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.
Melampsora larici-populina is a macrocyclic rust, and the haploid stage with two nuclei and the diploid of mononuclear sequentially occur annually. During the preservation of dry urediniospores at −80°C, we found that one isolate, ΔTs06, was different from the usual wild-type isolate Ts06 at −20°C because it has mixed polykaryotic urediniospores. However, the other spores, including the 0, I, III, and IV stages of a life cycle, were the same as Ts06. After five generations of successive inoculation and harvest of urediniospores from the compatible host Populus purdomii, the isolate ΔTs06 steadily maintained more than 20% multiple nucleus spores. To test the pathogenesis variation of ΔTs06, an assay of host poplars was applied to evaluate the differences between ΔTs06 and Ts06. After ΔTs06 and Ts06 inoculation, leaves of P. purdomii were used to detect the expression of small secreted proteins (SSPs) and fungal biomasses using quantitative real-time PCR (qRT-PCR) and trypan blue staining. ΔTs06 displayed stronger expression of five SSPs and had a shorter latent period, a higher density of uredinia, and higher DNA mass. A transcriptomic comparison between ΔTs06 and Ts06 revealed that 3,224 were differentially expressed genes (DEGs), 55 of which were related to reactive oxygen species metabolism, the Mitogen-activated protein kinase (MAPK) signaling pathway, and the meiosis pathway. Ten genes in the mitotic and meiotic pathways and another two genes associated with the “response to DNA damage stimulus” all had an upward expression, which were detected by qRT-PCR in ΔTs06 during cryopreservation. Gas chromatography–mass spectrometry (GC-MS) confirmed that the amounts of hexadecanoic acid and octadecadienoic acid were much more in ΔTs06 than in Ts06. In addition, using spectrophotometry, hydrogen peroxide (H2O2) was also present in greater quantities in ΔTs06 compared with those found in Ts06. Increased fatty acids metabolism could prevent damage to urediniospores in super-low temperatures, but oxidant species that involved H2O2 may destroy tube proteins of mitosis and meiosis, which could cause abnormal nuclear division and lead to multinucleation, which has a different genotype. Therefore, the multinuclear isolate is different from the wild-type isolate in terms of phenotype and genotype; this multinucleation phenomenon in urediniospores improves the pathogenesis and environmental fitness of M. larici-populina.
Corydalis acuminata Franch., C. edulis Maxim. and C. racemosa (Thunb.) Pers. of family Papaveraceae are rich in multiple alkaloids and widely used as Chinese medicinal herbs, for treating cough, pruritus, sores tinea and snake venom (Zhang et al. 2008, Iranshahy et al. 2014). In April 2021, orange rust pustules were observed on C. acuminata, C. edulis and C. racemosa in Shaanxi Province (34°4’56’’ N, 108°2’9’’ E, alt. 770 m), China. Samples were collected and voucher specimens were preserved in the Herbarium Mycologicum Academiae Sinicae (nos. HMAS249947–HMAS249949), China. Consequent geospatial investigations revealed that diseased plants can be observed at an altitude of 400–1000 m, and show an incidence from 40% to 80% varied by altitude. Spermogonia epiphyllous, subcuticular, densely grouped, oval or round, 0.14–0.36 × 0.09–0.30 mm, pale orange-yellow, and type 3 of Cummins and Hiratsuka (1963). Aecia mostly hypophyllous, subepidermal without peridia, Caeoma-type, erumpent, densely grouped, oval or round, 0.27–0.85 × 0.15–0.43 mm, and orange-yellow; hyaline peridial cells produced in a periphery of the sorus under the ruptured epidermis of host plants. Aeciospores globoid or broadly ellipsoid, catenulate with intercalary cells, 15.7–20.1 × 10.8–15.7 μm, yellow to pale orange; walls hyaline, verrucose, 1.7–3.1 μm thick. This fungus was morphologically identified as Melampsora (Melampsoraceae). The rDNA-28S and the internal transcribed spacer (ITS) regions were amplified using primers NL1/NL4 and ITS1/ITS4 (Ji et al. 2020; Wang et al. 2020). Bi-directional sequences were assembled and deposited in GenBank (accession nos. MW990091–MW990093 and MW996576–MW996578). Phylogenetic trees were constructed with the ITS+rDNA-28S dataset based on maximum-likelihood (ML), maximum-parsimony (MP) and Bayesian Inference (BI). ML and MP bootstrap values were calculated by bootstrap analyses of 1,000 replicates using MEGA-X (Kumar et al. 2018), while BI posterior probabilities (Bpps) were calculated using MrBayes ver. 3.1.2 (Ji et al. 2020; Wang et al. 2020). Phylogenetic analyses grouped our specimens and Melampsora ferrinii Toome & Aime into one clade, highly supported by bootstrap values of ML, MP, and Bpps of 100%/100%/1. Inoculations were conducted with 1-year-old plants of original host, Salix babylonica L. (Toome & Aime 2015). Aeciospores suspension with a concentration of 106 spores/ml were sprayed on 20 healthy leaves, with another 20 healthy leaves sprayed with sterile water as the control. The inoculated plants were kept in darkness at 20–25 °C for 2 days and then transferred into greenhouse at 23°C with 16 h light per day. After 8–10 days of inoculation, yellow pustules of uredinia appeared on abaxial surfaces of the inoculated leaves, which were identical to Toome & Aime (2015) reported, while the control leaves remained healthy. Inoculations with the same method were conducted by spraying urediniospores, and the same rust symptoms developed after 8 days. Genus Corydalis was verified as the alternate host of M. chelidonii-pierotii Tak. Matsumoto, M. coleosporioides Dietel, M. idesiae Miyabe and M. yezoensis Miyabe & T. Matsumoto (Shinyama & Yamaoka 2012; Okane et al. 2014; Yamaoka & Okane 2019), and C. incisa (Thunb.) Pers. was speculated as the potential alternate host of M. ferrinii (Toome & Aime 2015). Based on morphology, phylogeny and pathogenicity, we firstly report M. ferrinii in mainland China and verify C. acuminata, C. edulis and C. racemosa instead of C. incisa as its alternate hosts.
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