Background: Plants maintain the steady-state balance of the mutually beneficial symbiosis relationship with their endophytic fungi through secondary metabolites. Meanwhile endophytic fungi can serve as biological inducers to promote the biosynthesis and accumulation of valuable secondary metabolites in host plants through a variety of ways. The composition and structure of endophytic fungal community are affected by many factors, including tissues, seasons and so on. In this work, we studied the community diversity, temporal and spatial pattern of endophytic fungi detected from the roots, stems and leaves of Huperzia serrata in different seasons. The correlation between endophytic fungi and huperzine A (HupA) content in plants was analyzed. Results: A total of 7005 operational taxonomic units were detected, and all strains were identified as 14 phyla, 54 classes, 140 orders, 351 families and 742 genera. Alpha diversity analysis showed that the diversity of endophytic fungi in stem and leaf was higher than that in root, and the diversity in summer (August) was lower than that in other months. NMDS analysis showed that the endophytic fungal communities of leaves, stems and roots were significantly different, and the root and leaf communities were also different between four seasons. Through correlation analysis, it was found that 33 genera of the endophytic fungi of H. serrata showed a significant positive correlation with the content of HupA (p < 0.05), of which 13 genera (Strelitziana, Devriesia, Articulospora, Derxomyces, Cyphellophora, Trechispora, Kurtzmanomyces, Capnobotryella, Erythrobasidium, Camptophora, Stagonospora, Lachnum, Golubevia) showed a highly significant positive correlation with the content of HupA (p < 0.01). These endophytic fungi may have the potential to promote the biosynthesis and accumulation of HupA in plant. Conclusions: This report is the first time to analyze the diversity of endophytic fungi in tissues of H. serrata in different seasons, which proves that there is variability in different tissues and seasonal distribution patterns. These findings provide references to the study of endophytic fungi of H. serrata.

Tree peonies (Paeonia suffruticosa Andr. and hybrids) are well-known ornamental and medicinal plants cultivated in temperate and subtropical regions around the world. From June to September 2021, severe leaf spot disease was observed on 3 tree peony cultivars (‘Luoyanghong’, ‘Moyushenghui’, ‘Roufurong’) in Xinxiang (35º29´N, 113º95´E) and Luoyang (34º64´N, 112º49´E), Henan Province, China. Leaf spot incidence was as high as 28% (‘Luoyanghong’), 45% (‘Moyushenghui’) and 67% (‘Roufurong’), respectively. Symptoms appeared initially as small purple spots less than 1 mm in diameter in the middle and upper parts of the leaves, and then evolved to coalescent lesions, causing brown scorch ultimately. From each cultivar, 5 diseased leaves were collected. Leaflet tissues (3-4 mm2) cut from spot margins were surface sterilized in 75% alcohol for 45 s, washed 5 times with sterile distilled water, and then cultivated on potato dextrose agar (PDA) medium at 28 °C in the dark. Eleven isolates were obtained, and colonies grown from single conidia on PDA were 80-85 mm in diameter after 10 d, with scattered small, dark-based spikes on the surface of the colonies. The aerial mycelium was cottony, dense, and dark gray near the center on the reverse side. Conidia were cylindrical to clavate, with rounded apex and rounded base, and the conidia contents were granular, 8.44-14.06×3.60-4.31 μm (mean=11.28×3.69 μm, n=40). Appressoria were mostly subglobose or with a few broad lobes, pale to medium brown, 3.36-6.72×3.35-5.60 μm (mean=5.02×4.55 μm, n=20). Based on the culture representation and conidial morphology, the isolates were characterized as Colletotrichum gloeosporioides species complex (Weir et al. 2012; Fu et al. 2019). To further identity the species, the actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the ribosomal internal transcribed spacers (ITS) loci of isolates PSW0002, PSW0008 and PSW0009 were amplified using ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF/GDR, and ITS1/ITS4, primers (Weir et al. 2012; Schena et al; 2014; Kim et al. 2021; Li et al. 2021). Fifteen sequences were deposited in GenBank (ACT for OP225605, OP225606, and OP225607, CAL for OP225608, OP225609 and OP225610, CHS for OP225611, OP225612 and OP225613, GAPDH for ON321897, OP225614, and OP225615, and ITS for ON323473, OP214349 and OP214350 ), which showed 100% sequence similarity to Colletotrichum aenigma (JX009443 and JX009519 for ACT, JX009683 and JX009684 for CAL, JX009774 and JX009903 for CHS-1, JX010244 and JX009913 for GAPDH, JX010243 and JX010148 for ITS). Three isolates clustered with C. aenigma (ex-holotype culture ICMP 18608) in the multi-locus phylogenetic tree with a bootstrap value of 100%. To achieve Koch's postulates, pathogenicity was tested on 5-year-old healthy potted plants (‘Luoyanghong’). Thirty leaves were inoculated with 10 µL conidial suspension (isolate PSW0002, 1×106 conidia/mL) and the controls were inoculated with sterile water. Plants were placed in a greenhouse at 28°C under conditions with 12 h photoperiod and 90% relative humidity. After 5 to 10 days, distinct spots were observed on the inoculated leaves, while the control leaves showed no symptoms. C. aenigma was reisolated from all inoculated leaves, but not from the control. C. aenigma has been reported to cause anthracnose on Pyrus pyrifolia (Weir et al. 2012), Camellia sasanqua (Chen et al. 2019), Juglans regia (Wang et al. 2020), Paeonia ostii (Ren et al. 2020), and Capsicum annuum (Sharma et al. 2022). A previous study reported C. gloeosporioides as a pathogen of anthracnose in tree peonies in China (Xuan et al. 2017), the typical symptoms were big necrotic lesions (5-10 mm diam) on leaves，which were significantly different from those caused by C. aenigma. To our knowledge, this is the first report of C. aenigma causing anthracnose in tree peonies in China. This finding may help to take effective control of anthracnose in tree peonies.

Paeonia ludlowii is a threatened and valuable germplasm in the cultivated tree peony gene pool, with distinctive traits such as tall stature, pure yellow flowers, and scarlet foliage in autumn. However, the crossability barrier limits gene transfer from P. ludlowii to cultivated tree peony. Therefore, our study investigated the reasons for the lack of crossability between P. ludlowii and Paeonia ostii ‘Fengdan’. Distant cross pollination (DH) resulted in the formation of many calloses at the ends of the pollen tubes, which grew non-polar, twisted, entangled, and often stopped in the style. Pollen tubes elongated the fastest in self-pollination (CK), and pollen tubes elongated faster and fewer pollen tube abnormalities were observed in stigmas treated with KCl solution before pollination (KH) than in DH. During pollen–pistil interactions, the absence of stigma exudates, high levels of H₂O₂, O₂⁻, MDA, ^•OH, ABA, and MeJA, and lower levels of BR and GA₃ may negatively affect pollen germination and pollen tube elongation in the pistil of P. ostii ‘Fengdan’. Pollen tubes in CK and KH penetrated the ovule into the embryo sac at 24 h after pollination, whereas only a few pollen tubes in DH penetrated the ovule at 36 h after pollination. Pre-embryo abnormalities and the inhibition of free nuclear endosperm division resulted in embryo abortion in most of the fruits of DH and many fruits of KH, which occurred between 10 and 20 days after pollination, whereas embryos in CK developed well. Early embryo abortion and endosperm abortion in most of the fruits of DH and KH led to seed abortion. Seed abortion in KH and DH was mainly due to an insufficient supply of auxins and gibberellins and lower content of soluble protein and soluble sugars. The cross failure between P. ludlowii and P. ostii ‘Fengdan’ is mostly caused by a pre-fertilization barrier. KH treatment can effectively promote pollen tube growth and facilitate normal development of hybrid embryos. These findings provide new insights into overcoming the interspecific hybridization barrier between cultivated tree peony varieties and wild species.