Yingqing Luo

Penicillium astrolabium is a primary pathogenic fungus that causes grape blue mold during postharvest, leading to substantial losses in the grape industry. Nevertheless, hypovirulence-associated mycoviruses can attenuate the virulence of postharvest grape-rot pathogens, thereby offering a promising biocontrol tool. Characterizing the mycovirus repertoire of P. astrolabium is imperative for grape protection, yet remains largely unexplored. Here, we screened six strains harboring viruses in 13 P. astrolabium isolates from rotted grapes. Using high-throughput sequencing, four novel dsRNA viruses and two +ssRNA viruses were identified from the six P. astrolabium strains. The dsRNA viruses belonged to two families—Chrysoviridae and Partitiviridae—and were designated to Penicillium astrolabium chrysovirus 1 (PaCV1), Penicillum astrolabium partitivirus 1′ (PaPV1′), Penicillum astrolabium partitivirus 2 (PaPV2), and Penicillum astrolabium partitivirus 3 (PaPV3). For the +ssRNA viruses, one was clustered into the Alphaflexiviridae family, while the other one was clustered into the Narnaviridae family. The two +ssRNA viruses were named Penicillium astrolabium alphaflexivirus 1 (PaAFV1) and Penicillium astrolabium narnavirus 1 (PaNV1), respectively. Moreover, several viral genomic contigs with non-overlapping and discontinuous sequences were identified in this study, which were probably representatives of five viruses from four families, including Discoviridae, Peribunyaviridae, Botourmiaviridae, and Picobirnaviridae. Taken together, our findings could expand the diversity of mycoviruses, advance the understanding of mycovirus evolution in P. astrolabium, and provide both potential biocontrol resources and a research system for dissecting virus–fungus–plant interactions.

Cirsium arvense (L.) Scop. var. integrifolium Wimm. & Grab. (syn. Cirsium setosum (Willd.) MB.), a perennial herbaceous plant in the Asteraceae family, is widely distributed across Europe, Australia, Asia, North Africa, North and South America (Nischwitz and Newcombe 2004; Yang et al. 2024). In China, its tender stems and leaves are utilized as a vegetable and also a traditional medicine because of its heat-clearing efficacy (Jiang et al. 2013). In July 2023, symptoms of powdery mildew were observed in C. arvense var. integrifolium populations in Deyang city (N30°35', E104°37'), Sichuan Province, China. Approximately 40.38% (63/156) of the surveyed plants showed symptoms. The disease manifested as irregular small white, powdery patches, typically on the upper surface of leaves, which progressed to extensive hyphal growth covered over 90% of the leaf surface. With the disease advanced, the infected leaves wilted and senesced. Voucher specimens of symptomatic leaves (CaPM-ZL) as representative samples have been deposited at China West Normal University. The conidiophores were unbranched, straight, and measured 78.47 to 246.69 µm (avg. 154.60±49.59 μm) × 4.98 to 29.50 µm (avg. 13.58±8.06 μm) (n= 30). The conidia, produced singly, were hyaline, ellipsoid-ovoid to nearly cylindrical with fibrosin bodies, measuring 53.12 to 72.47 μm (avg. 59.82±6.41 μm) in length and 25.86 to 41.72 μm (avg. 33.85±4.87 μm) in width (n=30). Scanning electron microscopy revealed the conidia were turgid and smooth, with gentle contractions or bulges at both poles. The pathogen was provisionally identified as Podosphaera xanthii based on morphological characteristics (Braun and Cook 2012). To confirm the identification, the internal transcribed spacer region (ITS) and nuclear ribosomal large subunit (LSU) for CaPM-ZL was amplified using primers ITS4/PM5 and LSU1/LSU2, and analyzed using the BLASTn search of GenBank, respectively. The ITS (PP951890) and LSU (PQ644087) sequences were deposited into GenBank and showed 100% similarity with P. xanthii sequences (ITS: MT260063, KY388504; LSU: OP765401, PP897382). Based on the concatenated sequences of ITS and LSU, the phylogenetic analysis was performed in MEGAX with maximum likelihood method (Kumar et al. 2016) clustered CaPM-ZL into the Podosphaera xanthii clade with a 99% bootstrap support value. Combining morphological and phylogenetic analyses, CaPM-ZL was conclusively identified as Podosphaera xanthii. To evaluate pathogenicity, leaves of 3 healthy potted C. arvense var. integrifolium plants (3 leaves per plant) were inoculated by gently pressing them with diseased leaves, while 3 no-contact plants served as controls. The inoculated plants were isolated from the controls in a greenhouse maintained at 27±1°C, with a 14-hour light period and 80% relative humidity. After 7 days, the inoculated plants exhibited powdery mildew symptoms, while the control remained asymptomatic. Morphological characteristics and sequencing of inoculated pathogen were matched those of naturally infected plants. P. xanthii has been previously reported on Acalypha indica (Wu et al. 2024), Siraitia grosvenorii (Pan et al. 2024), and Vigna unguiculata (Zhang et al. 2024) in China. To the best of our knowledge, this is the first report of powdery mildew caused by P. xanthii on C. arvense var. integrifolium in China. This species as a new host of P. xanthii suggests that controlling the spread of this disease may be more challenging, and will contribute to an increased understanding of the host range. Our finding will provide the fundamental knowledge for future powdery mildew diagnosis and control.

Symphyotrichum subulatum (Michx.) G. L. Nesom (syn. Aster subulatus), an annual herb in the Asteraceae family, is native to North America. Nowadays, it has become an invasive weed in several provinces of China, including Jiangsu, Zhejiang, and Sichuan (Li and Xie, 2002). Despite being invasive, this species holds significance in Chinese medicine, where it is used for the external treatment of eczema and swollen sore poison (Hu, 2020). In June 2023, symptoms of powdery mildew were observed in S. subulatum populations in Deyang and Nanchong, Sichuan Province, China. About 32.73% among 55 surveyed S. subulatum plants showed signs of infection. Symptoms initially appeared as small, scattered white powdery patches on the leaves, which enlarged and coalesced over time. Subsequently, hyphal growth forming extensive conidia covered up to 90% of the leaf area on both surfaces (Fig. S1A, B), and the infected leaves withered and fell off (Fig. S1A). A specimen was archived at China West Normal University (SsPM-ZL). Conidiophores were cylindrical and erect, 66.4 to 183.2 µm (avg. 108.2±40.8 μm) in length (n=30) (Fig. S1C). Conidia, produced singly, were ellipsoid-ovoid to nearly cylindrical, measuring 29.5 to 36.7 μm in length (avg. 32.9±2.6 μm) and 16.0 to 19.9 μm in width (avg. 17.4±1.3 μm), lacking distinct fibrosin bodies (n=30) (Fig. S1D). Under a scanning electron microscope, turgid conidia displayed reticulate wrinkles on the surface, with gentle contractions or bulges at both poles (Fig. S1E, F). Based on these characteristics, the powdery mildew fungus was consistent with the genus Golovinomyces (Bradshaw et al. 2022a). To confirm the identity of the causal fungus of specimen (SsPM-ZL), the calmodulin (CAM), RNA polymerase II subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and the internal transcribed spacer (ITS) were amplified using PMCAMF/R, PMRPB2F/R, PMGAPDH1/3R, PMGSF/R, and PM5/ITS4 primers (Bradshaw et al. 2022b), and deposited in GenBank (CAM: OR761878; RPB2: OR761881; GAPDH: OR761879; GS: OR761880; ITS: OR758452). BLAST analysis showed 99 to 100% identity with the sequences of Golovinomyces ambrosiae (FH00941234) for CAM (ON101658, 99.65%), RPB2 (ON119165, 100%), GS (ON075690, 99.78%), and ITS (ON073876, 99.47%). Phylogenetic analysis was performed in MEGAX with maximum likelihood method (Kumar et al. 2016) and clustered SsPM-ZL into the G. ambrosiae clade with a 100% bootstrap support value based on the concatenated sequences of CAM, RPB2, GAPDH, GS and ITS (Fig. S2). Combining morphological and phylogenetic analyses, SsPM-ZL was identified as Golovinomyces ambrosiae. To evaluate pathogenicity, leaves of 3 healthy potted S. subulatum plants (3 leaves per plant) were inoculated by gently pressing them with diseased leaves, while 3 non-contact plants were used as control. Plants in two groups were incubated in separate greenhouses maintained at 27±1°C, with a photoperiod of 14 hours and a relative humidity of 80%. After 7 days, the inoculated plants exhibited symptoms of powdery mildew (Fig. S1H, J), while the control plants remained asymptomatic (Fig. S1G, I). Morphological characteristics of the artificially induced powdery mildew were consistent with those on naturally infected plants. Powdery mildew caused by G. ambrosiae has been reported affecting Helianthus tuberosus (Huang et al. 2017) and Bidens pilosa (Mukhtar et al. 2022) in China. To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on S. subulatum in China. Our finding will provide the fundamental knowledge for future powdery mildew diagnosis and the development of potential control strategies.