Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are among the world’s most serious and widespread orchid viruses; they often infect orchids, causing devastating losses to the orchid industry. Therefore, it is critical to establish a method that can rapidly and accurately detect viruses in the field using simple instruments, which will largely reduce the further spread of viruses and improve the quality of the orchid industry and is suitable for mass promotion and application at grassroots agrotechnical service points. In this investigation, we established a rapid amplification method for virus detection at 39 °C for 35 min to detect the presence of CymMV and ORSV simultaneously, sensitively, and specifically in orchids. Primers for the capsid protein (CP)-encoding genes of both viruses were designed and screened, and the reaction conditions were optimized. The experimental amplification process was completed in just 35 min at 39 °C. There were no instances of nonspecific amplification observed when nine other viruses were present. The RPA approach had detection limits of 10⁴ and 10³ copies for pMD19T-CymMV and pMD19T-ORSV, respectively. Moreover, the duplex RT-RPA investigation confirmed sensitivity and accuracy via a comparison of detection results from 20 field samples with those of a gene chip. This study presents a precise and reliable detection method for CymMV and ORSV using RT-RPA. The results demonstrate the potential of this method for rapid virus detection. It is evident that this method could have practical applications in virus detection processes.

The rose industry in China faces significant challenges due to viral pathogens such as Prunus necrotic ringspot virus (PNRSV), apple stem grooving virus (ASGV), rose virus C (RVC), and Citrus tatter leaf virus (CTLV). This study aims to establish a rapid, efficient, and accurate detection method for these viral diseases to aid in their prevention and control. Based on morphological and microscopic observations, coat protein and polyprotein gene sequences of these viruses were retrieved from the NCBI database, and specific primers were designed based on highly conserved regions. Primer concentration, cDNA concentration, and annealing temperature were optimized to develop a multiplex RT-PCR detection system. The optimal reaction system was established with a total volume of 50 μl, with upstream and downstream primer concentrations of 0.3 μmol/liter for ASGV and CTLV, and 0.2 μmol/liter for PNRSV and RVC, with template cDNA at 2.0 ng/μl. Using touchdown PCR, we performed 10 cycles with an annealing temperature gradient of 65 to 55°C, decreasing by 1°C per cycle, followed by 20 cycles with a constant annealing temperature of 55°C. This method was successfully applied to 23 cut rose samples, yielding clear and distinct bands. These results demonstrate that the method can efficiently and accurately detect single or combined infections of the four rose viruses, offering a valuable tool for the rose industry in China.

Fusarium wilt (caused by Fusarium oxysporum f. sp. Lilii) is one of the most damaging diseases in lily (Lilium sargentiae Wilson). Although some F. oxysporum-resistant lily varieties have been identified and are being utilized in resistant breeding, the regulation network of the resistance-associated mechanisms is yet to be studied due to the lack of reliable reference genes for qRT-PCR (quantitative reverse transcription PCR) normalization. The reliability of results by qRT-PCR relies mainly on the stability of the reference genes. This study investigated the reliability of nine candidate reference genes (CYP, EF1-α, GAPDH, TUB, UBQ, AQP, HIS, PGK, and RPL13) for qRT-PCR analysis of F. oxysporum-resistant genes. Expression stability analysis via common programs GeNorm, BestKeeper, and NormFinder, at different time points post-inoculation of F. oxysporum, revealed that all nine genes met the basic requirements of reference genes. Amongst them, HIS and GAPDH displayed the highest and the lowest expression stability, respectively. The reliability of HIS was further validated by analyzing the expression levels of four resistance-related candidate genes. The expression patterns of the four target genes were consistent with their responses to pathogenetic fungi in other plants. Our results show that HIS is the most suitable reference gene for accurately normalizing F. oxysporum-resistant genes’ expressions in L. sargentiae.

Marigold (Tagetes spp.), a widely cultivated popular ornamental plant that is highly susceptible to fungal diseases, particularly leaf spot and stem wilt, which can substantially reduce its ornamental and economic value. This study aimed to identify the causal pathogens responsible for these diseases and evaluate the efficacy of selected antifungal agents against them. The causal agent of leaf spot was identified as Alternaria alternata, whereas stem wilt was caused by the Fusarium solani species complex (FSSC), based on Koch’s postulates, morphological characterization, and molecular identification. The antifungal activities of five commercially available fungicides were subsequently evaluated against both pathogens. The results showed that isomycete fluridine suspension (40%), metalaxyl–oxametrin suspension (32%), eugenol liquid (0.3%), pyrazolesterin methyl thiobacillin suspension (41%), and pyrazolesterin tebuconazole suspension (30%) demonstrated varying degrees of inhibitory activity. The EC50 values for A. alternata ranged from 10.588 to 49.604 mg/L, while those against FSSC ranged from 12.273 to 27.921 mg/L. These findings provide insights into the potency of these fungicides and offer a scientific basis for developing integrated disease management strategies for marigold cultivation, although further research is required to validate and evaluate their long-term efficacy and environmental safety.

Objective: This study aimed to elucidate the regulatory mechanism of an auxin amide hydrolase gene (IAA-Leucine Resistant1-like Hydrolase, RhILL1) in the petal pigmentation of rose (Rosa hybrida), providing theoretical insight into the hormonal regulation of flower coloration at the molecular level. Methods: Using petals at Stage 3 (S3) of the cut rose cultivar ‘Pink Floyd’ as experimental material, we cloned the rose auxin amide hydrolase gene RhILL1 and validated its function via virus-induced gene silencing (VIGS). The expression levels of anthocyanin biosynthetic genes, anthocyanin content, and auxin (IAA) levels were analyzed to assess the role of RhILL1 in petal pigmentation. Results: The full-length open reading frame (ORF) of RhILL1 was cloned, spanning 1326 bp and encoding a 441-amino-acid protein harboring two conserved domains, Peptidase_M20 and M20_dimer, characteristic of the ILL1 protein family. Functional characterization was performed using VIGS. Quantitative real-time PCR (qRT-PCR) revealed that RhILL1 expression progressively increased from the Green (G) stage to S3, correlating with intensified petal coloration. Silencing RhILL1 resulted in visibly lighter petals, the reduced expression of anthocyanin biosynthetic genes, and a significant decrease in endogenous indole-3-acetic acid (IAA) levels compared with controls. Moreover, exogenous application of 10 μM naphthaleneacetic acid (NAA) to petals significantly preserved petal pigmentation. Conclusion: These findings suggest that RhILL1 contributes to the development and maintenance of petal coloration in rose, likely by modulating IAA levels, thereby influencing the expression of anthocyanin biosynthesis-related genes.