Detection of microorganisms with antibacterial activities from different industrial wastes and GC-MS analysis of crude microbial extract

Many soil microorganisms’ i.e., bacteria and fungi produce secondary metabolites called antibiotics. These are used for the treatment of some of the bacterial, fungal and protozoal diseases of humans. There is a need for isolation of a broad spectrum of antibiotics from microorganisms due to the emergence of antibiotic resistance. In the present study two antibiotic producing bacteria Klebsiella pneumoniae and Bacillus cereus were isolated from pharmaceutical and poultry feed industry of Hattar, Haripur Pakistan. Total 10 waste samples were collected from different industries (Marble, Ghee, Soap, Mineral, Steel, Poultry Feed, Pharmaceutical, Qarshi, Cosmetic and Glass). Thirty-three bacterial strains were isolated from industrial wastes of these ten different industries. Fourteen out of thirty-three bacterial strains exhibited antimicrobial activities against at least one of the test microbes considered in this study including Escherchia coli, Staphylococcus aureus and Salmonella typhi. The bacteria were isolated by standard serial dilution spread plate technique. Morphological characterization of the isolates was done by Gram staining. Nine bacterial isolates out of fourteen were initially identified as B. cereus and five as K. pneumoniae through biochemical characterization. The antibacterial activities were tested by well diffusion method. Maximum number of antibiotic producing bacteria were isolated from pharmaceutical and poultry feed industry based on the results of primary screening, the most potential isolates S9, S19, S20, S22 and S23 were selected for secondary screening. The maximum activity against E. coli and S. aureus was recorded by bacterial isolate S19 i.e zones of inhibition of 6.5mm and 9mm while S20 showed 7.5mm and 6mm zones respectively. Molecular identification was carried out on the basis of 16S rRNA sequence analysis. Finally, the isolates were identified as B. cereus accession number LC538271and K. pneumoniae accession number MT078679. Analysis of bacterial extract S20 through GC-MS indicated the presence of 8 compounds of diverse nature and structure. Present study suggests that wastes of pharmaceutical and poultry feed industry may have antibiotic producing bacteria. These bacteria could be utilized for the production of antibiotics. B. cereus and K. pneumoniae isolated from wastes of poultry feed and pharmaceutical industries have the potential to produce antibiotics and could be used to control the microbial growth.

Occurrence of Grapevine Leafroll-Associated Virus-3 (GLRaV-3), Complete Nucleotide Sequence and Cultivar Susceptibility to a GLRaV-3 Isolate from Shaanxi Province of China

Grapevine (Vitis spp.) is globally one of the most economically important fruit crops. China is the largest grapevine-growing country of the world and Shaanxi province is one of the major grapevine-growing provinces in the country. A survey of GLRaV-3 found it widespread, with 57–100% infection frequencies, in both wine and table grapevine cultivars of three grapevine-growing regions of Shaanxi province. The virus infection frequencies varied with cultivars and regions. In order to obtain the full genomic length of a new GLRaV-3 isolate, GLRaV-3-Sau (accession number MK988555), was sequenced. This isolate has a genome of 18026 nucleotides, and 14 open reading frames (ORFs). The full-genome of the isolate GLRaV-3-Sau shared 85.88% nucleotide identity to GLRaV-3-LN, another isolate found in China. Coat protein (CP) genes of GLRaV-3 isolates were identical (99%) to the Vitis vinifera isolate (accession number HQ185608.1) from the USA. Immunohistochemistry for virus localization found that distribution patterns were similar in red-berried cultivar ‘Cabernet Sauvignon’ and white-berried cultivar ‘Chardonnay’, and GLRaV-3 is restricted in phloem tissue of vascular bundles. Virus transmission by micrografting found virus transmission efficiency was higher in ‘Chardonnay’ and ‘Thompson Seedless’ than in ‘Hunan-1’, indicating that ‘Hunan-1’ was less sensitive to GLRaV-3. As far as we know, these are the most comprehensive comparisons on the genome and CP genes of GLRaV-3 worldwide and the first to have found that the grapevine ‘Hunan-1’ is less susceptible to GLRaV-3.

First Report of New Bacterial Leaf streak of Rice Caused by Pantoea ananatis in China

Rice (Oryza sativa L.) is the largest grain crop, accounting for about 40 % of the total grain production in China. In mid-July 2021, bacterial leaf streak-like disease emerged in rice varieties Chunyou584 and Yongyou2604 in Linyi city, Shandong Province, China. Disease incidences of the disease ranged from 80% to 90% in the surveyed fields. Infected rice leaves displayed dark green to yellowish-brown water-soaked thin streaks, and a large amount of beaded yellow oozes were observed on the lesions. After drying, there were gelatinous granules that were not easy to fall off and spread between leaf veins (Fig.S1A). According to the field symptoms of this disease, it was preliminarily suspected to be rice bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc), which is a guaranteed disease in China. To isolate the causal agent, leaf discs (~1 cm2) of diseased leaves were collected from the margins of the lesions, surface sterilized and ground into pieces in sterile double distilled water. The 10-3, 10-4 and 10-5 dilutions were spread onto peptone sugar agar (PSA) and incubated at 28°C for 36 hours. Yellow mucous bacterial colonies were consistently obtained on PSA medium. To identify the pathogen, fragments of the 16S rDNA, leuS and rpoB were amplified and sequenced using the primers previously reported (Yu et al. 2021). Three strains (LY01, LY02 and LY03) showed identical colony morphology and LY01 was used for further analyses. Sequence analyses showed that the fragments of 16S rDNA (955 bp, GenBank accession number: OK261898), leuS (755 bp, GenBank accession number: OK298387) and rpoB (926 bp, GenBank accession number: OK298388) of strain LY01 shared 99.16%, 99.46% and 100% similarities with those of Pantoea ananatis TZ39 (GenBank accession numbers: CP081342.1 for 16S rDNA, MW981338.1 for leuS and MW981344.1 for rpoB), respectively, which suggest the pathogenic bacterial strain LY01 isolated is P. ananatis. In addition, the single colony of P. ananatis LY01 was shown as Fig. S2B. Furthermore, pathogenicity tests were also performed according to the following steps. Bacterial suspension at OD600=0.1 was inoculated into eight rice leaves of four healthy rice plants (Chunyou 584) at 25-33°C and 60%-80% relative humidity in the field using a clipping method (Yang et al. 2020) or spraying methods, and sterile distilled water was as negative control. The clipped leaves (Fig. S1B) and spray-inoculated leaves (Fig. S1C) showed dark green water-soaked streaks at 14 days after inoculation, respectively, which showed similar symptoms with those samples collected from the fields (Fig. S1A). On contrary, the control rice leaves remained healthy and symptomless (Fig. S2A). The bacterium was re-isolated in the inoculated rice leaves and the re-isolated bacterial isolates, which was confirmed by sequencing 16S rDNA, leuS and rpoB, incited the same symptoms as in fields, which fulfills Koch’s postulates. In the past decade, P. ananatis was reported to result in grain discoloration and leaf blight in China (Yan et al. 2010; Xue et al. 2020, Yu et al. 2021), which could result in 40% - 60% yield losses. To our best knowledge, this is the first report of the bacterial leaf streak-likely disease occurred in Shandong Province caused by P. ananatis, so we named it as Pantoea leaf streak of rice. Although P. ananatis was also reported in Zhejiang province and Jiangxi province, which caused leaf streak lesions on rice, the disease symptoms are completely different from those of Pantoea leaf streak of rice. To the best of our knowledge, this is the first report of Pantoea leaf streak of rice caused by P. ananatis. This study provides sloid evidence that Pantoea leaf streak of rice in Eastern China can be caused by the new pathogen, P. ananatis, rather than Xoc as traditionally assumed. Disease development and quarantine of the new Pantoea leaf streak of rice disease caused by P. ananatis on rice need more attention in the near future.

An investigation on the chloroplast and nuclear genomes of taxa belong to the subgenus Dracunculus (Bess.) Rydb. of Artemisia L. (Asteraceae) in Turkey

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasn’t been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.

Optimization of Chlorella Culture Conditions with Response Surface Methodology to Increase Biomass

Microalgae is gaining popularity as a major ingredient in nutrition supplements. To mass cultivate, it is imperative to improve the biomass yield hence optimization of cultures conditions becomes paramount. In this work, an attempt has been made to optimize the microalgal production using response surface methodology (RSM) and validate further the optimized parameters. The optimum conditions for the cultivation of Chlorella sp. KPU016 under optimized nutrient conditions were pH 8.2, the light intensity of 3100 lx, glycerol 1.44 g.L-1 (under pre-set conditions of 12 h lighting, the temperature at 27±1°C. With these RSM-driven optimum conditions, the yield of microalgal biomass achieved was 282.50 mg.L-1. For larger-scale microalgal harvesting, the validated optimal conditions can be inferred as the best for enhanced microalgal production. The isolate was partially sequenced and submitted to the NCBI database and the GenBank accession number is MZ348364.

Association between a SLC23A2 Gene Variation, Plasma Vitamin C Levels, and Risk of Different Diseases

Background: Vitamin C is an important plasma water-soluble antioxidant that plays an essential role in the absorption of iron, detoxification of exogenous compounds, and remaking vitamin E for the protection of lipid membranes. In addition, vitamin C is essential in the synthesis of collagen. Vitamin C concentrations of plasma are determined by dietary intake and genetic factors. Ascorbic acid is the functional form of vitamin C, which is transported into the cell through sodium vitamin C transporters (SVCTs). There are two forms of SVCTs which are SVCT1 encoded by the SLC23A1 gene and SVCT2 encoded by the SLC23A2. The SLC23A2 gene locus on human chromosome 20P12. It expresses in most human tissues, except lung and skeletal muscle that it is important in regulating the intracellular concentration of ascorbic acid to protect the cell from oxidative stress and promote type 1 collagen maturation. Maintaining proper concentrations of plasma and cellular vitamin C concentration is important for the normal metabolic function of the body and preventing several diseases. In the contrast, a low concentration of vitamin C caused by SLC23A2 variation can cause several chronic diseases. Our systematic review discusses four diseases related to the variation of SLC23A2 gene and plasma vitamin C levels which are glaucoma, acute coronary syndrome among women, gastric cancer, and HPV16-associated head and neck cancer. Methods: By using NCBI databases, specifically GenBank to analyze DNA sequence and mRNA sequence of SLC23A2 gene. GenBank file format was helpful to extract an accession number of the gene, number of amino acids, number of exons and introns, and length of nucleotides. FASTA format was also useful to retrieve the nucleotide sequence and get the function of the protein. BLAST was used to compare the protein product of the SLC23A2 gene between humans and Macaca mulatta (Rhesus monkey). Results: the accession number of the SLC23A2 gene was NC_000020.11, the number of exons found was 18, and the gene was located in chromosome 20. This gene encodes one of the two required transporters, and the encoded protein accounts for tissue-specific uptake of vitamin C. This gene had an official symbol of SLC23A1. And they found a significant association between the single-nucleotide polymorphism (SNP) rs1279683 (A > G) in SLC23A2 and an increased risk of POAG in homozygous G allele (GG) carriers. Also, POAG patients with this SNP appear to have a significantly lower level of plasma vitamin C compared to other genotypes. Finally, many organisms have the same gene, such as dogs, mice, rats, and chickens. Conclusion: there is a significant association between SLC23A2 gene mutation, increased risk for vitamin C deficiency, and several diseases. SNP in the SLC23A2 gene was significantly associated with a higher risk of POAG in GG allele carriers as well as lower plasma vitamin C concentration.

KARAKTERISASI BERBASIS MARKA MOLEKULER ITS2 TERHADAP SUB-SPESIES KOMPLEKS Anopheles vagus vagus DAN Anopheles vagus limosus

The presence of intraspecies variations of An. vagus later categorized as the subspecies of An. vagus vagus and An. vagus limosus, could be an obstacle to the identification process, which is an important step for malaria vector’s competence characterization. Based on morphological identification, those subspecies could be distinguished by the presences of pale scales in prehumeral and pale bands in proboscis. The objective of this research was to compare subspecies complexes of An. vagus morphologically and molecularly using Internal Transcribed Spacer 2 (ITS2). Anopheles samples were collected from Bangsring, Banyuwangi. Their phylogenetic tree was constructed by using NJ method based on their ITS2 sequences. BLAST result showed that An. vagus vagus and An. vagus limosus were similar to An. vagus FJ654649.1 from East Java Indonesia and East Timor based on its 99% homology and their molecular distance. The Neighbour Joining (NJ) tree grouped those subspecies in one clade with a boostrap value of 82%. This subspeciation might be due to the different rates of evolution. ITS2 sequences of An. vagus vagus and An. vagus limosus were submitted to GenBank with the accession number of MW314227.1 and MW319822.1, respectively. Kemunculan variasi intraspesies An. vagus yang kemudian dikategorikan sebagai subspesies An. vagus vagus dan An. vagus limosus menjadi kendala dalam proses identifikasi yang merupakan langkah penting dalam menentukan kompetensi vektor malaria. Berdasarkan karakter morfologi, subspesies tersebut dibedakan dengan adanya sisik pucat pada bagian prehumeral dan pita pucat pada probosis. Penelitian ini bertujuan untuk membandingkan subspesies An. vagus secara morfologis dan molekuler menggunakan Internal Transcribed Spacer 2 (ITS2). Nyamuk Anopheles didapatkan dari Bangsring, Banyuwangi. Konstruksi pohon filogeni dilakukan berdasarkan sekuen ITS2 yang dianalisis menggunakan metode NJ. Hasil BLAST menunjukkan, ITS2 An. vagus vagus dan An. vagus limosus memiliki tingkat homologi 99% dan jarak evolusi molekuler terendah dengan An. vagus FJ654649.1 dari Jawa Timur Indonesia dan Timor Timur. Pohon NJ mengelompokkan subspesies tersebut dalam satu klade dengan nilai boostrap 82%. Hal ini dapat terjadi karena perbedaan kecepatan evolusi yang memungkinkan terjadinya subspesiasi. Urutan basa ITS2 dari An. vagus vagus dan An. vagus limosus telah didaftarkan ke GenBank dengan nomor aksesi MW314227.1 dan MW319822.1.

Structural characterization of a MAPR-related archaeal cytochrome b5M protein

We recently reported that the membrane associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b5 (cytb5) domain proteins, called cytb5M (MAPR-like). Relative to classical cytb5 proteins, MAPR and ctyb5M proteins shared unique sequence elements and a distinct heme binding orientation at an approximately 90⁰ rotation relative to classical cytb5, as demonstrated in the archetypal crystal structure of a cytb5M protein (PDB accession number 6NZX). Here, we present the second crystal structure of an archaeal cytb5M domain (Methanococcoides burtonii WP_011499504.1, PDB:6VZ6). It exhibits similar heme-binding to the 6NZX cytb5M, supporting the deduction that MAPR-like heme orientation was inherited from the prokaryotic ancestor of the original eukaryotic MAPR gene.

First Report of Pectobacterium aroidearum Causing Soft Rot in Olecranon Honey Peach (Prunus persica L.) in China

Olecranon honey peach (Prunus persica L.) is a popular fruit tree cultivated in Guangdong Province of China. Due to its excellent economic values and popularity, it has recently been widely adopted and planted in several other southern Provinces and Autonomous Region in China, including Yunnan, Hunan, Jiangxi, Guizhou, and Guangxi. In Lianping County of Guangdong Province alone, the annual peach fruit production was about 78,800 tonnes (Xie et al. 2017). In July 2021, peach fruits showing soft rot symptoms were collected from an olecranon honey peach plantation in Lechang, Guangdong, China. Symptoms included tissue disintegration with bacterial oozes and rotting smells. To isolate the causal agent of soft rot in the peach fruits, the bacterial oozes from various rotted fruits were streaked on the modified YEB agar plate (Huang et al. 2021), and 21 bacterial colonies were selected for PCR amplification using the primers targeting the conserved region of 16S rDNA gene (Wei et al. 2020). A blastN analysis of the DNA sequences of the obtained PCR fragments in NCBI website indicated that 17 isolates named as ZL strains were potential bacterial species of Pectobacterium with about 99% similarity (Genbank accession number of ZL1: OK189602) to Pectobacterium aroidearum SCRI 109T (Genbank accession number: NR_159926). Three of them (ZL1, ZL2 and ZL3) were selected for assay of pathogenicity. The bacterial suspensions (10 μl, 1×106 CFU/ml) of strains ZL1, ZL2 and ZL3 were injected into olecranon honey peach fruits by using a syringe. A portion of peach fruits were similarly injected with sterile distilled water as the negative control. After 18 h incubation at 25 °C, the typical symptom of soft rot, i.e., tissue decay, became visible on the peach fruits inoculated with the bacterial suspensions. After inoculation for 42 h, bacterial oozes were exuded from rotting tissues. Peach fruits without injuries were also sprayed with the bacterial suspensions under the same conditions, but decay symptoms were not observed, suggesting that the bacterial infection needs the wounding or injuries. To fulfill the Koch’s postulates, bacterial colonies were re-isolated from bacterial oozes, and their conserved region of 16S rDNA fragments were amplified and sequenced. Bioinformatics analysis of the DNA sequence data confirmed that all the isolated colonies were Pectobacterium strains. Using the Biolog Gen III system, the representative strain ZL1 was identified as Pectobacterium (SIM 0.56). Transmission electron microscopy analysis showed that the bacterial cells of strain ZL1 were rod-shaped with peripheral flagella. To further determine the species of ZL strains, eight housekeeping genes (acnA, gapA, icd, mdh, mtlD, pgi, proA and rpoS) were analyzed by the methods described previously (Nabhan et al. 2013). The amplified DNA sequences analyzed by the blastN program in NCBI showed that the sequences of eight housekeeping genes from strains ZL1, ZL2 and ZL3 were identical to each other (Genbank accession number: OK274248 to OK274255), and most of the gene sequences shared over 99% similarity to their counterparts in P. aroidearum L6 (Genbank accession number: NZ_CP065044) (Xu et al. 2021), except that the acnA and proA genes showed about 98% and 96% similarity respectively to the corresponding genes of P. aroidearum L6. In addition, the multi-locus sequence analysis (MLSA) using DNA sequences of above eight housekeeping genes showed that ZL strains were grouped with other P. aroidearum strains. Taken together, the results of molecular and biochemical assays confirmed that ZL strains isolated from olecranon honey peach fruits were P. aroidearum. To our knowledge, this is the first report of P. aroidearum causing soft rot disease in olecranon honey peach in China. P. aroidearum is a relatively newly described soft rot pathogen (Nabhan et al. 2013). More recently, the pathogen was found causing soft rot infections in lettuce, Chinese cabbage, pepper (Capsicum annuum) fruits, konjac, carrot and Syngonium podophyllum (Barroso et al. 2019; Moraes et al. 2020; Sun et al. 2019; Tang et al. 2020; Xu et al. 2021). The results of this study add a new plant species to the host range of P. aroidearum.

Molecular and Morphological Data Confirmed First Record of Abbreviata kazakhstanica Markov and Paraskiv, 1956 (Spirurida: Physalopteridea) in Iran

Background: The genus Abbreviata (Spirurida: Physalopteridea) currently contains 47 species. Physalopteridae nematodes infect a large number of vertebrates, including mammals, birds, reptiles and amphibians. The current study is a report of the first morphological and molecular identification of A. kazakhstanica (Spirurida: Physalopteridea) in Pseudopus apodus in Iran. Methods: Eleven road-killed P. apodus, were collected from, Iran during 2016-2018. The nematodes were isolated from stomach. After morphological study, the genomic DNA of the parasites was extracted using CTAB method. The DNA was used for PCR amplification of cytochrome c oxidase subunit I (cox1). The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. Results: After detailed microscopic examination, the A. kazakhstanica was identified. The cox1 sequences confirmed the species of helminth. The new sequences of A. kazakhstanica were submitted to GenBank under the accession number MK578751-2. Conclusion: Regarding the limited data on parasitological status of Iranian reptiles, more specific and comprehensive investigations are needed to identify the parasitic fauna.