First report of powdery mildew caused by Erysiphe euonymicola on wintercreeper (Euonymus fortunei) in China.

Wintercreeper (Euonymus fortunei) is an evergreen shrub, a semi climbing plants with very strong vitality. It can tolerate a broad range of environmental conditions varying from full sun to deep shade. So, wintercreeper is a good groundcover plants and tree species of vertical greening. It is widely used in urban greening in China. In May 2021, severe powdery mildew signs were found on wintercreeper plant in a garden of Luohe cities of Henan province, China. Approximately 40% of leaves on a plant were symptomatic, and about 60% of the plants were infected. Powdery mildew colonies appeared as white spots on the upper surface of the leaf and stem of the plants in the initial stage. Later, mycelial growth was amphigenous, thick, forming irregular white patches, effused to cover the entire leaf surface. At last, leaves turned yellow and senescence. One representative voucher specimen was deposited at the herbarium of Shangqiu Normal University (SQNU), Shangqiu, China, under the accession number of EF02. Conidiophores arising terminally from the mother cell, mostly central, erect, straight, 40.7 to 67.7µm (average 49.1µm) 6.1 to 8.5µm (average 7.5µm) (n=30) composed of 3 to 4 cells and produced conidia singly. Conidia were obovoid-ellipsoid, apex rounded, base subtruncate, ends truncate or subtruncate, long 26.1 to 36.4µm (average 30.7µm) and width 9.9 to 16.0µm (average 11.8µm) (n=30). No chasmothecia were observed. The morphological characteristics and measurements were consistent with those of Erysiphe euonymicola (Braun and Cook 2012). The internal transcribed spacer regions of EF02 were amplified using primers ITS1/ITS4, and sequences directly. The obtained sequence of 648 bp was deposited in GenBank (accession no. OM304857). The isolate (EF02) was 99.83% similarity with E. euonymicola on E. fortunei var. radicans (KM361621 from Korea) (Lee et al. 2015), 99.23% with E. euonymicola on E. fortunei (MT510003 from USA), 99.23% with E. euonymicola on E. japonicus (LC270841and LC270834 from Azerbaijan, AB250228 from Japan ). The domains D1 and D2 of the 28S rDNA obtained 619 bp sequences from the powdery mildew of isolate EF02 with primer NL1/NL4, deposited in GenBank (OM302187). A NCBI BLAST search of the EF02 isolate showed 100% similarity with 28S rDNA sequence of E. euonymicola on E. japonicus (AB250230 from Japan) (Limkaisang et al. 2006), and 28S phylogenetic tree analysis EF02 isolate is located in the same branch as E. euonymicola. The pathogenicity was confirmed by gently pressing the infected leaves onto five healthy plants. Five uninoculated plants served as controls. Ten inoculated and non-inoculated plants were placed in different growth chambers with 14-h photoperiod at 22±2°C and 60% of relative humidity. After 11 to 13 days, powdery mildew colonies developed on inoculated plants. Non-inoculated control plants did not show powdery mildew symptoms. The fungus on inoculated leaves was morphologically identical to that first observed in the field. Although E. euonymi-japonici (synonym E. euonymicola) has been recorded on E. japonicus in China (Li et al. 2011), this is the first report of powdery mildew caused by E. euonymicola on E. fortunei in China. It could become a threat to the widespread planting of wintercreeper, Similar report has been in Korea (Lee, C. K., et al. 2015).

Powdery Mildew of tall verbena Caused by Podosphaera xanthii in China.

Tall verbena (Verbena bonariensis) is widely used in landscape layout in sparse forests, botanical gardens and villa areas, and it is also a kind of Chinese herbal medicine. The flower and bud use as Chinese herbal medicine causing heat-clearing and detoxification. In August 2019, observed on tall verbena in Shangqiu, Henan Province, China. Symptoms white fungal growth observed on both sides of fresh and mature leaves of tall verbena grown on campus at Shangqiu Normal University. More than 60% plants were infected by this fungus and infected plants were observed to shed leaves early. One representative voucher specimen was deposited at the herbarium of Shangqiu Normal University (SQNU), Shangqiu, China, under the accession number of Vb01. Conidiophores of the fungus contained 5-6cells and measured 102.0-142.9µm (mean 126.2µm, n = 30) long, with foot-cells of conidiophores measured 36.5-46.9µm (mean 42.9µm, n = 30). Conidia produced in chains were variable in shape, ellipsoid to ovoid, and measured 24.8-30.7×12.0-17.9 µm (mean 27.35×15.08µm, n = 50), with a length/width ratio of 1.4 to 2.3. Fibrosin bodies were observed in the conidia. No chasmothecia were found. The morphological characteristics were consistent with the genus Podosphaera. To identify above powdery mildew fungus at species level using molecular markers, the complete ITS region of rDNA from the representative specimen was amplified with universal primer pair ITS1/ITS4 and directly sequenced. The resulting sequence of 567 bp was deposited in GenBank (Accession No. MT864007). A BLAST search of this sequence showed more than 99% sequence identity with P. xanthii isolates (AB046985, AB462804, AB936277 and KM260741) on various hosts (Hirata T, 2000; Meeboon, J., and Takamatsu, S. 2015; Tam L. T. T. 2016). Phylogenetic analysis revealed that ITS sequence from Vb01 isolate was grouped into a clade with P. xanthii s. lat.. A pathogenicity test was conducted through inoculation by gently pressing a diseased leaf onto five healthy leaves of a potted tall verbena. Five non-inoculated seedlings were used as controls. The plants were maintained in a greenhouse at 26 to 28 ℃, and relative humidity of 60 to 65%. Inoculated leaves developed symptoms after nine days, whereas the control plants remained symptomless. The fungus presented on the inoculated plant was morphologically identical to that originally observed on diseased plants, fulfilling Koch's postulates. P. xanthii s. lat. has been reported on several hosts in China (Braun & Cook, 2012). This is the first report of P. xanthii s. lat. on Verbena bonariensis outside of Japan (Meboon, 2015).

First report of chili pepper fruit rot caused by Fusarium incarnatum in China.

Pepper (Capsicum annuum L.), with annual production over 1 million tons, is ranked the first vegetable crop in Hainan Province, China. In December 2018, fruit rot of chili pepper , with yield loss of up to 15%, was found in 10 fields (12 hm2) in Yacheng (18°N, 109°E), Hainan Province, China. Water-soaked and soft lesions developed on fruit, with white to light gray fungal mycelium present inside. The diseased fruit turned soft and decayed at the later stages. Diseased tissue was cut into 12 pieces of 0.5×0.5 cm, surface-disinfected with 2% sodium hypochlorite for 2 min, followed by 70% ethanol for 30 s, rinsed with sterile distilled water five times, and plated onto potato dextrose agar (PDA). After growing on PDA for 2 to 3 days at 28°C in an incubator without light, 10 pure culture isolates were obtained. All isolates had abundant dense white aerial mycelia that became beige with age. The macroconidia were slightly curved with four to seven septa, 29.51 to 42.15 × 4.29 to 6.22 µm. Spindle-shaped mesoconidia with three to four septa were abundantly produced, 20.34 to 24.54 × 4.58 to to 11.70 × 2.35 to 3.20 µm. Chlamydospores were absent. Based on the morphological characteristics, the fungus was tentatively identified as Fusarium incarnatum (Leslie and Summerell 2006). An isolate SQHP-01 was chosen for molecular identification and pathogenicity test. Two DNA fragments of the isolate, the internal transcribed spacer (ITS) and translation elongation factor genes (EF-1α) were amplified for sequencing. BLAST analysis showed that sequences of ITS (GenBank acc. no. MN317371) and EF-1α (acc. No. MN928788) had 99.61 to 100% identity with those of known F. incarnatum (MN480497 and KF993969). Phylogenetic analysis was conducted using neighbor-joining algorithm based on ITS and EF-1a genes separately, and the isolate was well clustered with F. incarnatum both with 100% bootstrap support. Pathogenicity test of the isolate were carried out twice on five healthy chili pepper fruit. After surface-disinfection, fruit were wounded at three different points and 20 µl of conidial suspension (106 conidia/ml) were deposited on each wound. Unwounded inoculation was conducted by spreading 100 µl of the suspension on each fruit surface including the pedicel and calyx. The fruit spread with sterile distilled water represented the negative control. All fruit treatments were placed on the moist sterile cotton in moist chambers at 25°C with 16 h light and 8 h darkness. After 4 to 6 days, water-soaked necrotic lesions appeared on the wounded fruit, the symptoms identical to those observed in the field. Water-soaked necrotic lesions developed on the pedicel and calyx of unwounded fruit. No symptoms were observed on the control fruit. The morphology and sequences of re-isolated fungal isolates from the tested peppers were the same as the original isolate. To our knowledge, this is the first report of F. incarnatum (synonym of F. semitectum) causing fruit rot on chili pepper in China. F. incarnatum has been reported to cause root rot of greenhouse pepper in China (Li et al. 2018), fruit rot of bell pepper in Trinidad (Ramdial et al. 2016) and Pakistan (Tariq et al. 2018). Effective control strategies need to be developed to prevent the economic losses caused by the disease in chili pepper.

A novel basic helix-loop-helix transcription factor, ZjICE2 from Zoysia japonica confers abiotic stress tolerance to transgenic plants via activating the DREB/CBF regulon and enhancing ROS scavenging.

KEY MESSAGE: ZjICE2 works as a positive regulator in abiotic stress responses and ZjICE2 is a valuable genetic resource to improve abiotic stress tolerance in the molecular breeding program of Zoysia japonica. The basic helix-loop-helix (bHLH) family transcription factors (TFs) play an important role in response to biotic or abiotic stresses in plants. However, the functions of bHLH TFs in Zoysia japonica, one of the warm-season turfgrasses, remain poorly understood. Here, we identified ZjICE2 from Z. japonica, a novel MYC-type bHLH transcription factor that was closely related to ICE homologs in the phylogenetic tree, and its expression was regulated by various abiotic stresses. Transient expression of ZjICE2-GFP in onion epidermal cells revealed that ZjICE2 was a nuclear-localized protein. Also, ZjICE2 bound the MYC cis-element in the promoter of dehydration responsive element binding 1 of Z. japonica (ZjDREB1) using yeast one-hybrid assay. A phenotypic analysis showed that overexpression of the ZjICE2 in Arabidopsis enhanced tolerance to cold, drought, and salt stresses. The transgenic Arabidopsis and Z. japonica accumulated more transcripts of cold-responsive DREB/CBFs and their downstream genes than the wild type (WT) after cold treatment. Furthermore, the transgenic plants exhibited an enhanced Reactive oxygen species (ROS) scavenging ability, which resulted in an efficient maintenance of oxidant-antioxidant homeostasis. In addition, overexpression of the ZjICE2 in Z. japonica displayed intensive cold tolerance with increases in chlorophyll contents and photosynthetic efficiency. Our study suggests that ZjICE2 works as a positive regulator in abiotic stress responses and the ICE-DREB/CBFs response pathway involved in cold stress tolerance is also conserved in Z. japonica. These results provide a valuable genetic resource for the molecular breeding program especially for warm-season grasses as well as other leaf crop plants.

Distribution and biosynthesis of 20-hydroxyecdysone in plants of Achyranthes japonica Nakai.

There is increasing interest in phytoecdysteroids (PEs) because of their potential role in plant defense against insects. To understand the mechanism regulating their levels in plants, the fluctuation, distribution, and biosynthesis of PE 20-hydroxyecdysone (20E) examined in Achyranthes japonica. The total amount of 20E per individual plant initially remained at a constant level, and increased markedly after the first leaf pair (LP) stage, while the concentration of 20E in a given plant decreased rapidly during vegetative growth. In addition, the incorporation of [2-(14)C]-mevalonic acid into 20E did not differ significantly depending on plant organs and developmental stages, suggesting that biosynthesis of 20E is not restricted to particular organs or growth stages.