As an important nonwood bioresource, fishscale bamboo (Phyllostachys heteroclada Oliver) is widely distributed in the subtropical region of China. Rhombic-spot disease, caused by Neostagonosporella sichuanensis, is one of the most serious diseases that threatens fishscale bamboo health. However, there is limited knowledge about how rhombic-spot disease influences the diversity and structures of phyllosphere fungal communities. In this study, we investigated the phyllosphere fungal communities from stems, branches, and leaves of fishscale bamboo during a rhombic-spot disease outbreak using 18S rRNA sequencing. We found that only the phyllosphere fungal community from stems was significantly affected by pathogen invasion in terms of community richness, diversity, and structure. FUNGuild analysis revealed that the major classifications of phyllosphere fungi based on trophic modes in stems, branches, and leaves changed from symbiotroph-pathotroph, no obvious dominant trophic mode, and symbiotroph to saprotroph, saprotroph–pathotroph–symbiotroph, and saprotroph–symbiotroph, respectively, after pathogen invasion. The fungal community composition of the three tissues displayed significant differences at the genus level between healthy and diseased plants. The associations among fungal species in diseased samples showed more complex co-occurrence network structures than those of healthy samples. Taken together, our results highlight the importance of plant pathological conditions for the assembly of phyllosphere fungal communities in different tissues.

Thaumatin-like proteins (TLPs) are involved in the plant defense response against pathogens, and most of them exhibit antifungal activity. However, the role of TLPs in pathogen-induced defense responses in spruce is not fully understood. In this study, four TLP genes encoding thaumatin-like protein, designated as PlTLP1–4, were isolated and identified from Picea likiangensis needles. Sequence analysis showed that PlTLP1, PlTLP3, and PlTLP4 contained 16 conserved cysteine residues, while PlTLP2 had only 10 conserved cysteine residues. qPCR analysis showed that PlTLPs were expressed in all tissues tested, PlTLP1, PlTLP3, and PlTLP4 had the highest expression levels in young fruits, while PlTLP2 had the highest expression levels in roots. In addition, the expression levels of four PlTLPs were significantly upregulated during infection by Lophodermium piceae. Four recombinant PlTLPs expressed in Escherichia coli exhibited obvious β-1,3-glucanase activity. The antifungal activity assay showed that four recombinant PlTLPs had significant inhibitory effects on the mycelial growth of L. piceae, Fusarium proliferatum, Botrytis cinerea, and Roussoella doimaesalongensis. Microscopic observation revealed that the recombinant PlTLP1–4 induced the morphological changes of the mycelia of L. piceae, and the recombinant PlTLP2 and PlTLP3 induced the morphological changes of the mycelia of F. proliferatum and R. doimaesalongensis, while all the recombinant PlTLPs had no obvious negative effect on the morphology of B. cinerea mycelium. These results suggest that PlTLP genes may play an important role in the defense response of P. likiangensis against L. piceae invasion.

Phenylalanine ammonia-lyase (PAL) is the crucial enzyme of the phenylpropanoid pathway, which plays an important role in plant disease resistance. To understand the function of PAL in Picea asperata, in this study, the full-length cDNA sequence of the PAL gene from this species was isolated and named PaPAL. The gene contains a 2160-bp open reading frame (ORF) encoding 720 amino acids with a calculated molecular weight of 78.7 kDa and a theoretical isoelectric point of 5.88. The deduced PaPAL protein possesses the specific signature motif (GTITASGDLVPLSYIA) of phenylalanine ammonia-lyases. Multiple alignment analysis revealed that PaPAL has high identity with other plant PALs. The tertiary structure of PaPAL was predicted using PcPAL from Petroselinum crispum as a template, and the results suggested that PaPAL may have a similar function to that of PcPAL. Furthermore, phylogenetic analysis indicated that PaPAL has a close relationship with other PALs from the Pinaceae species. The optimal expression condition of recombinant PaPAL in Escherichia coli BL21 (DE3) was 0.2 mM IPTG (isopropyl β-D-thiogalactoside) at 16 °C for 4 h, and the molecular weight of recombinant PaPAL was found to be approximately 82 kDa. Recombinant PaPAL was purified and exhibited high PAL activity at optimal conditions of pH 8.6 and 60 °C. Quantitative real-time PCR (qRT-PCR) showed the PaPAL gene to be expressed in all tissues of P. asperata tested, with the highest expression level in the needles. The PaPAL gene was induced by the pathogen (Lophodermium piceae), which caused needle cast disease, indicating that it might be involved in defense against needle cast disease. These results provide a basis for understanding the molecular mechanisms of the PAL gene in the process of P. asperata disease resistance.

Plant chitinase, a member of the pathogenesis-related proteins and glycoside hydrolase family 19, plays a crucial role in plant resistance to pathogenic fungi. In this study, we isolated a chitinase gene encoding PaChi from Picea asperata, containing an open reading frame of 768 bp that encodes 255 amino acid residues. PaChi includes the chitin-binding domain, catalytic domain, and active site characteristic of class IV chitinases. Homologous modeling and phylogenetic analysis indicated that PaChi exhibits the highest structural similarity and closest genetic relationship with the chitinase of Norway spruce, suggesting potential functional similarities. RT-qPCR analysis demonstrated that PaChi expression is relatively high in the roots of P. asperata. Additionally, PaChi transcription levels were significantly upregulated during the early stages of Lophodermium piceae infection, indicating active involvement in the defense response of P. asperata against L. piceae infection. SDS-PAGE analysis revealed that the recombinant PaChi has a molecular weight of approximately 27 kDa. The purified recombinant PaChi exhibited a chitinase activity of 4.61 U/mg, with optimal pH and temperature at 7.0 and 40 °C, respectively. Notably, purified recombinant PaChi significantly affected the mycelial morphology of L. piceae, inclusion body concentration and cell swelling. These findings lay the groundwork for further understanding the molecular mechanisms by which PaChi is involved in biological stress.