNA band for the putative five M. medusae specimens, with clc3a2f/clc3a2r primer 502 bps and clc3a3f/clc3a3r primer 594 bps, respectively, and the positive of M. larici-populina and negative controls of ddH2O without products (Figure 6), which implied the 5 specimens were M. medusae f.sp. deltoidae.

4. Discussion

Two of the five fully characterized samples of M. medusae were collected from Populus deltoides cv. ‘Zhonghua hongye’, a bud mutation propagated from Populus deltoides in China. ‘Zhonghua hongye’ has been regarded as fast-growing, rust-resistant and ornamental, so it has been widely planted in inland China since 2000. We have sampled the rust disease on ‘Zhonghua hongye’ from 2017–2018 in Shaanxi, and from 2015 through 2018 in Hennan, and 2016–2018 in Sichuan. Its new susceptibility to introduced M. medusae may change its trajectory of expansion and commercialization. The susceptibility to M. medusae of species in section Tacamahaca (i.e., P. yunnanensis, P. simonii and P. szechuanica) is not surprising, as North American species in this section of the genus are also susceptible to varying extents [8]. The susceptibility of these same Asian species in Tacamahaca has also been reported before from New Zealand [42]. The host specificity of M. medusae as reported in its original description [10] was limited to P. deltoides; it did not include P. tremuloides nor any other species of section Populus. Thus, the confirmation here of the absence of M. medusae on surveyed P. alba and P. tomentosa in China is not surprising.
The aecial host of M. medusae in its native range in Eastern North America is Larix laricina, but Pseudotsuga menziesii can also be a host [43]. In China, the aecial host is not yet known. The most likely hosts in China would be species of Larix, Pseudotsuga and possibly Cathaya. These three genera belong to subfamily Laricoideae of Pinaceae, although Cathaya may be closer to Pinus than to Larix/Pseudotsuga [44]. No rust fungi have ever been reported on Cathaya [11], although this absence is likely a function of a lack of attention. For example, only recently was the first pathogen of Cathaya described as a new species [45].
The involvement of M. medusae in inter-specific hybridization complicates its introduction into China. In our survey, we frequently found Melampsora larici-populina in close proximity to M. medusae. We have not yet surveyed for the hybrid M. medusae-populina, but its existence in China is now a distinct possibility that should be researched.

5. Conclusions

Our combined morphology- and sequence-based approach led to the identification of five species of Melampsora in surveys in China. The most important discovery was that of the introduction of North American M. medusae that is now both widespread and persistent from year to year in China.

Author Contributions

Materials collection: Y.Z., P.Z. and C.Z.; experiments: Y.Z. and Z.W.; investigation: H.D. and P.Z.; writing-original draft preparation: Z.W. and Y.Z.; writing-review and editing: G.N.

Funding

This research was funded by “the national key research projects, grant number 2017YFD0600103-4-2” and National natural science committee, grant number “31670650”.

Acknowledgments

We thank Liu Xiaoyong from the Institute of Microbiology, Chinese Academy of Sciences for help with morphological identification and voucher specimen reserving. We thank Zhang Chunni for help collecting in Yulin City.

Conflicts of Interest

The authors declare no conflict of interest.

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Forests 10 00182 g001 550
Figure 1. Sites (red dots) for rust sampling of P. deltoides cv. ‘Zhonghua hongye’. Dark dots represent the potential distribution of this formerly rust-free cultivar.
Figure 1. Sites (red dots) for rust sampling of P. deltoides cv. ‘Zhonghua hongye’. Dark dots represent the potential distribution of this formerly rust-free cultivar.
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Forests 10 00182 g002 550
Figure 2. Echinulation patterns of urediniospores and associated paraphyses of Melampsora medusae and Melampsora larici-populina (the two species of Melampsora found on sections Tacamahaca and Aigeiros in our surveys in China) as observed in the SEM. (a–d) Melampsora medusae; (e–h) Melampsora larici-populina; (a, e) abundant paraphyses under SEM; (b, f) Uredinium under SEM; (c, j) urediniospores with echinulate spines under SEM; (d, h) spines on the surface of urediniospores under SEM.
Figure 2. Echinulation patterns of urediniospores and associated paraphyses of Melampsora medusae and Melampsora larici-populina (the two species of Melampsora found on sections Tacamahaca and Aigeiros in our surveys in China) as observed in the SEM. (a–d) Melampsora medusae; (e–h) Melampsora larici-populina; (a, e) abundant paraphyses under SEM; (b, f) Uredinium under SEM; (c, j) urediniospores with echinulate spines under SEM; (d, h) spines on the surface of urediniospores under SEM.
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Figure 3. Germination of urediniospores of M. medusae and M. larici-populina on a medium of 2% agar. (a–c) M. medusae; (d–f) M. larici-populina; (a) the flowing nuclei (arrows) after 6 h; (b) the swollen top of the germ tube (arrows) after 10 h; (c) germ nucleates (arrows) with 4 nucleates after 16 h; (d) the flowing nuclei of MLP (arrows) after 8 h; (e) the germ tube with 2 nuclei (arrows) after 10 h; (f) the germ tube with 2 nuclei (arrows) after 16 h.
Figure 3. Germination of urediniospores of M. medusae and M. larici-populina on a medium of 2% agar. (a–c) M. medusae; (d–f) M. larici-populina; (a) the flowing nuclei (arrows) after 6 h; (b) the swollen top of the germ tube (arrows) after 10 h; (c) germ nucleates (arrows) with 4 nucleates after 16 h; (d) the flowing nuclei of MLP (arrows) after 8 h; (e) the germ tube with 2 nuclei (arrows) after 10 h; (f) the germ tube with 2 nuclei (arrows) after 16 h.
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Figure 4. Maximum Likelihood (ML) phylogenetic tree of nrDNA-ITS sequences of species of Melampsora from HMAS collections (red triangles) made during poplar leaf rust surveys in China, 2015–2018. Bootstrap values >50% are shown.
Figure 4. Maximum Likelihood (ML) phylogenetic tree of nrDNA-ITS sequences of species of Melampsora from HMAS collections (red triangles) made during poplar leaf rust surveys in China, 2015–2018. Bootstrap values >50% are shown.
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Figure 5. Maximum likelihood (ML) phylogenic tree of 28S rDNA(D1/D2) regions. Note: Bootstrap values >50% are shown and red marks are specimens we collected.
Figure 5. Maximum likelihood (ML) phylogenic tree of 28S rDNA(D1/D2) regions. Note: Bootstrap values >50% are shown and red marks are specimens we collected.
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Figure 6. Amplification with primer clc3a2f/clc3a2r and primer clc3a3f/clc3a3r. Lanes 1 and 10: Marker DM2000; lanes 2–6: Amplification from uredinia of the five specimens of M. medusae (HMAS 247969-71, HMAS 247972-73) using primers clc3a2f/clc3a2r; Lanes 11–15: Amplification from uredinia of the five specimens of M. medusae (HMAS 247969-71, HMAS 247972-73) using primers clc3a3f/clc3a3r; Lanes 7–8 and 16–17(HMAS247968, HMAS247978): The positive control with DNA of M. larici-populina; Lanes 9 and 18: The negative control with ddH2O.
Figure 6. Amplification with primer clc3a2f/clc3a2r and primer clc3a3f/clc3a3r. Lanes 1 and 10: Marker DM2000; lanes 2–6: Amplification from uredinia of the five specimens of M. medusae (HMAS 247969-71, HMAS 247972-73) using primers clc3a2f/clc3a2r; Lanes 11–15: Amplification from uredinia of the five specimens of M. medusae (HMAS 247969-71, HMAS 247972-73) using primers clc3a3f/clc3a3r; Lanes 7–8 and 16–17(HMAS247968, HMAS247978): The positive control with DNA of M. larici-populina; Lanes 9 and 18: The negative control with ddH2O.
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Table
Table 1. Fifteen fully characterized samples of Melampsora in China identified as species, with host plants, sampling locations, years, and voucher and GenBank accession numbers. The two samples of rust on P. deltoides are both from cv. ‘Zhonghua hongye’.
Table 1. Fifteen fully characterized samples of Melampsora in China identified as species, with host plants, sampling locations, years, and voucher and GenBank accession numbers. The two samples of rust on P. deltoides are both from cv. ‘Zhonghua hongye’.
Host PlantsLocality a/YearVoucher Specimen no. bGenBank Accession Accession no.Species
ITSD1/D2
P. szechuanicaLuding Sichuan/2016HMAS247968MK028576MK064523M. larici-populina
P. yunnanensisJinyang Sichuan/2016HMAS 247969MK028588MK064536M. medusae
P. simoniiXichang Sichuan/2017HMAS 247970MK028589MK064537M. medusae
P. szechuanicaKangding Sichuan/2018HMAS 247971MK028590MK064535M. medusae
P. cathayanaWutaishan Shanxi/2018HMAS 247974MK028577MK064526M. larici-populina
P. cathayanaLiupanshan Ningxia/2017HMAS 247975MK028578MK064527M. larici-populina
P. wilsoniiQinling Shaanxi/2018HMAS 247978MK028579MK064529M. abietis-populi
P. simoniiHuangyuan Qinghai/2018HMAS 247976MK028584MK064525M. larici-populina
P. simoniiHaixi Qinghai/2018HMAS 247977MK028583MK064524M. larici-populina
P. alba var. pyramidalisYanqing Beijing/2018HMAS 247979MK028581MK064531M. magnusiana
P. alba var. pyramidalisYulin Shaanxi/2018HMAS 247980MK028580MK064532M. magnusiana
P. tomentosaTianshui Gansu/2018HMAS 247981MK028582MK064530M. magnusiana
P. euphraticaInner Mongolia/2018HMAS 247982MK028585MK064533M. pruinosae
P. deltoidesLinyou Shaanxi/2017HMAS 247973MK028586MK064528M. medusae
P. deltoidesYihai Sichuan/2015HMAS 247972MK028587MK064534M. medusae
a: Locality with name of provinces and prefectures; b: HMAS: State Key Laboratory of Mycology Institute of Microbiology Chinese Academy of Sciences.
Table
Table 2. Primer sequences for M. medusae.
Table 2. Primer sequences for M. medusae.
Primer NamePrimer SequenceAnnealing Temperature (℃)Size of Amplified Product (bp)Sampling Locus/Year
clc3a2f5′-GGGGGTCTTTAGGACAAA-3′54502Shaanxi/2017,2018
Sichuan/2016
Henan/2015,2018
Qinhai/2018
Ningxia/2017
clc3a2r5′-TTCGAGCCAGCATGA AACAC-3′
clc3a3f5′-TTCGAGCCAGAAGTTTGTTTC-3′52594
clc3a3r5′-TTCGAGCCAGGATCACTT-3′
Table
Table 3. Morphology of the five Melampsora species found on Populus in China during 2015–2018 surveys.
Table 3. Morphology of the five Melampsora species found on Populus in China during 2015–2018 surveys.
SpeciesUrediniospores
ShapeSize
(average) (μm)		Wall Equatorial Part Thickness (average) (μm)Bald Spot
M. larici-populinaEllipsoid or Oblong35.13–48.11 × 20.02–25.31
(40.93) × (22.70)		3.40–11.63
(5.60)		Apical
M. medusaeObovate or Oval20.64–31.45 × 14.39–20.38
(26.56) × (18.66)		1.76–3.62
(3.14)		Equatorial
M. magnusianaGlobose or Ovate17.02–29.34 × 14.16–24.22
(23.93) × (18.70)		1.20–4.65
(2.48)		Absent
M. pruinosaeGlobose or Ellipsoid23.18–31.11 × 23.59–28.53
(27.88) × (25.87)		0.84–1.57
(1.26)		Absent
M. abietis-populiGlobose or Ovate20.77–26.34 × 15.14–21.52
(22.92) × (17.93)		1.14–1.83
(1.59)		Absent

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MDPI and ACS Style

Zheng, W.; Newcombe, G.; Hu, D.; Cao, Z.; Yu, Z.; Peng, Z. The First Record of a North American Poplar Leaf Rust Fungus, Melampsora medusae, in China. Forests 2019, 10, 182. https://doi.org/10.3390/f10020182

AMA Style

Zheng W, Newcombe G, Hu D, Cao Z, Yu Z, Peng Z. The First Record of a North American Poplar Leaf Rust Fungus, Melampsora medusae, in China. Forests. 2019; 10(2):182. https://doi.org/10.3390/f10020182

Chicago/Turabian Style

Zheng, Wei, George Newcombe, Die Hu, Zhimin Cao, Zhongdong Yu, and Zijia Peng. 2019. "The First Record of a North American Poplar Leaf Rust Fungus, Melampsora medusae, in China" Forests 10, no. 2: 182. https://doi.org/10.3390/f10020182

APA Style

Zheng, W., Newcombe, G., Hu, D., Cao, Z., Yu, Z., & Peng, Z. (2019). The First Record of a North American Poplar Leaf Rust Fungus, Melampsora medusae, in China. Forests, 10(2), 182. https://doi.org/10.3390/f10020182

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