16S rDNA Sequence Analysis Research Articles

Diversity and spatial distribution of endophytes community composition in noni (Morinda citrifoliaL.)

The objective of this study was to investigate the endophytic microbial communities in noni (Morinda citrifolia L.) whole plants, including the roots, branches, leaves, seeds and fruits. By using microbial culture-dependent methods, a research on the diversity of endophytic bacterial communities in Noni samples was conducted in this study. Partial sequence analysis of 16S rDNA, 26S rDNA D1/D2 region and ITS rDNA was used for the endophytic strains identification. The results indicated that 6 strains of bacteria were highly similar with 13 known genera which belong to α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The 13 genera were Asaia sp., Methylobacterium sp., Rhizobium sp., Sphingomonas sp., Burkholderia sp., Enterobacter sp., Pantoea sp., Bacillus sp., Brachybacterium sp., Curtobacterium sp., Microbacterium sp., Streptomyces sp. and Flavobacterium sp., respectively. Further, 12 strains of yeast were highly similar with genus of Cryptococcus sp., Rhodotorula sp. belonging to Basidiomycota and 13 strains of mould were in a high similarity with genera Talaromyces sp., Cladosporium sp., Chaetomium sp. and Mucor sp. belonging to Ascomycota. According to available literature, this was the first investigational report in the world on the diversity of endophytic communities in noni plants. This study has laid a solid foundation for further study in beneficial microbes, which would help to improve the growth and efficacy of noni component production.

Characterization of an Endophytic Bacterium G062 Isolate with Beneficial Traits

An endophytic bacterium isolate G062 was characterized base on its molecular genetic potents, morphology, physiology, and biochemistry reactions. Analysis of 16S rDNA sequences of G062 showed the highest similarity to Paracoccus halophilus (98%). Detection of the phlD and prnC genes occurrence indicated that the bacterium had this antibiotic-like genes of Diacethylphloroglucinol (DAPG) and pyrrolnitrin. The cells are rod shaped (0.59-0.89 × 1.85-3.3 μm), aerobic, Gram negative, non motile, non spore forming, positive catalase, positive oxydase, could reduce NO3 to N2, nitrogen fixing, producing siderophore and plant growth hormones-like compounds (IAA, Gibberellin, and zeatin), and solubilizing phosphate. The G062 isolate could grow on media containing 2.5% NaCl. Range of the temperature and pH growth were 15-40 and 5.0-9.5 °C, respectively. The bacterium did not cause red blood cells lysis. There was no hypersensitive response when it was injected into tobacco leaves, and it was not pathogenic against potato plantlets. Moreover, the bacterium promoted the growth of the potato plant and had high colonization ability. These results suggested that the bacterium had beneficial and good traits as biological agent candidate to promote potato plant growth.

Babesia behnkei sp. nov., a novel Babesia species infecting isolated populations of Wagner's gerbil, Dipodillus dasyurus, from the Sinai Mountains, Egypt.

BackgroundAlthough a number of new species of Babesia/Theileria have been described recently, there are still relatively few reports of species from Africa. In this study based on the evaluation of morphology and phylogenetic relationships, we describe a novel species from Wagner’s gerbil, Babesia behnkei n. sp.MethodsRodents (n = 1021) were sampled in four montane valleys (wadies) in 2000, 2004, 2008 and 2012 in the Sinai Mountains, Egypt. The overall prevalence of Babesia spp. was highest in the Wagner’s gerbil (Dipodillus dasyurus; 38.7%) in comparison to the prevalence in the spiny mice species, Acomys dimidiatus and A. russatus. Morphological investigations were conducted for the comparison of trophozoites of the novel species of Babesia with the B. microti King’s 67 reference strain. Thirty-two isolates derived from D. dasyurus over a 9 year period (2004-2012) from two wadies (29 isolates from Wadi Gebel and 3 from Wadi El-Arbaein) were investigated by microscopic, molecular and phylogenetic analysis. A near-full-length sequence of the 18S rRNA gene and the second internal transcribed spacer (ITS2) region were amplified, sequenced and used for the construction of phylogenetic trees.ResultsA novel species of Babesia was identified in two isolated populations of D. dasyurus. Phylogenetic analysis of 18S rDNA and ITS2 sequences revealed that B. behnkei n. sp. is most closely related to B. lengau from cheetahs from South Africa and to Nearctic species found only in North America (the pathogenic B. duncani and B. conradae) and that it is more distant to the cosmopolitan rodent parasite B. microti. Trophozoites of B. behnkei were smaller and less polymorphic than trophozoites of B. microti.ConclusionBabesia behnkei n. sp. is a novel species of the ‘Duncani group’ maintained in isolated populations of Dipodillus dasyurus occurring in the Sinai Mountains of Egypt.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0572-9) contains supplementary material, which is available to authorized users.

The occurrence of coliform bacteria in the cave waters of Slovak Karst, Slovakia.

The diversity and abundance of coliform bacteria (taxonomically enterobacterias), an important quality water indicator, were determined for four representative caves in Slovak Karst: Domica Cave, Gombasecká Cave, Milada Cave and Krásnohorská Cave. Three hundred and fifty-two enterobacterial isolates were successfully identified by biochemical testing (commercial ENTEROtest 24) and selected isolates confirmed by molecular techniques (PCR, 16S rDNA sequence analysis). A total of 39 enterobacterial species were isolated from cave waters, with predominance of Escherichia coli, Serratia spp. and Enterobacter spp. PCR amplification of lacZ gene is not specific enough to provide a reliable detection of coliform bacteria isolated from the environment. Sequence analysis of 16S rDNA confirmed that all of the selected isolates belong to the family Enterobacteriaceae. In general, physical and chemical parameters of cave waters in Slovak Karst corresponded to national drinking water quality standards.

Identification of antibiotic-producing Bacillus sensu lato isolated from national parks of Hoang Lien and Phu Quoc in Vietnam

Many lipopeptide antibiotics produced from Bacillus sensu lato (Bacillus s. l.) against drug-resistant bacteria have been recently reported. To explore the potential production of the antibiotics from this group of bacteria in Vietnam, we collected 38 soil samples from two national parks of Hoang Lien and Phu Quoc and isolated 411Bacillus s. l. strains. Of those, 22 strains had antagonistic activity against both susceptible S. aureus and E. coli. The strains were further tested on drug-resistant bacteria collected at Bach Mai hospital and 20 strains demonstrated antagonistic activity against at least 2 of 18 drug-resistant bacteria K. pneumoniae, E. coli, A. baumannii and S. aureus. Analysis of 16S rDNA sequence showed that most of the broad spectrum antibiotic producers were Paenibacillus species whereas narrow spectrum antibiotic producers were Bacillus species. Strains PQH 0103 and PQH 0410 were probably produced novel antibiotic agents as they suspected to be novel taxa in Paenibacillus genus. Moreover, strains PQH 1509 and PQH 1702 produced broad spectrum antibiotics were identified as P. chitinolyticus. To our best knowledge, there is no report on antibiotic production from this species. Further elucidation of chemical structure of antibiotics produced from Bacillus s. l. isolated in soils of Vietnam is needed.
 Gần đây, nhiều kháng sinh lipopeptide từ vi khuẩn Bacillus sensu lato (Bacillus s. l.) có khả năng sinh kháng sinh kháng vi khuẩn kháng thuốc đã được phát hiện và công bố. Để tìm hiểu khả năng sinh kháng sinh của nhóm vi khuẩn này ở Việt Nam, chúng tôi tiến hành thu thập 38 mẫu đất tại vườn Quốc gia Hoàng Liên và vườn Quốc gia Phú Quốc và phân lập được 411 chủng vi khuẩn Bacillus s. l.. Trong số đó, 22 chủng có khả năng sinh kháng sinh kháng 2 chủng vi khuẩn S. aureus và E. coli nhậy cảm thuốc. 20 trong số 22 chủng này có hoạt tính kháng ít nhất từ 2 đến 18 chủng kiểm định kháng thuốc thuộc các loài K. pneumoniae, E. coli, A. baumannii và S. aureus. Dựa trên kết quả phân loại bằng trình tự gene 16S rRNA, các chủng Bacillus s. l. sinh kháng sinh phổ rộng là các loài Paenibacillus trong khi đó các loài sinh kháng sinh phổ hẹp là Bacillus. Trên cây phát sinh chủng loại, chủng PQH 0103 và PQH 0410 có thể là những loài mới và có tiềm năng sinh các chất kháng sinh mới. Hơn nữa, chủng PQH 1509 và PQH 1702 sinh kháng sinh phổ rộng được phân loại vào loài P. chitinolyticus. Đây là loài chưa có công bố nào về khả năng sinh chất kháng sinh và có thể là loài sinh chất kháng sinh mới. Nghiên cứu giải mã cấu trúc chất kháng sinh từ các loài Bacillus s. l. phân lập tại Việt Nam cần được làm rõ trong tương lai.

Characterization and quantification of the fungal microbiome in serial samples from individuals with cystic fibrosis.

BackgroundHuman-associated microbial communities include fungi, but we understand little about which fungal species are present, their relative and absolute abundances, and how antimicrobial therapy impacts fungal communities. The disease cystic fibrosis (CF) often involves chronic airway colonization by bacteria and fungi, and these infections cause irreversible lung damage. Fungi are detected more frequently in CF sputum samples upon initiation of antimicrobial therapy, and several studies have implicated the detection of fungi in sputum with worse outcomes. Thus, a more complete understanding of fungi in CF is required.ResultsWe characterized the fungi and bacteria in expectorated sputa from six CF subjects. Samples were collected upon admission for systemic antibacterial therapy and upon the completion of treatment and analyzed using a pyrosequencing-based analysis of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA sequences. A mixture of Candida species and Malassezia dominated the mycobiome in all samples (74%–99% of fungal reads). There was not a striking trend correlating fungal and bacterial richness, and richness showed a decline after antibiotic therapy particularly for the bacteria. The fungal communities within a sputum sample resembled other samples from that subject despite the aggressive antibacterial therapy. Quantitative PCR analysis of fungal 18S rDNA sequences to assess fungal burden showed variation in fungal density in sputum before and after antibacterial therapy but no consistent directional trend. Analysis of Candida ITS1 sequences amplified from sputum or pure culture-derived genomic DNA from individual Candida species found little (<0.5%) or no variation in ITS1 sequences within or between strains, thereby validating this locus for the purpose of Candida species identification. We also report the enhancement of the publically available Visualization and Analysis of Microbial Population Structures (VAMPS) tool for the analysis of fungal communities in clinical samples.ConclusionsFungi are present in CF respiratory sputum. In CF, the use of intravenous antibiotic therapy often does not profoundly impact bacterial community structure, and we observed a similar stability in fungal species composition. Further studies are required to predict the effects of antibacterials on fungal burden in CF and fungal community stability in non-CF populations.

Phylogenentic and enzymatic characterization of psychrophilic and psychrotolerant marine bacteria belong to γ-Proteobacteria group isolated from the sub-Antarctic Beagle Channel, Argentina.

The phylogenetic and physiological characteristics of cultivable-dependent approaches were determined to establish the diversity of marine bacteria associated with the intestines of benthonic organisms and seawater samples from the Argentina's Beagle Channel. A total of 737 isolates were classified as psychrophlic and psychrotolerant culturable marine bacteria. These cold-adapted microorganisms are capable of producing cold-active glycosyl hydrolases, such as β-glucosidases, celulases, β-galactosidases, xylanases, chitinases, and proteases. These enzymes could have potential biotechnological applications for use in low-temperature manufacturing processes. According to polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S ribosomal DNA (ARDRA) and DNA gyrase subunit B (gyrB-RFLP), 11 operational taxonomic units (OTU) were identified and clustered in known genera using InfoStat software. The 50 isolates selected were sequenced based on near full sequence analysis of 16S rDNA and gyrB sequences and identified by their nearest neighbors ranging between 96 and 99% of identities. Phylogenetic analyses using both genes allowed relationships between members of the cultured marine bacteria belonging to the γ-Proteobacteria group (Aeromonas, Halteromonas, Pseudomonas, Pseudoalteromonas, Shewanella, Serratia, Colwellia, Glacielocola, and Psychrobacter) to be evaluated. Our research reveals a high diversity of hydrolytic bacteria, and their products actuality has an industrial use in several bioprocesses at low-temperature manufacturing.

English

A bacterial strain, which was able survive Pb, Zn and Cr -heavy metal compound culture was isolated from the soils of Shanjia Villiage, Qufu City, Shandong Province. An observation of the morphological and biochemical characteristics of the compound and the analysis of 16SrDNA sequence revealed that it was Bacillus. Standing liquid culture was used to study the tolerance of Pb Zn Cr -heavy metals compound and the ability to absorb and clean the heavy metal compound. Further study was made to compare the absorbing ability of Cu, Cd and Hg. &nbsp; Key words: Bacillus, heavy metal, Pb, Zn, Cr.

제주연안으로부터 분리한 Paenibacillus sp. MK-11의 어류 질병 세균에 대한 항균활성 탐색

In this study, we isolate and identify bacteria from seawater collected from Jeju coast, to evaluate the antimicrobial activity against the fish pathogenic bacteria. 14 bacterial strains were isolated and identified using physiological, biochemical and molecular tools. Antibacterial activity of all the 14 isolates were screened against four major fish pathogens namely, two Gram-positive: Streptococcus iniae, Streptococcus parauberis and two Gram-negative: Vibrio anguillarum, Edwardsiella tarda. Results revealed that among the 14 isolates, MK-11 was found to have antibacterial activity against S. iniae, S. parauberis, V. anguillarum Particularly, S. iniae was susceptibility with the MIC value of 250 μg/ml. The biochemical and physio-chemical results reveal that MK-11 had the sugar-alcohol disassemble ability of the D-sorbitol and D-mannitol. Also the utilization of the yeast extract, sorbitol and di-potassium phosphate were noted to be high. The optimum culture condition such as pH and temperature was recorded as pH 6.0, 25°C and along with 1% NaCl which differs from the previous reports particularly in nutrient resolutions. As results of the analysis of 16S rDNA sequences, MK-11 show the high similarity with Paenibacillus polymyxa, P. jamilae, P. brasilensis 99.78%, 99.43%, 99.39%, repectively. Hence, in the present study, the isolated Paemibacillus sp. MK-11 from Jeju seawater possesses the antibacterial activity against fish pathogens and it could be used as a new antibiotic agents against the gram positive fish pathogens.

Redescription and molecular diagnosis of Hepatozoon theileri (Laveran, 1905) (Apicomplexa: Adeleorina: Hepatozoidae), infecting Amietia quecketti (Anura: Pyxicephalidae)

Blood smears prepared from the peripheral blood of 20 wild caught Amietia quecketti (Boulenger) from the North-West University Botanical Gardens, North West Province, South Africa, were examined for the presence of haemogregarines. A haemogregarine species comparative in morphology, host and geographical locality to that of Haemogregarina theileri Laveran, 1905 was detected. The original description of H. theileri was based solely on frog peripheral blood gamont stages. Later, further parasite stages, including trophozoites and merogonic liver stages, were recorded in a related Amietia sp. from equatorial Africa. This species was originally classified as a member of the genus Haemogregarina Danilewsky, 1885, but due to the close life cycle and morphological resemblance to those of Hepatozoon species, H. theileri was later transferred from Haemogregarina to Hepatozoon Miller, 1908. In the present study, meront and merozoite stages not described before, along with previously observed trophozoite, immature and mature gamont stages, are described from the peripheral blood of hosts. In addition, comparative phylogenetic analysis of the partial 18S rDNA sequence of Hepatozoon theileri to those of other haemogregarine species, including those of species of Hepatozoon and a Haemogregarina, support the taxonomic transfer of H. theileri to Hepatozoon, nesting H. theileri within a clade comprising species parasitising other amphibians. This is the first molecular and phylogenetic analysis of an African anuran species of Hepatozoon.

Isolation and characterization of an electrochemically active and cyanide-degrading bacterium isolated from a microbial fuel cell

A facultative anaerobic bacterium capable of generating electricity and degrading cyanide was isolated from a microbial fuel cell and was designated as MC-1. According to its morphological characteristics and the sequence analysis of 16S rDNA, the isolated bacterium was identified as a strain of Klebsiella sp. Cyclic voltammetry showed that the strain MC-1 exhibited high electrochemical activity. The preliminary electricity-production experiment showed that the strain MC-1 could use glucose–cyanide mixtures for electricity production in a microbial fuel cell (MFC). The maximum voltage was 412 mV, and the chemical oxygen demand (COD) removal rate and cyanide degradation rate were 88.34% and 99.51%, respectively, when the MFC was fed with glucose–cyanide mixtures. The results demonstrated that the strain MC-1 was promising for the bioremediation of cyanide-containing wastewater in MFCs.

Isolating, screening and applying chromium reducing bacteria to promote growth and yield of okra (Hibiscus esculentus L.) in chromium contaminated soils

Hexavalent chromium [Cr (VI)], extensively used in different industries, is one of the most toxic heavy metals. The Cr (VI) reducing bacteria could be helpful in decreasing its toxic effects. The present study was conducted to evaluate the potential of Cr (VI) reducing bacteria to improve growth and yield of okra (Hibiscus esculentus L.) in Cr-contaminated soils. Most of the selected bacterial isolates significantly increased the growth and yield of okra. Maximum response was observed in the plants inoculated with the isolate K12 where plant height, root length, fruit weight and number of fruits per plant increased up to 77.5 percent, 72.6 percent, 1.4 fold and 2.9 fold, respectively. Moreover, inoculation with bacteria caused significant decrease in Cr (VI) concentration in soil and plant parts across all treatments. The maximum decrease of 69.6, 56.1 and 40.0 percent in Cr (VI) concentrations in soil, plant vegetative parts and plant reproductive parts, respectively, was observed in the treatment inoculated with the strain K12. Based on amplification, sequencing and analysis of 16S rDNA sequence, the strain K12 was found belonging to genus Brucella and was designated as Brucella sp. K12. These findings suggest that the strain K12 may serve as a potential bioresource to improve crop production in Cr-contaminated soils.

Dermacentor everestianus Hirst, 1926 (Acari: Ixodidae): phylogenetic status inferred from molecular characteristics.

Dermacentor everestianus Hirst, 1926, is only reported in Northwestern China and Nepal. Few researches about this species have been involved, especially for molecular characteristics. The taxonomy studies of D.everestianus are mainly based on morphological features, and its taxonomic status is an ongoing controversy. To clarify the molecular characteristics and phylogenetic status of D.everestianus and other related species, the sequences of mitochondrial 16S ribosomal DNA (rDNA) and cox1 fragments were analyzed in the present study. Analysis of 16S rDNA and cox1 sequences showed 99.3-100% identity within D.everestianus individuals, with the genetic divergence among them was 0-0.0086. The interspecific distance of 16S rDNA and cox1 between D.everestianus and some other Palaearctic species including D. silvarum, D. nuttalli, and D. marginatus was much smaller than that between D.everestianus and Nearctic Dermacentor ticks (D.albipictus, D.nitens, and D.variabilis). Such relationships of these ticks were also verified in the phylogenetic analysis. Two major clades were recovered within Dermacentor spp. with more than 90% bootstrap support in the phylogenetic trees. D.everestianus together with D.silvarum, D.nuttalli, and D.marginatus were included in the clade I (Eurasia lineage). Other analyzed tick species including D.variabilis, D.nitens, and D.albipictus formed clade II, which are distributed in Nearctic realm. These indicated that the genus Dermacentor was at least composed of two lineages. Thus, further researches including additionally molecular markers on all Dermacentor species globally should be taken to precisely resolve relationships within Dermacentor.

Molecular characterization and phylogenetic analysis of the causative agent of hemoplasma infection in small Indian Mongoose (Herpestes Javanicus).

Hemoplasmas are the trivial name for a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. This study is the first report of hemoplasma infection in Small Indian Mongoose (Herpestes Javanicus) based on molecular analysis of 16S rDNA. Whole blood samples were collected by sterile methods, from 14 live captured mongooses, in the south of Iran. Candidatus Mycoplasma turicensis (CMt)-like hemoplasma was detected in blood samples from one animal tested. BLAST search and phylogenetic analysis of partial 16S rDNA sequence (933bp) of the hemoplasma from Small Indian mongoose (KJ530704) revealed only 96-97% identity to the previously described CMt followed by 95% and 91% similarity with Mycoplasma coccoides and Mycoplasma haemomuris, respectively. Accordingly, the Iranian mongoose CMt isolate showed a high intra-specific genetic variation compared to all previously reported CMt strains in GenBank. Further molecular studies using multiple phylogenetic markers are required to characterize the exact species of Mongoose-derived hemoplasma.

Analysis of the bacterial community composition in acidic well water used for drinking in Guinea-Bissau, West Africa

Potable water is a resource out of reach for millions worldwide, and the available water may be chemically and microbiologically compromised. This is particularly acute in Africa, where water-networks may be non-existent or restricted to a small fraction of the urban population, as in the case of Guinea-Bissau, West Africa. This study was carried out seasonally in Bolama (11°N), where unprotected hand-dug wells with acidic water are the sole source of water for the population. We inspected the free-living bacterial community dynamics by automated rRNA intergenic spacer analyses, quantitative polymerase chain reaction and cloning approaches. The results revealed a clear seasonal shift in bacterial assemblage composition and microbial abundance within the same sampling site. Temperature, pH and turbidity, together with the infiltration and percolation of surface water, which takes place in the wet season, seemed to be the driving factors in the shaping and selection of the bacterial community and deterioration of water quality. Analysis of 16S rDNA sequences revealed several potential pathogenic bacteria and uncultured bacteria associated with water and sediments, corroborating the importance of a culture-independent approach in drinking water monitoring.

Lactic acid microflora of the gut of snail Cornu aspersum

The intestinal lactic acid microflora of the edible snail Cornu aspersum was studied by culture-based methods and was phenotypically and molecularly characterized. The antibacterial activity of lactic acid bacteria (LAB) isolates was investigated. Snails in different stages of development were collected from farms located in several regions of Bulgaria. One hundred twenty-two isolates, belonging to the group of LAB, were characterized morphologically and were divided into four groups. Representative isolates from each morphological type were subjected to phenotypic characterization and molecular identification. The snail gut lactic acid microflora was composed by Enterococcus (17 isolates), Lactococcus (12 isolates), Leuconostoc (7 isolates), Lactobacillus (18 isolates) and Weissella (1 isolate). The species affiliation of Lactococcus lactis (12), Leuconostoc mesenteroides (4) and Lactobacillus plantarum (2) was confirmed by species-specific primers. The Lactobacillus isolates were identified by sequence analysis of 16S rDNA as Lactobacillus brevis (12), L. plantarum (2), Lactobacillus graminis (1) and Lactobacillus curvatus (3). The species L. brevis, L. graminis and L. curvatus were found in snails in a phase of hibernation, whereas L. plantarum was identified both in active and hibernation phases. Antibacterial activity (bacteriocine-like) was shown only by one strain of L. mesentereoides P4/8 against Propionibacterium acnes. The present study showed that the LAB are a component of the microbial communities in the snail digestive system. This is the first report on Lactobacillus strains detected in the gut of C. aspersum.

Analysis of 16S rDNA sequences of endophytic bacteria associated with Huanglong disease of citrus plants in Guangning County, China

Analysis of 16S rDNA sequences of endophytic bacteria associated with Huanglong disease of citrus plants in Guangning County, China

Characterisation of a phytoplasma associated with Petunia witches' broom disease in Iran

Petunia hybrida (Solanaceae) is an economically important ornamental plant species that is used in residential and commercial landscapes. Varieties are diverse and available in a range of colours (Abu-Qaoud et al, 1; Hiuts et al, 3). In July 2012, symptoms of witches' broom, yellowing, little leaf, phyllody and virescence were observed in Petunia in Sistan and Baluchestan province, Iran (Fig. 1a, b). Total DNA was extracted separately from 0.5 g of shoot material of two symptom-bearing and one symptomless plant (Fig. 1c) using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 2). The samples were analysed for detection of phytoplasma DNA by both direct and nested PCR assays using phytoplasma universal 16S rDNA primer pairs P1/P7 and R16F2n/R16R2 that amplify fragments of 1800 bp and 1250 bp, respectively (Lee et al., 4). The 1800 and 1250 bp PCR products were obtained following direct and nested PCR from all symptom-bearing but not from symptomless plants. The 1250 bp PCR product was directly sequenced. BLAST analysis of the partial 16S rDNA sequence (GenBank Accession No. JX570935) revealed that the phytoplasma associated with Petunia witches' broom (PtWB) shared 100% identity with some phytoplasmas related to 16Sr-II phylogenetic group, ‘Candidatus Phytoplasma aurantifolia’, such as chickpea phyllody phytoplasma (FJ870549.1), tomato big bud phytoplasma (JQ868448), ‘Ca. P. aurantifolia’ (JX104335) and Echinacea witches' broom phytoplasma (JF340075.1). This result was further confirmed by the analysis using iPhyClassifier online tool (http://www.plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) where it was determined that PtWB phytoplasma is related to 16Sr group-II, subgroup D. The phylogenetic neighbour-joining tree (MEGA 5 software) based on the partial 16S rDNA sequences (1250 bp) of the PtWB phytoplasma and several reference phytoplasmas corroborated these results and showed that PtWB phytoplasma is closely related to ‘Candidatus Phytoplasma australasiae’ in the 16Sr-II phytoplasma clade (Fig. 2). Salehi et al. (unpublished) have recently detected a phytoplasma associated with phyllody symptoms in Petunia by PCR in Fars province, Iran. However, this is the first report of molecular characterisation of a phytoplasma associated with PtWB disease in Iran.

Screening, identification and characterization of antagonistic bacteria against Ralstonia solanacearum

Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial diseases of tobacco. As a recurring soil-borne disease, it is controlled mainly by chemical methods that are not only costly and less efficient but also have food safety and environment safety problems. Consequently, biological control methods that use antagonistic strains have gained strength in tobacco bacterial wilt disease research. To find new antagonist bacteria against R. solanacearum, 238 strains were identified and screened for in vitro antibiosis. By placing each strain on cultured pathogen plates with toothpicks 24 strains showed inhibition zones. Among the 24 strains, the width of the inhibition zones of 7 antagonist strains exceeded 10 mm. Then pathogen inhibitive ingredients of the 7 strains were extracted to determine bacteriostatic efficacy. Finally, 3 promising strains(H19, Y6, H34) were obtained. H19 and Y6 were identified as Bacillus amyloliquefaciens and the other strain(coded H34) identified as B. methylotrophicus. The strain classification was based on phylogenetic analysis of 16S rDNA sequence, gyrB sequence and fatty acid composition. Siderophores secreted by microorganisms to take up iron from the environment had an action mode of disease suppression that was solely based on competition with the pathogens for iron. Besides, indole-3-acetic acid(IAA) is a phytohormone known to be involved in root initiation, cell division and cell enlargement. In follow-up experiments, all the three strains showed the ability to produce siderophores and plant growth promotor IAA. However, productions of siderophores and IAA were different, with H19 and Y6 as the highest and lowest producers under improved CAS and Salkowski colorimetric methods. The three strains were further characterized for plant growth promoting traits and disease control effects. Compared with control treatment, the strains significantly improved the growth of tobacco with increases of 19%-24% in the maximum leaf length, 7%-12% in the maximum leaf width, 70%-115% in plant height, 2%-14% in stem diameter, 40%-49% in fresh weight and 32%-42% in dry weight. H19, H34 and Y6 reduced bacterial wilt incidence by 76.57%, 60.98% and 69.83%, respectively in greenhouse experiments. This was slightly lower than using 40% agricultural streptomycin which had disease incidence control efficacy of 78.66%. In conclusion, the research isolated new efficient antagonistic strains against tobacco bacterial wilt. It also proved the mechanism of disease control and plant growth promotion by H19, H34 and Y6. The study further showed that the three strains had great potential for plant growth promotion and biological control under greenhouse conditions, which developed the needed reference for follow-up studies.

Characterization of an algicidal bacterium Brevundimonas J4 and chemical defense of Synechococcus sp. BN60 against bacterium J4

As part of efforts to enhance the strategies explored to eliminate the adverse impacts of cyanobacterial blooms, we isolated an algicidal bacterium, J4, from Lake Taihu. Analysis of 16S rDNA sequence revealed that strain J4 belonged to the genus Brevundimonas. Bacterium J4 exhibited algicidal activity mainly through excretion of extracellular algicidal compounds that were further extracted with methanol and purified by silica gel chromatography and high performance liquid chromatography (HPLC). The compounds showed thermal stability, strong polarity and water solubility in J4 cultures. Study on the algicidal activity of J4 against two dominant cyanobacterial bloom-forming species in Lake Taihu showed that J4 exhibited lower algicidal rate against Synechococcus sp. BN60 (48.6%, t=6 days) than against Microcystis aeruginosa 9110 (91.8%, t=6 days). Additionally, rapid reduction in cell density of J4 was observed in co-cultures of Synechococcus sp. BN60 and bacterium J4 but not observed in co-cultures of M. aeruginosa 9110 and bacterium J4 during algicidal process, which was the main reason why the algicidal rate of J4 against BN60 was lower than against 9110. The reduction in cell density of J4 resulted from inducible production of antimicrobial-like compound secreted by Synechococcus sp. BN60 in co-cultures of Synechococcus sp. BN60 and bacterium J4, which reflected a kind of chemical defense from cyanobacteria (BN60) against algicidal bacteria (J4). However, M. aeruginosa 9110 had no chemical defense against J4, suggesting that whether cyanobacterial chemical defense occurs or not between cyanobacteria and algicidal bacteria depends on specific cyanobacteria–algicidal bacteria pairs. These results show that not only one-sided algicidal effect but also two-sided reciprocal inhibition interactions exist between algicidal bacteria and cyanobacteria, indicating the complexity of cyanobacteria–algicidal bacteria interactions in Lake Taihu and the need to take the cyanobacterial defensive responses into consideration when assessing potential use of algicidal bacteria.