Medicinal Plant Research 2024, Vol.14, No.6, 345-357 http://hortherbpublisher.com/index.php/mpr 349 4.2 Artificial inoculation and pest challenge assays Artificial inoculation and pest challenge experiments are usually conducted in a greenhouse or laboratory environment to ensure that plant materials are exposed to specific pathogens or pests under uniform and reproducible conditions. By controlling the inoculation concentration, time and environmental factors, this method can accurately compare the resistance performance of various varieties. For instance, standardized inoculation with Alternaria alternata or Fusarium oxysporum and uniform release of aphids have been widely used in the study of resistance mechanisms and the screening of resistant varieties (Ding et al., 2023; Miao et al., 2023a; Li et al., 2024a; Seliem et al., 2024). Transgenic and gene-edited materials are also often tested under artificial inoculation for pest and disease stress to verify the effect of specific resistance genes (Shinoyama et al., 2015; Pak et al., 2020). Standardized scoring systems are key to quantifying disease severity and insect damage. Common scoring methods include numerical scoring or grading, examining indicators such as lesion size, leaf yellowing, insect population density or plant growth potential. Based on the scoring results, resistance levels can be assigned, allowing objective comparisons and facilitating early identification of resistant materials (Fu et al., 2018; Seliem et al., 2024). For example, white rust resistance testing uses obvious susceptible/resistant phenotypes, while aphid resistance is quantified by damage index and population growth rate (Kos et al., 2014; Nabeshima et al., 2014; Fu et al., 2018). 4.3 Molecular and biochemical validation Molecular validation mainly studies the expression of candidate resistance genes through techniques such as RNA-Seq, qRT-PCR or transcriptome analysis. These methods can identify differentially expressed genes (DEGs) activated under pest and disease infestation conditions and their regulatory pathways. For example, overexpression or silencing of genes such as CmWRKY8-1, CmMYB15 and CmNAC083 have been shown to regulate the resistance of Hangbaiju to wilt, aphids and black spot, respectively (An et al., 2019; Ding et al., 2023; Miao et al., 2023a; Huang et al., 2023; Zhang et al., 2025). Expression profiling can also help reveal the key roles of plant hormone signaling, secondary metabolism synthesis and defense enzyme activity in resistance responses (Liu et al., 2020; 2021; Zhang et al., 2023; Li et al., 2024a; b). Marker-assisted selection (MAS) uses molecular markers (e.g., SSR, ISSR, and SNP) that are closely linked to resistance loci to achieve early screening of resistant materials and efficient breeding. ISSR markers have been used to distinguish leaf spot-resistant Hangbaiju varieties, and SNP markers have been associated with white rust resistance in parental populations (Fu et al., 2018; Sumitomo et al., 2021; 2022). Association analysis and genome-wide association analysis (GWAS) have also identified markers associated with aphid and white rust resistance, enabling the application of MAS in resistance breeding (Fu et al., 2018; Xu et al., 2021). Currently, MAS strategies are being combined with field and laboratory screening to accelerate the development and application of resistant varieties. 5 Case Studies 5.1 Analysis of CmWD40-mediated disease-resistant Hangbaiju In the process of screening disease- and insect-resistant Hangbaiju varieties, the study of immune regulation mechanisms at the molecular level provides precise targets for the breeding of superior germplasm. Zhang et al. (2025) reported an effector protein AaAlta1 derived fromAlternaria alternata, which can recognize and activate the WD40 repeat protein CmWD40 in Chrysanthemum morifolium, thereby activating the plant's jasmonic acid (JA) signaling pathway and inducing programmed cell death and defense responses. The study showed that in Hangbaiju expressing AaAlta1, JA pathway-related genes were upregulated, indicating that this effector has the potential to induce host disease resistance. At the same time, CmWD40 was expressed in a circadian rhythm under normal conditions, and its overexpression significantly enhanced the resistance of chrysanthemums to black spot disease, while gene silencing reduced resistance, verifying its positive regulatory role in the disease response (Figure 2). The study also used subcellular localization and bimolecular fluorescence complementation (BiFC) technology to confirm that AaAlta1 interacts with CmWD40 in the cell nucleus.
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