Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of β-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

Phellodendron amurense Rupr is a perennial herb in Rutaceae family, which used to treat jaundice and gastrointestinal diseases as the commonly traditional Chinese medicine. In the end of August 2013, typical signs and symptoms of leaf spot disease on P. amurense were observed at the Medicinal Herb Garden in Shenyang Agricultural University (SYAU), Shenyang, Liaoning, China. The fungus from symptomatic tissues was identified as Nothophoma quercina based on molecular phylogenetic analysis combined with morphological and cultural characteristics. To our knowledge, this is the first report of leaf spot on P. amurense caused by N. quercina in China.

Sweet cherry (Prunus avium L.) is becoming increasingly popular in China, but its postharvest quality deteriorates significantly during harvest storage and transport. Here, we investigated the efficiency of different modified atmosphere packaging (MAP) treatments on the quality and physiology of ‘Meizao’ sweet cherry during 60 days of cold storage (0 ± 0.5 °C). Fruits were sealed in four types of MAP low-density polyethylene (LDPE) liners (PE20, PE30, PE40, and PE50), with unsealed 20 μm LDPE packaging bags used as the control. Our findings demonstrated that PE30 packaging established an optimal gas composition (7.0~7.7% O₂ and 3.6~3.9% CO₂) that effectively preserved ‘Meizao’ sweet cherry quality. It maintained the fruit color, firmness, soluble solid content (SSC), titratable acidity (TA), and vitamin C (Vc) content while simultaneously delaying deteriorative processes such as weight loss, pedicel browning, and fruit decay. These results indicate that PE30 was the most suitable treatment for preserving the quality of ‘Meizao’ sweet cherries during cold storage. Furthermore, physiological research showed that significant inhibition of respiration rate was achieved by PE30, accompanied by maintained activities of antioxidant enzymes (CAT, POD, and SOD), which consequently led to reduced accumulations of ethanol and malondialdehyde (MDA) during cold storage. To date, no systematic studies have investigated the physiological and biochemical responses of ‘Meizao’ to different thickness-dependent LDPE-MAP conditions. These observations highlight the power of the optimized PE30 packaging as an effective method for extending the fruit storage life, delaying postharvest senescence, and maintaining fruit quality of ‘Meizao’ sweet cherry.

This study aimed to identify fungal species causing fruit rot of chestnut (Castanea mollissima Blume) in Hebei Province, China and analyze the resistance differences among major cultivars. A total of 220 fungal isolates were obtained from healthy and diseased kernels, which were classified into six distinct genera: Diaporthe (48.6%), Talaromyces (22.3%), Alternaria (10.5%), Mucor (9.5%), Fusarium (5.5%), and Rhizopus (3.6%). Based on both morphological and molecular analyses, six representative isolates of the six genera were identified as Diaporthe eres Nitschke, Talaromyces rugulosus Samson, N. Yilmaz, Frisvad & Seifert, Alternaria alternata (Fr.) Keissl., Mucor circinelloides Tiegh., Fusarium proliferatum (Matsush.) Nirenberg, and Rhizopus stolonifer (Ehrenb.) Vuill. Among these, D. eres was first reported to cause fruit rot on C. mollissima in China. Moreover, disease resistance evaluation of major cultivars showed significant differences: YG, YSSF, and DBH exhibited strong resistance under both natural conditions (with 1.67% to 5.27% DI after 180 days storage) and artificial inoculation (with 32.96 ± 0.64 to 52.61 ± 0.55 DI); while YJ was highly susceptible (with 47.71% decay incidence and 70.50 ± 7.22 DI). Correlation analysis revealed that the disease index was negatively correlated with sucrose and sorbitol contents, but positively correlated with stachyose and fructose contents. This study advances the understanding of postharvest chestnut fruit rot and provides a theoretical basis for breeding resistant cultivars and developing control strategies to mitigate losses and ensure food safety.

AT-hook motif nuclear-localized (AHL) proteins are pivotal in plant growth, development, and stress responses. Nevertheless, there is limited research on AHL proteins in Malus sieversii. Our study identified 25 AHL genes from the M. sieversii genome, named MsAHL1–MsAHL25. The encoded protein sequences had lengths ranging from 195 to 554 amino acids, molecular weights from 19.17 to 58.53 kDa, and isoelectric points from 4.67 to 10.09. Chromosomal mapping revealed that these 25 genes were unevenly distributed across 10 chromosomes. Collinearity analysis of AHL genes in M. sieversii implied that gene loss might have occurred during its evolution. The phylogenetic tree classified the AHL proteins of M. sieversii into two subfamilies, showing a close relationship with multiple proteins of M. domestica. Promoter analysis indicated that the AHL genes in M. sieversii harbored numerous stress- and hormone-responsive elements, suggesting their potential role in various stress responses. qRT-PCR analysis of six representative MsAHLs under biotic and abiotic stresses demonstrated that the expression of MsAHL13, MsAHL15, and MsAHL17 was significantly upregulated under salt, drought, and cold stresses, while MsAHL01 expression was inhibited under low-temperature stress. All six MsAHLs were induced by the pathogen Valsa mali. Subcellular localization analysis of the specifically expressed protein MsAHL13 showed its nuclear location. Furthermore, luciferase and yeast two-hybrid assays confirmed the in vitro physical interaction between the MsAHL13 and MsMYB1 proteins. This research offers an important theoretical basis for further exploration of the functional mechanisms of this gene family in responding to environmental stresses.

The red-necked longhorn beetle Aromia bungii is a destructive wood-borer difficult to control with insecticides. We evaluated a combined biocontrol strategy using the ectoparasitic mite Pyemotes zhonghuajia and the entomopathogenic fungus Beauveria bassiana. Fungal virulence was dose- and stage-dependent, causing up to 100% mortality at 10⁸ conidia mL⁻¹ within 5–7 days. Mites alone induced limited mortality (~44% in young larvae, ≤7% in older ones), whereas conidia-carrying mites achieved complete kill within 7–8 days, comparable to direct fungal application. The combination significantly accelerated mortality (P < 0.05), reflecting synergistic interaction between mite venom and fungal infection. These findings highlight the potential of P. zhonghuajia as both a parasitoid and a vector for B. bassiana, offering an innovative and eco-friendly approach to manage A. bungii and related xylophagous pests.