Tephritidae fruit flies (Diptera: Tephritidae) are regarded as important damage-causing species due to their ability to cause great economic losses in fruit and vegetable crops. Bactrocera minax and Bactrocera tsuneonis are two sibling species of the subgenus Tetradacus of Bactrocera that are distributed across a limited area of China, but have caused serious impacts. They share similar morphological characteristics. These characteristics can only be observed in the female adult individuals. The differences between them cannot be observed in preimaginal stages. Thus, it is difficult to distinguish them in preimaginal stages morphologically. In this study, we used molecular diagnostic methods based on cytochrome c oxidase subunit I and species-specific markers to identify these two species and improve upon the false-positive results of previous species-detection primers. DNA barcode sequences were obtained from 900 individuals of B. minax and 63 individuals of B. tsuneonis. Based on these 658 bp DNA barcode sequences of the cytochrome c oxidase subunit I gene, we successfully designed the species-specific primers for B. minax and B. tsuneonis. The size of the B. minax specific fragment was 422 bp and the size of the B. tsuneonis specific fragment was 456 bp. A series of PCR trials ensured the specificity of these two pairs of primers. Sensitivity assay results demonstrated that the detection limit for the DNA template concentration was 0.1~1 ng/μL for these two species. In this study, we established a more reliable, rapid, and low-cost molecular identification method for all life stages of B. minax and B. tsuneonis. Species-specific PCR can be applied in plant quarantine, monitoring and control of B. minax and B. tsuneonis.

Several tephritid fruit fly species that are damaging to citrus in China are world-wide quarantine pests. Two field tests were conducted in China to evaluate the effectiveness of fruit bagging (i.e., fruits were grown in bags for at least 1.5 mo until harvest) and culling for risk mitigation of these pests during the fruit harvest season of 2017. The first test was conducted in Pinghe County of Fujian Province. The purpose of this test was to assess the effectiveness of fruit bagging on risk mitigation of fruit flies affecting pomelo, Citrus maxima (Burm. fil.) Osbeck (Rutaceae). External inspection and internal fruit cutting of 3,000 bagged and 3,040 unbagged fruits revealed few oviposition marks and absence of living flies in the bagged fruits, compared to 129 fruit fly-infested fruits containing 634 live larvae and 4 pupae in the unbagged fruits. Later molecular and morphological identification concluded that these larvae and pupae were Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). The second test was conducted in Shimen County of Hunan Province. The purpose of this test was to assess the effectiveness of packing house culling on risk mitigation of fruit flies in Satsuma mandarin, Citrus unshiu (Swingle) Marcov. (Rutaceae). A total of 20,000 fruits were cut (10,000 fruits before culling, and another 10,000 after culling). In the fruits that did not receive the culling treatment, 1 infested fruit with 7 third instar larvae of Bactrocera minax (Enderlein) (Diptera: Tephritidae) and 25 fruits with fruit fly oviposition marks were found. In the fruits that received the culling treatment, fruit flies were absent and 10 fruits with oviposition marks were found. These results suggest that fruit bagging and packinghouse culling could contribute to risk mitigation of fruit flies in citrus in China. This is a preliminary report, with further work necessary to develop a systems approach for risk mitigation of fruit flies in the commodities.

Maize stalk rot is a soilborne disease that poses a serious threat to maize production worldwide, with the most significant cause being fungal stalk rot. The development of a visual and rapid detection method for the maize stalk rot pathogen is significant for its prompt and accurate identification, enhancing agricultural production efficiency, and implementing timely preventive measures. These measures will help safeguard the maize yield and quality, ultimately reducing agricultural losses. In this study, we aimed to develop an efficient method to detect maize stalk rot pathogens. We focused on three pathogenic fungi commonly found in maize-producing regions worldwide: Fusarium verticillioides, F. proliferatum, and F. graminearum. Based on translation elongation factor 1-α, we developed a rapid detection technique using recombinase polymerase amplification–CRISPR/Cas12a, combined with test strips to develop an on-site rapid visual detection test for these pathogens. The method showed detection sensitivity for F. verticillioides, F. proliferatum, and F. graminearum within 20 min at concentrations of 7.8 pg/μl, 0.11 ng/μl, and 0.13 ng/μl, respectively. The sensitivity increased with increasing reaction time. Testing of field disease samples indicated that the method is effective in detecting nucleic acids obtained through crude extraction methods. In conclusion, we developed a visually rapid detection technology that does not rely on complex instruments and equipment for the on-site early detection of F. verticillioides, F. proliferatum, and F. graminearum in the field to implement effective control measures, ensuring stable and high maize yields.