Mehrdad Abbasi
curator of fungus collection
Department of Botany
Plant Pests and Diseases Research Institute
P.O.Box 1454, Tehran 19395
IRAN
Email:puccinia_2000@yahoo.com
Title of dissertation: Taxonomic investigation of Puccinia species parasitic on Poaceae in Iran
Mehrdad Abbasi
SUMMARY
Rust fungi (Uredinales) are one of the largest natural taxa within the kingdom Fungi. More than 7000 species belonging to 100-125 genera and 14 families are accepted currently. Although species infecting gramineous hosts are economically important and belong to the best studied fungi, little or nothing is known about the life cycle and taxonomic placement of many species. The largest genus, Puccinia, contains ca. 4000 spp., 650 of which occur on Poaceae. There are 17 genera and 450 species known from Iran, 45 of which are graminicolous belonging to the genera Puccinia and Uromyces.
Species concepts and concepts below the species level in graminicolous rust fungi differ considerably. Some authors prefer a narrow species concept emphasizing biological specialization (host range) as the main criterion for species delimitation. Other authors used a broad species concept with morphological characters as the main criteria for species delimitation. This broad concept made it easier to identify species using the light microscope but it also led to polyphyletic groups like the Puccinia recondita aggregate. Both narrow and broad species concepts are still used and, consequently, many different and sometimes confusing scientific names occur in the literature.
Taxa should be natural, i.e. express phylogenetic relationships. For this reason scientists considered additional features for the identification and classification of organisms including graminicolous rust fungi; biochemical and molecular markers were used in particular. To obtain further data for the classification of Poaceae rusts in general and the species complexes of P. graminis, P. recondita , P. hordei, P. striiformis, and P. coronata in particular, we studied numerous specimens using morphological and molecular techniques. In the molecular studies we compared ribosomal DNA sequences of the variable internal transcribed spacer (ITS I+II) region and the more conservative 5.8S ribosomal RNA gene.
The same sequences were used to analyze relationships between aecial stages and unknown telial stages and vice versa in heteroecious species. Sequences of our own database and GenBank were used for this study. Life cycle and anamorph-teleomorph connections of some species could be identified.
MATERIAL AND METHODS
Spores from dried specimens were mounted mostly in lactophenol and examined with an Olympus BH2 Nomarski Differential Interference Contrast Microscope (DIC) at a magnification of x 400 or x 1000. Specimens were photographed using a PM-10 AD photomicrographic system. Fifty spores per specimen were randomly selected and measured. Specimens were deposited in the Herbarium Ministerii Iranici Agriculturae (IRAN) in Tehran and duplicates were sent to the Arthur Herbarium, Purdue University (PUR). Spore state and life cycle terminology follow Cummins & Hiratsuka (1983). For Scanning Electron Microscopy (SEM) urediniospores were mounted on a specimen stub and coated with gold (3-3.5 minutes at 29 mA). The specimens were observed and photographed using a Leitz AMR/1000 Scanning Electron Microscope. Cluster analyses were done with NTSYS software using SAHN clustering. The clustering method used was UPGMA (unweighted, pair-group method with arithmetic average). For DNA extraction less than 30-year-old herbarium specimens from IRAN and PUR were used. DNA was extracted by the crushing and the grinding methods. In the latter method spores (mostly aeciospores) were suspended in 50 µl of low EDTA TE (0.089 M Tris base, 0.045 M boric acid, 0.05 µM EDTA) + 1% 2-mercaptoethanol buffer in 1.5 ml plastic tubes and ground with a mini-pestle mounted in an electric drill. The ITS region of each isolate was amplified with primers ITS4 and ITS5. Amplification cycling parameters were: 40 cycles of 94 C for 30 sec, 50 C for 1 min, 72 C for 2 min, and a final extension of 10 min at 72 C. The size and quantity of amplification products were verified on 1% agarose gels and extracted with the GeneClean spin kit (Carlsbad, CA) according to the manufacturer’s instructions. Purified products were quantified with a Hoefer DyNAQuant 2000 fluorometer (Hoefer Scientific Instruments, San Francisco, CA) and cloned with the TA cloning kit (Invitrogen Corp., Carlsbad, CA). The presence of inserts was confirmed by digestion with EcoRI and agarose electrophoresis. Plasmid DNA was prepared with the Wizard miniprep kit (Promega, Madison, WI) and DNA concentration was estimated with a fluorometer. DNA samples were sequenced with the ThermoSequenase fluorescent-labeled primer cycle sequencing kit (Amersham Pharmacia Biotech) by mixing 8 pmole of CY-5 labeled primer with approximately 500 ng plasmid DNA in a total volume of 26 µl. Six µl of the DNA solution were added to each of four tubes containing 2 µl of A, C, G or T termination mix and stirred by vortexing. DNA was amplified in a thermal cycler at 94 C for 3 min, followed by 25 cycles of 55 C for 30 s, 72 C for 2 min and 94 C for 30 s. After adding 6 µl of stop dye, 6 µl of each reaction was sequenced on an ALFexpress automated DNA sequencer (Amersham Pharmacia Biotech). Each clone was sequenced in both directions with the M13 reverse and M13-40 primers. In the majority of specimens more than one clone were sequenced to minimize errors caused by PCR amplification.
DNA sequences were aligned with the profile mode of Clustal X and were edited manually when necessary. Following alignment, genetic distances among all isolates were calculated and neighbor-joining trees were prepared with the Draw N-J Tree option of Clustal X. Bootstrap analyses (1000 replications) were performed on the resulting trees with the Bootstrap N-J Tree option of Clustal X, and the final trees were visualized and printed with Njplot. In the second analysis, the ITS database was assembled by combining DNA sequences downloaded from GenBank with new data from different specimens of leaf rusts. Because sequences downloaded from GenBank only include the last 42 bases at the 3’ end of ITS1, we therefore used only the alignable data common to all specimens for drawing the tree (i.e., the last 42 bp of ITS1, and all of the 5.8S and ITS2 sequences).
RESULTS and DISCUSSION
1. Puccinia graminis
P. graminis is a macrocyclic heteroecious species with aecial hosts in the Berberidaceae and telial hosts in the Poaceae. Morphological features of 44 specimens of P. graminis were evaluated. Cluster analysis by the unweighted pair group method using arithmetic average (UPGMA) was performed for urediniospore morphology. As a result of this analysis three infra-specific taxa were recognized for stem rust of wheat and grasses in Iran. These taxa are as follows:
- P. graminis subsp. graminis var. graminis, on Triticum aestivum and other closely related genera belong to Triticeae.
- P. graminis subsp. graminis var. stakmanii, on grasses belong to Triticeae, Bromeae, Poeae and Avenae.
- P. graminis subsp. graminicola, on Poa spp.
A first phylogram, based on the entire ITS1+2 and 5.8S, using neighbor-joining analysis and Puccinia hordei, P. recondita and P. striiformis as an outgroup showed three clades. Clade no. 1 includes specimens on different hosts from Iran only. Some specimens form a small not highly supported clade within this group. Clade no. 2 includes specimens on Elymus and Eremopyrum from Iran and four specimens from other countries (3 specimens on Triticum from the USA and one specimen on Secale from Germany). A single specimen on Avena sp. from Iran forms the third clade. The second set of analyses based on the last 42 bp of the ITS1 plus the entire 5.8S and ITS2 region resulted in a similar phylogram. The second tree also showed three clades, and differs from the former one only in the position of specimen no. 26 on Aegilops crassa which clustered within clade no. 2 in the first phylogram.
Infraspecific delimitation of P. graminis is based on urediniospore morphology. The morphological data do not correspond with data obtained from DNA sequence analyses. Consequently, morphological characters of urediniospores in P. graminis s.l. do not reflect phylogenetic relationships within this species, confirming previous studies by Zambino and Szabó (1993) and Swertz (1994). Our results indicate that P. graminis is genetically more diverse in Iran than elsewhere. In some parts of Iran, in particular in the Elburz Mts., Berberis spp. and various grasses are growing together and it seems that ancestral P. graminis most likely developed in similar plant communities. Our investigation revealed that P. graminis has great infraspecific variablity and diversity in Iran, especially in the Elburz area. This result suggests that Iran is one of the primary evolutionary gene centers of P. graminis.
2. P. coronata s. l.
P. coronata is a macrocyclic heteroecious species with aecial hosts in the Rhamnaceae and telial hosts in the Poaceae. P. mesneriana is considered a related microcylic species on Rhamnus. Based on morphological characters only, four P. coronata varieties were recognized for Iran: P. coronata var. coronata, P. coronata var. avenae, P. coronata var. hordei, and P. coronata var. golestanica var. nov.
The phylogram for the last 42 bp of ITS1, and all of the 5.8S and ITS2 sequences of P. coronata in combination with ITS sequence data of selected graminicolous rust species from GenBank and our own databank shows that P. coronata s.l. specimens including P. mesneriana form a monophyletic group. Within this clade the following subclades were formed: Clade A with specimens of P. coronata on Avena spp., Lolium perenne, Alopecurus aequalis and Festuca elatior. The ITS sequences of P. coronata on these hosts were identical. Clade B with the aecial stage on Rhamnus pallasii and P. mesnieriana on R. palestina. Clade C consisted of P. coronata on Agropyron repens and Hordeum sp. The only isolate of P. coronata on Calamagrostis canadensis formed clade D. These results clearly confirm the existence of different taxa in P. coronata s.l.
3. P. hordei s. l.
P. hordei is a macrocyclic heteroecious species with aecial hosts mainly in the Liliaceae and telial hosts in the Poaceae. Phylogenetic analysis based on the ITS sequence data confirmed that P. hordei is a complex species. On the basis of these molecular and morphological features, three taxa were recognized: P. hordei s. str. on Hordeum vulgare; P. holcina on Hordeum bulbosum, H. glaucum, H. spontaneum, Lolium temulentum, L. rigidum, Lophochloa phleoides, Trisetum flavescens and Vulpia myuros; and P. taeniatheri sp. nov. on Taeniatherum spp.
4. P. striiformis s. l.
P. striiformis is a species forming uredinia and telia on various species of Poaceae. It is assumed to be heteroecious, but the aecial host is not known. ITS sequence data showed that there is considerable difference between specimens on Triticum, Dactylis glomerata and Poa pratensis. This corresponds with a previous isozyme phenotype study. P. striiformis on Triticum and Hordeum geniculatum are identical. Based on the above molecular and on morphological data, we suggest dividing P. striiformis as follows:
- P. striiformis s. str. mainly on Triticeae.
- P. pseudostriiformis sp. nov. on Poa pratensis (Poeae)
- P. striiformoides nom. et stat. nov. on Dactylis glomerata (Poeae)
5. P. recondita s. l.
P. recondita is a macrocyclic heteroecious species with aecial hosts on various families in the Dicotyledonae and telial hosts in the Poaceae. Former (molecular) studies have already indicated that the species is polyphyletic consisting of morphologically similar but taxonomically unrelated species. The phylogram including also two specimens with aecial stages which were assumed to belong to the P. recondita complex and some other grass rusts as an outgroup contains three distinct clades. Branching was supported by high bootstrap values. One clade is formed by two specimens on Bromus spp. (nos. 194II & 99) with 98% identity in the ITS. This confirms that P. bromina, which is considered synomymous with P. recondita by most authors, is a “good” species causing brown leaf rust on Bromus. An additional clade is formed by specimens on Aegilops taushii (no. 101), Triticum aestivum (no. 109) and Elymus hispidus (no. 206) and the two aecial stageson Thalictrum spp. (nos. 232 & 194I). Within this clade the rust specimens on Triticum and Aegilops were identical and related to specimen no. 206 on E. hispidus. Based upon these results and morphological studies, we consider using the name Puccinia persistens for members of this large clade and also taking up the name P. persistens subsp. triticina for wheat leaf rust and P. persistens subsp. agropyrina for leaf rust on Elymus hispidus. Our results support the taxonomic concept for the brown leaf rust complex suggested by Markova & Urban (1998). However, further sequence data, e.g., of material on the P. persistens type host (Agropyron repens), are required for final statements. The remaining specimens on Secale segetale (no. 195) and Elymus sp. (no. 215) form another clade with specimen no. 229 an aecial stage on Cerinthe minor (Boraginaceae). The ITS sequences of these specimens are identical and clearly support the use of P. recondita s. str. for members of this clade with aecial hosts in the Boraginaceae.Our analysis confirms that P. recondita s.l. is polyphyletic and that P. recondita s. str. is more closely related to P. hordei s.l. than to other groups of the P. recondita complex.
6. Puccinia spp. on Arundineae
A morphological study of the species of Puccinia parasitizing Arundineae (Poaceae) in Iran is provided. Five species, viz. P. akiyoshidaensis, P. isiacae, P. magnusiana, P. phragmitis,and P. trabutii were accepted. Of these, P. akiyoshidaensis was recorded for the first time in Iran. The taxonomic position of this species and P. moriokaensis were discussed.
7.Additional species
- Puccinia ariorum sp. nov. on Bromus dantoniae.
This new rust species is characterized by verrucose teliospores.
- P. australis on Sedum hispanicum (0, I) and Cleistogenes serotina (II, III).
This heteroecious species is a first record for Iran.
- P. fragosoana on Imperata cylindrica
This species is new for the Iranian rust flora.
- P. stipae var. stipina on Stipa pennata, S. pennata subsp. zalesskii, S. capillata, S. holosericea
This taxon is recorded for the first time on a telial host in Iran.
- P. schoenanthi on Cymbopogon olivieri and C. schoenanthus.
This is the second record worldwide.
8. Connecting telial and aecial stages of selected rusts using ITS sequence analyses
The ITS region from unknown aecial stages on eight hosts was sequenced. Comparison of these sequences with those in our own database and in GenBank could connect them with species whose aecial stage was not known at all or with stages on so far unknown host plants, or the anamorph-teleomorph connection could be confirmed. The alignment revealed that an aecial stage on Clematis orientalis (Aecidium orbiculare; no. 228) and P. wolgensis on Stipa, and an aecial stage on Cerinthe minor (no. 229) and P. recondita s. str. were identical. Cerinthe minor is a matrix nova for P. recondita s.str. Aecidium sp. on Berberis (no. 231) and Puccinia brachypodii from GenBank (accession L08730) are almost identical (98% agreement). Aecial stages on Thalictrum spp. (nos. 232 & 194I) are related to P. persistens s.l. as indicated by previous authors. The Aecidium sp. on T. minus (no. 194I) was closely related to P. persistens subsp. agropyrina and the aecial stage on T. sultanabadense (no. 232) was related to P. persistens on Agropyron smithii from the USA (GenBank no. L08702) (98% and 97% identity, respectively). The aecial stage on Rhamnus pallasii (no. 250) was related to P. coronata s.l. We could not connect the aecial stage on Zygophyllum sp. (Zygophyllaceae) (no.239) with another species. This fungus was distantly related to P. sorghi (GenBank no. L08734). An aecial stage on Ornithogalum sp. (Liliaceae) was closely related to P. hordei s.str. (96% identity) and Uromyces scillarum (GenBank no. L08697) (97% identity).
All identified specimens in this study are described in detail and are documented with drawings and microphotographs. Host range and distribution data and an identification key are provided as well.