Identification of Berberis spp. as Alternate Hosts for Puccinia achnatheri-sibirici Under Controlled Conditions and Morphologic Observations of Sexual Stage Development of the Rust Fungus

Abstract

Gramineous grasses are a large group of species, many of which act as accessory (secondary) host for a large number of rust fungi, including devastating rust pathogens of cereals. Among the rust fungi, some are known to be heteroecious and have a complete macrocyclic life cycle with five types of spores on distinct plant species, but for many others the complete life cycle is unknown. Puccinia achnatheri-sibirici, a rust fungus infecting grasses in the genus Achnatherum, has been known for only its uredinial and telial stages; however, the other (pycnial and aecial) stages have not been identified. In this study, we demonstrate that P. achnatheri-sibirici is a heteroecious, macrocyclic fungus with the sexual stage on barberry (Berberis spp.) through inoculation. Pycnia and aecia were successively produced on the inoculated barberry plants. Inoculation of Achnatherum extremiorientale leaves with aeciospores produced yellow-orange uredinia with high infection types, whereas inoculation of wheat variety Mingxian 169, highly susceptible to Puccinia striiformis f. sp. tritici which causes stripe rust on wheat, produced chlorotic flecks but no uredinia. The ITS sequence analysis of P. achnatheri-sibirici did not match with any sequence in the NCBI database and had the highest homology with 94% compared to Puccinia brachypodii and Puccinia aizazii. Observations of the uredinial, telial, and basidial stages on A. extremiorientale and pycnial and aecial stages on Berberis shensiana using light and scanning electron microscopes revealed its characteristics. Morphological characteristics of urediniospores and teliospores are most similar with those of P. achnatheri-sibirici described in the literature. This study proved (1) the life cycle of P. achnatheri-sibirici as heteroecious and macrocyclic and the alternate host as barberry; (2) the description of life stages in the sexual cycle, especially the morphologies of aecial, pycnial, and basidial stages; and (3) the expansion of knowledge on the rust flora on barberry.

Keywords: Achnatherum; Berberis spp.; Puccinia achnatheri-sibirici; alternate host; gramineous grass.

FIGURE 1

Morphology of gramineous grass, Achnatherum extremiorientale, and natural rust infection on the grass species by Puccinia achnatheri-sibirici, observed in Xinjie, Baoji, Shaanxi Province, in October 2013. (A–C) Plants, heads, and seeds of Achnatherum extremiorientale. (D–F) Uredinial and telial symptoms of natural infection of P. achnatheri-sibirici on grass A. extremiorientale, respectively.

FIGURE 2

Pycnial and aecial stages of Puccinia achnatheri-sibirici on barberry (Berberis shensiana) after infection by basidiospores and responses of Achnatherum extremiorientale and wheat cv. Mingxian 169 to aeciospores. (A) Pycnial stage of P. achnatheri-sibirici infecting the barberry (B. shensiana Ahrendt) after inoculation with basidiospores. (B) Aecial formation on the infected barberry. (C) The grass, Achnatherum extremiorientale, presenting orange uredinia on leaves after inoculation using P. achnatheri-sibirici aeciospores produced on the barberry in Figure 2B. (D) Black stripes formed and restricted between leaf veins of the grass. (E) Wheat cv. Mingxian 169 showed obvious chlorotic lesions after inoculation with aeciospores of P. achnatheri-sibirici. (F) Non-inoculated leaves of Mingxian 169 wheat used as control.

FIGURE 3

Phylogenetic tree of Puccinia achnatheri-sibirici and Puccinia species based on the sequence of nuclear ribosomal internal transcribed spacer (ITS) amplified with primer ITS1RustF10d/ITS4 (White et al., 1990; Barnes and Szabo, 2007). Clade robustness was assessed using a bootstrap analysis with 1,000 replicates. The numbers on the branches are bootstrap values from MP. Reference sequences used in this analysis are listed in Table 1.

FIGURE 4

Scanning electron microscopy observations of Puccinia achnatheri-sibirici infecting Berberis shensiana Ahrendt at the pycnial stage. (A) A young pycnium at the early stage with eruption from epidermis (E) of the host, producing paraphyses (P) and flexuous hyphae (F). Bar = 50 μm. (B) Paraphyses (P) and flexuous hyphae (F) of a mature pycnium radiated on the ostiole of the pycnium with production of numerous pycniospores congesting around the opening. Bar = 50 μm. (C) Enlargement of the paraphyses (P) and flexuous hyphae (F). Bar = 5 μm. (D) Mature pycniospores (PS). Bar = 5 μm.

FIGURE 5

Scanning electron microscopy observations of the development of aecia and aeciospores of Puccinia achnatheri-sibirici infecting Berberis shensiana at the aecial stage. (A) An initial aecial cup (aecium, A) erupted from the epidermis (E) of the host. Bar = 100 μm. (B,C) A mature aecial cup (AC) broke and released aeciospores (A) embraced by a layer peridium (P) that was formed by peridial cells. Bar = 100 μm. (D,E) A mature aeciospore (A) was spherosome-shaped and has germ pores (G) and the numerous, column-shaped ornaments (verrucae, V) on the surface. Bar = 10 μm. (F) Close-up of verrucae (V). Bar = 1 μm.

FIGURE 6

Light and scanning electron microscopy observations of urediniospores of Puccinia achnatheri-sibirici. (A–C). A young urediniospore (U) developing at the top of a long sporophore (SP) formed in a uredinium based on observations using light and scanning electron microscopy. Bar = 20 μm. (D) Light microscopy observation showing orange and near sphere-shaped urediniospores. Bar = 20 μm. (E) Scanning electron microscopy (SEM) observation presenting urediniospore production in a uredinium. Bar = 20 μm. (F) SEM observation showing a mature urediniospore with numerous cones on the surface. Bar = 10 μm. (G) Enlargement of the cones (C) on the surface of a urediniospore. Bar = 1 μm.

FIGURE 7

Morphologic observation of Puccinia achnatheri-sibirici teliospores by light microscopy. (A,B) Production of a teliospore (T) in a uredinium (U). Bar = 20 μm. (C) Mature teliospores with/without a short stalk cell. Bar = 20 μm.

FIGURE 8

Puccinia achnatheri-sibirici teliospores (TS) germinated to produce basidiospores on the water agar medium. (A) Migration of nucleus (N) and cellular contents from the top cell of a two-celled teliospore (TS) into the top of a metabasidium (M) at the early stage of teliospore germination. (B) Formation of a septum (S) separated the top of a metabasidium (M) containing the nucleus (N) from the vacant basal cell of the metabasidium developed from a two-celled teliospore (TS). (C) Meiosis I showing formation of two daughter nuclei (N) at the top of a metabasidium (M) derived from a teliospore (TS). (D) Meiosis II showing formation of a four-celled metabasidium (M) separated by four speta (S). (E) A basidiospore (BS) growing at the tip of a sterigmata (ST) developed from a metabasidium (M). (F) Morphological single-celled and ovoid-shaped basidiospores (BS). Bars = 50 μm.

FIGURE 9

A Puccinia achnatheri-sibirici teliospore (TS) showing each of two cells could germinate to produce a metabasidium. Panel (A) showing that each cell of a two-celled teliospore (TS) of Puccinia achnatheri-sibirici can germinate to produce a metabasidium (M). Bars = 50 μm. (B) Formation of secondary basidiospores (SB) at the tips of primary basidiospore (PB). Bars = 50 μm.