First report of Fusarium ussurianum causing leaf spot on Hemerocallis citrina in Sichuan Basin of China.

Hemerocallis citrina is a popular vegetable crop in China, due to abundant nutrients in its edible flower buds. In March 2021, serious symptoms of leaf spot were observed on nearly 90% cultivated H. citrina seedlings in the fields of Dazhou city (31°17'56″ N, 107°31'59″ E), Sichuan, China. Symptomatic leaves were collected from 15 seedlings in five different sampling sites (3 seedlings per site). Small pieces (5 × 3 mm) of lesion margin were excised, surface disinfected in 70% ethanol for 20 s and 1% sodium hypochlorite (NaClO) for 40 s, washed, dried, placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/L) and incubated in dark at 25 ℃ for two days. Finally, eight purified isolates, HHC-FL22, HHC-FL23, HHC-FL25, HHC-FL26, HHC-FL27, HHC-FL28, HHC-FL29 and HHC-FL30, showing similar morphology were obtained through transferring hyphal tips to fresh PDA plates. On PDA plates, mycelia were initially white but gradually became light yellow, and scarlet diffusible pigments were also produced with time. On carnation leaf agar, our isolates produced slightly curved macroconidia with 4 to 8 septa that measured 3.1 to 5.7 × 36.8 to 69.3 µm (n = 30). Microconidia and chlamydospores were not observed. Our isolates were initially identified as Fusarium species based on morphological features (Leslie and Summerell 2006). To further confirm accurate identity, primers EF1/EF2 (O'Donnell et al. 2010), TRI1015B/TRI1013E (Hao et al. 2017), RPB1-F5/RPB1-G2R (O'Donnell et al. 2010), and fRPB2-5F/fRPB2-11aR and RPB2-5f2/RPB2-7cr (O'Donnell et al. 2012) were used to amplify gene sequences of translation elongation factor-1 alpha (TEF1), 3-O-acetyltransferase (Tri101), and DNA-directed RNA polymerase II largest (RPB1) and second largest subunit (RPB2), respectively. Our sequences were deposited in GenBank under accession numbers OQ860946 to OQ860953 (TEF1), OR393245 to OR393252 (Tri101), OP131893 to OP131900 (RPB1), and OQ860954 to OQ860961 and OP131885 to OP131892 (RPB2), respectively. BLASTN searches of our sequences showed 99 ~ 100% identity with TEF1 (FJ240301.1), Tri101 (FJ240345.1), RPB1 (MW233297.1) and RPB2 (KM361666.1) of F. ussurianum NRRL 45681, and 99.05 ~ 100% identity with TEF1 (FJ240305.1) and Tri101 (FJ240349.1) of F. ussurianum NRRL 45833, respectively. Two independent maximum-likelihood phylogenetic trees based on different combined datasets of TEF1, Tri101, RPB1 and RPB2 of Fusarium species confirmed that our isolates were F. ussurianum. To test pathogenicity, conidial suspension from HHC-FL23 (106 conidia / mL) were sprayed to seedlings of cultivar "chuanhuanghua No.1" (n = 3) and incubated in a greenhouse (25°C under 90% relative humidity, 16/8 h light/dark cycle). Controls were treated with ddH2O. Ten days post-inoculation, natural symptoms appeared on leaves inoculated with HHC-FL23, but control group seedlings remained disease-free. This experiment was repeated three times. All re-isolated pathogens from diseased leaves were molecularly and morphologically identified using methods described above. Consequently, the re-isolated fungi were identical to these inoculated. The leaf spot disease could cause foliar damage and even drastic yield loss of flower buds under severe conditions. To our knowledge, this is the first report of F. ussurianum causing leaf spot in H. citrina worldwide. Our study will assist in monitoring causal agent diversity of leaf spot and breeding new resistant varieties in H. citrina.

First report of leaf spot caused by Epicoccum latusicollum on Hemerocallis citrina in China.

Hemerocallis citrina is a popular vegetable crop. Its eatable flower buds contain abundant nutrients, especially lecithin (Guo et al., 2022). In March 2021, leaf spot disease was observed on 90% cultivated H. citrina seedlings in Dazhou city (31°17'56″ N, 107°31'59″ E), Sichuan, China. Totally, 15 diseased seedlings were sampled (three samples per 666 m2). The symptomatic leaves were cut into pieces (5 × 3 mm), superficially disinfected with 70% ethanol for 20 s and 1% Sodium hypochlorite (NaClO) for 40 s, and washed with sterile distilled water six times. The disinfected tissues were incubated on PDA amended with streptomycin sulfate (50 mg/L) in dark at 25 ℃. Two days later, hyphal tips from the edges of growing colonies were transferred to fresh PDA plates. Finally, 40 purified isolates were obtained. Using primer pairs ITS1/ITS4 (Glass & Donaldson, 1995), amplified rDNA internal transcribed spacer (ITS) regions indicated that these isolates belonged to different genera, mainly including Epicoccum, Fusarium and Colletotrichum. Six isolates of Epicoccum genus similar in morphology, named HHC46, HHC47, HHC491, HHC492, HHC51 and HHC58, were selected for identification. Cultured on oatmeal agar for 7 days, colonies were initially white and villose. Fourteen days later, mycelia started to secrete scarlet pigment. The NaOH spot test showed color changed from green to red, identical to that in Epicoccum species (Boerema et al., 2004). Meanwhile, colonies produced abundant conidia. Conidia were ellipsoidal, aseptate, and 4.1 to 6.5 × 1.3 to 2.9 µm (n = 30). Chlamydospores were also observed, globose to subglobose. The morphological features were similar to those of Epicoccum latusicollum (Xu et al., 2022). The DNA sequences of Beta-tubulin (TUB2) and DNA-directed RNA polymerase II second largest subunit (RPB2) of six isolates were amplified and sequenced, using primer pairs Bt2a/Bt2b (Glass & Donaldson, 1995), and RPB2-5f2/RPB2-7cr (O'Donnell et al., 2012), respectively. BLASTN searches indicated our ITS (OP107240 - OP107245), TUB2 (OP131865 - OP131870) and RPB2 (OP131871 - OP131876) sequences except one TUB2 (OP131867), showed 100% identity to the corresponding sequences of E. latusicollum CGMCC:3.18346 (KY742101, KY742343 and KY742174, respectively). There was a nucleotide divergence between OP131867 and reference sequence. Based on concatenated ITS, TUB2 and RPB2 sequences, the constructed phylogenetic tree of Epicoccum species, confirmed that our isolates were E. latusicollum. To test pathogenicity, 2-year-old healthy seedlings of cultivar "chuanhuanghua No.1" were sprayed with conidial suspension of HHC51 (105 conidia/mL), with controls treated with sterile distilled water. Each treatment (biological replicates = 3) was incubated in a greenhouse (at 25°C under 90% relative humidity, 16/8 h light/dark cycle). The experiment was repeated twice. After 18 days, leaf spot symptom in inoculated seedlings appeared. Whereas, non-inoculated controls showed no symptom. The pathogens were re-isolated from diseased leaves and identified as E. latusicollum, based on morphology and molecular methods described above. E. sorghinum was previously reported as causal agent of leaf spot in H. citrina (Ma et al., 2021). To our knowledge, this is the first report of E. latusicollum causing leaf spot in H. citrina worldwide. Our study will assist with monitoring disease distribution in H. citrina and host diversity of E. latusicollum (Chen et al., 2017).

The comparative studies of complete chloroplast genomes in Actinidia (Actinidiaceae): novel insights into heterogenous variation, clpP gene annotation and phylogenetic relationships.

The genus Actinidia, also called kiwifruit, is characterized with abundant balanced nutritional metabolites, including exceptionally high vitamin C content. However, the traditional classification could not fully reflect the actual Actinidia species' relationships, which need further revision through more accurate approaches. Compared to the nuclear genome, the chloroplast genome has simple heredity characteristics, conserved genome structure and small size, suitable for deciphering complicated species' phylogenetic relationships. Here, the genome-wide comprehensive comparative analyses were performed over 29 independent chloroplast genomes' sequences derived from 25 Actinidia taxa. The average genome size is 156,673.38 bp, with an average 37.20% GC content. The long repeat sequences rather than SSRs (simple sequence repeats) in Actinidia were revealed to be the causal agent leading to the chloroplast genome size expansion. The clpP gene sequences with exon merge and intron deletion were annotated in all the 29 chloroplast genomes tested, which has been previously reported to be lost in Actinidia species. Comprehensive sequence analyses indicated the distinct variation at the clpP gene locus was Actinidiaceae-specific, emerging after the Actinidiaceae-other Ericales species divergence. Four highly divergent sequences (i.e., rps16 ~ trnQ-UUG, rps4 ~ trnT-UGU, petA ~ psbJ, and rps12 ~ psbB) evolved in the LSC (large single-copy) and SSC (small single-copy) regions embodying rps12 ~ psbB (including clpP gene and its up/downstream noncoding sequence) were identified as variation hot spots in Actinidia species. Based on either LSC region alone, combined sequences of LSC and SSC or the whole chloroplast genome sequences, three identical phylogenetic trees of the 25 Actinidia taxa with relatively improved resolution were reconstructed, consistently supporting the reticulate evolutionary lineage in Actinidia. Our findings could help to better understand the evolution characteristics of chloroplast genomes and phylogenetic relationships among Actinidia species.

First report of Nigrospora sphaerica causing fruit dried-shrink disease in Akebia trifoliata from China.

Akebia trifoliata, a recently domesticated horticultural crop, produces delicious fruits containing multiple nutritional metabolites and has been widely used as medicinal herb in China. In June 2020, symptoms of dried-shrink disease were first observed on fruits of A. trifoliata grown in Zhangjiajie, China (110.2°E, 29.4°N) with an incidence about 10%. The infected fruits were shrunken, colored in dark brown, and withered to death (Figure S1A, B). The symptomatic fruits tissues (6 × 6 mm) were excised from three individual plants, surface-disinfested in 1% NaOCl for 30s and 70% ethanol solution for 45s, washed, dried, and plated on potato dextrose agar (PDA) containing 50 mg/L streptomycin sulfate in the dark, and incubated at 25℃ for 3 days. Subsequently, hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, five pure cultures were obtained, including two identical to previously reported Colletotrichum gloeosporioides causing leaf anthracnose in A. trifoliata (Pan et al. 2020) and three unknown isolates (ZJJ-C1-1, ZJJ-C1-2, and ZJJ-C1-3). The mycelia of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 were white, and formed colonies of approximate 70 mm (diameter) in size at 25℃ after 7 days on potato sucrose agar (PSA) plates (Figure S1C). After 25 days, conidia were formed, solitary, globose, black, shiny, smooth, and 16-21 µm in size (average diameter = 18.22 ± 1.00 µm, n = 20) (Figure S1D). These morphological characteristics were similar to those of N. sphaerica previously reported (Li et al. 2018). To identify species of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3, the internal transcribed spacer (ITS) region, ß-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (Vilgalys and Hester 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF-2 (Zhou et al. 2015), respectively. Multiple sequence analyses showed no nucleotide difference was detected among genes tested except ITS that placed three isolates into two groups (Figure S2). BLAST analyses determined that ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 had 99.73% to N. sphaerica strains LC2705 (KY019479), 100% to LC7294 (KY019397), and 99.79-100% to LC7294 (KX985932) or LC7294 (KX985932) based on sequences of TUB2 (MW252168, MW269660, MW269661), TEF-1α (MW252169, MW269662, MW269663), and ITS (MW250235, MW250236, MW192897), respectively. These indicated three isolates belong to the same species of N. sphaerica. Based on a combined dataset of ITS, TUB2 and TEF-1α sequences, a phylogenetic tree was constructed using Maximum likelihood method through IQ-TREE (Minh et al. 2020) and confirmed that three isolates were N. sphaerica (Figure S2). Further, pathogenicity tests were performed. Briefly, healthy unwounded fruits were surface-disinfected in 0.1% NaOCl for 30s, washed, dried and needling-wounded. Then, three fruits were inoculated with 10 µl of conidial suspension (1 × 106 conidia/ml) derived from three individual isolates, with another three fruits sprayed with 10 µl sterilized water as control. The treated fruits were incubated at 25℃ in 90% humidity. After 15 days, all the three fruits inoculated with conidia displayed typical dried-shrink symptoms as those observed in the farm field (Figure S1E). The decayed tissues with mycelium and spores could be observed on the skin or vertical split of the infected fruits after 15 days' inoculation (Figure S1F-H). Comparably, in the three control fruits, there were no dried-shrink-related symptoms displayed. The experiment was repeated twice. The re-isolated pathogens were identical to N. sphaerica determined by sequencing the ITS, TUB2 and TEF-1α. Previous reports showed N. sphaerica could cause postharvest rot disease in kiwifruits (Li et al. 2018). To our knowledge, this is the first report of N. sphaerica causing fruits dried-shrink disease in A. trifoliata in China.