The hyperparasitic strain CPA001 was isolated from Pst urediniospores in Northwest A&F University, Yangling, Shaanxi, China. Firstly, urediniospores of Pst were propagated on the susceptible wheat cultivar Mingxian 169 as previously described (Cao et al., 2008). Wheat seedlings inoculated with urediniospores of Pst were kept in a growth chamber at about 16°C and 80–90% relative humidity (Lu et al., 2011). In total, 151 seedling plants were inoculated. Fourteen to twenty days after inoculation, more than half of the uredinia changed color from fresh yellow orange to gray or dark gray. Gray urediniospores were then transferred onto potato dextrose agar (PDA) medium with a sterilized needle. After incubation at 25° for 3 days, mycelia from the colony margins were transferred to fresh PDA plate and singe-spore purified to obtain a pure culture. The purified culture was stored on PDA slants at 4–8°C.

For the morphological observation, mycelial disks of 5 mm in diameter were taken from the growing margins of 3-day old PDA culture, transferred to potato carrot agar (PCA) plates and incubated at 25°C in a 12-h photoperiod for 1 week to induce conidia production. In addition, microscope slide cultures were prepared by placing a small amount of mycelia on PDA medium blocks (5 mm diameter) overlaid by a cover slip (Wang et al., 2015). Examination of the morphological characteristics of hyphae, conidiophores and conidia were conducted using an Olympus BX51T-32P01 optical microscope.

To further observe the ultrastructure of the parasitic fungus, wheat leaves bearing uredinia with abnormal colors were cut into pieces approximately 0.5 cm × 0.5 cm in size for scanning electronic microscope (SEM). Samples were immersed in 4.0% glutaraldehyde (pH 6.8) and fixed at 4°C for 4 h. Then samples were washed four times with 0.1 M phosphate buffer for 15 min each. Subsequently, samples were dehydrated for 30 min each in 30, 50, 70, 80, and 90% ethanol series, and finally 3 repeats in 100% ethanol. Samples were dried in a CO₂ vacuum, and sputter coated with gold (E-1045, Hitachi, Japan) for SEM examination (S-4800, Hitachi, Japan).

The susceptible wheat cultivar Mingxian 169 was used for propagating Pst urediniospores. When the first leaf had successfully expanded after 10 days, seedlings were inoculated with CYR32, a predominant race of Pst in China. Approximately 14 days later, the diseased leaves bearing urediniospores were inoculated with the mycoparasite A. alternata strain CPA001. The pure culture of CPA001 was formulated into spore suspension (1.0 × 10⁶ spores/mL in concentration) for spray inoculation. Healthy wheat leaves receiving A. alternata inoculation represented control check1 (CK1). Wheat leaves infected by Pst but not treated with the A. alternata conidia suspension represented CK2. Each treatment was carried out with wheat seedlings growing in three independent pots, with each pot containing about 24 plants. All treatments were placed in the same growth chamber, and observation of the symptoms was performed at the same time.

Simultaneously, Pst urediniaspores were directly inoculated with the A. alternata strain CPA001. The A. alternata conidia suspension (1.0 × 10⁶ spores/mL in concentration) was mixed with the Pst urediniospores, the spore mixture was sprayed on PDA medium, and incubated at 25° for 24 h. During co-cultivation, samples were collected to observe the dynamic infection process with SEM. Ultrastructural sample treatments were the same as ones described above.

The phenotype of disease was quantitively assessed by counting the number of uredinia pustules within a 5 cm² area at 9 days post CPA001 inoculation, using I_MAGEJ¹. To avoid bias among leaf samples, 35 random leaves were selected for each treatment and the entire experiment was repeated for three times.

Freshly collected urediniospores were cultured on sterile water at 9°C for 6 h, then placed on slides to count the numbers of germinated urediniospores using an Olympus BX51T-32P01 optical microscope. A germ tube length up to the one-half spore diameter was defined as germination. The germination rate was expressed as a percentage based on 100 urediniospores. One hundred urediniospores were selected randomly, and all experiments were performed at least three times.

Reference sequences from other Alternaria spp. were retrieved from GenBank (Table 1). Sequences were aligned with Clustal X (Thompson et al., 1997), and the final alignment was inspected with BioEdit 5.0.9.1 (Saitou and Nei, 1987). On the basis of the aligned sequences, a phylogenetic tree was constructed with the Maximum Likelihood (ML) method in the Molecular Evolutionary Genetics Analysis (MEGA) software version 6.0 with 1000 bootstrap replicates (Efron et al., 1996; Tamura et al., 2013).

Wheat leaves bearing normal yellow-colored uredinia and leaves bearing gray-colored uredinia resembling hyperparasite infection were examined under SEM (Figure 1). Yellow-colored uredinia contained round-shaped urediniospores, and contained no mycelium or spore of other fungal organism (Figures 1A,B). On the other hand, gray-colored uredinia were made up of shriveled or ruptured urediniospores, these spores were intertwined by dense filamentous hyphae, which become increasingly prevalent over time (Figures 1C–F). The dramatic shape change of urediniospores indicated their loss of cell viability. The prevalence of intertwined hyphae strongly suggested hyperparasitic colonization events.

Through in vitro culture, several candidate mycoparasitic fungal strains were obtained. Most strains resembled the previously reported C. cladosporioides in morphological appearance (Zhan et al., 2014), which were not characterized further. One strain, named CPA001, was characterized further and reported here. On PCA medium, the CPA001 culture initially developed light-gray colony and the center turned dark gray after 7 days (Figure 2A). The vegetative hyphae were brown, branched, septate, and 4 μm in diameter (Figure 2D). Conidia were typically obpyriform, dark brown, 20.2 to 35.2 μm × 8.0 to 12.6 μm in size, with 1–3 transverse and 0–1 longitudinal septate. Most conidia also had a short beak with a dimension of 1.6 to 9.4 μm × 2.9 to 5.0 μm (Figures 2B–D). SEM observation obtained more detailed morphological characteristics of the conidia and conidiophores (Figures 3A–F). SEM observations indicated that the fungus produced abundant conidia in long chains on short conidiophores (Figures 3A–C). Conidiophores were single, straight or slightly curved, and ranged from 12.3 to 60.6 μm × 2.2 to 4.0 μm (Figures 3E,F). These morphological features resembled Alternaria spp.

CPA001 was further identified to be A. alternata based on phylogenetic analysis with the ITS, GAPDH and RPB2 genetic markers (Figure 4). A range of Alternaria spp. reference isolates were selected for the phylogenetic tree construction. CPA001 was found to be most closely related to CBS121456, CBS127334, CBS126910,CBS795.72, CBS620.83, CBS107.53 and CBS115200, all belonging to the A. alternata species. The entire A. alternata clade was well-separated from other clades with a bootstrap value of 85%.

Pathogenicity testing showed that the obtained CPA001 strain could efficiently hyperparasitize Pst (Figure 5). Wheat leaves inoculated with Pst alone produced abundant orange-colored uredinia after 21 days post inoculation (Figure 5B). On the other hand, Pst pre-inoculated wheat leaves receiving subsequent A. alternata treatment showed a typical sign of mycoparasitic colonization, namely fewer rust pustule formation and abundant gray-colored hyphae covering the uredinia (Figures 5C–F). Such gray-colored hyphae were never observed with wheat leaves treated with A. alternata conidia suspension alone (CK1, Figure 5A). Reisolated strain from the parasitized uredinia showed the same morphological characteristics CPA001. At 9 days post CPA001 inoculation, the frequency of Pst pustule formation was merely 10% whereas that of the control treatment was 70% (Figure 6A). Urediniospores collected from CPA001-treated pustules also showed dramatically reduced viability (∼25% vs. 80%), indicated by germination rate (Figure 6B).

Scanning electronic microscope observations further confirmed that the A. alternata CPA001 strain could efficiently parasitize Pst. The A. alternata germ tube contacted with and penetrated into Pst urediniospores at 24 hpi, and caused complete urediniospore collapse at 36–48 hpi (Figures 7A–D).