Multilocus Sequence Analysis Research Articles

Functional analysis and comparative genomics of Rahnella perminowiae S11P1 and Variovorax sp. S12S4, two plant growth-promoting rhizobacteria isolated from Crocus sativus L. (saffron) rhizosphere.

Rahnella perminowiae S11P1 and Variovorax sp. S12S4 are two plant growth-promoting rhizobacteria that were previously isolated from the rhizosphere of Crocus sativus L. (saffron), and have demonstrated interesting PGP activities and promising results when used as inoculants in field trials. To further elucidate the molecular mechanisms underlying their beneficial effects on plant growth, comprehensive genome mining of S11P1 and S12S4 and comparative genomic analysis with closely related strains were conducted. Functional annotation of the two strains predicted a large number of genes involved in auxin and siderophore production, nitrogen fixation, sulfur metabolism, organic acid biosynthesis, pyrroloquinoline quinone production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, volatile organic compounds production, and polyamine biosynthesis. In addition, numerous genes implicated in plant-bacteria interactions, such as those involved in chemotaxis and quorum sensing, were predicted. Moreover, the two strains carried genes involved in bacterial fitness under abiotic stress conditions. Comparative genomic analysis revealed an open pan-genomic structure for the two strains. COG annotation showed that higher fractions of core and accessory genes were involved in the metabolism and transport of carbohydrates and amino acids, suggesting the metabolic versatility of the two strains as effective rhizosphere colonizers. Furthermore, this study reports the first comparison of Multilocus sequence analysis (MLSA) and core-based phylogenies of the Rahnella and Variovorax genera. The present study unveils the molecular mechanisms underlying plant growth promotion and biocontrol activity of S11P1 and S12S4, and provides a basis for their further biotechnological application in agriculture.

Diversity of Strains in the Pseudomonas syringae Complex Causing Bacterial Stem Blight of Alfalfa (Medicago sativa) in the United States.

Alfalfa growers in the Intermountain West of the United States have recently seen an increased incidence in bacterial stem blight (BSB), which can result in significant herbage yield losses from the first harvest. BSB has been attributed to Pseudomonas syringae pv. syringae and P. viridiflava; however, little is known about the genetic diversity and pathogenicity of these bacteria or their interaction with alfalfa plants. Here, we present a comprehensive phylogenetic and phenotypic analysis of P. syringae and P. viridiflava strains causing BSB on alfalfa. A multilocus sequence analysis found that they grouped exclusively with P. syringae PG2b and P. viridiflava PG7a. Alfalfa symptoms caused by both bacterial groups were indistinguishable, although there was a large range in mean disease scores for individual strains. Overall, PG2b strains incited significantly greater disease scores than those caused by PG7a strains. Inoculated plants showed browning in the xylem and collapse of epidermal and pith parenchyma cells. Inoculation with a mixture of PG2b and PG7a strains did not result in synergistic activity. The populations of PG2b and PG7a strains were genetically diverse within their clades and did not group by location or haplotype. The PG2b strains had genes for production of the phytotoxin coronatine, which is unusual in PG2b strains. The results indicate that both pathogens are well established on alfalfa across a wide geographic range and that a recent introduction or evolution of more aggressive strains as the basis for emergence of the disease is unlikely.

Occurrence of 'Candidatus Phytoplasma asteris'-Related Strains infecting Chinaberry (Melia azedarach) Showing Chlorotic Leaf Symptoms on Hainan Island of China.

Chinaberry (Melia azedarach), belonging to the family of Meliaceae, is an ornamental tree distributes across southern of China. In the autumn of 2021, In an area of 400 acres located in Wanning city of Hainan Province, a tropical island in China, with coordinates of 110°28'42.72″E, 19°2'9.96″N, about 20 % (100) of the chinaberry trees showed disease symptoms included chlorotic leaves. The disease symptoms were consistent with infections by a phloem-limited prokaryotic pathogen phytoplasma. The samples of six symptomatic and three asymptomatic were collected for pathogen detection. To identify the pathogen, total nucleic acids were extracted from 0.10 g fresh leaf tissues from the diseased and healthy plant using CTAB DNA extraction method based on Doyle and Doyle. Three primer pairs of R16mF2/R16mR1, secAfor1/secArev3 and fTuf1/rTuf1 were used for specific identification of phytoplasma conserved gene fragments of 16S rDNA, secA and tuf, PCR amplification. Target PCR bands were amplified from the DNA of six diseased chinaberry samples, but not from the DNA of the healthy samples. The products of amplified were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The phytoplasma gene sequences of 16S rRNA, secA and tuf were obtained and all the sequences were identical with the length of 1336 bp, 710 bp and 955 bp, respectively. Representative sequence data for strain MaCL-hn were deposited in Genbank under accession Nos. OR438638 (16S rDNA), OR513089 (secA) and OR860415 (tuf). The phytoplasma strain identified in the study was described as chinaberry chlorotic leaf (MaCL) phytoplasma, MaCL-hn strain. BLAST search based on 16S rRNA genes showed that 43 strains in 16SrI group 'Candidatus Phytoplasma asteris' showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on secA genes showed that 9 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on tuf genes showed that 21 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. RFLP analysis based on iPhyClassifier indicated that the MaCL-hn strain was a member of 16SrI-B subgroup with a similarity coefficient 1.00 to the reference phytoplasma strain (AP006628). Phylogenetic tree was constructed based on 16S rRNA by MEGA 11.0 using neighbor-joining (NJ) method with 1000 bootstrap value. The results showed that the MaCL-hn strains were clustered into one clade with 16SrI group 'Ca. Phytoplasma asteris' related strains with 99 % bootstrap value. Multilocus sequence analysis (MLSA) based on the concatenated sequences with the length of 3001 bp including the sequences of 16S rRNA, secA and tuf showed that the MaCL-hn strains were clustered into one clade with the phytoplasma strains in the group with 100 % bootstrap value. To our knowledge, this is the first report that chinaberry can be infected by 'Ca. Phytoplasma asteris'-related strains belonging to 16SrI-B subgroup on Hainan Island of China. This finding in the study will contribute to the epidemic monitoring and the preventive management of the phytoplasmas and their related diseases.

First Report of Leaf Spot on Taraxacum mongolicum Caused by Alternaria solani in China.

Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27'17.65″N, 101°47'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 μm, and 80.7 × 3.9 μm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3，TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch's postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.

Multi-locus sequence analysis of ‘Candidatus Mycoplasma haematomacacae’ in free-ranging macaques from Thailand suggestive of a closer relationship to hemotropic mycoplasmas in capuchins and potential origin from bats

Although ‘Candidatus Mycoplasma haematomacacae’ (formerly known as ‘Candidatus Mycoplasma haemomacaque’) has been reported on extensively in macaques from Thailand, the USA, Japan, and Brazil, its genetic characterization has primarily been restricted to the 16S rRNA sequences with no exploration on multi-locus sequence analysis. The primary goal of this study was to characterize ‘Ca. M. haematomacacae’ among Thai macaques based on multiple genetic markers. Between April 2018 and November 2021, blood samples were taken from 580 free-ranging macaques (560 Macaca fascicularis and 20 Macaca nemestrina) in 15 locations encompassing 10 provinces throughout Thailand. Using the conventional PCR assay targeting the 16S ribosomal RNA (16S rRNA) gene, 338 out of 580 macaques (58.27 %) tested hemoplasma-positive. Of these, 40 positive samples were further subjected to DNA sequencing, and all were identified as ‘Ca. M. haematomacacae’. Subsequently, the partial nucleotide sequences of 23S ribosomal RNA (23S rRNA) and RNase P RNA (rnpB) genes of this particular hemoplasma species were amplified through nested PCR assay. The analysis of multi-locus genetic markers revealed that the 23S rRNA and rnpB sequences exhibited higher levels of genetic diversity than the 16S rRNA sequences. Furthermore, the 16S rRNA analyses demonstrated that ‘Ca. M. haematomacacae’ infecting Old World monkeys (Macaca spp.) was most closely related to hemotropic Mycoplasma spp. in black-capped capuchins (Sapajus apella) and Marcgrave's capuchins (Sapajus flavius) from Brazil, as well as establishing a common ancestor clade with hemotropic Mycoplasma spp. from the Neotropical bats in Belize and Peru and an Old World bat in Spain. The 23S rRNA analyses likewise evidenced that ‘Ca. M. haematomacacae’ formed a sister clade with hemotropic Mycoplasma spp. in Neotropical bats from Belize and Panama. Thus, the present findings, based on multi-locus sequence analysis, suggest a potential origin of ‘Ca. M. haematomacacae’ from Neotropical and Old World bats. To the best of the authors’ knowledge, this study provided the largest dataset so far of multi-locus genetic sequences of ‘Ca. M. haematomacacae’ isolated from Thai macaques and enhanced the accuracy of phylogenetic analyses, providing insights into their origins among hemotropic Mycoplasma spp. discovered worldwide.

Genomic characterization of Pseudomonas syringae pv. syringae from Callery pear and the efficiency of associated phages in disease protection.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.

Bartonella in bat flies from the Egyptian fruit bat in the Middle East

In the family of fruit bats, Pteropodidae Gray, 1821, as in the third most diverse group of bats (Chiroptera), the bacterium of the genus Bartonella was detected in several species as well as in a few species of their insect ectoparasites in some tropical and sub-tropical regions of the Old World. The Egyptian fruit bat, Rousettus aegyptiacus (Geoffroy, 1810), is one of the most widespread fruit bats, occurring between South Africa, Senegal, and Pakistan. In this bat species, Candidatus Bartonella rousetti has been detected in three African populations in Nigeria, Kenya, and Zambia. This fruit bat, however, also occurs in the Palaearctic, an area isolating the species geographically and phylogenetically from the Afrotropical part of its distribution range. We screened the blood-sucking bat flies (family Nycteribiidae) from R. aegyptiacus for the presence of the Bartonella bacteria. A rich material of bat fly Eucampsipoda aegyptia (Macquart, 1850), a monoxenous ectoparasite of the Egyptian fruit bats, was collected at 26 localities in seven countries (Egypt, Iran, Jordan, Lebanon, Oman, United Arab Emirates, and Yemen) of the Middle East in 2007–2013. The DNA isolates from the bat flies were subjected to a three-marker (gltA, ssrA, and intergenic spacer region, ITS) multilocus sequence analysis. Based on the amplification of the fragment of ssrA gene by a real-time PCR, 65 E. aegyptia samples from 19 localities in all seven countries were positive for the bacteria. One to five Bartonella-positive individuals of E. aegyptia were collected per one individual of R. aegyptiacus. An analysis of the ITS and gltA genes indicated the presence of an uncultured Bartonella sp., belonging to the Cand. B. rousetti genogroup, identified from populations of the Egyptian fruit bat in Africa. These results support the hypothesis that Bartonella’s diversity corresponds to its host’s diversity (and phylogenetic structure). Specific lineages of pathogens are present in specific phylogenetic groups of bats.

Analyses of Multilocus Sequences and Morphological Features Reveal Ilyonectria Species Associated with Black Rot Disease of Gastrodia elata.

Black rot is a common disease of Gastrodia elata, causing serious threats to G. elata production. In this study, a total of 17 Cylindrocarpon-like strains were isolated from G. elata black rot tissues. Multilocus sequence analyses based on ITS, HIS, TEF, and TUB combined with morphological characterizations were performed to identify six Ilyonectria species, including four new species, Ilyonectria longispora, I. sinensis, I. xiaocaobaensis, and I. yunnanensis, and two known species, I. changbaiensis and I. robusta. The pathogenicity of 11 isolates comprising type strains of the four new species and representative isolates from each of the six species was tested on healthy tissues of G. elata. All isolates were pathogenic to G. elata tissues, and symptoms were identical to black rot disease, confirming that our isolates were the causal agents of black rot disease of G. elata.

Biochemical and Molecular Identification of a <i>Native Bacillus thuringiensis gv. cytolyticus</i> Isolate with Insecticidal Effect Against the Pod Borer Larvae (<i>Helicoverpa armigera</i>)

The aim of this study is to isolate and identify autochthonous bacterial strains with insecticidal effect against the larvae of the tomato moth Helicoverpa armigera. Dead and diseased larvae were collected from chili plots in the region of Gotraniya from the governorate of Kairouan, Tunisia. These larvae were used to isolate eight bacterial strains named Hr1 to Hr8. The bacterial isolates were characterized by macroscopic and microscopic observations and 16S rRNA sequencing. Hr1, Hr2, Hr4, Hr5, Hr6, and Hr8 were identified as Bacillus spp., Hr3 as Staphylococcussp., and Hr7 as Enterobacter sp. Their insecticidal activity was evaluated against third-instar larvae of H. armigera. A Hr1 bacterial isolate showed an insecticidal effect against H. armigera larvae, causing 60% larval mortality four days post-treatment at a dose of 1.8 × 109 CFU/ml. Based on further characterization studies, Hr1 was identified as Bacillus thuringiensis gv. cytolyticus following Biolog biochemical tests and multi-locus sequence analyses studies based on sequencing of seven housekeeping genes.

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.

Multi-locus sequence analysis reveals phylogenetically segregated Entamoeba histolytica population.

Amoebiasis, caused by the enteric parasite, Entamoeba histolytica, is one of the major food- and water-borne parasitic diseases in developing countries with improper sanitation and poor hygiene. Infection with E. histolytica has diverse disease outcomes, which are determined by the genetic diversity of the infecting strains. Comparative genetic analysis of infecting E. histolytica strains associated with differential disease outcomes from different geographical regions of the world is important to identify the specific genetic patterns of the pathogen that trigger certain disease outcomes of Amoebiasis. The strategy is able to elucidate the genealogical relation and population structure of infecting E. histolytica strains from different geographical regions. In the present study, we have performed a comparative genetic analysis of circulating E. histolytica strains identified from different parts of the world, including our study region, based on five tRNA-linked short tandem repeat (STR) loci (i.e., D-A, NK2, R-R, STGA-D and A-L) and evaluated their potential associations with differential disease outcomes of Amoebiasis. A number of regional-specific, emerging haplotypes of E. histolytica, significantly associated with specific disease outcomes have been identified. Haplotypes, which have a significant positive association with asymptomatic and amoebic liver abscess outcomes, showed a significant negative association with diarrheal outcome, or vice versa. Comparative multi-locus analysis revealed that E. histolytica isolates from our study region are phylogenetically segregated from the isolates of other geographical regions. This study provides a crucial overview of the population structure and emerging pattern of the enteric parasite, E. histolytica.

Diesel−biodegradation and biosurfactant−production by Janthinobacterium lividum AQ5-29 and Pseudomonas fildesensis AQ5-41 isolated from Antarctic soil

Given the substantial diesel demand in Antarctic operations, the means of addressing ecological restoration following its inappropriate release are attracting attention from researchers. The Madrid Protocol mandates the use of indigenous microbes in bioremediation. Recent studies have proposed many native isolates with biodegradation temperatures exceeding 20 °C, which are impractical for Antarctic contexts. Therefore, harnessing psychrophilic, native degraders with biosurfactant−producing traits presents an advantage for implementation in the harsh Antarctic environment. In this study, effective consortia/isolates demonstrated robust growth and biodegradation rates at 10 °C with diesel as the sole carbon source. Two primary bacterial members, Janthinobacterium lividum and Pseudomonas fildesensis, were identified from the most effective consortium SI 20 using 16S rRNA and multilocus−sequence−analysis (MLSA) clustering. The degraders were characterised as being psychrophilic, Gram-negative, rod−shaped, and catalase− and oxidase−positive. Despite the observed antagonistic effects during co-cultivation, strains J. lividum AQ5-29 and P. fildesensis AQ5-41 demonstrated effective diesel removal (2.91 & 4.20 mg mL−1) with biodegradation of C10 to C30 hydrocarbons (40−100%) at 10 °C in less than 8 days. Both strains also were identified as biosurfactant producers with varying emulsification activities (32−92%) and cell surface hydrophobicity (52−58%). These findings highlight the potential of both strains for restoring diesel-related substrates, particularly in Antarctica.

Syncephalastrum massiliense sp. nov. and Syncephalastrum timoneanum sp. nov. Isolated from Clinical Samples.

Mucormycosis is known to be a rare opportunistic infection caused by fungal organisms belonging to the Mucorales order, which includes the Syncephalastrum species. These moulds are rarely involved in clinical diseases and are generally seen as contaminants in clinical laboratories. However, in recent years, case reports of human infections due to Syncephalastrum have increased, especially in immunocompromised hosts. In this study, we described two new Syncephalastrum species, which were isolated from human nails and sputum samples from two different patients. We used several methods for genomic and phenotypic characterisation. The phenotypic analysis relied on the morphological features, analysed both by optical and scanning electron microscopy. We used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, energy-dispersive X-ray spectroscopy, and BiologTM technology to characterise the proteomic, chemical mapping, and carbon source assimilation profiles, respectively. The genomic analysis relied on a multilocus DNA sequence analysis of the rRNA internal transcribed spacers and D1/D2 large subunit domains, fragments of the translation elongation factor-1 alpha, and the β-tubulin genes. The two novel species in the genus Syncephalastrum, namely S. massiliense PMMF0073 and S. timoneanum PMMF0107, presented a similar morphology: irregular branched and aseptate hyphae with ribbon-like aspects and terminal vesicles at the apices all surrounded by cylindrical merosporangia. However, each species displayed distinct phenotypic and genotypic features. For example, S. timoneanum PMMF0107 was able to assimilate more carbon sources than S. massiliense PMMF0073, such as adonitol, α-methyl-D-glucoside, trehalose, turanose, succinic acid mono-methyl ester, and alaninamide. The polyphasic approach, combining the results of complementary phenotypic and genomic assays, was instrumental for describing and characterising these two new Syncephalastrum species.

Characterization of Emerging Xanthomonas campestris Isolates on the Nonnative Weed Garlic Mustard (Alliaria petiolata).

Nonnative plant infestations provide unique opportunities to investigate pathogen emergence with evolutionarily recent plant introduction events. The widespread distribution of invasive plants and their proximity to genetically related crops highlights the risks of nonnative plants acting as ancillary hosts and fostering microbial recombination and pathogen selection. Garlic mustard (Alliaria petiolata) is a widespread, nonnative cruciferous weed that grows throughout North America and along the forested edges of diverse agricultural fields. The recent identification of a novel Xanthomonas campestris pv. incanae strain isolated from a diseased A. petiolata population led to the current investigation of the distribution and diversity of X. campestris isolates from naturally infected A. petiolata. A total of 14 diseased A. petiolata sites were sampled across three states, leading to the identification of diverse X. campestris pathotypes and genotypes. Pathogenicity assays and multilocus sequence analyses identified pathogenic X. c. pv. incanae and X. c. pv. barbareae strains collected from disparate A. petiolata populations. Moreover, independently collected X. c. pv. incanae strains demonstrated a broad cruciferous host range by infecting cabbage (Brassica oleracea var. capitata), garden stock (Matthiola incana), and the cover crop yellow mustard (Guillenia flavescens). This study highlights the genetic variability and host potential of natural X. campestris populations and the potential risks to Brassica crops via widespread, dense garlic mustard reservoirs.

Comamonas resistens sp. nov. and Pseudomonas triclosanedens sp. nov., two members of the phylum Pseudomonadota isolated from the wastewater treatment system of a pharmaceutical factory.

Five strains of two novel species were isolated from the wastewater treatment systems of a pharmaceutical factory located in Zhejiang province, PR China. Strains ZM22T and Y6 were identified as belonging to a potential novel species of the genus Comamonas, whereas strains ZM23T, ZM24 and ZM25 were identified as belonging to a novel species of the genus Pseudomonas. These strains were characterized by polyphasic approaches including 16S rRNA gene analysis, multi-locus sequence analysis, average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), physiological and biochemical tests, as well as chemotaxonomic analysis. Genome-based phylogenetic analysis further confirmed that strains ZM22T and Y6 form a distinct clade closely related to Comamonas testosteroni ATCC 11996T and Comamonas thiooxydans DSM 17888T. Strains ZM23T, ZM24 and ZM25 were grouped as a separate clade closely related to Pseudomonas nitroreducens DSM 14399T and Pseudomonas nicosulfuronedens LAM1902T. The orthoANI and isDDH results indicated that strains ZM22T and Y6 belong to the same species. In addition, genomic DNA fingerprinting demonstrated that these strains do not originate from a single clone. The same results were observed for strains ZM23T, ZM24 and ZM25. Strains ZM22T and Y6 were resistant to multiple antibiotics, whereas strains ZM23T, ZM24 and ZM25 were able to degrade an emerging pollutant, triclosan. The phylogenetic, physiological and biochemical characteristics, as well as chemotaxonomy, allowed these strains to be distinguished from their genus, and we therefore propose the names Comamonas resistens sp. nov. (type strain ZM22=MCCC 1K08496T=KCTC 82561T) and Pseudomonas triclosanedens sp. nov. (type strain ZM23T=MCCC 1K08497T=JCM 36056T), respectively.

Diaporthe species on palms – integrative taxonomic approach for species boundaries delimitation in the genus Diaporthe, with the description of D. pygmaeae sp. nov.

The application of traditional morphological and ecological species concepts to closely related, asexual fungal taxa is challenging due to the lack of distinctive morphological characters and frequent cosmopolitan and plurivorous behaviour. As a result, multilocus sequence analysis (MLSA) has become a powerful and widely used tool to recognise and delimit independent evolutionary lineages (IEL) in fungi. However, MLSA can mask discordances in individual gene trees and lead to misinterpretation of speciation events. This phenomenon has been extensively documented in Diaporthe, and species identifications in this genus remains an ongoing challenge. However, the accurate delimitation of Diaporthe species is critical as the genus encompasses several cosmopolitan pathogens that cause serious diseases on many economically important plant hosts. In this regard, following a survey of palm leaf spotting fungi in Lisbon, Portugal, Diaporthe species occurring on Arecaceae hosts were used as a case study to implement an integrative taxonomic approach for a reliable species identification in the genus. Molecular analyses based on the genealogical concordance phylogenetic species recognition (GCPSR) and DNA-based species delimitation methods revealed that speciation events in the genus have been highly overestimated. Most IEL identified by the GCPSR were also recognised by Poisson tree processes (PTP) coalescent-based methods, which indicated that phylogenetic lineages in Diaporthe are likely influenced by incomplete lineage sorting (ILS) and reticulation events. Furthermore, the recognition of genetic recombination signals and the evaluation of genetic variability based on sequence polymorphisms reinforced these hypotheses. New clues towards the intraspecific variation in the common loci used for phylogenetic inference of Diaporthe species are discussed. These results demonstrate that intraspecific variability has often been used as an indicator to introduce new species in Diaporthe, which has led to a proliferation of species names in the genus. Based on these data, 53 species are reduced to synonymy with 18 existing Diaporthe species, and a new species, D. pygmaeae, is introduced. Thirteen new plant host-fungus associations are reported, all of which represent new host family records for Arecaceae. This study has recognised and resolved a total of 14 valid Diaporthe species associated with Arecaceae hosts worldwide, some of which are associated with disease symptoms. This illustrates the need for more systematic research to examine the complex of Diaporthe taxa associated with palms and determine their potential pathogenicity. By implementing a more rational framework for future studies on species delimitation in Diaporthe, this study provides a solid foundation to stabilise the taxonomy of species in the genus. Guidelines for species recognition, definition and identification in Diaporthe are included.

Genetic and phenotypical diversity of Pseudomonas syringae population in the Russian Federation.

Proteobacteria comprising species of Pseudomonas syringae group cause diseases of many plants around the world. The phytopathogen has a complex taxonomic structure, which is constantly being revised due to the emergence of new molecular and biochemical diagnostic methods. Here for the first time, we describe the genetic and phenotypic diversity of 57 strains of Pseudomonas syringae isolated from affected soybeans, cereals, sunflowers, and other plants in the Russian Federation from 1950 to 2019. Genetic diversity was assessed by Multi Locus Sequence Analysis (MLSA) using fragments of the genes of glyceraldehyde-3-phosphate dehydrogenase (gapdh), the DNA-directed RNA polymerase subunit D (rpoD), gyrase (topoisomerase) B subunit (gyrB), and citrate synthase I (gltA). The synthesis of syringomycin and coronatine by bacteria was assessed by the reaction of susceptible yeast culture, seedlings of barley, tomato, and sunflower, and by presence of toxin genes confirmed by PCR test. The pathogenicity of the strains was confirmed on seedlings of dicotyledonous and monocotyledonous plants of peas, soybean, sunflowers, barley and wheat, as the most affected crops. The sensitivity of bacteria to 10 antibiotics of the main mechanisms of activity and two bactericidal commercial products was tested by standard disc method. The obtained results showed a high genetic homogeneity of the Russian population of P. syringae, which infects various agricultural crops, and an increase in the proportion of antibiotic-resistant strains over the years.

Multilocus Sequence Analysis of Aspergillus Species from Clinical Samples and the Surge of Cryptic Aspergillosis in Southern India: A Descriptive Cross-sectional Study

Introduction: Aspergillus species cause a broad range of infections, from Allergic Bronchopulmonary Aspergillosis (ABPA) to invasive aspergillosis. Accurate species-level identification is crucial for diagnosis and treatment, especially since some rare Aspergillus species exhibit increased resistance to common antifungal agents. Aim: To identify Aspergillus species at the species complex level and determine the emergence of cryptic Aspergillus species using Multilocus Sequence Analysis (MLSA). Materials and Methods: This descriptive cross-sectional study was conducted over eight months, from January 2023 to August 2023, in three tertiary care centres in Chennai, India. Sixty Aspergillus strains were isolated from various clinical samples. The primary inclusion criteria were patients with suspected aspergillosis, categorised based on the European Organszation for Research and Treatment of Cancer Mycoses Study Group (EORTC-MSG) criteria. Isolates were grown on Oatmeal Agar or Sabouraud Dextrose Agar (SDA) and phenotypically identified using tease mount and slide culture techniques. Genomic fungal Deoxyribonucleic acid (DNA) was extracted and amplified using specific primers for Internal transcribed spacer (ITS), beta-tubulin, hydrophobin, and calmodulin genes, followed by nucleic acid sequencing. Results: Among the 60 Aspergillus isolates, the Aspergillus flavus complex comprised 26 (43.3%), the Aspergillus fumigatus complex 16 (26.7%), the Aspergillus niger complex 12 (20%), and the Aspergillus terreus complex 6 (10%). Initial speciation relied on microscopic and cultural characteristics. Isolates were recovered from various clinical specimens: bronchial wash 6 (10%), Bronchoalveolar lavage (BAL) fluid 10 (16.7%), sputum 3 (5%), ear swab 20 (33.3%), endotracheal aspirate 8 (13.3%), pus and tissue from the nasal cavity and paranasal sinus 11 (18.3%), and tissue from other sites 2 (3.3%). MLSA identified common species such as A. flavus, A. fumigatus, A. terreus, and A. niger, as well as rare species including A. awamori, A. carneus, A. lentulus, A. oryzae, A. tamarii, A. tubingensis, and A. welwitschiae. Sequence identity ranged from 99% to 100%, confirming species diversity. Phylogenetic analysis using ITS, beta-tubulin, hydrophobin, and calmodulin genes supported species identification with robust bootstrap support (1000 replications). Conclusion: Cryptic Aspergillus species are prevalent in clinical samples from the South Indian population, with a notable presence from the section Nigri and the first-time isolation of Aspergillus carneus in clinical samples from India. Molecular identification is essential for determining the invasive potential and guiding appropriate antifungal therapy.

Comparative Phylogenetic Analysis and Protein Prediction Reveal the Taxonomy and Diverse Distribution of Virulence Factors in Foodborne Clostridium Strains.

Clostridium botulinum and Clostridium perfringens, 2 major foodborne pathogenic fusobacteria, have a variety of virulent protein types with nervous and enterotoxic pathogenic potential, respectively. The relationship between the molecular evolution of the 2 Clostridium genomes and virulence proteins was studied via a bioinformatics prediction method. The genetic stability, main features of gene coding and structural characteristics of virulence proteins were compared and analyzed to reveal the phylogenetic characteristics, diversity, and distribution of virulence factors of foodborne Clostridium strains. The phylogenetic analysis was performed via composition vector and average nucleotide identity based methods. Evolutionary distances of virulence genes relative to those of housekeeping genes were calculated via multilocus sequence analysis. Bioinformatics software and tools were used to predict and compare the main functional features of genes encoding virulence proteins, and the structures of virulence proteins were predicted and analyzed through homology modeling and a deep learning algorithm. According to the diversity of toxins, genome evolution tended to cluster based on the protein-coding virulence genes. The evolutionary transfer distances of virulence genes relative to those of housekeeping genes in C. botulinum strains were greater than those in C. perfringens strains, and BoNTs and alpha toxin proteins were located extracellularly. The BoNTs have highly similar structures, but BoNT/A/B and BoNT/E/F have significantly different conformations. The beta2 toxin monomer structure is similar to but simpler than the alpha toxin monomer structure, which has 2 mobile loops in the N-terminal domain. The C-terminal domain of the CPE trimer forms a "claudin-binding pocket" shape, which suggests biological relevance, such as in pore formation. According to the genotype of protein-coding virulence genes, the evolution of Clostridium showed a clustering trend. The genetic stability, functional and structural characteristics of foodborne Clostridium virulence proteins reveal the taxonomy and diverse distribution of virulence factors.

Pan-Genomic Analysis Identifies the Chinese Strain as a New Subspecies of Xanthomonas fragariae.

Xanthomonas fragariae is classified as a quarantine pathogen by the European and Mediterranean Plant Protection Organization. It commonly induces typical angular leaf spot (ALS) symptoms in strawberry leaves. X. fragariae strains from China (YL19, SHAQP01, and YLX21) exhibit ALS symptoms in leaves and more severe symptoms of dry cavity rot in strawberry crowns. Conversely, strains from other countries do not cause severe dry cavity rot symptoms in strawberries. After employing multilocus sequence analysis (MLSA), average nucleotide identity (ANI), and amino acid identity (AAI), we determined that Chinese strains of X. fragariae are genetically distinct from other strains and can be considered a new subspecies. Subsequent analysis of 63 X. fragariae genomes published at NCBI using IPGA and EDGAR3.0 revealed the pan-genomic profile, with 1,680 shared genes present in all 63 strains, including 71 virulence-related genes. Additionally, we identified 123 genes exclusive to all the Chinese strains, encompassing 12 virulence-related genes. The qRT-PCR analysis demonstrated that the expression of XopD, XopG1, CE8, GT2, and GH121 out of 12 virulence-related genes of Chinese strains (YL19) exhibited a constant increase in the early stages (6, 24, 54, and 96 hours postinoculation [hpi]) of strawberry leaf infected by YL19. So, the presence of XopD, XopG1, CE8, GT2, and GH121 in Chinese strains may play important roles in the early infection process of Chinese strains. These findings offer novel insights into comprehending the population structure and variation in the pathogenic capacity of X. fragariae.