Mycoscience: Advance Publication doi: 10.47371/mycosci.2021.03.006 Short communication (Received January 13, 2021; Accepted March 16, 2021) J-STAGE Advance Published Date:May 1, 2021 Short communication A new desert-dwelling oomycete, Pustula persica sp. nov., on Gymnarrhena micrantha (Asteraceae) from Iran Mohammad Reza Mirzaee1, Sebastian Ploch2, Marco Thines2,3,* 1 Plant Protection Research Department, South Khorasan Agricultural and Natural Resources Research and Education Center, AREEO, Birjand, Iran 2 Senckenberg Biodiversity and Climate Research Centre, Senckenberganlage 25, 60325 Frankfurt am Main, Germany 3 Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Max-von-Laue-Str. 13, 60483 Frankfurt am Main, Germany * Corresponding author. Senckenberg Biodiversity and Climate Research Centre, Senckenberg Gesellschaft für Naturforschung, Mertonstr. 17-21, 60325 Frankfurt am Main, Germany E-Mail address: [email protected] (M.Thines) ABSTRACTAdvance Publication The obligate biotrophic oomycete genus Pustula is one of the four major linages of white blister rusts (Albuginaceae) identified so far. Species of the genus Pustula cause white blister rust on numerous genera in the asterids, represented by several phylogenetically distinct genus-specific lineages, most of which still await formal description. Thus, the observation of the species of Pustula on the Asteraceae subfamily Gymnorhenoideae, pointed out to the existence of a hitherto undescribed species. By the morphological and molecular phylogenetic investigation conducted in this study it is concluded that the pathogen on - 1 - Mycoscience: Advance Publication Gymnarrhena micrantha from Iran indeed represents a hitherto unknown species and is described as P. persica. This species has apparently adapted to desert condition and is, after Albugo arenosa, the second species of white blister rust from Iranian deserts, highlighting the adaptability of white blister rusts to hot and dry habitats. Keywords: Albuginales, cox2, one new species, phylogeny Despite similarities, such as filamentous growth and osmotrophic nutrient uptake, the phylum Oomycota is unrelated to fungi of the kingdom Mycota, but instead belong to the kingdom Straminipila, which also contains diatoms and brown seaweeds (Beakes & Thines, 2017). Organisms in the Oomycota have adapted to a wide range of climate conditions and lifestyles (Thines, 2014) and can be found in both arctic habitats (Hassett, Thines, Buaya, Ploch, & Gradinger, 2019) and hot deserts (Mirzaee et al., 2013). The highest diversity has so far been found among the two independently-evolved, obligate biotrophic lineages parasitizing angiosperm plants, the downy mildews and white blister rusts (Thines, 2014; Wijayawardene et al., 2020). The white blister rusts have evolved to sporulate below the epidermis of their hosts and to liberate their spores by enzymatic digestion of the epidermal layer covering the pustules (Heller & Thines, 2009). The family Albuginaceae contains the three genera that cause white blister disease of angiosperms, Albugo, Pustula, and Wilsoniana. The latter two have been segregated from Albugo based on their largely different cytology, differences in sporangia and oospore morphology, as well as deep phylogenetic divide (Thines & Spring, 2005). In total, there are four major lineages in the Albuginaceae (Voglmayr & Riethmüller, 2006), each with a specific host range. Albugo s.str. parasitizes mostly Brassicales, but a few lineages are present on other orders (Choi, Shin, Ploch, & Thines, 2011b; Ploch, Choi, & Thines, 2018). Albugo s.l. parasitizes members of the Convolvulaceae and is distinguishable from Albugo s.str. by a pronounced oogonium ornamentation (Voglmayr & Riethmüller, 2006; Thines & Voglmayr, 2009). Wilsoniana is parasitic to caryophyllids and features broadly pear-shaped sporangia and densely ridged or reticulate oospores (Thines & Spring, 2005; Thines & Voglmayr, 2009). Pustula parasitizes various asterids, in particular Asteraceae, and is characterised by usually densely reticulate oospores and sporangia with an equatorial wall thickening (Thines & Spring, 2005; Choi, Thines, Tek, & Shin, 2012). Traditionally, it has been assumed that species causing white blisterAdvance rust disease are specific mostly on thePublication host family level (Wilson, 1907; Biga, 1955; Choi & Priest, 1995). However, phylogenetic investigations have revealed that in Albugo, besides the generalist species, A. candida (Pers.) Roussel, several distinct, host-specific species exist, which seem to be specific below the host genus level (Choi, Shin, Hong, & Thines, 2007; Choi, Shin, Ploch, & Thines, 2008; Choi, Shin, & Thines, 2009; Thines et al., 2009; Ploch et al., 2010; Choi & Thines, 2011). Also in the genus Pustula, species seem to be specific on at least the host genus level (Ploch et al., 2011), leading to the description of P. - 2 - A new desert-dwelling oomycete, Pustula persica sp. nov., on Gymnarrhena micrantha (Asteraceae) from Iran helianthicola C. Rost & Thines affecting sunflower (Rost & Thines, 2011), and the re- appraisal of several species previously thought to be synonyms of P. obtusata (Link) C. Rost (syn. P. tragopogonis (Pers.) Thines) (Choi et al., 2012). In line with this, three new species of Pustula were recently introduced from the Junggar Basin in China (Xu, Song, Xi, & Jiang, 2016; Xu et al., 2018). During field trips in Iran, the occurrence of Pustula on Gymnarrhena micrantha Desf. was noticed. Gymnarrhena micrantha is a hardy member of Asteraceae growing in dry, mostly bare and sandy areas in the deserts of Iran. It is an ephemeric, amphicarpic, dwarf desert annual herb which mainly distributed in the drier parts of Mediterranean biome of North Africa and the Middle East. Although, some variation across the distribution range has been noticed in collections, there is one species recognized in the genus. In a study of the tribe Inuleae using the cpDNA gene ndhF, it was found that Gymnarrhena did not belonging to Asteroideae as previously thought, but rather to the paraphyletic Cichorioideae complex or sister to the entire Asteroideae, and was, thus, proposed as the sole member of the subfamily Gymnarrhenoideae (Anderberg, Eldenäs, Bayer, & Englund, 2005; Funk & Fragman-Sapir, 2009). Given the host specificity previously observed for the genus Pustula (Ploch et al., 2011; Xu et al., 2016, 2018), it seemed plausible that the Pustula species occurring on Gymnarrhena does not belong to any Pustula species described so far. Therefore, it was the aim of this study to clarify the phylogenetic relationships of the potential new species and to investigate its morphology. Specimens sequenced in this study have been deposited in the Herbarium Senckenbergianum in Frankfurt (international herbarium code FR). The collection details are given in Table 1. Thin cross sections using a razor blade were done on wetted herbarium specimens with white blister symptoms. Sections were transferred to 60% lactic acid or 5% aqueous chloral hydrate solution on a slide. The preparations for microscopy were warmed up covered with coverslips and screened in bright-field using a compound light microscope (VWR TR 500 PH, VWR International, Darmstadt, Germany). Subsequently, suitable preparations were investigated in differential interference contrast (DIC) using a using a compound light microscope (Zeiss Imager2, Carl Zeiss, Jena, Germany) for measurements and photographs. Measurements were performed at ×1,000 magnification. Measurements are presented as (minimum–)mean minus standard deviation–mean–mean plus standard deviation(Advance–maxiumum), with all values apart fromPublication the mean rounded to the nearest 0.5 µm increment, followed by the number of measurements done for the respective organ. Genomic DNA was extracted from small pieces of leave tissue with pustules of Pustula from dried specimens. DNA extraction and PCR were performed as reported before (Mirzaee et al., 2013). In short, the innuPREP plant DNA extraction kit (Analytik Jena GmbH, Jena, Germany) was used for DNA extraction and PCR was performed using cox2 primers reported previously (Hudspeth, Nadler, & Hudspeth, 2000). PCR products were sequenced - 3 - Mycoscience: Advance Publication by the laboratory centre of the Senckenberg Biodiversity and Climate Research Centre, with the primers used in PCR. GenBank accession numbers for the sequences obtained in this study are given in Table 1. The partial cox2 sequences from the specimens were edited using the DNASTAR computer package version 8 (Lasergene, Madison, WI, USA), and Geneious version 5.3.4 (Biomatters Ltd., Auckland, New Zealand). Subsequently, they were added to the dataset of Ploch et al. (2011). In addition, the sequences of two Pustula species recently described were added (Xu et al., 2016, 2018). Sequences were aligned on the Mafft webserver (Katoh, Rozewicki, & Yamada, 2019) using default settings. Phylogenetic analyses were done on the TrEase webserver (http://thines-lab.senckenberg.de/trease/) using FastTree2 (Price, Dehal, & Arkin, 2010) for Minimum Evolution inference, RAxML (Stamatakis, 2014) for Maximum Likelihood inference with 1,000 bootstrap replicates, and Bayesian inference using MrBayes, version 3.2 (Ronquist et al., 2012) with 5 Million generations, while other parameters were set to default. In the phylogenetic reconstructions (Fig. 1), Pustula sp. from infecting Gymnarrhena micrantha is occupying an isolated position, with no clear