Short Communication
DNA barcode sequencing discovered the aecial host of Puccinia chunjiei
2022 Volume 64 Issue 1 Pages 35-39
Details
2022 Volume 64 Issue 1 Pages 35-39
The telial stage of Puccinia chunjiei was described in 2012 from a single specimen collected in China (DAOM 240982). This species is the closest relative of P. graminis but differs in telial morphology on their grass hosts and in DNA sequences. As part of a DNA-barcoding project for rust herbarium specimens, collections of the aecial stage of P. graminis on Berberis were processed. For one specimen, BPI 1103856, the ITS sequence matched that of P. chunjiei and the aecial morphology differed from P. graminis. An expanded description of P. chunjiei is presented with photographs of the aecial stages of both species.
A long term study was initiated in 2006 to generate reference DNA sequences from rust herbarium specimens accessioned in the Canadian National Mycological Herbarium in Ottawa (DAOM) or in other international herbaria. Puccinia chunjiei M. Liu, C.J. Li & Hambl. [as ‘chunjii’] was recognized during our sampling of species causing diseases of cereal crops and other grasses. It was described in 2012 based on a single collection from Gansu Province in China (DAOM 240982), from a grass host in the genus Elymus (Liu & Hambleton, 2012). That specimen was collected in 1996 by Chun-Jie Li and generously contributed to ML for study. Our DNA sequences for the nuclear rDNA internal transcribed spacer (ITS, NR_111548 / HQ012446) and mitochondrial cytochrome oxidase I (COI, HQ012473) loci demonstrated that P. chunjiei was closely related to stem rust, Puccinia graminis Pers., but represented a distinct lineage. Teliospore characteristics were identified that differentiate P. chunjiei from P. graminis and other morphologically similar species. No other spore states were observed.
The rusts found on Berberis L. have also been a major focus of our DNA barcoding efforts because of the role of this plant genus in the lifecycle of P. graminis on wheat and also P. striiformis Westend. A specimen identified as P. graminis, collected in the Ningxia Hui Autonomous Region of China in 1961 on an unidentified Berberis species, and accessioned in the U.S. National Fungus Collection (BPI 1103856), was sampled for study in 2007 but sequencing failed. Over time, our protocols for generating ITS barcode sequences from herbarium specimens improved resulting in an increase in sequencing success rates. Recently DNA sequencing was attempted again for BPI 1103856 and the sequence obtained was an exact match to our data for P. chunjiei. We propose a revised description of the species, incorporating features and illustrations of the aecial stage of its lifecycle, and adding a longer sequence for the telial specimen and host sequences. For comparison, we also sequenced and examined the aecial stage of several herbarium specimens of P. graminis, selected to represent the three ITS subclades sensu Liu et al. (2015) and all collected on Berberis vulgaris L.
Two separate extractions, using small pieces (20 mm × 3 mm or 8 mm × 5 mm) of heavily infected leaf covered with aecia from herbarium specimen BPI 1103856, were processed for DNA extraction using a Macherey-Nagel NucleoMag® 96 Trace kit (Macherey Nagel GmbH & Co. KG, Düren, Germany) and a KingFisher ML magnetic particle processor (Thermo Fisher Scientific Oy, Yantaa, Finland). Both extractions resulted in the same ITS sequence. Portions of other leaves were used to examine aecial morphology and obtain measurements for BPI 1103856 and six P. graminis specimens (Table 1). Images were captured using a Zeiss Discovery V12 stereomicroscope and AxioCam HRc camera or a Zeiss Imager M2 and Axiocam 503 color camera, and analysed by ZEN (blue edition) 2.6 (Carl Zeiss Canada Limited, Ontario, Canada). Cross sections were cut with a Riechert-Jung Cryocut E (Leica Microsystems, Vienna, Austria) or a Starlet Portable Cryostat 11/120VAC 60HZ (Rose Scientific, Alberta, Canada) and mounted on microscope slides in a drop of water. Small pieces of the specimen were temporarily softened in a moist chamber, then clumps of spores were removed with tweezers and mounted in lactic acid. A minimum of 25 spores were measured for length and width (Table 1). Colours were recorded according to Kornerup and Wanscher (1978). For scanning electron microscopy (SEM), clumps of spores excised from aecia were mounted on carbon double sticky tape on aluminum stubs and coated with a 6.5 nm layer of platinum in an Emitech K550V sputter coater (EM Technologies Ltd., Ashford, Kent, England). The samples were imaged on a Hitachi SU7000 FESEM (Hitachi, Tokyo, Japan).
| Speciesa | Voucher No.b | Provenance | Year Collected | Aeciospore Width-Length (µm)c | GenBank Accession No. | |
| ITS +/- 28S (rust) | rbcL (Berberis host) | |||||
| Puccinia chunjiei Puccinia graminis |
BPI 1103856 | China, Ningxia Hui Aut. Reg. | 1961 | 17.5-20 (n= 75) | ON505610 | ON540786 |
| Clade 1 | PUR F17143 | Turkey, Gumusane | 1960 | 20-22 (n= 35) | ON505611 | - |
| Clade 2 | DAOM 225323 | Canada, Quebec | 1997 | 17-18 (n= 50) | HQ012438 | ON540782 |
| (S)reg.nr. F40524 | Sweden, Uppland | 2004 | 17-19 (n= 40) | MT965583 | ON540784 | |
| (S)reg.nr. F35775 | Sweden, Narke | 1997 | 18-20.5 (n= 50) | ON505609 | - | |
| Clade 3 | DAOM 150117 | Canada, Ontario | 1975 | 17-18 (n= 40) | ON505608 | - |
| (S)reg.nr. F29311 | Sweden, Gastrikland | 2003 | 18-20 (n= 25) | MT965582 | ON540783 | |
a Sub-specific clades of P. graminis sensu Liu et al. 2015 (Table 1, Fig. 1).
b Herbaria: BPI: US National Fungus Collections, BELTSVILLE, Maryland, USA; DAOM: Canadian National Mycological Herbarium, Agriculture and AgriFood Canada, OTTAWA, Ontario, Canada; PUR: Arthur Herbarium, Purdue University, WEST LAFAYETTE, Indiana, USA; S: Swedish Museum of Natural History, STOCKHOLM, Sweden.
c n = number of spores measured
Methods for polymerase chain reaction (PCR) amplification and sequencing of the ITS2 plus partial 28S region (when possible) for the rusts, and a portion of the ribulose‐1,5‐bisphosphate carboxylase/oxygenase large subunit (rbcL) gene for selected hosts, were as described previously (Léveillé-Bourret, Eggertson, Hambleton, & Starr, 2021). Additional sequences for the telial specimen, DAOM 240982, were also generated using the same DNA extract as in the original publication (Liu & Hambleton, 2012) to add partial 28S (the GenBank record was updated) and host rbcL data. The sequence for the host psbA-trnH intergenic spacer discussed in Liu and Hambleton (2012) has since been deposited in GenBank. Sequences were edited using Geneious Prime 2021.2 (Biomatters, Auckland, New Zealand). Collection information, sequence accession numbers and aeciospore sizes are summarized in Table 1 for the specimens examined.
Puccinia chunjiei M. Liu, C.J. Li & Hambl. [as ‘chunjii’], Mycologia 104(5): 1060 (2012). MycoBank no.: MB 565191.
Holotypus: CHINA, PR, Gansu Province, farm field, Xiahe county, on Elymus sp., 20 Sep 1996, Chun-Jie Li (DAOM 240982). Gene sequences: 1) rust, NR_111548 (ITS, 554 bp), HQ012446 (ITS-28S, updated to 839 bp), HQ012473 (COI, 335 bp); 2) host, ON540785 (rbcL, 553 bp), ON540787 (psbA-trnH intergenic spacer, 227 bp). Fig. 1A.
For the detailed description of telia and teliospores, see Liu and Hambleton (2012). Uredinia were not seen.
Spermogonia not seen. Aecia (Fig. 1B-E) light yellow to pale orange (3A5-5A3), cup-shaped, 230-280 µm diam at the opening, hypophyllous, in dense clusters covering the leaf surface; peridial cells (Fig. 1F, G) hyaline, verrucose, elongated polygonal, 15-28 × 12-18 µm; aeciospores hyaline, with slightly yellowish plasma, ovoid to globose, slightly irregularly angulate when free (Fig. 1H), polygonal in aecia arranged more or less hive-like (Fig. 1I), 16-23 × 14-20 (20±1.0 × 17.5±1.0 µm), wall finely verrucose to verrucose in a bizonate pattern, with small and large plugs present in the coarser area (verrucose, Fig. 3A, B), thickened apically (2.5-)4-9(-11) μm. On Berberis sp.
Aecial specimen examined: CHINA, Ningxia Hui Aut. Reg., Helanshan, on Berberis sp., 23 Jun 1961, Shu-jin Han (2205) (BPI 1103856). Gene sequences: 1) rust, ON505610 (ITS2-28S, 552 bp); 2) host, ON540786 (rbcL, 553 bp). Original identification: P. graminis (Detr: Jian-yun Zhuang). Fig. 1B.
Other herbaria: Herbarium Mycologicum Instituti Microbiologici Academiae Sinicae 55157 (not seen).
Notes: Teliospores of P. chunjiei differ from P. graminis mainly in having 1-2 apical projections, longer and more slender pedicels and narrower hila. The two species also differ in aecial morphology. The examined aecial specimens of P. graminis had pale yellow aecia (Fig. 2A-D) in separate discrete clusters of varying size (Fig. 2A, C). These features match the description of Aecidium berberidis Pers. ex J.F. Gmel. (Persoon 1801): “Cespitosum orbiculare parvum convexum, peridiis subelongatis flauis” (translated as: Growing in small convex clusters, subelongate with yellow peridia) and subsequent descriptions of the P. graminis aecia (Arthur & Cummins, 1962; Cummins, 1971; Savile, 1984; Schumann & Leonard, 2000). In contrast, aecia of P. chunjiei completely cover the abaxial leaf surface and are a deeper orange-yellow colour in the herbarium specimen. The two species share similar shape of the aecia and peridial cells (Figs. 1F, G, 2G, L). The aeciospores of both species are also similar (Figs. 1H, I, 2H-K), with overlapping size ranges amongst the specimens examined (Table 1), except slightly larger for Clade I P. graminis (PUR F17143). They both produce small and large plugs that may become detached leaving a clear area or tonsure (Fig. 3), corresponding to type four aeciospores sensu Savile (1984). The apical walls of P. chunjiei can be thicker than P. graminis Clade II (2-5.5 µm) and III (2.5-7 µm), but has similar range with Clade I ((1-)4-11 μm).
The host DNA-barcoding of selected specimens was performed to confirm identifications at the genus level with rbcL sequences, given that rust herbarium collections typically include very limited host material. Roy et al. (2010) demonstrated that molecular identifications of Berberis species is challenging because even applying multiple commonly used markers provided limited taxonomic resolution. According to a recently published flora for the region, Helan Mountain Flora (Hu, Duan, & Xu, 2021), there are five Berberis spp. reported in the area, i.e. Berberis amurensis Rupr., B. caroli C.K. Schneid, B. dubia C.K. Schneid., B. poiretii C.K. Schneid and B. sibirica Pall. Based on the presence of net-like leaf veins and at least one branched thorn, the host of BPI 1103856 is most similar to B. poiretii.
Our discovery that P. chunjiei shares the same alternate host genus as P. graminis is unsurprising, given the close phylogenetic relationship of the two rusts. To date, only two specimens have been identified as P. chunjiei, one on each host in its lifecycle, Elymus (telia) and Berberis (aecia). Both were collected in northern China, in neighbouring regions, and were originally identified as P. graminis. Berberis poiretii, present in seven provinces in Northern China (GRIN https://npgsweb.ars-grin.gov/gringlobal/taxon/taxonomydetail?id=6935, accessed on Oct 11, 2022), including Qinghai province bordering Xiahe county (Gansu province) to the east, could also have contributed to the dispersal of the rust. Our analyses (Liu & Hambleton, 2012) demonstrated that ITS sequences are diagnostic for differentiating the two species. Sequencing more specimens from East Asia may find more collections and even perhaps a uredinial stage.
The authors declare no conflicts of interest.
We thank the herbaria cited in Table 1 for providing specimens, Quinn Eggertson and Julie Carey for technical assistance with DNA sequencing, Keith Hubbard for assistance with SEM, and Ms. Dihong Zheng for facilitating morphological identification of the aecial host. We appreciate the constructive suggestions for improvement made by two anonymous reviewers to an earlier version of this manuscript. This study was supported by Agriculture and Agri-Food Canada Project ID J-002272.
