First Report of Leaf Spot on Stephania tetrandra Caused by Albifimbria verrucaria in China.

Stephania tetrandra S. Moore belongs to the family Menispermaceae and is a Chinese medicinal plant widely distributed in tropical and subtropical regions of Asia and Africa. The root can be used for a variety of treatments (Jiang et al. 2020). In August 2021, leaf spot symptoms were observed on S. tetrandra cultivated in Jiangxi (114.456E, 27.379N, southern China). The disease symptoms included a slight constriction of the leaves, with irregularly shaped brown to black spots with well-defined borders. Severely affected leaves were shed by the plant. In order to determine the cause, symptomatic leaves were surface-disinfested with 0.6% NaOCl for 2 min, and rinsed twice in sterile water, then incubated on moist paper towels at 26°C in the dark for 2 days. Cream-colored sporodochia were observed within the leaf spots, turning dark green to black within 16 hours. A slow-growing white fungus was isolated from 95% of the samples (n = 30) on PDA. Dark green sporodochia emerged after 7 to 10 days of incubation, and released tip-end oval, non-septate, hyaline conidia measuring 6.7 to 8.5 µm (mean 7.5 µm, n = 50) by 2.0 to 3.3 µm (mean 2.7 µm, n = 50). Concentric rings were interspersed with sporodochia on the continually incubated mycelium. The morphological characteristics of the isolates matched the description of Albifimbria (Lombard et al. 2016). Nucleotide sequences, amplified from isolate FJL5C using primers of the internal transcribed spacer (ITS) (White et al. 1990), calmodulin (cmdA; Carbone and Kohn 1999), and RNA polymerase II second largest subunit (rpb2; O'Donnell et al. 2007), were deposited in GenBank under accession numbers OM317911, OM386815, and OM386816. A BLASTn analysis of the sequences showed 100% identity with the type strain CBS 328.52 (Lombard et al. 2016) of Albifimbria verrucaria (syn. Myrothecium verrucaria) for ITS, and 99% for cmdA and rpb2 (KU845893, KU845875, and KU845931, respectively). A phylogenetic tree generated using the three sequences showed that the isolate from S. tetrandra grouped with the A. verrucaria isolates, but away from other species of Albifimbria. These results together with the lack of a pale luteus exudate produced by A. viridis (Lombard et al. 2016) implied that the isolate was A. verrucaria. The culture was deposited in Guangdong Microbial Culture Collection Center (GDMCC 3.716). To verify pathogenicity, conidial suspension (106 conidia/mL in 0.05% Tween 20 solution) was sprayed onto six healthy plants. Six other plants sprayed with the Tween 20 solution alone served as controls. All plants were incubated in the dark at 26°C and 95% humidity for 30 hours, then transferred to a greenhouse at 26°C and 12 hours of illumination per day for 2 to 3 days. Inoculated leaves developed similar symptoms to those described above, whereas control plants remained healthy. The same pathogen was isolated from the diseased leaves, with the same morphological and molecular traits as those from the field plants. This experiment fulfilled Koch's postulates and confirmed that A. verrucaria causes leaf spots on S. tetrandra. This pathogen has been reported to cause disease in a wide range of weeds, legumes, and crop plants (Herman et al. 2020). To our knowledge, this is the first report of A. verrucaria causing leaf spots on S. tetrandra in natural or controlled environments. The disease can seriously threaten S. tetrandra on growth and yield loss.

First Report of Sclerotium rolfsii Causing Southern Blight on Stephania tetrandra in China.

Stephania tetrandra S. Moore is a perennial liana and is widely cultivated in southern China for traditional Chinese medicine as a diuretic, anti-inflammatory, and antirheumatic treatment (Jiang et al. 2020). In August 2021, it was observed that a severe stem rot disease affected St. tetrandra cultivated in Anfu, Jiangxi province, China (114°27'26" E, 27°22'46" N). The disease symptoms included constriction and rot at the base of the stem, and covered with a layer of white mycelia. The plants above-ground finally wilted and dried with a disease incidence ranging from 8% to 16%. Lots of dried plants formed withered patches of field. Sections (1.0~2.0 cm) from browning stem tissues were surface-disinfected with 75% ethanol for 15 s, followed by 60 s in 4% NaClO, rinsed twice in sterile water, dried on sterilized filter paper, placed on potato dextrose agar (PDA), and incubated at 26°C in the dark for 3 days. A white rhizomorphic fungal mycelium, that is similar to the mycelium of strain FJSR0 on the surface of an infected plant in the field, was isolated from the cultured tissues with 67% frequency. When incubated on PDA, white and fluffy mycelia with even margins and a slight halo formed. Mycelia-produced clamp connections were observed. Colonies grew quickly and covered the dish (diameter: 9 cm) in 5 or 6 days. After that, sclerotia were initially white, then turned yellow, and chestnut brown at maturity. Spherical and subspherical sclerotia were observed after 8 days, with each plate containing 448 to 634 sclerotia (0.8 to 1.4 mm diameter; mean = 0.94 mm; n = 50). On the basis of morphology, the pathogen was similar to Sclerotium rolfsii Sacc. [teleomorph: Athelia rolfsii (Curzi) Tu & Kimbrough] (Sun et al. 2020; Ling et al. 2021). For molecular confirmation, the internal transcribed spacer (ITS) region with approximately 680 bp was amplified from strains FJRS0 and FJRS1 using primers ITS1/ITS4 (White et al. 1990). Two distinct types (different in one SNP and one 1-bp InDel) of ITS sequences were obtained from each isolate, and all isolates contain the two types (FJSR0: ON972516, ON972517; FJSR1: ON972520, ON972518). BLAST analysis of each type found that the hits, with identities >99%, are A. rolfsii except for two Sc. delphinii sequences (GU567775.1 and MK073010.1). Phylogenetic analysis placed strains FJSR0 and FJSR1 in the same clade as Sc. rolfsii but away from Sc. delphinii based on the previous method (Sun et al. 2021). Both morphological and molecular characteristics confirmed that the strains were Sc. rolfsii. For pathogenicity tests, PDA plugs (8 mm in diameter) covered with 5-day-old fungal mycelium were inoculated at the stem bases of three healthy St. tetrandra seedings and incubated at 26℃ and relative humidity of 80%. On the fifth day, inoculated plants were wilting. The infected stem bases turned brown to black and constricted as previously observed in the field. Some leaves, infected by the mycelium expanded from the PDA plugs, developed an orange and irregular spot. Sclerotia were observed 20 days post inoculation. In contrast, the leaves and stems of non-inoculated control plants remained symptomless. Pathogenicity tests were repeated three times. The fungus was reisolated consistently from each symptomatic tissue, thus completing Koch's postulates. Although Sc. rolfsii has been previously reported to cause a southern blight symptoms on vegetables, ornamentals, grass, and medicinal and leguminous crops (Sun et al. 2020; Ling et al. 2021), this is the first report of Sc. rolfsii causing similar symptoms of southern blight on St. tetrandra in China.

Assembly of the viroplasm by viral non-structural protein Pns10 is essential for persistent infection of rice ragged stunt virus in its insect vector.

Rice ragged stunt virus (RRSV), an oryzavirus, is transmitted by brown planthopper in a persistent propagative manner. In this study, sequential infection of RRSV in the internal organs of its insect vector after ingestion of virus was investigated by immunofluorescence microscopy. RRSV was first detected in the epithelial cells of the midgut, from where it proceeded to the visceral muscles surrounding the midgut, then throughout the visceral muscles of the midgut and hindgut, and finally into the salivary glands. Viroplasms, the sites of virus replication and assembly of progeny virions, were formed in the midgut epithelium, visceral muscles and salivary glands of infected insects and contained the non-structural protein Pns10 of RRSV, which appeared to be the major constituent of the viroplasms. Viroplasm-like structures formed in non-host insect cells following expression of Pns10 in a baculovirus system, suggesting that the viroplasms observed in RRSV-infected cells were composed basically of Pns10. RNA interference induced by ingestion of dsRNA from the Pns10 gene of RRSV strongly inhibited such viroplasm formation, preventing efficient virus infection and spread in its insect vectors. These results show that Pns10 of RRSV is essential for viroplasm formation and virus replication in the vector insect.