First Report of Powdery Mildew Caused by Leveillula lappae on Tricholepis stewartei in Pakistan

Abstract

Tricholepis stewartei C.B.Clarke ex Hook.f. is an Asian plant belonging to family Asteraceae Bercht. & J. Presl, whose members are mostly herbs and subshrubs. During the plant pathogenic survey of Ayubia National Park, district Abbottabad, Khyber Pakhtunkhwa Province, Pakistan, and its adjacent areas conducted in October 2017 to 2018, plants of T. stewartei with powdery mildew symptoms were collected to be checked for infection with powdery mildew fungus. Disease symptoms (i.e., white powdery mycelium with blackish spots immersed in these mycelium patches covering the entire leaf) were observed. Microscopic observations showed hyaline, thin, and smooth walled, 3- to 5-μm-wide hyphae. Conidiophores were erect to bent, with one following cell, and 5 to 12 × 76 to 92 μm. Conidia were formed singly, primary conidia broadly lanceolate, apically narrowed, obtuse to pointed, and 14 to 30 × 35 to 60 μm; secondary conidia were ellipsoid or cylindrical and 12 to 30 × 39 to 55 μm. Germ tubes were subterminal or near the base, short, cylindrical, straight to sinuous, apex simple. Chasmothecia were scattered, globose to subglobose, light brown to dark brown or blackish brown, and 124 to 217 μm in diameter. Peridium cells were irregularly shaped and 9 to 18 × 8 to 18 μm. Appendages were numerous, mycelioid, simple to irregularly branched, thin walled, thicker at the base, smooth, yellowish below, hyaline above (toward tip), mostly continuous, and 4 to 8 × 50 to 160 μm. Asci were numerous, stalked to sessile, 26 to 41 × 41 to 109 μm, and (1- to) 2-spored. Ascospores were subglobose, hyaline to light yellow, and 17 to 24 × 37 to 48 μm. The morphology of the asexual and sexual morphs of the powdery mildew samples found on T. stewartei in Pakistan agrees well with the concept and description of Leveillula lappae in Braun and Cook (2012). Khodaparast et al. (2010) examined the morphology of Leveillula on composites in Iran in detail and differentiated six species. Leveillula collections on a wide range of hosts, agreeing with L. lappae, were assigned to L. taurica sensu lato. To confirm morphological identification, genomic DNA was extracted directly from mycelia and chasmothecia using a Gene Jet Plant Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, Waltham, MA) (Andersson et al. 2017). The internal transcribed spacer (ITS) region of the isolate was amplified using primers PMITS1 (5′-TCGGACTGGCC[T/C]AGGGAGA-3′) and PMITS2 (5′-TCACTCGCCGTTACTGAGGT-3′) as forward and reverse primers, respectively (Cunnington et al. 2003). The sequence (ITS) retrieved from this collection (568 bp) supports its identification as a species of the genus Leveillula, but a further identification to species level is not possible owing to lacking reference sequence data for L. lappae in GenBank. There is one difference between the new sequence and a sequence retrieved from L. taurica on Carthamus L. (AB667864), identity 99% (561/562), and two differences to L. duriaei on Phlomis persica Boiss. (AB667859), identity 99% (609/611) including one gap and one substitution. However, phylogenetic analyses just based on ITS data are not sufficient within Leveillula to distinguish between closely allied species (Braun and Cook 2012). Pathogenicity was confirmed by pressing a diseased leaf onto young leaves of three asymptomatic potted plants. Three noninoculated plants were used as controls. Plants were maintained in a greenhouse. Inoculated leaves developed symptoms of powdery mildew after 7 days, whereas the control plants remained symptomless. The fungus on the inoculated leaves was morphologically identical to the fungus on the original infected leaves. Furthermore, L. lappae is still a heterogeneous complex of morphologically similar Leveillula spp. on composites that is in need of phylogenetic characterization and circumscription (Braun and Cook 2012). The whole genus Leveillula, above all the L. taurica complex within this genus, requires a phylogenetic multilocus approach that is currently not available. Therefore, the present collections can currently only be assigned to L. lappae. To our knowledge, this is the first report of L. lappae on T. stewartei from Pakistan. T. stewartei is also reported as new host of L. lappae worldwide.