A New Report of Uropyxis daleae in the Andes Range Mountains of Atacama Desert, Chile

A rust disease caused by Uropyxis daleae (Dietel & Holw.) Magnus was registered on the natural population of Dalea pennelli (Mac Bride) Mac Bride var. chilensis Barneby in the highland near the Andes in the northernmost of Chile. This pathogen shows uredinia with the presence of paraphyses and features, larger urediniospores and teliospores than U. daleae (Dietel & Holway) Magn. var. daleae. Based on morphological combined with 28S sequence analysis, we propose this pathogen as a new fungal variety: U. daleae var. socoromensis. This is the first report of Uropyxis daleae var. socoromensis attacking D. pennelli var. chilensis in Northern Chile.


Introduction
Dalea pennellii (J. F. Macbr.) J. F. Macbr. var. chilensis Barneby (Fabaceae), commonly known as "retamilla," is a small endemic shrub distributed in the highlands in northern Chile and southern Peru, above 3500 m above sea level (asl) [1]. It is a typical plant component of the native flora of arid landscapes [2]. In Chile, there is no information regarding the fungal species associated with this host plant. However, a rust fungus has been found attacking D. pennellii var. chilensis developing dusty black sori on the leaflets and stems. The rust fungi are a monophyletic, highly specialized group of obligate parasites in the phylum Basidiomycota, with a complex life cycle. Most of the rust fungi exhibit a high degree of host specificity [3].
Nowadays, the identification of rust fungi includes morphological and molecular studies [3] [4] [5]. The cell wall thickness, ornamentation, color, size, and shape of the telia and teliospores, and the pedicel characteristics and number and position of the germinative pores in the teliospores are important taxonomical features in the identification of rust fungi.
The Uredinales associated with native plants have been scarcely studied in Northern Chile but, some rust fungi have been described in coastal range and coastal valleys [6]. This study aims to report and describe a rust fungus associated with the canopy of D. pennellii var. chilensis, in Socoroma, Chile.

Sample Collection
Samples of D. pennellii var. chilensis were collected in Socoroma (18˚16'38''S, 69˚35'28''W) in the highlands of the Andes mountains at 3200 m asl. D. pennellii var. chilensis were growing in a slope with a northern exposure, 125 km northeast of Arica city and 3 km from Socoroma, in the north of Chile ( Figure 1). A total of 10 entire plants of D. pennellii var. chilensis showing rust, symptoms, and signs on the leaflets were collected in April and June 2017. Each sample was identified, labeled, and taken to the Plant Pathology Laboratory at Universidad de Tarapacá, where morphological and molecular studies were conducted. Besides, ten plant samples were desiccated at room temperature (20˚C -22˚C) and kept in a herbarium.

Morphological Studies
Morphometric studies were conducted on water mount urediniospores and teliospores collected from rust sori developed on leaflet samples of D. pennellii var. chilensis. The identification included studies of wall thickness, ornamentation, color, size, and shape of the 30 urediniopores and 30 teliospores. The characteristics of the teliospore pedicels and the number and position of the germinative pores and spore size were determined. An aqueous solution of chloral hydrate 450 g/L was used to facilitate seeing the germinal pores of the teliospores [7]. For the observation of morphological details of the spores, the Bruzesse and Hassan dye [8] was used. The preparations were sealed with nail polish, labeled, and stored at room temperature until used. Measurements and digital photographs were made with the aid of an Olympus BX51 microscope and the Q Capture-Pro version 6 Software (Surrey, BC, Canada).
For scanning electron microscope (SEM) EVO LS 10 (Carl Zeiss, Bonn, Germany) observations, where sections of leaflets of (0.2 cm 2 ) with telia were obtained. The specimens were mounted on aluminum sample holders with double-contact carbon adhesives and analyzed in a low vacuum and in variable pressure mode, where the chamber pressure was 150 Pa (under vacuum) and in the column 2 × 10 −5 Torr (high vacuum). The working distance used (WD) varied depending on the type of sample, and what was needed to observe, the acceleration voltage used was 15 KV and a tilt (tilt) from 0˚ to 90˚, while the images were taken with a resolution of 3024 × 2304 pixels at a scanning speed of 12 minutes with 54 seconds.
The specimens were deposited in the mycological collection of Universidad de Concepción Herbarium (CONC-F 1894).

Molecular and Phylogenetic Analysis
Due to rust cannot be grown in culture media, teliospores were used for molecular analysis, which was removed and collected from telia on leaves with a sterile scalpel. DNA from three independent infected plants was extracted using EZNA® Insect DNA Kit (Omega Bio-Tek Inc, Norcross, GA, USA) according to the manufacturer's instructions. Subsequently, the D1/D2 domain of the large subunit ribosomal DNA 28S (LSU rDNA) was amplified using primers LR6 (5'-CGCCAGTTCTGCTTACC-3') and Rust2inv (5'-GATGAAGAACACAGTGAAA-3') [9]. Polymerase chain reactions of LSU rDNA were conducted in an Amplitronyx®6 thermal cycler (Nyx-Technik, San Diego, California, USA) using a final volume of 20 μl. Each reaction contained 1 μl of DNA extract, 5 pmoles of each primer, 2.5 mM of each dNTP, 2 mM MgCl 2 , 1X PCR buffer (KCl), 1 unit of Taq DNA polymerase (Thermo Scientific, Waltham, MA, USA), and sterile distilled water. Cycling conditions were: 5 min at 94˚C; 35 cycles of 1 min at 94˚C; 1 min at 55˚C; 1 min at 72˚C and a final elongation step of 2 min at 72˚C. PCR blank reaction control without template DNA was always incorporated. The PCR products were visualized on 1.0% agarose gel and stained with GelRed (Biotium Inc., Hayward, CA, USA). The amplified PCR products were purified and sequenced by Macrogen (Macrogen, Geumcheon-gu, South Korea). The nucleotide sequences were visualized and edited using the 4 Peaks software (http://nucleobytes.com). A BLASTn analysis was used to compare the DNA sequence obtained of the rust fungus isolates from Socoroma with rDNA D1/D2 data sequences available in GenBank (http://www.ncbi.nlm.nih.gov/).
Phylogenetic reconstruction was performed using the maximum likelihood algorithm, using Molecular Evolutionary Genetics Analysis (MEGA version 6.0) software [10], and the robustness of the branches was assessed by bootstrap analysis [11] of 1000 replicates. The sequence of Encyclometra sp. (KY781819) was used as an outgroup. Etymology. From the Andean village of Socoroma, in reference to the collection site (Figure 1).

Specimens Studied
To confirm the morphological identification, a PCR fragments of LSU rDNA of isolates from Socoroma were successfully amplified and sequenced, obtaining fragments of LSU of 1446, 1456 and 1443 bp, which were deposited in the Gen- Bank (Accession number MG969964, MN337264 and MN337263, respectively).
The phylogenetic analysis using sequences of the LSU rDNA grouped the isolates Socoroma in a different cluster from other rust fungi, and it was phylogenetically close to the reference isolate of U. daleae (KY798364) (Figure 4).

Discussion
In this study, a rust fungus attacking D. pennelli ("retamilla") was characterized and identified as Uropyxis daleae, and based on morphological and molecular differences, a new variety, U. daleae var. socoromensis, is proposed. Rust fungi are very specific obligate parasites. To our knowledge, U. daleae var. socoromensis was only found on "retamilla" growing above 3500 m asl in the Andes Mountains in Northern Chile, where a high frequency of endemic plant species, animals, and fungi have been described [13]. Therefore, it is possible that this new variety of U. daleae may have coevolved in situ with its plant host. At present, "retamilla" grows as a wild shrub; however, it has a high potential as an ornamental plant.
The genus Uropyxis J. Schröt. (Basidiomycota, Pucciniales, Uropyxidaceae), was first distinguished from Puccinia spp. in 1875 by the presence of teliospores with two germination pores in each cell [11]. Nowadays, 30 spe- The presence of uredinia distinguishes U. daleae var. socoromensis from U. farlowii (Arthur) J.W. Baxter, a rust fungus that is also parasite Dalia spp., but apparently lacks of the uredinial stage. Therefore, Uropyxis daleae var. socoromensis is described for the first time attacking D. pennelli var. chilensis in Northern Chile. Previously, only U. naumanniana has been reported in southern Chile [14].