Neighbor-joining Method Research Articles

Microsatellite Markers & Mitochondrial D-Loop Based Phylogenetic And Diversity Analysis In Gabrali Cattle.

The Gabrali cattle is a multipurpose breed, native to Khyber Pakhtunkhwa, Pakistan. Despite its economic importance, scientific data about its phylogeny and genetic diversity is scarce. To address this issue, the present study was conducted on thirty (30) unrelated Gabrali male and female animals, from which blood samples were collected and DNA was extracted. Twelve (12) microsatellite loci and 1159bp of D-loop region were amplified via PCR and visualized on a 2% agarose gel. Sanger sequencing technique was used to sequence the D-loop amplicons. The microsatellite loci revealed 83 alleles with MNA (mean no of alleles per locus) = 8.8, the Ho (observed heterozygosity) and He (expected heterozygosity) of all loci were 0.58 and 0.50 respectively, mean PIC (Polymorphic Information content) = 0.59 and FIS (Inbreeding coefficient) = 0.056 using GeneAlEx® software. Microsatellite analysis revealed a normal allelic distribution across the breed, consistent with Hardy-Weinberg equilibrium. D-loop sequencing revealed eight haplotypes with 30 SNPs using DNA SP 6.0 software. High AT content was observed than GC content i.e. 55.9% and 44.1% respectively, while the transition to transversion ratio was R = 10:1. The value of Haplotype and Nucleotides diversity was Hd = 0.8601 ± SD = 0.0895 and Π = 0.0136 ± SD = 0.00197 respectively. Phylogenetic analysis done using Neighbor-joining method with bootstraps value of 1000, revealed the monophyletic clade of both Gabrali and Bos indicus haplotypes. Furthermore, the introgression of genes from national cattle breeds into Gabrali cattle was observed. It is concluded that Gabrali cattle have common ancestry with Bos indicus having no threats of genetic bottleneck having substantial genetic diversity.

Septic shock and death due to Dolosigranulum pigrum bacteremia: a case report.

We report the case of a 77-year-old Japanese woman with neuromyelitis optica spectrum disorder, treated with 15 mg of prednisolone daily, who presented with acute septic shock and altered consciousness due to Dolosigranulum pigrum bacteremia. Intravenous extracellular fluids and broad-spectrum antibiotics were administered as an initial treatment. However, the patient died shortly after admission. We performed 16S rRNA sequencing and analyzed the murJ gene. A phylogenetic tree constructed using the neighbor-joining method showed that the strain belonged to clade C3, according to the classification proposed by Flores Ramos et al. Although D. pigrum, a commensal bacterium of the upper respiratory tract, is rarely associated with infections, it occasionally causes serious infections. Accumulation of clinical and bacteriological data is crucial for understanding the pathogenicity and infections associated with this organism.

First report of basal stem rot on sugarcane (var. Badila) caused by Sclerotium rolfsii in China.

Badila (Saccharum officinarum) is one of the important chewing cane in south China. During the year 2019-2020, as much as 60.2%-87.5% of sugarcane plants stem showed red rot developments were observed in the fields of Yongning District, Nanning city, Guangxi province. Symptomatic plants showed red rot at basal stem nodes and sheath, when the disease serious, the epidermis and aerial roots decomposed and exfoliated, then formed sclerotiums, the upper stem also occurred the symptom. Infected plant tissues were dissected into small pieces with 0.1 × 0.1cm in size and surface sterilized in 0.1% HCl2 for 2 min, followed by 75% ethanol for 30 s, rinsed three times with sterile distilled water. Then the tissues were placed onto potato dextrose agar (PDA) plates and incubated at 25 °C for 3 days. Numerous white globoid sclerotia were formed on PDA after 5 days of growth. The sclerotia (2 to 3 mm in diameter) were white at first and then gradually turned dark brown. Aerial mycelia usually formed many narrow hyphal strands 4 to 9 μm wide. Five uniform isolates were obtained from diseased sugarcane plants. Pathogenicity of representative strain W1 was confirmed by inoculating 120-day-old Badila plants grown in field. Five plants were inoculated with colonized agar discs (6mm in diameter) by applying toothpick tips to the lower part of the stem. Five non-inoculated plants served as control. The inoculated and non-inoculated plants were sprayed sterile water then incubated with plastic film for maintained high moisture. All the plants were placed inside of a growth chamber at 26 ± 2°C with a 14-h photoperiod and 80% relative humidity. All inoculated plants showed red rot at stem and sheath after 2 weeks, whereas the control plants were symptomless. By the third week, mycelium and sclerotia developed on the crown on the inoculated plants. The fungus was re-isolated from the artificially inoculated plants. To confirm the species-level identification, partial of the ribosomal DNA internal transcribed spacer (ITS), mitocondrial small subunit (SSU), and nuclear ribosomal large subunit (LSU) regions of representative strain W1 were amplified and sequenced using the primers pairs ITS1/ITS4 (White et al. 1990), ITS-Fu-F /ITS-Fu-R and SRLSU1//SRLSU2 (Kumar et al., 2016), respectively. The resulting ITS, SSU and LSU sequences were deposited in GenBank (GenBank accession no. MW620994, MW617878, and MW617872) and shared 99.42%, 100% and 100% sequence identity with Athelia rolfsii isolate (JN017199, OM319631, and MT225781). Phylogenetic analysis conducted with neighbor-joining (NJ) method using MEGA6.0 revealed that the isolate share a common clade with reference sequence of A. rolfsii in GenBank Data Library. Based on morphological and molecular characteristics, the fungus was identified as A. rolfsii (anamorph: Sclerotium rolfsii) (Paul et al. 2017; Paparu et al. 2020). Although S. rolfsii has been reported causing sugarcane sett rot in Australia (Bhuiyan et al., 2019) and seedlings of sugarcane in Indian (Gopi et al., 2023), as we know, this is the first report of sugarcane basal stem rot disease caused by this fungus in China. This study will be helpful for the prevention and control sugarcane basal stem rot in the future.

First Report of Bacterial Leaf Spot Disease on Ginger Caused by Enterobacter quasiroggenkampii in China.

In autumn 2023, an unknown leaf spot disease has occurred on ginger (Zingiber officinale Roscoe) in two fields of approximately 1800 m2 in Yongning District (22°49'N; 108°48'E), Nanning, China, with a incidence of 20-30%. The symptoms began as yellow spots on the leaves, expanding into elliptical to irregular lesions with yellow edges, the middle of the lesion turning grey-white in dry weather. Finally, multiple spots caused necrosis of the whole leaf. Twelve diseased leaves from six plants of two fields were collected, surface disinfected and ground. The ground samples were diluted and plated on nutrient agar (NA) medium at 28 °C for 48-72 h. The purified colonies appeared milky white and round, with smooth edges. Three isolates (GL1, GL2 and GL3) were selected for identification and pathogenic determination. They were gram negative, could utilize sorbitol, mannitol, inositol, raffinose, melibiose, disaccharides, and citrate; negative for methyl red, phenylalanine decarboxylase, hydrogen sulfide, urease; positive for voges-proskauer test and ornithine decarboxylase. These characteristics were consistent with Enterobacter genus (Wu et al., 2020). Genomic DNA was extracted from three isolates. The 16S rDNA region was amplified using 27F/1492R primers (Weisburg et al. 1991) and sequenced (accession no. PP837703-PP837705). Blastn analysis revealed that 16S rDNA sequences for GL1 was 99% identical (1373/1387 nt), GL2 96% (1364/1422 nt) and GL3 95% (1365/1435 nt) to Enterobacter quasiroggenkampii WCHECL1060 (NR_179166). To determine the species, the sequences of gyrB, rpoB and atpD genes were amplified using primers gyrB 01-F/gyrB 02-R, rpoB CM7/rpoB CM31b, and atpD 01-F/atpD 02-R, respectively (Lin et al. 2015; Zhu at al. 2010; Zhang et al. 2013). The GenBank accession numbers for the sequences were PP857680-PP857688. A multilocus phylogenetic tree was constructed with the concatenated sequence of 16S rDNA-gyrB-rpoB-atpD by using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA6 software. The three isolates clustered with E. quasiroggenkampii. Fifteen Darou ginger variety plants at the 4-5 leaf stage were tested for pathogenicity. Two to three leaves of each ginger plant were pricked with a syringe needle of 0.36mm in diameter or not and inoculated by spraying the bacterial suspension (108 CFU/mL), sterile water was used as a control. Five plants were inoculated with each isolate and the test was repeated three times. After 3-4 days of inoculation, all wounded leaves and about 10% of the unwounded leaves showed symptoms similar to those observed in the field. Control plants did not develop symptoms. Enterobacter quasiroggenkampii isolates were re-isolated from the inoculated leaves with symptoms, and their identity was confirmed by gyrB sequencing and colony morphology, completing Koch's postulates. Enterobacter quasiroggenkampii is a pathogen of humans that can cause nosocomial infections (Wu et al., 2020). In Guangxi, E. quasiroggenkampii was identified as one of the pathogens causing mulberry wilt (Jiao, 2022). To our knowledge, this is the first report of E. quasiroggenkampii causing bacterial leaf spot disease of ginger. The results of this study not only have practical significance for the control of ginger leaf spot, but also can provide excellent materials for the study of the differentiation and pathogenic mechanism of the genus Enterobacter, which has important academic value.

First Report of Rhizopus arrhizusi causing rot of Dictyophora rubrovolvata in China.

Dictyophora rubrovolvata, as an edible fungus with high medicinal value, is widely cultivated in several provinces in China (Hang et al. 2012). However, between December 2023 and March 2024, a rot disease occurred in the main production area in Fengxian District, Shanghai, China (N30°93', E121°49'). The disease incidence was 25% in the affected 1.33-ha growing area. High temperatures (>25℃) and poor ventilation provide favorable conditions for the spread of this disease. The disease mainly occurs at the stage of fruiting bodies formation of D. rubrovolvata. When the epidermis is damaged and broken, it becomes infested with mold, which then produces a layer of moldy rot with pus. The infected D. rubrovolvata tissues at the edge of the lesions were isolated, surface sterilized and cultured on potato dextrose agar (PDA) at 30 ℃ under dark conditions. Pure cultures were obtained by single-spore isolation. After 3 days, isolates were transferred to Czapek Yeast agar (CYA) (Samson et al, 2014). On CYA, the fungal colony consisted of white flocculent hyphae. Scanning electron microscopy analysis showed that the mycelium was white, and the internodes of the stolons formed characteristic pseudoroots, from which upwardly clustered erect, unbranched sporocarp peduncles expanded apically to form rounded sporocarp sacs, within which sporocarp spores were produced. (Hariprasath P, 2019). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS) and intergenic spacer (IGS) gene of the isolate were amplified by PCR (White et al. 1990; Liu XY. 2008). The resulting sequence was deposited in GenBank with accession PP951880 and PQ001670, respectively. PCR results and morphological observations indicated the isolated strain was a pure culture and the strain was designated as DIC01. Comparative results showed that the sequences with accession numbers MT603964.1 and DQ990323.1 showed high homology of 99.15% and 98.96% to the ITS and IGS sequences of Rhizopus arrhizusi DIC01, respectively. Phylogenetic analysis with ITS and IGS genes of the isolated strain and 7 Rhizopus spp. strains were performed using MEGAX with Neighbor-Joining (NJ) method. Based on the results of growth habits, morphological observations, and phylogenetic analysis, the pathogen was identified as R. arrhizusi. A spore suspension of the R. arrhizusi DIC01 (1 x107 conidia/mL) was inoculated back to healthy D. rubrovolvata. Five healthy fruit bodies of D. rubrovolvata were injected, and another five healthy morels were treated with potato dextrose broth (PDB) as controls. D. rubrovolvata was incubated at 25°C and 90% relative humidity without ventilation for 5 days. The pathogen successfully infected the D. rubrovolvata, which developed white moldy lesions similar to those of natural diseases. The controls remained healthy without any symptoms. The pathogen was reisolated from the affected lesions and identified as R. arrhizusi DIC01 based on its morphological characteristics and phylogenetic marker genes. R. arrhizusi has been reported to cause endothelial cell damage and mycelial invasion into blood vessels, leading to thrombosis and tissue necrosis. (Hariprasath P, 2019). To our knowledge, this is the first report of R. arrhizusi causing rot disease of D. rubrovolvata. This study confirmed that R. arrhizusi is the pathogenic fungus responsible for rotting disease in D. rubrovolvata farms in Fengxian, Shanghai.

Genome-Wide Identification of the Geranylgeranyl Pyrophosphate Synthase (GGPS) Gene Family Associated with Natural Rubber Synthesis in Taraxacum kok-saghyz L. Rodin.

Taraxacum kok-saghyz Rodin (TKS) is a recognized alternative source of natural rubber comparable to the rubber tree. The geranylgeranyl pyrophosphate synthase (GGPS) catalyzed the synthesis of geranylgeranyl pyrophosphate (GGPP), which is an important enzyme in the secondary metabolism pathway. In this study, we present the first analysis of the GGPS gene family in TKS, where a total of seven TkGGPS family members were identified. Their core motifs, conserved structural domains, gene structures, and cis-acting elements were described. In addition, two phylogenetic trees were constructed based on the Neighbor-Joining and Maximum-Likelihood methods, and the TkGGPSs were highly conserved and exhibited good collinearity with the other species. Transcriptome data showed that seven TkGGPS gene members were expressed in all the 12 tissues measured, and TkGGPS1, TkGGPS3, and TkGGPS6 were highly expressed in latex, suggesting that they may be associated with natural rubber synthesis. Meanwhile, quantitative real-time PCR (qRT-PCR) showed that the expression levels of the TkGGPS genes were regulated by the ethylene and methyl jasmonate (MeJA) pathways. Subcellular localization results indicated that all the TkGGPS proteins were also located in chloroplasts involved in photosynthesis in plants. This study will provide valuable insights into the selection of candidate genes for molecular breeding and natural rubber biosynthesis in TKS.

First Report of Leaf Rot on Catalpa bungei caused by Fusarium verticillioides in China.

Catalpa bungei originates from China. Because of its well-developed root system and strong resistance to wind and soil, it is one of the top ten recommended species of ecological management (Jian et al., 2022). In September 2023, a severe leaf rot of C. bungei was observed at Lanlake farm (500 acres) in Nanyang (33°3'23" N, 112°28'50" E), China. The incidence rate of leaf rot reached 45% (n = 100). The pale-yellow spots initially appeared on the adaxial surface of leaf margins, subsequently enlarging to form irregular black rot lesions, with the yellow halos around the necrotic area of the lesion, ultimately causing the entire leaves to wither. Diseased leaves (20) were collected, cut into pieces, sterilized, and then placed on potato dextrose agar (PDA). A total of 25 purified fungal strains were isolated, and three strains (QS2-1, QS2-2, QS2-3) from distinct areas were selected for further analysis. Each strain produced abundant aerial mycelium, initially white, which later developed purple pigments. The aerial conidiophores were sparsely branched, ending with verticillate phialides. The strains generally produced many more microconidia than macroconidia on PDA media. Microconidia were clavate and measured 3.9 to 6.6 × 1.1 to 2.4 μm (n = 50). To produce macroconidia, we used YPG liquid medium (0.3% yeast extract, 1% peptone, and 2% glucose) with shaking (200 r.p.m.) for 5 days. Macroconidia were slender, straight, and measured 19.5 to 27.1 × 1.9 to 3.5 μm, with 3 to 5 septa (n = 50). The morphological characteristics matched the species description of Fusarium verticillioides (Sacc.) Nirenberg 1976 (Leslie and Summerell, 2006). The rDNA internal transcribed spacer (ITS), β-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase II largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified for molecular identification (O'Donnell et al., 2022). The sequences were submitted to GenBank with accession numbers OR741762, OR741763, OR741765 (ITS), OR762222, OR762223, OR939807 (tub2), OR939799, OR939800, PQ035927 (tef1), OR778611, OR939808, OR939809 (cmdA), PQ035921, PQ035922, PQ035923 (rpb1), and PQ035924, PQ035925, PQ035926 (rpb2). BLASTn analysis of QS2-1 sequences exhibited 99% similarity with F. verticillioides sequences (strains CBS 576.78) MT010888 of cmdA (711/713, 99%), MT010956 of rpb1 (1790/1791, 99%), and MT010972 of rpb2 (868/870, 99%). A phylogenetic tree was constructed using concatenated sequences along with the sequences of the type strains employing the neighbor-joining method, showing the three strains formed a clade with the type strain CBS 576.78. Pathogenicity was tested on 10 healthy potted seedlings by spraying them with a conidial suspension (106 conidia ml-1), while 5 seedlings were sprayed with sterilized water as a control. The plants were placed in climate incubators. Ten days after inoculation, typical lesions were observed on the treated plants, but not on the control group. The reisolated strains were identified as F. verticillioides through morphological characterization, thus fulfilling Koch's postulates. F. verticillioides is known to cause Fusarium ear rot on maize, and other plants including Brassica rapa (Akram et al., 2020) and Schizonepeta tenuifolia (Li et al., 2024). This is the first documented instance of F. verticillioides causing leaf rot on C. bungei globally. Identifying the pathogen is critical to implementing effective disease management strategies, especially in choosing proper pesticide agents and screening disease-resistant varieties.

The herd-level prevalence of caprine arthritis-encephalitis and genetic characteristics of small ruminant lentivirus in the Lithuanian goat population

Caprine arthritis-encephalitis (CAE) is a progressive disease of goats caused by small ruminant lentivirus (SRLV) and is considered as one of the most important threats for goat farming in developed countries. The disease prevalence has never been investigated in the Lithuanian goat population. Therefore, a descriptive cross-sectional study was carried out in 2021-2022 to determine if SRLV infection was present in the Lithuanian goat population and, in the case of a positive result, to estimate the true herd-level prevalence of SRLV infection and specify genotypes and subtypes of SRLV responsible for the infection. Thirty goat herds counting >5 adult goats were randomly selected and, in each herd, a representative sample of adult goats was blood-sampled and tested serologically for SRLV infection using a commercial ELISA. The herd was considered infected if at least one goat tested positive and the true herd-level prevalence of SRLV infection was estimated using the Bayesian approach. Seropositive animals were found in 17 / 30 herds (57%; 95% confidence interval: 39%, 73%). The true herd-level prevalence was 56% (95% credible interval: 36%, 76%). In 10 / 17 seropositive herds whose owners consented for resampling of seropositive goats, 1 to 5 seropositive goats were tested using the nested real-time PCR (nRT-PCR). Goats from 9 seropositive herds tested positive in the nRT-PCR: in 4 herds for genotype A, in 4 herds for genotype B, and in 1 herd – 2 goats for genotype B and 1 goat for genotype A. From each of 9 nRT-PCR-positive herds, 1 PCR product of each genotype was sequenced using Sanger method and the phylogenic tree was constructed using the neighbor-joining method in the Molecular Evolutionary Genetics Analysis software. Four herds turned out to be infected with B1 subtype (91% identity with the prototypic strain), 3 herds with A2 subtype (90%-92% identity), and a herd with mixed infection was infected with B1 (91% identity) and A2 subtype (90% identity). In one herd, the only seropositive goat was found to be infected with the strain most closely related to the A1 subtype (80% identity). This study shows for the first time that SRLV infection is present and widespread in the Lithuanian goat population and both classical SRLV genotypes, represented by quite typical subtypes A2 and B1, appear to be responsible for the infection.

Assessment of Genetic Parameters and Diversity Organization of Ginger Accessions Cultivated in Burkina Faso as Revealed by Microsatellite Markers

Rhizomes of Ginger (Zingiber officinale Rosc.) are widely used as a spice across the world. It has originated from South-east Asia and introduced to many parts of the world, and has been cultivated as a spice and for medicinal purpose. Ginger is a high value crop in Burkina Faso. The genetic diversity organization in ginger species is not well-known in Burkina Faso. Genetic resources are imperative for varietal selection. The importance of genetic resources depends on the levels of their diversity. The present investigation aims to assess the diversity of ginger accessions collected from Burkina Faso. Forty-seven ginger accessions collected from three provinces, Comoé (03), Léraba (14), Kénédougou (30), located in the southwestern zone of Burkina Faso were genotyped for their molecular traits using microsatellite markers. A total of 24 alleles were revealed by electrophoresis. All the simple-sequence repeats (SSR) set primers used revealed high polymorphic rate (100%) with the expected heterozygosity (0.499), showing their ability in assessment of the diversity levels and genetic organization of ginger accessions due to their codominance. The dendrogram obtained by the Neighbor-Joining method revealed three main genetic groups. The genetic differentiation index value (0.313) showed that the accessions of the three provinces were different, with an interesting diversity observed from kénédougou ginger accessions. Information from this study could be used in the development of effective management strategies for the conservation and effective use of the genetic resources within ginger accessions grown in Burkina Faso.

Occurrence of Phytoplasmas Belonging to 16SrII Group Associated with Cyanthillium cinereum Witches'-Broom Diseases in China.

Cyanthillium cinereum, which belongs to the family of Asteraceae, is an annual or perennial herbaceous plant with significant medicinal uses for treating colds and fever. During September to November of 2020, C. cinereum showing symptoms of witches'-broom were found in economic forests distributed in Ding'an, Hainan Province of China, with 20% incidence. The symptoms of the plant were consistent with infections by 'Candidatus Phytoplasma' species. To identify the pathogen, five symptomatic and three asymptomatic C. cinereum samples were collected. Total DNAs were extracted using 0.10 g fresh leaf tissues of symptomatic and asymptomatic C. cinereum through a CTAB DNA extraction method according to Doyle and Doyle (1990). PCR amplification were performed employing the primer pairs of R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008) specific for the conserved gene fragments of 16S rRNA and secA from phytoplasma. The PCR products were purified and sequenced through Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China), and the obtained sequences were deposited in GenBank. The phytoplasmal 16S rRNA and secA gene fragments obtained in the study were all identical with the length of 1325 bp (GenBank accession: PP098738) and 741 bp (PP072217), respectively. The phytoplasma strain was described as CcWB-hnda. A BLAST search based on 16S rRNA genes indicated that CcWB-hnda strain was identical to phytoplasmas belonging to 16SrII group like peanut witches'-broom phytoplasma strain T48 (OR239773) and 'Ca. Phytoplasma aurantifolia' strain TB2022 (CP120449). Virtual RFLP profiles based on 16S rRNA gene fragments obtained by iPhyClassifier (Zhao et al., 2009) showed that CcWB-hnda strain was a member of 16SrII-A subgroup with 1.00 similarity coefficient to the reference phytoplasma strain (L33765). A BLAST search based on secA genes indicated that CcWB-hnda had 100% sequence identity with phytoplasmas belonging to 16SrII group such as 'Ca. Phytoplasma aurantifolia' isolate TB2022 (CP120449), Vigna unguiculata witches'-broom phytoplasma (OR661282) and Emilia sonchifolia witches'-broom phytoplasma (MW353710). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing Neighbor-Joining method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) demonstrated that CcWB-hnda was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value respectively. To our knowledge, this is the first report of C. cinereum infected by phytoplasmas belonging to 16SrII-A subgroup in China. Identification of the vector insects of the pathogens is necessary in future, revealing the epidemiology of the related diseases. Phytoplasmas belonging to same 16Sr group or subgroup can infect different plants and spread through them in nature. The finding in this study will be beneficial to epidemic monitoring and early warning of C. cinereum witches'-broom disease and the related plant diseases caused by the phytoplasmas belonging to 16SrII group.

First Report of Xanthomonas campestris pv. raphani Causing Leaf Spot Disease on Broccoli in China.

Broccoli (Brassica oleracea L. var. italica) is one of the important cruciferous vegetables in China, known for its considerable economic and nutritional value (Li et al. 2021). In September 2023, leaf spot disease was observed on broccoli seedlings in the commercial fields in Weifang City, Shandong Province, China (119°15'E, 36°70'N). Further investigation revealed that the disease incidence was approximately 60% in 4.5 square hectometers (hm2) broccoli field, resulting in substantial economic losses. This disease primarily affects the leaves, manifesting distinct symptoms such as circular, dark necrotic spots that gradually lighten and are encircled by a chlorotic halo. Some lesions further develop a black or purplish border, exhibit concentric zonation, and eventually fall off, leaving behind holes, as shown in Figure S1B and C. In severe cases, decay originates from the central perforation and spreads outwards, as shown in Figure S1A. To identify the causal agent of this disease, infected leaf tissues were collected and surface disinfected by immersing in 75% ethanol for 30 seconds, followed by three rinses with sterile water. The samples were grinded in sterile deionized water, and the extract was plated on NA. After incubation at 28°C for 48 h, individual colonies were transferred to fresh NA plates. A total of 12 strains with the similar morphological characteristics were isolated from diseased samples collected from the three plots. After 48 hours of growth on NA medium at 28℃, each colony attained a diameter ranging from 3 to 5 mm. These colonies appeared yellow, slightly elevated, nearly circular in shape, with a smooth and moist edge. Three representative strains were selected for further investigation. All strains were Gram-negative, aerobic, and rod-shaped. The analysis of BIOLOG GENIII microplate system revealed the capability of three isolates using cellobiose, trehalose, glucose, mannose, galactose, and sucrose. Furthermore, the isolates were unable to hydrolyze arginine and utilize rhamnose and inositol. The 16S rRNA gene was amplified and sequenced to confirm that three isolated bacteria belong to the genus Xanthomonas (OR772321-OR772323), followed by PCR amplification for 4 housekeeping genes atpD, dnaK, gyrB, and rpoD (Young et al. 2008; Saux et al. 2015). The obtained sequences were submitted to the GenBank under accessions OR789628-OR789630, OR785471-OR785473, OR789631-OR789633, and OR785468-OR785470. Gene sequences were aligned, concatenated, and used to generate a phylogenetic tree using the neighbor-joining method in MEGA11 (Tamura et al. 2021). The phylogenetic analysis revealed that all the three isolates were clustered with Xanthomonas campestris pv. raphani strains 5055 and 576, respectively (Figure S2). (Dubrow et al. 2022). These results were consistent with those of the reported X. campestris pv. raphanin (Cruz et al. 2015). To verify the pathogenicity of these strains, we used a spray inoculation method. In detail, bacterial suspensions (30 mL per treatment) containing approximately 108 CFU/ml were sprayed onto healthy, four-week-old broccoli plants and incubated in a phytotron at 28°C and above 90% RH. Negative controls were performed using sterile distilled water. Each isolate underwent three trials and each treatment included 12 broccoli seedlings. Leaf spot symptoms were observed on 5 days post inoculation, as shown in Figure S1E, F and G. Negative control plants showed no symptoms (Figure S1D). We further re-isolated the bacterium from the symptomatic plants and verifying the bacteria as X. campestris pv. raphanin with the aforementioned sequence analysis, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of X. campestris pv. raphani causing leaf spot disease on broccoli in China. This study enhances our understanding of the pathogenic bacterium on broccoli and lays the groundwork for developing targeted management strategies.

First Report of Klebsiella variicola Causing Tomato Pith Necrosis in Yunnan Province, China.

Tomato (Solanum lycopersicum L.) is a key vegetable crop in China. In August 2023, an outbreak of bacterial pith necrosis in tomato occurred in Lufeng County, Yunnan Province, China, affecting over 40% of the tomato plants in a greenhouse. The stems of infected plants developed a waterlogged soft rot and the disease progressed, the lower leaves and lateral branches of infected plants gradually wilted and died. A longitudinal cut of the stem revealed hollow pith with brown vascular tissue. To isolate the pathogen, the plant surface was disinfested with 75% ethanol. Then, a piece of infected tissue from the base of the stem was excised and immersed in sterile water for 2 min. A small amount of liquid was streaked onto TTC (2,3,5-triphenyltetrazolium chloride) agar medium using an inoculation loop, and plates were incubated at 28℃ for 24 h. Colonies on the TTC plate were white, indicating that the pathogen was not Ralstonia solanacearum. Colonies grown on LB (Luria-Bertani) agar medium were randomly selected and subjected to preliminary pathogenicity tests. Based on the results, a colony named Kv4 was selected and purified through six subcultures in LB agar medium. Biochemical tests showed the strain utilized D-sorbitol, raffinose and citrate but not adonitol, and was positive for methyl red, D-glucose (acid), urea hydrolysis, lysine decarboxylase, and motility, and negative for phenylalanine deaminase, H2S production, indole production, and ornithine decarboxylase. These characteristics align with Klebsiella species (Garrity et al. 2007). To determine the species of strain Kv4, partial sequences of the 16S rDNA, phoE, leuS, and rpoB genes were amplified (Barrios-Camacho et al. 2019) and sequenced. Through BLASTn analysis, strain Kv4 sequences of 16S rDNA (OR888750) had 99.47% identity (1488/1496 bp), phoE (OR899599) had 98.69% (605/613 bp) identity, leuS (OR899598) had 99.07% identity (959/968 bp), and rpoB (OR899597) had 97.69% (633/648 bp) identity with Klebsiella variicola strain FF0907. Using the ClustalW algorithm in MEGA11 for nucleotide sequence alignment, phylogenetic trees were constructed with 16S and phoE, leuS, and rpoB via the neighbor-joining method, confirming strain Kv4 as K. variicola. To test pathogenicity, the roots of 25 'Moneymaker' tomato plants with four to five true leaves were wounded, then each plant inoculated with a 15 mL bacterial suspension (OD600=0.6) of strain Kv4, while the control plants received sterile water. Plants were incubated at 28℃ with a 16 h photoperiod. Experiments were done twice. At 15 days after inoculation (DAI), all plants inoculated with Kv4 showed yellowing, unevenly distributed small black necrotic spots on the leaf surface, and purple-brown soft rot at the stem base. By 18 DAI, there was a gradual transformation of the stem bases from green to purplish brown. At 21 DAI, 60% of the inoculated plants displayed brownish soft rot at the stem base. In contrast, the control plants remained symptom-free. The pathogen was re-isolated from the stem and identified as K. variicola via sequence analysis of 16S, phoE, leuS, and rpoB. In recent years, several new bacterial pith necrosis diseases were reported in tomato (Guo et al. 2023; Ivić et al. 2023). This is the first study documenting K. variicola causing bacterial pith necrosis in tomato. Once considered a benign plant endophyte, Sun et al. (2023) reported K. variicola causing banana sheath rot in Guangdong and Guangxi Provinces, China. Malik et al. (2023) reported that K. variicola caused leaf streak in sorghum in India. This report of bacterial pith necrosis in tomato caused by K. variicola strain Kv4 underscores the escalating threat posed by emerging pathogens to agricultural production. The emergence of K. variicola as a tomato pathogen complicates plant disease management strategies.

Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023

Background Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java. Methods Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT–qPCR confirmed the presence of H9 and N2 genes in the samples. RT–PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate’s similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal. Results RT–qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal. Conclusion In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

Molecular genetic monitoring of HIV-1 variants circulating in St. Petersburg

The aim of the study: to assess the genetic diversity of HIV-1 variants circulating in St. Petersburg.Materials and methods. The study included 289 patients with virological ART failure in 2022 in the St. Petersburg AIDS Center. Fragments of the pol gene encoding integrase, reverse transcriptase and protease were analyzed by polymerase chain reaction and Sanger sequencing. Phylogenetic tree created by the Neighbor-joining method with 1000 repeats of nucleotide sequences, bootstrap values >70. To assess the circulation of HIV genovariants in dynamics in St. Petersburg, sequences and clinical and laboratory parameters obtained from 544 patients since 2018. The total sample included 833 samples (289 were collected in 2022), compared with the results of a study from 1104 HIV-infected patients in 2006–2011.Results and discussion. Monovariants of HIV dominated in the examined patients (95.1%, 275 people), subtype A6 — 88.2% (255 people); subtype B — 5.9% (17 people); C — 0.3% (1 person); G — 0.7% (2 people), the proportion of recombinant forms — 4.9% (14 people). In the sample collection, the proportion of new HIV cases of non-A subtype was 13.3% in 2006–2011, and 11.1% in 2018–2022. A significant increase in the incidence of recombinant forms of HIV-1 was revealed from 1.6% to 3.5% (c2=6.111; p=0.014). In the group (2018–2022), the recombinant form of CRF63_02A6 was more common (15/29 people).Conclusion. Phylogenetic analyses makes it possible to determine HIV subtypes, but also to establish the potential geographical origin of the virus, to identify transmission clusters taking into account the socio-demographic indicators of HIV-infected patients. Molecular epidemiological monitoring can be used to develop and implement programs to counter the spread of HIV among the population. The dominant genetic variant of HIV circulating in St. Petersburg is sub-subtype A6, as it was 10 years ago. The proportion of new cases of infection with non-A subtype of HIV remains stable, with a downward trend. The increase in the frequency of detection of recombinant forms of HIV-1 is probably related to the migration processes of the population.

Machine learning can be as good as maximum likelihood when reconstructing phylogenetic trees and determining the best evolutionary model on four taxon alignments

Phylogenetic tree reconstruction with molecular data is important in many fields of life science research. The gold standard in this discipline is the phylogenetic tree reconstruction based on the Maximum Likelihood method. In this study, we present neural networks to predict the best model of sequence evolution and the correct topology for four sequence alignments of nucleotide or amino acid sequence data. We trained neural networks with different architectures using simulated alignments for a wide range of evolutionary models, model parameters and branch lengths. By comparing the accuracy of model and topology prediction of the trained neural networks with Maximum Likelihood and Neighbour Joining methods, we show that for quartet trees, the neural network classifier outperforms the Neighbour Joining method and is in most cases as good as the Maximum Likelihood method to infer the best model of sequence evolution and the best tree topology. These results are consistent for nucleotide and amino acid sequence data. We also show that our method is superior for model selection than previously published methods based on convolutionary networks. Furthermore, we found that neural network classifiers are much faster than the IQ-TREE implementation of the Maximum Likelihood method. Our results show that neural networks could become a true competitor for the Maximum Likelihood method in phylogenetic reconstructions.

First Report of Rhizoctonia solani AG1-IB Causing Tobacco Target Spot in China.

Tobacco target spot, caused by Rhizoctonia solani Kühn, induces shot-hole lesions on leaves that that significantly reduce yield and quality of tobacco. In July 2022, samples (n=5) with target spot were collected from three tobacco fields, one each in Puer (22.63°N, 100.72°E, cv. Yunyan87) and Mengzi (23.26°N, 103.36°E, cv. Yunyan87) of Yunnan province and one in Dandong (40.63°N, 124.18°E, cv. Liaoyan17) of Liaoning province, China; disease incidence in these fields was approximately 30%~40%. Initial symptoms (2- to 3-mm-diameter lesions) appeared on the middle to lower leaves, then expanded to 2 to 3 cm in diameter and developed the shot-hole appearance. Pieces of tissue (5×5 mm) were cut from the edge of lesions, surface sterilized, rinsed in sterile water, then placed on the surface of water agar (WA) and incubated at 25℃ for 2 days in the dark. Single hyphal tips were taken from fungal isolates identified as R. solani based on the morphological traits (Tsror 2010), then transferred onto potato dextrose agar (PDA) and cultured for 3 d as described above. A total of 15 pure cultures were obtained. With the exception of YN-3 (isolated from Puer), YN-62 (isolated from Mengzi) and LN-95(isolated from Dandong) strains, which exhibited hyphal fusion reaction with AG1-IB standard strain, all the other strains demonstrated hyphal fusion with AG-3 standard strain (Ogoshi 1987). Genomic DNA of these three strains were extracted by the CTAB method and ITS regions of rDNA were sequenced (White et al. 1990). The sequences were deposited in GenBank with accession No. OR770079, OR770080 and OR770082. All the three rDNA-ITS sequences exhibited 99.85% similar to AG1-IB found in GenBank, and a phylogenetic tree using a neighbor-joining method grouped the three strains within the R. solani AG-1 IB clade. Therefore, based on the hyphal fusion reaction and molecular methods, these isolates were identified as R. solani AG1-IB. To determine pathogenicity of the isolates, the healthy leaves of tobacco plants (cv. Yunyan 87) were used. Five-mm-diameter mycelial plugs of the strain on PDA were inoculated on leaves that had been previously wounded with a sterile needle, and cotton balls moistened with sterile water were used for moisturizing the inoculation sites. Ten leaves were inoculated for each strain and leaves inoculated with PDA plugs were as control. The experiment was conducted twice. All plants were incubated for 2 d at 15℃ to 25℃ and 90% relative humidity with a 12 h photoperiod/day. Irregularly shaped lesions appeared on the leaves around each of the inoculated sites, but not on control leaves. The pathogens were reisolated and confirmed be R. solani AG1-IB by hyphal fusion and molecular identification tests as previously described, thereby fulfilling Koch's postulates. It has been reported that AG-3, AG-2 (Mercado Cardenas et al. 2012), AG-5 (Wang et al. 2023) and AG-6 (Sun et al. 2022) of R. solani could cause tobacco target spot, but AG-3 is considered the main causal agent (Marleny Gonzalez et al. 2011). To our knowledge, this is the first report of AG1-IB causing tobacco target spot in China and worldwide. The AG1-IB strain has a wide host range including cabbage, mint, lettuce, beans, and rice (Gonzalez et al. 2006). The discovery poses a new challenge for the prevention and control of tobacco target spot, especially when contemplating disease management strategies such as crop rotation and fungicide treatments.

First report of wheat leaf spot disease caused by Curvularia inaequalis in China.

Wheat (Triticum aestivum) is an economically important crop widely cultivated in China. In August 2022, brown oval leaf spots with yellow halos were observed on approximately 10% wheat seedlings over an area of about 1 hectare in Xining City, Qinghai Province, which adversely affected wheat growth and production. Six diseased leaves were collected from the field in Huangyuan county (101°69' E, 37°04' N). The 0.5 cm × 0.5 cm pieces were cut from the border between healthy and diseased regions of the sampled leaves, surface sterilized for 10 s in 75% ethanol, followed by a 1% NaClO for 90 s, and rinsed three times with distilled sterile water. The pieces of leaf tissue were dried with sterile tissue, and plated on potato dextrose agar (PDA) amended with streptomycin (0.02 g/L) and ampicillin sulfate (0.05 g/L) to eliminate bacterial contamination. The dishes were placed in an incubator at 25°C for 72 h in dark. Three isolates, WGC201, WGC202 and WGC203, were obtained by a single-spore culture method. Fungal colonies on PDA media were dark green (Fig. 1A and 1B). Conidiophores were septate and geniculate terminals, while conidia exhibited straight or slightly curved forms with four transverse septa, the central cell being notably longer and wider than the others. The size of such conidia were 27.34 µm to 40.62 µm× 11.61 µm to 15.97 µm (number = 50) (av. 32.71 µm× 13.11 µm) (Fig. 1C and 1D) (Moubasher et al. 2010). The internal transcribed spacer (ITS) region of nuclear ribosomal DNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced using universal primers ITS1/ITS4 and GPDF/GPDR (White et al. 1990; Berbee et al. 1999). DNA sequences were deposited into the NCBI database (ITS, PP789629, PP801333, and PP801574; GAPDH, PP849124, PP849125, and PP849126). Phylogenetic analysis with a neighbor-joining method based on the concatenated sequences of ITS and GAPDH genes showed that the three isolates clustered within a C. inaequalis branch (Fig. 2). Based on morphological and molecular identification, the fungal isolates were identified as C. inaequalis. The pathogenicity test was conducted in a greenhouse at 25°C using a spore suspension method and three isolates were used. Conidia were produced on PDA media (25℃) for 14 days. Plates were washed with sterilized distilled water and filtered with cheese cloth. Conidial suspension was adjusted to a concentration of 1×107 conidia/mL. Fifteen healthy seedlings of a wheat cultivar Xiaoyan-6 at a 3-4 leaf stage were inoculated by evenly spraying a 100mL spore suspension. Plants inoculated with sterile water served as a control. All plants were covered with plastic bags for 3 days. At 7 days after inoculation, all pathogen-inoculated plants showed similar symptoms (brown leaf oval spots with yellow halos) with those observed in the field, while all plants inoculated with sterile water showed no symptoms (Fig. 1E and 1F). The pathogen was reisolated from the symptomatic leaves and proved to be C. inaequalis. Morphological, molecular and pathogenic results indicated that C. inaequalis is the pathogen causing wheat leaf disease in China. The results are consistent with a previous report in Azerbaijan (Özer et al. 2020). To our knowledge, this is the first report of C. inaequalis causing spot disease on wheat in China. The occurrence, spread and economic importance to different wheat cultivars of the emerging disease in China will be further investigated and evaluated.

Two new species of Macropelopia (Diptera, Chironomidae) from Oriental China, delineated with morphology and COI sequences.

Two new species, Macropelopia (Macropelopia) excavata Xu & Fu, sp. nov. and Macropelopia (Macropelopia) quadrimacula Xu & Fu, sp. nov., are described as male adults. A key to identify the males of Macropelopia from China is provided. Furthermore, in order to ascertain the genetic distance between these species and their morphological characteristics, mitochondrial cytochrome c oxidase subunit I gene sequences were uploaded to the National Center for Biotechnology Information. These COI sequences were then utilized to infer the relationships between the species, employing the neighbor-joining method.

Characterization of Longidorus pisi (Nematode) from South Africa: Morphological and molecular insights utilizing 18S rDNA analysis

Longidorus is a species of nematode that can be harmful to plants. Some of these nematodes are plant pests and can also transmit plant viruses such as potato virus U and cacao necrosis virus. Accurate identification of these nematodes is imperative due to their detrimental impact on agricultural crops. This study was conducted in 2022 at Limpopo University to and determine the plant-parasitic nematodes. The nematode was found and extracted using the tray method. Then, its DNA was extracted using the Chelex method. 18S rDNA was amplified using specific primers to identify the nematode. The Nblast analysis based on the small subunit ribosomal DNA showed a 99% similarity with a population of Longidorus pisi (MK172049) from Bulgaria. Additionally, phylogenetic analysis was performed using the neighbor-joining method implemented in mega x software. The analysis indicated that the South African populations of L. pisi are similar to other L. pisi, with a 99-bootstrap value. In conclusion, there is a need to investigate the diversity of this species to assess the impact of this nematode on crop yields.

Radiation-Tolerant Fibrivirga spp. from Rhizosphere Soil: Genome Insights and Potential in Agriculture.

The rhizosphere of plants contains a wide range of microorganisms that can be cultivated and used for the benefit of agricultural practices. From garden soil near the rhizosphere region, Strain ES10-3-2-2 was isolated, and the cells were Gram-negative, aerobic, non-spore-forming rods that were 0.3-0.8 µm in diameter and 1.5-2.5 µm in length. The neighbor-joining method on 16S rDNA similarity revealed that the strain exhibited the highest sequence similarities with "Fibrivirga algicola JA-25" (99.2%) and Fibrella forsythia HMF5405T (97.3%). To further explore its biotechnological potentialities, we sequenced the complete genome of this strain employing the PacBio RSII sequencing platform. The genome of Strain ES10-3-2-2 comprises a 6,408,035 bp circular chromosome with a 52.8% GC content, including 5038 protein-coding genes and 52 RNA genes. The sequencing also identified three plasmids measuring 212,574 bp, 175,683 bp, and 81,564 bp. Intriguingly, annotations derived from the NCBI-PGAP, eggnog, and KEGG databases indicated the presence of genes affiliated with radiation-resistance pathway genes and plant-growth promotor key/biofertilization-related genes regarding Fe acquisition, K and P assimilation, CO2 fixation, and Fe solubilization, with essential roles in agroecosystems, as well as genes related to siderophore regulation. Additionally, T1SS, T6SS, and T9SS secretion systems are present in this species, like plant-associated bacteria. The inoculation of Strain ES10-3-2-2 to Arabidopsis significantly increases the fresh shoot and root biomass, thereby maintaining the plant quality compared to uninoculated controls. This work represents a link between radiation tolerance and the plant-growth mechanism of Strain ES10-3-2-2 based on in vitro experiments and bioinformatic approaches. Overall, the radiation-tolerant bacteria might enable the development of microbiological preparations that are extremely effective at increasing plant biomass and soil fertility, both of which are crucial for sustainable agriculture.