First Report of Black Leaf Spot Caused by Epicoccum latusicollum on Chinese Yam in China.

Chinese yam (Dioscorea polystachya Turczaninow cv. Tiegun), which belongs to the family Dioscoreaceae, is widely cultivated throughout China due to its high economic and medicinal value. In June 2023, black leaf spots on Chinese yam (cv. Purple 1) were observed in Nanchang city (28.45° N, 115.49° E) of Jiangxi province, southeastern China. The incidence of the disease ranged between 70 and 85% of plants, and up to 30% of the leaves per plant were affected in the field over a 2-week period of study. Infected foliage displayed brown necrotic lesions, elliptical or irregular, with yellow halo at the edge of the lesion (0.5 to 3 cm diam.). To identify the causal agent, 32 symptomatic leaves of eight symptomatic plants were collected. Small pieces from the margin of necrotic leaf tissue (about 3 x 3 mm) were surface sterilized in 75% ethanol for 30 s followed in 0.1% HgCl2 for 1 min, and washed three times with ddH2O. Then, the pieces were transferred onto potato dextrose agar (PDA) plates and incubated at 26°C for 3 days with a 12-h light-dark cycle. From the 32 isolates, 21 exhibited similar morphology after hyphal tipping resulting in an isolation frequency of 65.6%. Colonies on PDA were initially white aerial hyphae but became grayish with age, and a reddish orange pigment on the underside. After 16 days of incubation, pycnidia were observed, which were dark, spherical or flat spherical, and 64.1 to 172.5 µm (n = 25) in diameter. Conidia were ellipsoidal, aseptate, hyaline, and 4.1 to 5.6 × 1.8 to 2.7 µm (n = 80). In addition, a blackish green discoloration was produced on malt extract agar (MEA) using the NaOH spot test. The isolates were tentatively identified as Epicoccum spp. based on morphological characteristics (Chen et al. 2017). Isolate AYZ-1 was randomly selected for identification and pathogenicity testing. Genomic DNA of the isolate (AYZ-1) was extracted and amplified by polymerase chain reaction (PCR) using ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990), Btub2Fd/Btub4Rd for the ß-tubulin (TUB) region (Woudenberg et al. 2009), LROR/LR7 for the large ribosomal RNA gene (LSU) region (Rehner and Samuels 1994), and RPB2-5F2/fRPB2-7cR for RNA polymerase II second largest subunit (RPB2) region (Liu et al. 1999), respectively. The concatenated sequences (GenBank Accession No. OR574165, OR567827, OR574166, OR567828, respectively) shared 99.8 to 100% identity with Epicoccum latusicollum (OP788080, MN329871, OR428532, and OL422485, respectively). A neighbor-joining phylogenetic tree was generated based on the concatenated sequences in MEGA7, placed isolate (AYZ-1) within E. latusicollum. To fulfill Koch's postulates, healthy leaflets from three one-year-old Chinese yam (cv. Purple 1) were used as inoculation materials, using isolate AYZ-1. Two sites of each leaf were wounded with a sterile needle and covered with a piece of cotton drenched with 200 µL spore suspension (106 conidia/mL) on the left sides, while sterilized water served as the control on the right sides of leaves. All inoculated leaves were covered with clear polyethylene bags for 24 h. Plants were grown outdoors at a daily average temperature of 26°C with relative humidity over 45%. After 7 days of incubation, the leaves showed the same symptoms as the original diseased leaves. The E. latusicollum isolate was re-isolated from diseased leaves and confirmed by morphology and sequencing analysis, fulfilling Koch's postulates. E. latusicollum has been previously reported to cause black root on yam in China's south-western province of Sichuan (Han et al. 2019). Meanwhile, leaf spot have been reported on many plants by this genus, such as tobacco (Guo et al. 2020) and banana (Liu et al. 2023). According to our knowledge, this is the first report of E. latusicollum causing black leaf spot on Chinese yam in China. This finding will provide an important reference for understanding the biology of E. latusicollum and the distribution of the disease, but more research is needed to determine if management is warranted.

First report in China of Fusarium humuli as a causative agent of Chinese yam wilt.

Wilt is one the most serious soil-born fungal diseases of Chinese yam (Dioscorea polystachya Turczaninow cv. Tiegun), affecting plant production in many growing regions in Jiangxi province, China. The average annual incidence of wilt is 45-80%. In 2020, affected plants with wilt symptoms including withered and dried leaves, vascular discoloration, and brown necrotic stem lesions at the soil line or in the crown of the plant (Figure 1A-D) were collected from the Ruichang, Yongfeng and Taihe production areas (four fields per location) in Jiangxi province. A total of fifteen isolates were recovered from the infected stem tissues of Chinese yam and purified by single spore cultures on PDA growth medium. The fifteen isolates were similar in morphology so isolate JXRC11 was selected to be representative of the group. Pure fungal colonies of JXRC11were found to be round, white, with margin entire (Figure 1E). Macroconidia with 3-5 septations were straight to slightly curved, 23.8-40.3 µm in length and 2.6-3.9 µm in width, with predominantly 5-septate macroconidia on carnation leaf agar (CLA) (Figure 1F). However, neither microconidia or chlamydospores were observed on CLA. The morphological characteristics of the isolate were consistent with the description observed previously for Fusarium humuli species complex (Wang et al. 2019). To confirm morphological identification, ITS, CAM, TEF-1α, RPB1 and RPB2 were amplified using the primers ITS5/ITS4 (White et al. 1990), CL1/CL2A (O'Donnell et al. 2000), EF1/EF2 (O'Donnell et al. 1998), Fa/G2R (O'Donnell et al. 2010), and 5f2/11ar (O'Donnell et al. 2010), respectively. BLASTn analysis of the ITS sequence (GenBank accession no. MZ768912), EF-1α (MZ824669), CAM (MZ824670), RPB1 (MZ824672) and RPB2 (MZ824673) alignment showed 99.55%, 99.68%, 99.85%, 97.61% and 99.76% identity to those of F. humuli CQ1039 (MK280845, MK289570, MK289712, MK289840 and MK289724), respectively. Multilocus phylogenetic analyses showed that the sequences of ITS, CAM, EF-1α, RPB1, and RPB2 of the isolate belonged to the incarnatum clade (FIESC-33) of the F. incarnatum-equiseti species complex with an independent branch (Figure 2). Pathogenicity tests were conducted on one-month-old Chinese yam seedlings using a seedling root dip method (Li et al. 2013). The roots and rhizomes of seedlings grown to two meters in height were dipped into spore suspensions (1×106 spores/mL) of isolate JXRC11 for 30 min and then transferred into 20 cm diameter plastic pots containing steam-sterilized soil and placed in a greenhouse under 12 h photoperiod. After 15 d, the inoculated seedlings showed typical wilt symptoms similar to those observed in farm fields, whereas the control remained unaffected (Figure 1G-H). The pathogen was then re-isolated from the infected plants, the re-isolations were identified as F. humuli by sequencing EF-1α, fulfilling the Koch's postulates. It has been reported that the pathogen F. oxysporum Schlecht causes Fusarium wilt in five species of Dioscorea (Nwankiti and Arene, 1978). Moreover, at least 5 species of Fusarium were identified as a causative agent of Chinese yam wilt (Fang et al. 2020). To our knowledge, this is the first report of Fusarium wilt disease on Chinese yam caused by a member of the F. humuli in Jiangxi, China. This report will contribute to developing management strategies to control the disease.

Microbial regulation of nitrous oxide emissions from chloropicrin-fumigated soil amended with biochar.

The microbial mechanism underpinning biochar's ability to reduce emissions of the potent greenhouse gas nitrous oxide (N2O) is little understood. We combined high-throughput gene sequencing with a dual-label 15N-18O isotope to examine microbial mechanisms operative in biochar made from Crofton Weed (BC1) or pine wood pellets (BC2) and the N2O emissions from those biochar materials when present in chloropicrin (CP)-fumigated soil. Both BC1 and BC2 reduced N2O total emissions by 62.9-71.9% and 48.8-52.0% in CP-fumigated soil, respectively. During the 7-day fumigation phase, however, both BC1 and BC2 increased N2O production by significantly promoting nirKS and norBC gene abundance, which indicated that the N2O emission pathway had switched from heterotrophic denitrification to nitrifier denitrification. During the post-fumigation phase, BC1 and BC2 significantly decreased N2O production as insufficient nitrogen was available to support rapid population increases of nitrifying or denitrifying bacteria. BC1 and BC2 significantly reduced CP's inhibition of nitrifying archaeal bacteria (AOA, AOB) and the denitrifying bacterial genes (nirS, nirK, nosZ), which promoted those bacterial populations in fumigated soil to similar levels observed in unfumigated soil. Our study provided insight on the impact of biochar and microbes on N2O emissions.

Complete genome sequence of the sesame pathogen Ralstonia solanacearum strain SEPPX 05.

Ralstonia solanacearum is a soil-borne phytopathogen associated with bacterial wilt disease of sesame. R. solanacearum is the predominant agent causing damping-off from tropical to temperate regions. Because bacterial wilt has decreased the sesame industry yield, we sequenced the SEPPX05 genome using PacBio and Illumina HiSeq 2500 systems and revealed that R. solanacearum strain SEPPX05 carries a bipartite genome consisting of a 3,930,849 bp chromosome and a 2,066,085 bp megaplasmid with 66.84% G+C content that harbors 5,427 coding sequences. Based on the whole genome, phylogenetic analysis showed that strain SEPPX05 is grouped with two phylotype I strains (EP1 and GMI1000). Pan-genomic analysis shows that R. solanacearum is a complex species with high biological diversity and was able to colonize various environments during evolution. Despite deletions, insertions, and inversions, most genes of strain SEPPX05 have relatively high levels of synteny compared with strain GMI1000. We identified 104 genes involved in virulence-related factors in the SEPPX05 genome and eight absent genes encoding T3Es of GMI1000. Comparing SEPPX05 with other species, we found highly conserved secretion systems central to modulating interactions of host bacteria. These data may provide important clues for understanding underlying pathogenic mechanisms of R. solanacearum and help in the control of sesame bacterial wilt.