First report of branch dieback caused by Neoscytalidium dimidiatum on Styphnolobium japonicum (L.) Schott in China.

Styphnolobium japonicum (L.) Schott (family Fabaceae Juss.) also called pagoda tree, is widely planted in northern China in landscape plantings, for erosion control and forestry. In recent years, symptoms of branch dieback were observed on S. japonicum in the southern part of Xinjiang province, China. From 2019 to 2022, in total ca. 1000 ha area was surveyed in Korla (41.68°N, 86.06°E), Bohu (41.95°N, 86.53°E) and Alaer (41.15°N, 80.29°E). Typical symptoms were observed in 70% of the surveyed branches. To identify the cause, we collected 50 symptomatic branches. Symptoms were initially observed on green current-year twigs, which turned grayish white in color. In the later stages of disease development, a large number of nacked black conidia formed under epidermis of perennial branches, causing visible black protrusions (pycnidia) on branch surface. The disease occurred throughout the entire growing season of S. japonicum. Symptoms also occurred on the inflorescence, fruit, and twigs. In some cases, infection resulted in tree mortality. Isolations were made from the margin between healthy and diseased tissues. Small pieces were excised, surface disinfested (75% ethanol 30 s, 1% NaClO solution 5 mins), cut into pieces (5 to 10 mm2), and incubated on PDA medium at 28℃ for 3 days. A total of 16 isolates (GH01-GH16) with similar colony morphology were obtained. The colonies were initially white, gradually turning to olive-green on the surface and black on the underside after 7 days. Microscopically, the conidia were aseptate, 1-septate, two-septate, and muriform, 2.6-4.5 × 2.9-27.6 µm (n=50). Pycnidia ranged in size from 120.2 to 135.5 × 112.4 to 118.6 µm (n=20). Those morphological characters matched the descriptions of Neoscytalidium dimidiatum (previously N. novaehollandiae) (Alizadeh et al. 2022; Pavlic et al. 2008). For molecular identification, genomic DNA of GH01-GH16 were extracted from fresh mycelia. The internal transcribed spacer (ITS), large subunit ribosomal RNA gene (LSU), and translation elongation factor 1-alpha (EF1-α) gene were amplified using the primer sets ITS1/ITS4 (White 1990), LRoR/LR5 (Vilgalys and Hester 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The sequences were deposited in GenBank (accession No. OP379832, OQ096643-OQ096657 for ITS, OP389048, OQ127403-OQ127417 for LSU, and OQ136617, OQ586044-OQ586058 for EF1-α). The ITS sequence had 100% identity (505/505 bp) to MT362600. Similarly, the LSU and EF1-α sequences were found to be identical to MW883823 (100%, 821/821 bp) and KX464763(99%, 256/258 bp), respectively. Pathogenicity was tested on one-year-old healthy S. japonicum seedlings. Spores of representative isolate GH01 were produced on PDA by incubating for 7-days at 28℃. Conidia were washed with sterile water. Five trees were inoculated with 1 × 106 conidia/ml conidial suspensions and five trees were sprayed with sterile water. All trees were covered with plastic bags for 24 h and kept at 25°C in a greenhouse. Signs and symptoms were similar to those observed in field collections one month after inoculation, while no symptoms occurred on the controls. The original fungus was successfully reisolated from the inoculated trees and was identified as N. dimidiatum following the methods described above. N. dimidiatum has been reported in many Asian country such as Malaysia, India, Turkey, and Iran(Akgül et al. 2019; Alizadeh et al. 2022; Khoo et al. 2023; Salunkhe et al. 2023). To our knowledge, this is the first report of N. dimidiatum associated with branch dieback of S. japonicum in China. Our findings have expanded the host range of N. dimidiatum in China and provides a theoretical basis for the diagnosis and treatment of the disease.

Variations in the Components and Antioxidant and Tyrosinase Inhibitory Activities of Styphnolobium japonicum (L.) Schott Extract during Flower Maturity Stages.

Styphnolobium japonicum (L.) Schott is widely cultivated in China, and its flowers and flower buds (FFB-SJ) are commonly used as traditional Chinese medicine. This work aimed to assess variations in the chemical components and antioxidant and tyrosinase inhibitory activities of S. japonicum extract during five flower maturity stages (ES1-ES5). The results showed that the contents of total flavonoids, rutin, and narcissin were highest at ES1, whereas the contents of quercetin and isorhamnetin were highest at ES3. ES1 presented considerable antioxidant activities in terms of reducing power (RP) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH. ) and hydroxyl radical (. OH) scavenging capacity, whereas ES3 showed excellent tyrosinase inhibitory activity and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS.+ )- and O2 .- -scavenging capacity. Rutin and quercetin are the main bioactive components of FFB-SJ with antioxidant and tyrosinase inhibition, and the immature flower buds of S. japonicum (S2 and S3) with excellent biological activities and relatively high extract yields were the best for product development.

Identification of Ginkgo biloba supplements adulteration using high performance thin layer chromatography and ultra high performance liquid chromatography-diode array detector-quadrupole time of flight-mass spectrometry.

Ginkgo biloba is one of the most widely sold herbal supplements and medicines in the world. Its popularity stems from having a positive effect on memory and the circulatory system in clinical studies. As ginkgo popularity increased, non-proprietary extracts were introduced claiming to have a similar phytochemical profile as the clinically tested extracts. The standardized commercial extracts of G. biloba leaf used in ginkgo supplements contain not less than 6% sesquiterpene lactones and 24% flavonol glycosides. While sesquiterpene lactones are unique constituents of ginkgo leaf, the flavonol glycosides are found in many other botanical extracts. Being a high value botanical, low quality ginkgo extracts may be subjected to adulteration with flavonoids to meet the requirement of 24% flavonol glycosides. Chemical analysis by ultra high performance liquid chromatography-mass spectrometry revealed that adulteration of ginkgo leaf extracts in many of these products is common, the naturally flavonol glycoside-rich extract being spiked with pure flavonoids or extracts made from another flavonoid-rich material, such as the fruit/flower of Japanese sophora (Styphnolobium japonicum), which also contains the isoflavone genistein. Recently, genistein has been proposed as an analytical marker for the detection of adulteration of ginkgo extracts with S. japonicum. This study confirms that botanically authenticated G. biloba leaf and extracts made therefrom do not contain genistein, and the presence of which even in trace amounts is suggestive of adulteration. In addition to the mass spectrometric approach, a high performance thin layer chromatography method was developed as a fast and economic method for chemical fingerprint analysis of ginkgo samples.