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A key step in the study of tree pathology is the identification of an appropriate method for inoculating pathogens of diseases in branches and trunks. Pathogens of diseases in branches and trunks are commonly inoculated through punching, burning, and toothpick inoculation. However, there is a lack of comparative analyses of the inoculation outcomes of these three methods. In this work, six-year-old P. alba var. pyramidalis were inoculated with V. sordida using punching, burning, and toothpick techniques to investigate the differences in the effectiveness of these inoculation methods. Results reveal that the incidence rate was 93.55% in the toothpick inoculation group, significantly higher than the 80.65% in the burning inoculation group (chi-square, n = 90, p = 0.007), while punching inoculation exhibited significant pathological responses in the early stages, with spontaneous healing in the later stage. Additionally, toothpick inoculation was more efficient in inducing Valsa canker when inoculating the pathogen at the bottom of the tree, with lower intra- and inter-row spacing (stand density) providing better outcomes than higher intra- and inter-row spacing. The results of this study demonstrate that toothpick inoculation is an optimal option for studying the artificial inoculation of V. sordida in six-year-old P. alba var. pyramidalis, providing technical support for research on poplar diseases and offering a theoretical basis for the inoculation of other diseases in the branch and trunk.

Recently, a novel poplar mosaic disease caused by bean common mosaic virus (BCMV) was investigated in Populus alba var. pyramidalis in China. Symptom characteristics, physiological performance of the host, histopathology, genome sequences and vectors, and gene regulation at the transcriptional and posttranscriptional levels were analyzed and RT−qPCR (quantitative reverse transcription PCR) validation of expression was performed in our experiments. In this work, the mechanisms by which the BCMV pathogen impacts physiological performance and the molecular mechanisms of the poplar response to viral infection were reported. The results showed that BCMV infection decreased the chlorophyll content, inhibited the net photosynthesis rate (Pn) and stomatal conductance (Gs), and significantly changed chlorophyll fluorescence parameters in diseased leaves. Transcriptome analysis revealed that the expression of the majority of DEGs (differentially expressed genes) involved in the flavonoid biosynthesis pathway was promoted, but the expression of all or almost all DEGs associated with photosynthesis-antenna proteins and the photosynthesis pathway was inhibited in poplar leaves, suggesting that BCMV infection increased the accumulation of flavonoids but decreased photosynthesis in hosts. Gene set enrichment analysis (GSEA) illustrated that viral infection promoted the expression of genes involved in the defense response or plant-pathogen interaction. MicroRNA-seq analysis illustrated that 10 miRNA families were upregulated while 6 families were downregulated in diseased poplar leaves; moreover, miR156, the largest family with the most miRNA members and target genes, was only differentially upregulated in long-period disease (LD) poplar leaves. Integrated transcriptome and miRNA-seq analyses revealed 29 and 145 candidate miRNA−target gene pairs; however, only 17 and 76 pairs, accounting for 2.2% and 3.2% of all DEGs, were authentically negatively regulated in short-period disease (SD) and LD leaves, respectively. Interestingly, 4 miR156/SPL (squamosa promoter-binding-like protein) miRNA−target gene pairs were identified in LD leaves: the miR156 molecules were upregulated, but SPL genes were downregulated. In conclusion, BCMV infection significantly changed transcriptional and posttranscriptional gene expression in poplar leaves, inhibited photosynthesis, increased the accumulation of flavonoids, induced systematic mosaic symptoms, and decreased physiological performance in diseased poplar leaves. This study elucidated the fine-tuned regulation of poplar gene expression by BCMV; moreover, the results also suggested that miR156/SPL modules played important roles in the virus response and development of viral systematic symptoms in plant virus disease.

Insect infestations cause substantial changes in the leaves they attack, but the effects of insect infestation on photosynthesis and gene expression in the whole infestation period are rarely reported. In this study, the effects of poplar aphid (Chaitophorus populialbae Boyer de Fonscoloube) on photosynthetic rate and gene expression of Populus alba var. pyramidalis were studied, from 2 to 36 days after low-density aphid inoculation (DAI). The results revealed a dynamic change of photosynthesis in poplar after aphid infestation: compensatory increased at 8 DAI, was inhibited at 17 DAI, but repaired after 21 DAI. Aphid infestation promoted the expression of the majority of differentially expressed genes (DEGs) at 4, 6, 8, and 21 DAI. The DEGs were enriched into a plant–pathogen interaction, plant hormone signal transduction, and MAPK signaling pathway, suggesting a significant but transient resistance to disease or other stresses. Photosynthesis-related DEGs were downregulated at 8 DAI, which might result in photosynthetic inhibition at 17 DAI. The expression of flavonoid biosynthesis-related DEGs dynamic changed from all downregulated at 2 DAI to almost all upregulated at 21 DAI, suggesting a persistent resistance to insect infestation. This study reveals dynamic changes in photosynthesis, resistance to pathogens, and insects in poplar leaves after aphid infestation.

Fungal pathogens can induce canker lesions, wilting, and even dieback in many species. Trees can suffer serious physiological effects from stem cankers. In this study, we investigated the effects of Botryosphaeria dothidea (B. dothidea) on Populus bolleana (P. bolleana) leaves photosynthesis and stomatal responses, when stems were inoculated with the pathogen. To provide experimental and theoretical basis for preventing poplar canker early. One-year-old poplar stems were inoculated with B. dothidea using an epidermal scraping method. In the early stage of B. dothidea inoculation (2–14 days post inoculation, dpi), the gas exchange, stomatal dynamics, hormone content, photosynthetic pigments content, chlorophyll fluorescence parameters, and non-structural carbohydrate (NSC) were evaluated to elucidate the pathophysiological mechanism of B. dothidea inhibiting photosynthesis. Compared with the control groups, B. dothidea noteworthily inhibited the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), and other photosynthetic parameters of poplar leaves, but stomatal limit value (Ls) increased. Consistent with the above results, B. dothidea also reduced stomatal aperture and stomatal opening rate. In addition, B. dothidea not only remarkably reduced the content of photosynthetic pigments, but also decreased the maximum photochemical efficiency (Fv/Fm), actual photochemical efficiency (ΦPSII), electron transfer efficiency (ETR), and photochemical quenching coefficient (qP). Furthermore, both chlorophyll and ΦPSII were positively correlated with Pn. In summary, the main reason for the abated Pn under stem canker pathogen was that B. dothidea not merely inhibited the stomatal opening, but hindered the conversion of light energy, electron transfer and light energy utilization of poplar leaves. In general, the lessened CO2 and Pn would reduce the synthesis of photosynthetic products. Whereas, sucrose and starch accumulated in poplar leaves, which may be due to the local damage caused by B. dothidea inoculation in phloem, hindering downward transport of these products.