Well-supported Cluster Research Articles

Palaeognaths Reveal Evolutionary Ancestry of the Avian Major Histocompatibility Complex Class II.

The multigene family of the major histocompatibility complex (MHC) codes for the key antigen-presenting molecules of the vertebrate immune system. In birds, duplicated MHC class II (MHC-II) genes are highly homogenized by concerted evolution, and thus, identification of their orthologous relationships across long evolutionary timescales remains challenging. Relatively low evolutionary rate of avian MHC class IIA genes has been expected to provide a promising avenue to allow such inferences, but availability of MHC-IIA sequences in nonmodel bird species has been limited until recently. Here, taking advantage from accumulating genomic resources, we identified and analyzed MHC-IIA sequences from the most basal lineage of extant birds (Palaeognathae). Conserved region of the MHC-IIA membrane-proximal domain was used to search for orthologous relationships between palaeognath birds and nonavian reptiles. First, analyses of palaeognath sequences revealed the presence of a separate MHC-IIA gene lineage (DAA3) in kiwis, which did not cluster with previously described avian MHC-IIA lineages (DAA1 and DAA2). Next, phylogenetic reconstruction showed that kiwi DAA3 sequences form a single well-supported cluster with turtle MHC-IIA. High similarity of these sequences most likely reflects their remarkable evolutionary conservation and retention of ancient orthologous relationships, which can be traced back to basal archosauromorphs ca. 250 million years ago. Our analyses offer novel insights into macroevolutionary history of the MHC and reinforce the view that rapid accumulation of high-quality genome assemblies across divergent nonmodel species can substantially advance our understanding of gene evolution.

Analysis of fine-scale phylogeny of Burkholderia pseudomallei in relation to regional geography and drug susceptibility in Thailand

Melioidosis caused by Burkholderia pseudomallei (Bp) is a public health threat. Genomic-epidemiology research on this deadly disease is scarce. We investigated whole-genome sequences of Bp isolates in relation to environmental source and drug susceptibility. In total, 563 Bp isolates were collected from 11 Northeast Thai provinces during the period 2004–2021. Patients (n = 530 isolates), infected animals (n = 8), and environmental sources (n = 25) provided samples. Phylogenetic analysis revealed genetic diversity among the Bp isolates, including numerous well-supported clusters of varying sizes. Through in-depth analysis of 38 monophyletic clades (MCs), we found eleven associated with province of origin (p-value < 0.001). Closely related clusters (CRCs) within MCs resembled MLST-identified “sequence types” (STs). We found 102 known and 52 novel STs. ST-70 was the most prevalent in this area (n = 78; 13.85%). Sample type (human/environmental) and sampling time intervals were not correlated with genetic distance among clonal Bp isolates. Some members of 12 CRCs had acquired resistance to co-trimoxazole and one against amoxicillin-clavulanic acid. Within Northeast Thailand, there is an association between Bp genotype and geographical origin.

The pan-plastome of Prunus mume: insights into Prunus diversity, phylogeny, and domestication history.

Prunus mume in the Rosaceae and commonly referred to as mei or Chinese plum is widely used as a traditional ornamental flowering plant and fruit tree in China. Although some population and genetic analyses have been conducted for this species, no extensive comparisons of genetic variation from plastomes have yet been investigated. We de novo assembled a total of 322 complete P. mume plastomes in this study and did a series of comparative analyses to better resolve pan-plastomic patterns of P. mume. To determine the phylogeny and domestication history of this species, we reconstructed the phylogenetic tree of Prunus genus, and resolved the population structure of P. mume. We also examined the nucleotide variation of P. mume to find potential DNA barcodes. The assembled plastomes exhibited a typical quadripartite structure and ranged from 157,871 bp to 158,213 bp in total size with a GC content ranging from 36.73 to 36.75%. A total of 112 unique genes were identified. Single nucleotide variants (SNVs) were the most common variants found among the plastomes, followed by nucleotide insertions/deletions (InDels), and block substitutions with the intergenic spacer (IGS) regions containing the greatest number of variants. From the pan-plastome data six well-supported genetic clusters were resolved using multiple different population structure analyses. The different cultivars were unevenly distributed among multiple clades. We also reconstructed a phylogeny for multiple species of Prunus to better understand genus level diversity and history from which a complex introgressive relationship between mei and other apricots/plums was resolved. This study constructed the pan-plastome of P. mume, which indicated the domestication of P. mume involved multiple genetic origins and possible matrilineal introgression from other species. The phylogenetic analysis in Prunus and the population structure of P. mume provide an important maternal history for Prunus and the groundwork for future studies on intergenomic sequence transfers, cytonuclear incompatibility, and conservation genetics.

Kitasatospora cathayae sp. nov., a novel endophytic actinomycete isolated from the leaves of Cathaya argyrophylla.

Strain HUAS 3-15T was isolated from the leaves of Cathaya argyrophylla collected from Chenzhou, Hunan Province, PR China. The main fatty acids (>5.0 %) of the strain were anteiso-C15 : 0, C16 : 0, C18 : 1 ω9c, iso-C16 : 0, summed feature 5 (C18 : 2 ω6,9c/C18 : 0 ante), iso-C15 : 0 and anteiso-C17 : 0. MK-9(H6), MK-9(H8) and MK-9(H4) were detected as respiratory quinones. The diagnostic cell-wall diamino acid was meso-diaminopimelic acid. Galactose, glucose and ribose were also present in the cell wall. The major polar lipids consisted of diphosphatidylglycerol, phosphatidyl ethanolamine, phosphatidylinositol mannosides and unidentified phospholipids. The DNA G+C content of the genome sequence, consisting of 8 860 963 bp, is 72.4 mol%. blast analysis based on 16S rRNA gene sequences revealed that the strain belongs to the genus Kitasatospora, with 99.37, 99.03, 98.95, 98.68 and 98.67 % sequence similarity to Kitasatospora aureofaciens ATCC 10762T, Kitasatospora viridis DSM 44826T, Kitasatospora xanthocidica NBRC 13469T, Kitasatospora aburaviensis NRRL B-2218T and Kitasatospora kifunensis IFO 15206T, respectively. Phylogenetic trees based on 16S rRNA gene and whole-genome sequences demonstrated that strain HUAS 3-15T formed a well-supported cluster with K. aureofaciens ATCC 10762T. Further genomic characterization through average nucleotide identity (ANIb/m) and digital DNA-DNA hybridization analysis between strain HUAS 3-15T and K. aureofaciens ATCC 10762T showed values of 90.62/92.55 % and 45.3 %, respectively, lower than the 95-96 % ANI threshold and 70.0 % cutoff used as guideline values for species delineation in bacteria. Furthermore, the differences between the strain and its phylogenomic neighbour in terms of physiological (e.g. sole carbon source growth) and chemotaxonomic (e.g. cellular fatty composition) characteristics further supported this conclusion. Consequently, we concluded that strain HUAS 3-15T represents a novel species of the genus Kitasatospora, for which the name Kitasatospora cathayae sp. nov. is proposed. The type strain is HUAS 3-15T (=MCCC 1K08542T=JCM 36274T).

First report of Diaporthe eres causing leaf spot on Rosa roxburghii Tratt in Guizhou Province, China.

Rosa roxburghii Tratt is a plant from the Rosaceae family whose fruits are rich in vitamins, dietary fiber, flavonoids, phenolic acids, and other active components (Jiang, et al. 2024). In July 2023, about R. roxburghii 500 plants were investigated in a field of 6000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou province, China, and the results showed a leaf spot incidence of s 20 to 30%. . The affected leaves had irregular, black lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. Fifteen symptomatic leaves were collected from 10 plants washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were cut. After surface sterilization for30 s with 75% ethanol, 2 min with 3% NaOCl, three washes in sterilized distilled water, the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Three isolates (H3-Y-1-1, H3-Y-1-2, H3-Y-1-3) with identical morphology were obtained, and the isolate H3-Y-1-1was selected for further study. The colonies on PDA exhibited irregular growth patterns, with white felty aerial mycelium on the upper surface, and white mycelium on the lower surface. Conidiomata were irregularly distributed over the agar surface. The isolate H3-Y-1-1 produced darkly pigmented pycnidia on PDA after 30 days and oozed milky mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 4.74 - 5.96 × 1.52 - 2.24 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.13 - 25.74 × 0.86 - 1.29 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers PP411998 (ITS), PP502153 (TUB), PP502156 (TEF). BLAST searches of the sequences revealed (96%) (500/523nt), 97% (479/494 nt) and 99% (334/338 nt) homology with those of D. eres CBS 138594 from GenBank (OM698848, OM752196 and OM752197), respectively. Phylogenetic analysis using maximum-likelihood and Bayesian methods placed the isolate H3-Y-1-1 in a well-supported cluster with D. eres CBS 101742. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). To assess its pathogenicity, healthy R. roxburghii potted plants were inoculated with H3-Y-1-1 spore suspensions. Symptomatic leaves mirroring field symptoms were observed after XX days of incubations at XX°C, while control plants exhibited no symptoms. Diaporthe eres was consistently reisolated from the infected leaves showing brown irregular or round lesions at the initial stage of the disease, expanding and become more irregular over time ultimately causing leaf curling and plant death. To our knowledge, this is the first report of leaf spot on R. roxburghii caused by D. eres in China. The disease may become a serious threat to fruit of R. roxburghii production in China. Therefore, detection of this pathogen is very important to ensure timely disease management.

Reclassification of Aestuariicella albida as Pseudomaricurvus albidus comb. nov. and Aestuariicella hydrocarbonica as Pseudomaricurvus hydrocarbonicus comb. nov. Based on Comparative Genomics and Molecular Synapomorphies.

The genus Aestuariicella has been recently reclassified as a member of the family Cellvibrionaceae. However, the taxonomic position of the genus as a distinct member of the family has not been clarified. In the present study, we performed multilayered analyses anchored on genome sequences to clarify the relationship between the genera Aestuariicella and Pseudomaricurvus within the family Cellvibrionaceae. Phylogenetic analyses based on 16S rRNA gene, RNA polymerase beta subunit (RpoB) protein, and core gene sequences showed a well-supported tight cluster formed by the members of the two genera. Moreover, the analysis of the average amino acid identity (AAI) revealed that the members of the two genera shared 68.16-79.48% AAI, values which were within the range of observed AAI (≥ 67.23%) among the members of the same genus within the family Cellvibrionaceae. Members of the two genera also shared several common characteristics. Furthermore, molecular synapomorphies in a form of conserved signature indels were identified in six protein sequences that were exclusively shared by the members of the two genera. Based on the phylogenetic and molecular evidence presented here, we propose the reclassification of the species Aestuariicella albida and Aestuariicella hydrocarbonica as Pseudomaricurvus albidus comb. nov. and Pseudomaricurvus hydrocarbonicus comb. nov., respectively.

First Report of Calonectria ilicicola causing Leaf spot on Camellia sinensis in Yunnan province, China.

Tea (Camellia sinensis (L.) Kuntze) is among the most significant industrial crops due to its distinctive fragrance and flavor generated (Bag et al. 2022). From October to December in 2021, a leaf spot disease affected the quality and yield of tea (C. sinensis var. assamica cv. Yunkang 10), in Pu'er (100.57°E, 22.45°N), Yunnan province, China. Based on the survey, the incidence was approximately 15% in a plantation of 4500 m2 (2050 tea trees approximately). The symptoms on leaves were regular circular, dark brown lesions with black conidiomata in gray centers. Twenty symptomatic leaves were collected from 10 trees. After rinsing and surface sterilization (75% ethanol for 30 s and 3% NaClO for 90 s, rinsed 3 times with sterile distilled water), diseased tissues (5 × 5 mm) were cut at the junction of infected and healthy site and placed on potato dextrose agar (PDA) (3 pieces per plate) and incubated in the dark at 28℃ for 5 days (Mao et al. 2023). Three single-spore isolates 6a-H-1, 6a-H-2 and 6a-H-3 were obtained, which showed identical in morphology and molecular analysis. Therefore, the targeted isolate 6a-H-2 was used for further study. Fungal colonies were white, then gradually turning into goose yellow (Fig.2. A-C). Chlamydospores were dark brown and oval (Fig.2. G). Asci produced after 30 days approximately, were orange-red, nearly spherical, rough-surface, and measured as 470 µm ± 11.68 µm (n = 50) (Fig.2. H). Ascospores were released from the asci orifice (Fig.2. I) which were hyaline, fusoid with rounded ends, straight to slightly curved, two septate, slightly constricted at the septum, and ranged from 48.77 ± 2.76 µm × 6.22 ± 0.41 µm (n = 50) (Fig.2. D-F). Macroconidia were cylindrical (Fig.2. J), rounded at both ends, straight, with an average length of 63.5 ± 0.31 μm × 2.62 ± 0.03 μm without septa (n=50) (Fig.2. M-O). Stipe extension terminated in sphaero-pedunculate vesicles (Fig.2. K-L). The morphological features were consistent with the descriptions of Calonectria ilicicola (Pei et al. 2015; Polizzi et al. 2012). The pathogen was confirmed to be C. ilicicola by amplification and sequencing of the histone (HIS3), translation elongation factor 1-alpha (TEF1) and calmodulin (CAL) genes using primers H3-3F/H3-3R, EF1-728F/EF1-986R and CAL-228F/CAL-2Rd, respectively (Crous et al. 2004). The sequences of PCR products were deposited in GenBank with accession numbers OR188222 (HIS3), OR188223 (TEF1) and OR188221 (CAL). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides), 98.37% (241/245 nucleotides) and 99.58% (472/474 nucleotides) homology with those of C. ilicicola (CBS 190.50) in GenBank (AY725676, AY725726 and AY725764), respectively. Phylogenetic analysis (MEGA 7.0) using the Maximum Likelihood method placed the isolate 6a-H-2 in a well-supported cluster with C. ilicicola. The pathogenicity of 6a-H-2 was tested through a pot assay. Five healthy plants had their leaves scratched with a sterilized needle, then inoculated by spraying 20 mL of spore suspension (105 spores mL-1) of 6a-H-2. Five additional tea plants sprayed with sterile distilled water served as controls. All plants were placed in a growth chamber at 28℃, with 70% relative humidity. The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphology and molecular analyses (HIS3, TEF1 and CAL) from the infected leaves but not from the non-inoculated leaves. To our knowledge, this is the first report of leaf spot on tea caused by C. ilicicola in China. This study provides valuable information for the identification and control of the leaf spot on tea.

Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov., isolated from French cheese rinds.

Eight Gram-stain-negative bacterial strains were isolated from cheese rinds sampled in France. On the basis of 16S rRNA gene sequence analysis, all isolates were assigned to the genus Halomonas. Phylogenetic investigations, including 16S rRNA gene studies, multilocus sequence analysis, reconstruction of a pan-genome phylogenetic tree with the concatenated core-genome content and average nucleotide identity (ANI) calculations, revealed that they constituted three novel and well-supported clusters. The closest relative species, determined using the whole-genome sequences of the strains, were Halomonas zhanjiangensis for two groups of cheese strains, sharing 82.4 and 93.1 % ANI, and another cluster sharing 92.2 % ANI with the Halomonas profundi type strain. The strains isolated herein differed from the previously described species by ANI values <95 % and several biochemical, enzymatic and colony characteristics. The results of phenotypic, phylogenetic and chemotaxonomic analyses indicated that the isolates belonged to three novel Halomonas species, for which the names Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov. are proposed, with isolates FME63T (=DSM 113315T=CIRM-BIA2430T=CIP 111880T=LMG 32013T), FME64T (=DSM 113316T=CIRM-BIA2431T=CIP 111877T=LMG 32015T) and FME66T (=DSM 113318T=CIRM-BIA2433T=CIP 111876T=LMG 32014T) as type strains, respectively.

First report of Diaporthe eres causing leaf spot on Rhododendron latoucheae Franch. in China.

Rhododendron latoucheae Franch. is an evergreen shrub with charming fragrance and large and abundant flowers that make it highly attractive as an ornamental species. The species is distribution in southwest China covers several different habitats and environments (Zhang, et al. 2022). From May to July in 2023, symptoms of leaf spot were observed on R. latoucheae over a wide portion of the Baili Azalea Forest Area (27°10' to 27°20'N, 105°04' to 106°04'E), Guizhou Province, China. About 500 plants were surveyed, and the incidence of leaf spot on R. latoucheae leaves was 12%, significantly reducing their ornamental and economic value. The affected leaves had irregular, grey white lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar, and incubated at 25°C for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GULJ1-L7, GULJ1-L8, and GULJ1-L9) identical in morphology were obtained. The isolate GULJ 1-L7 was used for further study. Colonies on PDA irregular grew white felty aerial mycelium, becoming white felted aerial mycelium in the centre and grey-brown mycelium at the marginal area on the upper surface, while the lower surface presents alternating white, tan and taupe. Colony with conidiomata irregularly distributed over agar surface. In the representative isolate, darkly pigmented pycnidia (flask-shaped) were produced over the colony surface on PDA after about 30 days, and oozed milky or yellowish mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 5.15-10.29 × 1.54-3.33 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.34-31.91 × 1.01-1.90 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes and the calmodulin (CAL) using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, and CAL-228F/CAL-737R, respectively (Tao et al. 2020). Sequences from PCR amplification were deposited in GenBank with accession numbers OR740563 (ITS), OR754301 (TUB), OR754298 (TEF), and OR754295 (CAL) respectively. BLAST searches of the sequences revealed 99.07% (533/538 nt), 100% (490/490 nt), 99.69% (317/318 nt) and 98.95% (376/380 nt) homology with those of D. eres AR5193T from GenBank (KJ210529.1, KJ420799.1, KJ210550.1 and KJ434999.1), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum-likelihood method placed the isolate GULJ1-L7 in a well-supported cluster with D. eres CBS 138694T. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). The pathogenicity of GULJ1-L7 was tested through a pot assay. Due to the difficulty of artificial planting wild R. latoucheae, we conducted a pot essay to detect the pathogenicity of GULJ1-L7 using a closely related Rhododendron delavayi Franch. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying α and β spore mixture suspension (106 spores ml-1) of GULJ1-L7 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times. After 14 days, the treated leaves developed brown lesions similar to those in the field, whereas the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. latoucheae caused by D. eres in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. latoucheae.

First Report of Leaf Spot Caused by Clonostachys rosea on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis), which originated in southwest of China 60 - 70 million years ago, is widely consumed as a beverage for its potential enhancing effect on human health with rich polyphenol content (Pan et al. 2022).From October to December in 2021, a disease with symptoms similar to leaf spot affected the quality and yield of teaPuer (102°73 'E, 25°07' N), Yunnan province, China. Based on the survey, leaf spot symptoms were observed on approximately 60% of tea plants in a 5,700 m2 field. The symptoms initially appeared as shrinking, yellowing, and later became circular or irregular brown spots. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees, and portions of the diseased tissue (0.5×0.5 cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol and 2 min with 3% NaOCl, washed three times with sterilized distilled water), the disinfected pieces were dried and plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. Four single-spore isolates, FH-1, FH-5, FH-6 and FH-7, were obtained, these isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS] and translation elongation factor 1-alpha [TEF] genes. Therefore, the representative isolate FH-5 was used for further study. Fungal colonies were white or light yellow on PDA after 7 days incubated at 28ºC. Conidia were hyaline, round or oval, aseptate, occur singly or in clusters on hyphae or conidia stalks, and measured as 2.94 ± 1.79 × 1.82 ± 0.2 μm (n = 50). Primary conidiophores is Verticillium-like (Fig1.K,L), which generally formed first, 1-3-level verticillate, mostly with divergent branches and phialides, and measured as 16.67 ± 4.39 µm (n = 50). Secondary conidiophores is penicillate (Fig1.I,J), which generally appearing after one week, sometimes even more often branched, and with a length of 16.02 ± 3.83 µm (n = 50). The morphological features were consistent with the descriptions of Clonostachys rosea Schroers H.J. (Schroers et al. 1999). The pathogen was confirmed to be C. rosea by amplification and sequencing of the internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4 and EF1-728F/EF1-986R, respectively (Fu Rongtao 2019). The sequences of PCR products were deposited in GenBank with accession numbers ON332533 (ITS) and OP080234 (TEF). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides) and 98.37% (241/245 nucleotides) homology with those of C. rosea HQ-9-1 form GenBank (MZ433177 and MZ451399, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate FH-5 in a well-supported cluster with C. rosea. The pathogenicity of FH-5 was tested through a pot assay. Ten healthy tea plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (105 spores·mL-1) of FH-5 onto leaves until runoff, and the control leaves sprayed with sterile water. Inoculated plants were put in an artificial climate box at 25℃, 70% relative humidity. The pathogenicity test was replicated three times. Symptoms developed on all inoculated leaves but not on the control leaves. Lesions around the wound edge became pale yellow, and brown spots were first observed at 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after two weeks. The same fungus was reisolated and identifiedbased on themorphological characterization and molecular analyses (ITSand TEF)from the infected leaves but not from the noninoculated leaves. In addition, C. rosea has also been reported to cause diseases to broad bean (N. Afshari et al. 2017 ), garlic (Diaz et al. 2022), beet (Haque M.E et al. 2020) and other plants. To our knowledge, this is the first report of leaf spot on tea caused by C. rosea in China. This study provides valuable information for the identification and control of the leaf spot on tea.

First Report of Grey Spot on Tobacco caused by Alternaria alstroemeriae in China.

Tobacco (Nicotiana tabacum L.) is an important economic crop belonging to family Solanaceae and is widely cultivated in China (Basit 2021). From April to July in 2022, a foliar disease with symptoms similar to grey spot was extensively observed on tobacco in Guangxi Province (24°52' N, 111°23' E), China. Field surveys were conducted in 18 towns and the disease incidence was 0.89% to 6.95%. Symptomatic leaves displayed irregular, dark brown lesions surrounded by yellow halos and accompanied with black conidiomata in gray centers (Fig 1A-E). Symptomatic leaves were collected from 54 different tobacco plants. After surface sterilization (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on PDA and incubated at 25°C for 5 days (Fang 2007). Three single-spore isolates, GUCC BZ6-3, GUCC LJ3-4, and GUCC XH1-13 were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GUCC BZ6-3 was used for further study. The colonies on PDA were villiform, greyish (Fig 1F-G). Conidia were abundant, ovoid, with 2-6 transverse septa and 1-2 longitudinal septa 12.60 (9.43 to 14.76) × 4.30 (3.57 to 5.14) μm (n=50) (Fig 1H-S). The morphological features were consistent with Alternaria alstroemeriae E.G. Simmons & C.F. Hill (Simmons 2007; Nishikawa & Nakashima, 2013). The pathogen was confirmed to be A. alstroemeriae by amplification and sequencing of the ITS, GAPDH, LSU, TEF1, and RBP2 genes using primers ITS1/ITS4, gpd1/gpd2, LSU1Fd/LR5, EF1-728F/EF1-986R, and RPB2-5F2/fRPB2-7cR, respectively (Woudenberg 2013). The sequences of the PCR products were deposited in GenBank with accession numbers ON693856 (RBP2), ON714497 (ITS), ON694345 (GAPDH), ON931420 (TEF1) and ON714499 (LSU). BLAST searches of the obtained sequences revealed 99% (565/567 nucleotides), 99% (577/579 nucleotides), 99% (908/911 nucleotides), 99% (238/239 nucleotides), and 99% (751/753 nucleotides) homology with those of A. alstroemeriae in GenBank (MH863036, KP124154, MH874589, KP125072, and KP124765, respectively). Phylogenetic analyses of the sequence data consisted of Bayesian and Maximum likelihood analyses of the combined aligned dataset (MEGA 7.0 and PhyloSuite 1.2.2). The GUCC BZ6-3 in a well-supported cluster with A. alstroemeriae (Fig 2). The pathogen was thus identified as A. alstroemeriae based on morphological characterization and molecular analyses. The pathogenicity of GUCC BZ6-3 was tested through pot assay and carried out three times (Fang 2007). Ten healthy 30-day-old tobacco plants were inoculated by spraying a spore suspension (106 spores·ml-1) of strain GUCC BZ6-3 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 28°C with high relative humidity (95%) in a growth chamber. The symptoms developed on all inoculated leaves but not on the control. The lesions were first visible 48 h after inoculation, and typical lesions similar to those observed on field plants appeared after 7 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the noninoculated leaves. Results of pathogenicity experiments fulfilled Koch's postulates. To our knowledge, this is the first report of grey spot disease on tobacco caused by A. alstroemeriae in China. Our findings would be of great importance for the diagnosis and control of the emerging grey spot on tobacco.

First Report of Anthracnose on Tetrapanax papyriferus Caused by Colletotrichum fructicola in China.

Tetrapanax papyriferus is an evergreen shrub native to China and traditionally used as a herbal medicine (Li et al., 2021). In September 2021, a serious leaf spot disease with symptoms similar to anthracnose was extensively observed on T. papyriferus in Shibing county (E 127°12'0", N 25°11'60"), Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou province, China. Field surveys were conducted in about 1000 T. papyriferus plants in Shibing in September 2021. The incidence of the leaf spot on leaves was 45% to 60%, significantly reducing the quality of medicinal materials. The symptoms began as small yellow spots, developing a brown center and dark brown to black margin, and eventually the diseased leaves were wiltered and rotted. Symptomatic leaves were collected from 20 trees. Symptomatic tissue from diseased leaves was surface desinfected (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days (Fang. 2007). Three single-spore isolates were obtained (GUTC37, GUTC310 and GUTC311) and deposited in the collection of the Plant Pathology Deparment, College of Agriculture, Guizhou University, China (GUCC) (with the accession numbers, GUCC220241, GUCC220242, GUCC220243 respectively). These isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS], glyceraldehy-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], actin [ACT], and calmodulin [CAL] genes (White et al. 1990; Carbone and Kohn 1999; Templeton et al. 1992). Therefore, the representative isolate GUTC37 was used for further analysis. The pathogenicity of GUTC37 was tested through a pot assay. Plants were inoculated by spraying a spore suspension (106 spores·ml-1) of isolated strains onto leaves until runoff, and the control leaves sprayed with sterile water. The inoculated plants were incubated in a growth chamber at 28 ℃ and 95% relative humidity for 10 days. Pathogenicity tests were repeated three times (Fang. 2007). The symptoms developed on the inoculated leaves, while control remained asymptomatic. The lesions were first visible 72 h after inoculation, and typical lesions like those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the non-inoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white or greyish, aerial mycelium pale grey, dense, surface partly covered with orange conidial masses. The conidia were abundant, oval-ellipsoid, aseptate, and 13.89 (11.62 to 15.21) × 5.21 (4.39 to 5.65) µm (n=50). Appressorium were greyish green, nearly ovoid to cylindrical, 9.64 (6.62 to 14.61) × 6.33 (5.45-7.72) µm (n=50). The morphological features were consistent with the descriptions of Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Prihastuti et al. 2009). The pathogen was identified to be C. fructicola by amplification and sequencing of the five genes. The sequences of the PCR products were deposited in GenBank with accession numbers OP143657 (ITS), OP177868 (GAPDH), OP177865 (CHS-1), OP278677 (ACT) and OP177862 (CAL). BLAST searches of the obtained sequences revealed 100% (509/509 nucleotides), 99.63% (269/270 nucleotides), 99.31% (287/289 nucleotides), 99.29% (280/282 nucleotides), and 99.86% (728/729 nucleotides) homology with those of C. fructicola in GenBank (JX010165, JX010033, JX009866, FJ907426, and JX009676, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUTC37 in a well-supported cluster with C. fructicola. To our knowledge, this is the first report of anthracnose on T. papyriferus caused by C. fructicola in Guizhou, China. This study provides valuable information for the identification and control of the anthracnose on T. papyriferus.

First report of leaf spot on Rhododendron delavayi caused by Pestalotiopsis scoparia in China.

Rhododendron delavayi is an evergreen shrub with large scarlet flowers that make it highly attractive as an ornamental species. The species is native to southwest China (Cai et al. 2015). From May to July in 2022, symptoms of leaf spot were observed on R. delavayi over a wide portion of the Baili Azalea Forest Area (N 27°10'-27°20', E 105°04'-106°04'), Guizhou Province, China. About 500 plants were surveyed and the incidence of leaf spot on R. delavayi leaves was 20 to 30%, significantly reducing their ornamental and economic value. The affected leaves had irregular, dark brown lesions with a clear blackish brown boundary and black conidiomata in a grayish center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar and incubated at 25℃ for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GUDJ 61, GUDJ 62, GUDJ 63) that produced identical in morphology were obtained. The isolate GUDJ 61 was used for further study. Colonies on PDA grew velvety white on the upper surface and light yellow on the lower surface. Conidia were 5-celled, spindle- to ellipsoid-shaped, straight or slightly curved, 4-septate, and measured 39.0 ± 3.7 × 10.4 ± 0.79 µm (n=50). The morphological features were consistent with the description of Pestalotiopsis scoparia Maharachch., K.D. Hyde & Crous, (Maharachchikumbura et al. 2014). The pathogen was confirmed to be P. scoparia by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), and the partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b, and EF1-728F/EF-2, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers OP048045 (ITS), OP058111 (TUB) and OP058114 (TEF), respectively. BLAST searches of the sequences revealed 99% (549/552 nt), 99% (711/714 nt), and 82% (130/158 nt) homology with those of P. scoparia CBS 176.25T form GenBank (KM199330, KM199393, and KM199478), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUDJ 61 in a well-supported cluster with P. scoparia. The pathogen was thus identified as P. scoparia based on the morphological characterization and molecular analyses. The pathogenicity of GUDJ 61was tested through a pot assay. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying a spore suspension (106 spores mL-1) of GUDJ 61 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times (Fang 2007). After 12 days, the treated leaves developed brown lesions similar to those in the field, while the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. delavayi caused by P. scoparia in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. delavayi.

Ancylobacter mangrovi sp. nov., a novel endophytic bacterium isolated from mangrove plant

Two novel strains GSK1Z-4-2T and MQZ15Z-1 were isolated from branches of mangrove plants collected from Guangxi Zhuang Autonomous Region, China. Both strains were Gram-negative, aerobic, non-flagellated and non-spore-forming bacteria. The comparison of 16S rRNA gene sequences initially indicated that the two strains were assigned to the genus Ancylobacter with sharing the highest similarity to Ancylobacter pratisalsi DSM 102029T (97.3%). The 16S rRNA gene sequence similarity, average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between strains GSK1Z-4-2T and MQZ15Z-1 were 99.9%, 97.4% and 77.4%, respectively, which revealed that the two strains belonged to the same species. Phylogenetic analyses based on 16S rRNA gene sequences and the core proteome showed that the two strains formed a well-supported cluster with A. pratisalsi DSM 102029T. Moreover, the ANI and isDDH values between strain GSK1Z-4-2T and A. pratisalsi DSM 102029T were 83.0% and 25.8%, respectively, demonstrating that strain GSK1Z-4-2T was a previously undescribed species. Meanwhile, strains GSK1Z-4-2T and MQZ15Z-1 exhibited most of chemotaxonomic and phenotypic features consistent with the description of the genus Ancylobacter. Based on the polyphasic data, strains GSK1Z-4-2T and MQZ15Z-1 should represent a novel species of the genus Ancylobacter, for which the name Ancylobacter mangrovi sp. nov. is proposed. The type strain is GSK1Z-4-2T (=MCCC 1K07181T = JCM 34924T).

Leaf spot on Photinia × fraseri caused by Neopestalotiopsis asiatica in China.

Photinia × fraseri is a well-known evergreeen ornamental tree. Owing to its flower-like red leaves and its ability to tolerate stressful environments, P. fraseri is widely cultured as a fast-growing hedge in southern China. From JulytoSeptemberin2021, a disease with symptoms similar to leaf spot was extensively observed on P. fraseri in Daozhen county (28° 51 'N, 107° 57 'E), Zunyi, Guizhou province, China. About 500 plants were surveyed and the incidence of leaf spot on P. fraseri leaves was 35% to 70%, significantly reducing the ornamentalandeconomicvalue. The symptomatic leaves displayed irregular, watery dark brown lesions with black conidiomata in gray centers, and 10 symptomatic leaves were collected from 10 trees.After surface sterilization (0.5 min in 75% ethanol and 2 min in 3% NaOCl, washed three times with sterilized distilled water) (Fang 2007), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days. Three single-spore isolates, GZAAS21-0327, GZAAS21-0328 and GZAAS21-0329, were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GZAAS21-0328 was used for further study. The pathogenicity of GZAAS21-0328 was tested through a pot assay. Ten healthy plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (106spores mL-1) of GZAAS21-0328 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 28°C with high relative humidity (95%) in a growth chamber. The pathogenicity test was carried out three times (Fang 2007). The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after 15 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses (ITS, TUB and TEF) from the infected leaves but not from the noninoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white and sparse aerial mycelium on the surface with black, gregarious conidiomata. The conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate, septa darker than the rest of the cell, and 23.0 (21.0 to 27.0) × 6.0 (5.0 to 7.0) µm (n=50). The morphological features were consistent with the descriptions ofNeopestalotiopsis asiatica Maharachch. & K.D. Hyde (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014; Farr et al. 2022). The pathogen was confirmed to beN. asiaticaby amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b and EF1-728F/EF-2, respectively (Maharachchikumbura et al. 2014). The sequences of PCR products were deposited in GenBank with accession numbers OK563071 (ITS), OK584020 (TUB) and OK663023 (TEF). BLAST searches of the obtained sequences revealed 100% (482/482 nucleotides), 99.05% (419/421 nucleotides), and 99.33% (891/897 nucleotides) homology with those ofN. asiaticain GenBank (JX398983, JX399018 and JX399049, respectively). Phylogenetic analysis (MEGA 6.0) using the maximum likelihood method placed the isolate GZAAS21-0328 in a well-supported cluster withN. asiatica. The pathogen was thus identified asN. asiaticabased on the morphological characterization and molecular analyses. To our knowledge, this is the first report of leaf spot on P. fraseri caused by N. asiatica in China. This study provides valuable information for the identification and control of the leaf spot on Photinia × fraseri.

Genome-based analyses of the genus Acidovorax: proposal of the two novel genera Paracidovorax gen. nov., Paenacidovorax gen. nov. and the reclassification of Acidovorax antarcticus as Comamonas antarctica comb. nov. and emended description of the genus Acidovorax.

The genus Acidovorax is a genetically heterogeneous species clustering that comprises many environmental and plant-pathogenic taxa. To better understand the evolutionary relationships among the Acidovorax species, 22 available genome sequences of type strains including the genera Acidovorax and Comamonas were used to conduct the genome-based analyses. Three well-supported monophyletic clusters of the Acidovorax species were determined based on the phylogenomic tree reconstructed using core genes, while they were not grouped in the 16S rRNA gene-based phylogenetic tree. The species arrangements of the genus Acidovorax were further confirmed by the comparisons of the digital DNA-DNA hybridization and average nucleotide identity (ANI) values. The ANI, average amino acid identity, and the percentage of conserved proteins values among the inter-clusters were approximately 83, 81, and 61%, respectively, and thus were proposed as practical thresholds for genus delineation. Besides, Acidovorax antarcticus was much closer to members of the genus Comamonas rather than those of the genus Acidovorax based on the genome-based analysis. Taken together, we propose the division of the current genus Acidovorax into the emended genus Acidovorax and the two novel genera Paracidovorax gen. nov., Paenacidovorax gen. nov. and the transfer of Acidovorax antarcticus into the genus Comamonas as Comamonas antarctica comb. nov.

Multilocus genetic analysis indicates taxonomic status of “Candidatus Rickettsia mendelii” as a separate basal group

To date, the phylogeny of Rickettsia spp. from basal groups is based on the small number of identified species. Thus, the finding of “Candidatus Rickettsia mendelii” in 2016 is of great interest. In this study, “Ca. R. mendelii” was first identified in the Asian region in a new carrier, Ixodes pavlovskyi. “Candidatus R. mendelii”, along with “Candidatus Rickettsia tarasevichiae”, were found in Ixodes ticks collected on Russky Island (the Far East), where I. pavlovskyi coexists with I. persulcatus. To establish the taxonomic position of “Ca. R. mendelii”, a detailed genetic study was carried out. “Candidatus R. mendelii” was genotyped by five genetic fragments (16S rRNA, gltA, and ompB genes, groESL operon, and 23S–5S IGS region); among them, the ompB gene, groESL operon and 23S–5S IGS region were sequenced for the first time. In addition, “Ca. R. tarasevichiae” was genetically characterized by eight genetic loci (16S rRNA, gltA, ompA, ompB, sca4, htrA genes, groESL operon, and 23S–5S IGS region), of which the sca4 gene was first determined. Phylogenetic analysis indicated that regardless of analyzed genetic loci, “Ca. R. mendelii” formed a separate well-supported cluster on each phylogenetic tree. Phylogenetic analysis based on concatenated sequences of gltA, ompB, and groEL gene fragments (total length of 3191 bp) demonstrated that “Ca. R. mendelii”, like Rickettsia bellii, is a basal group of Rickettsia.

Taxogenomic status of phylogenetically distant Frankia clusters warrants their elevation to the rank of genus: A description of Protofrankia gen. nov., Parafrankia gen. nov., and Pseudofrankia gen. nov. as three novel genera within the family Frankiaceae.

The genus Frankia is at present the sole genus in the family Frankiaceae and encompasses filamentous, sporangia-forming actinomycetes principally isolated from root nodules of taxonomically disparate dicotyledonous hosts named actinorhizal plants. Multiple independent phylogenetic analyses agree with the division of the genus Frankia into four well-supported clusters. Within these clusters, Frankia strains are well defined based on host infectivity range, mode of infection, morphology, and their behaviour in culture. In this study, phylogenomics, overall genome related indices (OGRI), together with available data sets for phenotypic and host-plant ranges available for the type strains of Frankia species, were considered. The robustness and the deep radiation observed in Frankia at the subgeneric level, fulfilling the primary principle of phylogenetic systematics, were strengthened by establishing genome criteria for new genus demarcation boundaries. Therefore, the taxonomic elevation of the Frankia clusters to the rank of the genus is proposed. The genus Frankia should be revised to encompass cluster 1 species only and three novel genera, Protofrankia gen. nov., Parafrankia gen. nov., and Pseudofrankia gen. nov., are proposed to accommodate clusters 2, 3, and 4 species, respectively. New combinations for validly named species are also provided.

Filling a gap in the taxonomy of phyllachoroid fungi: Proposition of Neopolystigma, gen. nov., and the new family Neopolystigmataceae

ABSTRACT A Polystigma-like found on an herbaceous to shrubby species of Fabaceae (Andira humilis) in the Brazilian Cerrado was morphologically close to Polystigma pusillum, a leaf parasite on Andira inermis collected in Central America and Brazil. Phylogenetic analyses using a combination of the rDNA 28S, 18S, and internal transcribed spacer (ITS) regions placed both fungi in the Phyllachorales, and not in Polystigamataceae/Xylariales, where Polystigma species belong, and characteristically found on members of the Rosaceae, causing red leaf blotch containing bright-colored fungal stromata spread on the lesions. This disease prevails in orchards in the Northern Hemisphere, infecting Amygdalus, Cerasus, Padus, and Prunus species, but never in the Tropics. Polystigma species infecting other botanical families have been reallocated in different families, orders, and even classes in Ascomycota. In our phylogenetic analyses, the two species on Andira were allocated in Phyllachorales but separated in a well-supported cluster from Phyllachoraceae and Phaeochoraceae. In relation to Telimenaceae, the statistical support is not strong; however, considering that its type genus, Telimena, was never sequenced, we choose to accept Neopolystigma (type N. saraivae) as the type genus of a new family, Neopolystigmataceae. The sister species of N. saraivae, Polystigma pusillum found on A. inermis, was recombined into N. pusillum.

Rapid Multilateral and Integrated Public Health Response to a Cross-City Outbreak of Salmonella Enteritidis Infections Combining Analytical, Molecular, and Genomic Epidemiological Analysis.

On September 21, 2019, the Shenzhen and Dongguan Centers for Disease Control and Prevention received notification of a large cluster of suspected gastroenteritis involving primarily children who sought medical care at hospitals throughout two adjacent cities in China, Shenzhen, and Dongguan. A joint outbreak response was promptly initiated across jurisdictions in a concerted effort between clinical microbiologists, epidemiologists, and public health scientists. Concurrently, multiplex PCRs were used for rapid laboratory diagnosis of suspected cases; epidemiological investigations were conducted to identify the outbreak source, complemented by near real-time multicenter whole-genome analyses completed within 34 h. Epidemiological evidence indicated that all patients had consumed egg sandwiches served on September 20 as snacks to children and staff at a nursery in Dongguan, located near Shenzhen. Salmonella Enteritidis was isolated from case-patients, food handlers, kitchenware, and sandwiches with kitchen-made mayonnaise. Whole-genome single-nucleotide polymorphism (SNP)-based phylogenetic analysis demonstrated a well-supported cluster with pairwise distances of ≤1 SNP between genomes for outbreak-associated isolates, providing the definitive link between all samples. In comparison with historical isolates from the same geographical region, the minimum pairwise distance was >14 SNPs, suggesting a non-local outbreak source. Genomic source tracing revealed the possible transmission dynamics of a S. Enteritidis clone throughout a multi-provincial egg distribution network. The efficiency and scale with which multidisciplinary and integrated approaches were coordinated in this foodborne disease outbreak response was unprecedented in China, leading to the timely intervention of a large cross-jurisdiction Salmonella outbreak.