Intergenic Transcribed Spacer Research Articles

Characterization of atypical Mycoplasma anserisalpingitidis strains

Mycoplasma anserisalpingitidis is a waterfowl colonizing mycoplasma, mainly found in geese. In this study, we compared the whole genomes of five atypical M. anserisalpingitidis strains originating from China, Vietnam and Hungary, with the rest of the collection. Common methods used in the description of species are genomic analyses like the analysis of 16 S - intergenic transcribed spacer (ITS) - 23 S rRNA, of housekeeping genes, of the average nucleotide identity (ANI) and average amino acid identity (AAI) and phenotypic analyses like testing the growth inhibition and the growth parameters of the strains. The atypical strains showed notable genomic differences in all of the genetic analyses: on average ANI and AAI 95% (M. anserisalpingitidis ANI Minimum: 92.45, Maximum: 95.10; AAI Minimum: 93.34, Maximum: 96.37). The atypical strains formed a separate branch among the M. anserisalpingitidis strains in all phylogenetic studies. The small genome size and possibly higher mutation rate of the M. anserisalpingitidis species likely contributed to the observed genetic difference. Based on genetic analyses, the studied strains clearly represent a new genotype of M. anserisalpingitidis. The atypical strains showed slower growth in the medium containing fructose and three of the atypical strains showed diminished growth in the inhibition test. However, no definitive geno-phenotype associations were found regarding the fructose metabolism pathway in the atypical strains. The atypical strains are potentially at an early stage of speciation.

Cartusia hunanesis sp. nov. (Oculatellaceae, Oculatellales) from a Stream in China Based on Polyphasic Approach

Cartusia hunanesis sp. nov. was isolated from a stream in China, and two strains (ZJJ02 and ZJJ03) of which were inquired using morphological features, ecological evidence, and molecular data consisting of the 16S rRNA gene and 16S–23S rRNA intergenic transcribed spacer (ITS) region. Cartusia hunanesis varies from the type species Cartusia fontana by having only a single trichome in the sheath and large granules near the cross wall. The investigated strains of C. hunanesis were revealed to be a sister clade of C. fontana, according to the phylogenetic analysis based on the 16S rRNA gene. In addition, the Cartusia was clustered with the family Oculatellaceae members, genera Pegethrix and Elainella. These two strains displayed 97.6% similarity to C. fontana. The ITS region of C. hunanesis was found to be considerably distinct from that of C. fontana in terms of the secondary structure, which demonstrated that C. hunanesis is a novel species owing to the divergences in its morphological and genetic data compared with the related C. fontana.

Combining deep sequencing and conventional molecular approaches reveals broad diversity and distribution of fleas and Bartonella in rodents and shrews from Arctic and Subarctic ecosystems

BackgroundBartonella are intracellular bacteria that are transmitted via animal scratches, bites and hematophagous arthropods. Rodents and their associated fleas play a key role in the maintenance of Bartonella worldwide, with > 22 species identified in rodent hosts. No studies have addressed the occurrence and diversity of Bartonella species and vectors for small mammals in Arctic and Subarctic ecosystems, which are increasingly impacted by invasive species and climate change.MethodsIn this study, we characterized the diversity of rodent fleas using conventional PCR targeting the mitochondrial cytochrome c oxidase II gene (COII) and Bartonella species in rodents and shrews (n = 505) from northern Canada using conventional PCR targeting the ITS (intergenic transcribed spacer) region and gltA (citrate synthase) gene. Metagenomic sequencing of a portion of the gltA gene was completed on a subset of 42 rodents and four rodent flea pools.ResultsYear, total summer precipitation the year prior to sampling, average minimum spring temperature and small mammal species were significant factors in predicting Bartonella positivity. Occurrence based on the ITS region was more than double that of the gltA gene and was 34% (n = 349) in northern red-backed voles, 35% (n = 20) in meadow voles, 37% (n = 68) in deer mice and 31% (n = 59) in shrews. Six species of Bartonella were identified with the ITS region, including B. grahamii, B. elizabethae, B. washoensis, Candidatus B. rudakovii, B. doshiae, B. vinsonii subsp. berkhoffii and subsp. arupensis. In addition, 47% (n = 49/105) of ITS amplicons had < 97% identity to sequences in GenBank, possibly due to a limited reference library or previously unreported species. An additional Bartonella species (B. heixiaziensis) was detected during metagenomic sequencing of the gltA gene in 6/11 rodents that had ITS sequences with < 97% identity in GenBank, highlighting that a limited reference library for the ITS marker likely accounted for low sequence similarity in our specimens. In addition, one flea pool from a northern red-backed vole contained multiple species (B. grahamii and B. heixiaziensis).ConclusionOur study calls attention to the usefulness of a combined approach to determine the occurrence and diversity of Bartonella communities in hosts and vectors.Graphical

Porphyrosiphon annulatus sp. nov. (Oscillatoriales, Cyanobacteria) isolated on moist soil in Suwon, Republic of Korea

Porphyrosiphon annulatus sp. nov. belonging to Microcoleaceae, Oscillatoriales was collected from the moist soil under the rock in the Gwanggyo Mountain of Suwon City, Gyeonggi Province, Korea and cultured. Using the raw and cultured samples of the collected strains, we observed their morphological features through LM and TEM. Furthermore, their 16S rRNA gene and 16S–23S rRNA intergenic transcribed spacer (ITS) sequences were determined to be used phylogenetic analyses. We affirmed that thick sheath of the cells possessed transversely annular and longitudinal lamellations. The ultrastructure of thylakoids was a radial arrangement, which corresponds with a characteristic of the Microcoleaceae. In addition, P. annulatus displays the most similar morphology with type species P. notarisii; however, it differently holds the transversely annular lamellations with a shorter cell length and relatively dense trichome. Our 16S rRNA gene phylogeny showed that P. annulatus formed a clade with P. notarisii, and their DNA similarity was calculated to be 96.2%. The family Microcoleaceae was found to be paraphyletic with a distinct linage of the genus Porphyrosiphon. The ITS sequence of P. annulatus was unique in length and nucleotide composition, with different secondary structures of D1–D1ʹ and Box-B helices compared to those of close relatives. Overall, morphology and genetic traits of the Korean Porphyrosiphon suggest that P. annulatus should be considered to be a novel species.

The adult microbiome of healthy and otitis patients: Definition of the core healthy and diseased ear microbiomes.

Otitis media (OM) and externa (OE) are painful, recurrent ear conditions. As most otitis publications focus on the bacterial content of childhood ears, there remains a dearth of information regarding the adult ear microbiome including both bacteria and fungi. This study compares the outer ear microbiome of healthy adults to adults affected by OE and OM using both intergenic-transcribed-spacer (ITS) and 16S-rDNA sequencing. The adult ear core microbiome consists of the prokaryote Cutibacterium acnes and the eukaryotic Malassezia arunalokei, M. globosa, and M. restricta. The healthy ear mycobiome is dominated by Malassezia and can be divided into two groups, one dominated by M. arunalokei, the other by M. restricta. Microbiome diversity and biomass varied significantly between healthy and diseased ears, and analyses reveal the presence of a potential mutualistic, protective effect of Malassezia species and C. acnes. The healthy ear core microbiome includes the bacteria Staphylococcus capitis and S. capitis/caprae, while the diseased ear core is composed of known bacterial and fungal pathogens including Aspergillus sp., Candida sp., Pseudomonas aeruginosa, S. aureus, and Corynebacterium jeikeium. The data presented highlight the need for early detection of the cause of otitis to direct more appropriate, efficient treatments. This will improve patient outcomes and promote improved antimicrobial stewardship.

Endophytic fungi confirmed as entomopathogens of the new invasive pest, the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), infesting maize in South Sumatra, Indonesia

BackgroundThe fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), is a new invasive pest in Indonesia causing financial losses. S. frugiperda larvae hide in the leaf midrib all day. To overcome the hidden larvae, pathogenic endophytic fungi are needed. The objective of this research was to molecularly identify the endophytic fungal species from South Sumatra and determine the most pathogenic species against S. frugiperda larvae. Endophytic fungal identification was based on morphological and molecular characteristics. The molecular identification was based on gene sequential analysis of Intergenic Transcribed Spacer (ITS) 1 and ITS 4. Bioassay of the endophytic fungal species was treated against the 2nd larval instar of S. frugiperda.ResultThe results of molecular identification revealed endophytic fungal species consisted of Chaetomium sp. (GaTpeOi, JgTjPr, PiCrPga, and CaCjPga isolates), Aspergillus niger (L.) Van Tieghem (PsgTjPr, JgByU, and JaBuBys), Beauveria bassiana (Balsamo) Vuillemin (JgSPK, JaGiP, JaSpkPGA(2), JgCrJr, and JaTpOi (1) isolates), Curvularia lunata (Wakker) Boed. (JaGiPRB, CMTJP, JaMsBys, JaSpkPga(3), and JgTgSr isolates), Aspergillus flavus Link. (JgPWSR isolate), Penicillium citrinum Thom F 1539 (JaTpOi(2) isolate), and Metarhizium anisopliae (Metschn.) Sorokin (CaTpPGA isolate). All fungal isolates were deposited in the GenBank. The endophytic fungal species of B. bassiana (JgSPK isolate) caused up to (22.67%) mortality, C. lunata (JaSpkPga(3) isolate) caused (17.33%) mortality, and M. anisopliae (CaTpPga isolate) caused (8%) mortality of the pest’s larvae. First report of B. bassiana and C. lunata isolated from maize (Zea mays L.), while M. anisopliae was isolated from red chili (Capsicum annuum L.) as entomopathogenic endophytic fungi against S. frugiperda larvae in Indonesia.ConclusionThe endophytic fungi species of B. bassiana, C. lunata, and M. anisopliae had potentials as entomopathogens of S. frugiperda.

Development of a multiplex qPCR assay for the simultaneous detection of Mycoplasma bovis, Mycoplasma species, and Acholeplasma laidlawii in milk.

Contagious bovine mastitis caused by Mycoplasma bovis and other Mycoplasma species including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma canadense is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species Acholeplasma laidlawii. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between M. bovis and A. laidlawii as well as detect Mycoplasma spp. collectively, including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini and Mycoplasma alkalescens. In milk, the lower limit of detection of M. bovis, M. californicum, and A. laidlawii with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both M. bovis and A. laidlawii in milk when present in moderate (103 to 104 CFU/mL) to high (106 to 107 CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8–100]; n = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9–99.1]; n = 26) for Mycoplasma species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5–100]; n = 37) and Se was 93.3% (95% CI [68.1–99.8]; n = 15) for M. bovis. Our assay can detect and distinguish among M. bovis, other prevalent Mycoplasma spp., and non-pathogenic Acholeplasma laidlawii for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.

Microbial Diversity and Metabolite Profile of Fermenting Millet in the Production of Hausa koko, a Ghanaian Fermented Cereal Porridge.

Hausa koko is an indigenous porridge processed from millet in Ghana. The process involves fermentation stages, giving the characteristic organoleptic properties of the product that is produced largely at a small-scale household level and sold as a street food. Like many other indigenous foods, quality control is problematic and depends on the skills of the processor. In order to improve the quality of the product and standardize the process for large-scale production, we need a deeper understanding of the microbial processes. The aim of this study is to investigate the microbial community involved in the production of this traditional millet porridge and the metabolites produced during processing. High-throughput amplicon sequencing was used to identify the bacterial (16S rRNA V4 hypervariable region) and fungal [Intergenic Transcribed Spacer (ITS)] communities associated with the fermentation, while nuclear magnetic resonance (NMR) was used for metabolite profiling. The bacterial community diversity was reduced during the fermentation processes with an increase and predominance of lactobacilli. Other dominant bacteria in the fermentation included Pediococcus, Weissella, Lactococcus, Streptococcus, Leuconostoc, and Acetobacter. The species Limosilactobacillus fermentum and Ligilactobacillus salivarius accounted for some of the diversities within and between fermentation time points and processors. The fungal community was dominated by the genus Saccharomyces. Other genera such as Pichia, Candida, Kluyveromyces, Nakaseomyces, Torulaspora, and Cyberlindnera were also classified. The species Saccharomyces cerevisiae, Stachybotrys sansevieriae, Malassezia restricta, Cyberlindnera fabianii, and Kluyveromyces marxianus accounted for some of the diversities within some fermentation time points. The species S. sansevieria and M. restricta may have been reported for the first time in cereal fermentation. This is the most diverse microbial community reported in Hausa koko. In this study, we could identify and quantify 33 key different metabolites produced by the interactions of the microbial communities with the millet, composed of organic compounds, sugars, amino acids and intermediary compounds, and other key fermentation compounds. An increase in the concentration of organic acids in parallel with the reduction of sugars occurred during the fermentation process while an initial increase of amino acids followed by a decrease in later fermentation steps was observed.

Pinocchia daecheonga sp. nov. (Synechococcales, Cyanobacteria) isolated from a Daecheong Lake in Geum River, Republic of Korea

The collection of Pinocchia daecheonga sp. nov. was performed at the Daecheong Lake in Korea, five strains of which with four clones were investigated through light microscopy, transmission electron microscopy and molecular data comprising the 16S rRNA to 23S rRNA gene. P. daecheonga differed from type species P. polymorpha by absence of the polar aerotopes. Investigated stains and four clones of P. daecheonga turned out to be a sister clade to the P. polymorpha according to the phylogenetic analysis of 16S rRNA gene. In addition, the Pinocchia was clustered with the family Leptolyngbyaceae members, genera Leptothoe and Leptoelongatus. 16S–23S rRNA intergenic transcribed spacer (ITS) region of P. daecheonga was found to be substantially distinct to P. polymorpha in terms of the secondary structure and nucleotide sequence composition, which concludes that the Pinocchia daecheonga isolated from the Daecheong Lake is a unique species due to differences in morphology and genetic traits compared to the relative P. polymorpha.

Morpho-molecular identification and pathogenicity test on fungal parasites of guava root-knot nematode eggs in Lampung, Indonesia

Abstract. Swibawa IG, Fitriana Y, Solikhin, Suharjo R, Susilo FX, Rani E, Haryani MS, Wardana RA. 2020. Morpho-molecular identification and pathogenicity test on fungal parasites of guava root-knot nematode eggs in Lampung, Indonesia. Biodiversitas 21: 1108-1115. This study aimed to obtain and discover the identity of the species of fungal egg parasites of root-knot nematodes (RKN), which have a high pathogenic ability causing major losses in vegetable crops. The exploration of the fungi was carried out in 2016 and 2018 from Crystal guava plantations in East Lampung, Central Lampung, Tanggamus, and NirAma, a commercial product that has been used for controlling Meloidogyne sp. in Indonesia. Identification was carried out based on morphological characteristics and molecular-based gene sequential analysis of Intergenic Transcribed Spacer (ITS) 1 and ITS 4. A pathogenicity test was carried out in vitro and in a greenhouse using tomato plants as indicator plants. In the in vitro test, observations were made on the percentage of infected RKN eggs. The observations in the greenhouse test were carried out on RKN populations in the soil and roots of tomato plants, root damage (root knots), and damage intensity due to RKN infection. The exploration resulted in five isolates of fungal egg parasites of RKN from the guava plantations in East Lampung (2), Central Lampung (1), Tanggamus (1), and from the isolation results of commercial products (1). The isolates were given codes as B4120X (PT GGP PG1), B3010 (PT GGP PG4), B412G (PT GGP PG 4), B01TG (Tanggamus), and BioP (Commercial products). Based on their morphological characteristics, the isolates were classified into the genus of Paecilomyces. The results of molecular identification showed that the discovered fungi were Purpureocillium lilacinum (Thom.) Luangsa Ard. (Syn. Paecilomyces lilacinus (Thom.) Samson.). Based on the in vitro tests, the five fungal isolates were able to parasitize RKN eggs at 86.4-100%. In the greenhouse test, all isolates significantly suppressed nematode populations in the soil and tomato roots, inhibited the formation of root knots, and produced lower damage intensity compared to controls. Among all the isolates tested, B01TG had the best ability to infect nematode eggs (99.5%), suppressing the formation of root knots, nematode population in the soil and the roots of tomato plants, and the damage intensity compared to other isolates.

Bradyrhizobium frederickii sp. nov., a nitrogen-fixing lineage isolated from nodules of the caesalpinioid species Chamaecrista fasciculata and characterized by tolerance to high temperature in vitro.

The symbioses between legumes and nitrogen-fixing rhizobia make the greatest contribution to the global nitrogen input via the process of biological nitrogen fixation (BNF). Bradyrhizobium stands out as the main genus nodulating basal Caesalpinioideae. We performed a polyphasic study with 11 strains isolated from root nodules of Chamaecristafasciculata, an annual multi-functional native legume of the USA. In the 16S rRNA gene phylogeny the strains were clustered in the Bradyrhizobium japonicumsuperclade. The results of analysis of the intergenic transcribed spacer (ITS) indicated less than 89.9 % similarity to other Bradyrhizobium species. Multilocus sequence analysis (MLSA) with four housekeeping genes (glnII, gyrB, recA and rpoB) confirmed the new group, sharing less than 95.2 % nucleotide identity with other species. The MLSA with 10 housekeeping genes (atpD, dnaK, gap, glnII, gltA, gyrB, pnp, recA, rpoB and thrC) indicated Bradyrhizobium daqingense as the closest species. Noteworthy, high genetic diversity among the strains was confirmed in the analyses of ITS, MLSA and BOX-PCR. Average nucleotide identity and digital DNA-DNA hybridization values were below the threshold of described Bradyrhizobium species, of 89.7 and 40 %, respectively. In the nifH and nodC phylogenies, the strains were grouped together, but with an indication of horizontal gene transfer, showing higher similarity to Bradyrhizobium arachidis and Bradyrhizobium forestalis. Other phenotypic, genotypic and symbiotic properties were evaluated, and the results altogether support the description of the CNPSo strains as representatives of the new species Bradyrhizobiumfrederickii sp. nov., with CNPSo 3426T (=USDA 10052T=U686T=CL 20T) as the type strain.

Bradyrhizobium ripae sp. nov., a nitrogen-fixing symbiont isolated from nodules of wild legumes in Namibia.

Root-nodule bacteria were isolated from wild legumes growing in the Kavango region, Namibia. Using a polyphasic approach, four strains belonging to the genus Bradyrhizobium (WR4T, WR87, T10 and T12) were further characterized to clarify the taxonomic status of this group. On the basis of 16S rRNA gene sequences, the four strains showed highest similarity to Bradyrhizobium elkanii USDA 76T (99.9 %), Bradyrhizobium pachyrhizi PAC48T (identical) and to Bradyrhizobiumbrasilense UFLA03-321T (identical). Multilocus sequence analysis of concatenated glnII-recA-gyrB-dnaK-rpoB sequences and comparison of the intergenic transcribed spacer (ITS) sequences confirmed that the novel group belongs to a distinct lineage of the genus Bradyrhizobium, with <96.7 % (MLSA) and 97.25 % (ITS) nucleotide identity with B. elkanii USDA 76T. Results from the sequence-based analysis were validated by DNA-DNA hybridization experiments and suggested a novel species. Several phenotypic features including carbon compound utilization and growth characteristics supported the phylogenetic data, thus it is concluded that the strains represent a novel species, for which the name Bradyrhizobium ripae sp. nov. is proposed, with type strain WR4T [LMG 30283, DSM 105795, NTCCM 0019 (Windhoek)].

Microbiological analysis and assessment of biotechnological potential of lactic acid bacteria isolated from Tunisian flours

This work was carried out to identify lactic acid bacteria (LAB) from baking wheat flours and to evaluate their technological capabilities for potential incorporation in sourdough process. Six samples of wheat flours obtained from different geographical regions of Tunisia were microbiologically analyzed. Several technological features including acidification, antimicrobial, amylolytic, proteolytic, and antioxidant activities of six selected LAB strains were investigated for future in situ applications. Moreover, LAB were investigated for their ability to produce exopolysaccharides. A total of 45 autochthonous LAB were isolated and identified by genetic analysis of 16S–23S rRNA intergenic transcribed spacer (ITS)-generated patterns ITS-PCR. One of each ITS-PCR pattern was subjected to partial 16S rRNA gene sequencing, and strains were identified as Weissella cibaria, Lactobacillus plantarum, Lactobacillus brevis, Pediococcus pentosaceus, Pediococcus pentoseus, Pediococcus acidilactici, Enterococcus faecium, Enterococcus casseliflavus, and Enterococcus faecalis. All tested LAB showed good acidifying ability by decreasing significantly (p < 0.05) the pH of flour extract below 4.0 after 24 h and below 3.0 after 72 h. Pediococcus pentoseus and P. acidilactici presented fermentation quotient (FQ, ratio of lactic and acetic acids) close to the optimal range. All LAB isolates demonstrated extracellular proteolytic activity. Weissella cibaria S25 had the highest radical-scavenging activity with 25.57%. Lactobacillus plantarum S28 demonstrated the highest amylolytic activity (1386 U/mL) followed by P. acidilactici S16 (1086 U/mL). Although, L. plantarum S28 showed the highest production of exopolysaccharides (0.97 g/L). Moreover, varying halo of inhibition was detected against Escherichia coli, Staphylococcus aureus, Aspergillus niger, and Penicillium expansum. This study revealed that autochthonous flour LAB had interesting technological features and thus could be used in sourdough production.

Bacteria related to Bradyrhizobium yuanmingense from Ghana are effective groundnut micro-symbionts

The identification of locally-adapted rhizobia for effective inoculation of grain legumes in Africa’s semiarid regions is strategic for developing and optimizing cheap nitrogen fixation technologies for smallholder farmers. This study was aimed at selecting and characterising effective native rhizobia, from Ghanaian soils for groundnut (Arachis hypogaea L.) inoculation. From surface-disinfected root nodules of cowpea and groundnut plants grown on farmers’ fields, 150 bacterial isolates were obtained, 30 of which were eventually found to nodulate groundnut plants. After testing the symbiotic potential of these isolates on groundnut on sterilized substrate, seven of them, designated as KNUST 1001–1007, were evaluated in an open field pot experiment using 15N-labelled soil. Although 15N dilution analyses did not indicate differences among treatments in the proportion of nitrogen (N) derived from the atmosphere (%Ndfa), all seven strains increased total N derived from N2 fixation by inoculated groundnut plants as compared to the non-inoculated control. Inoculation with KNUST 1002 led to total N accumulation as high as that of the groundnut reference strain 32H1. Genetic characterisation of the isolates by sequence analysis of 16S rRNA gene, 16S – 23S rRNA intergenic transcribed spacer (ITS) region and nodC gene revealed that isolates KNUST 1003 and 1007 were related to Rhizobium tropici, a common bean symbiont. The other five isolates, including KNUST 1002 belonged to the Bradyrhizobium genus, being closely related to Bradyrhizobium yuanmingense. Therefore, this study revealed novel native Ghanaian rhizobia with potential for the development of groundnut inoculants.

English

The diversity of culturable bacterial and fungal communities was assessed from leaves, trunk and rhizosphere of coconut palms infected and non-infected by the Cote d’Ivoire lethal yellowing (CILY) phytoplasma. Bacterial and fungal microbes were isolated from leaves, trunk and rhizosphere samples collected from two villages of Grand-Lahou, Braffedon and Yaokro, by using a high-throughput 'dilution-to-extinction' cultivation method coupled with PCR and sequencing with primers that amplified both the 16S ribosomal RNA and intergenic transcribed spacer (ITS) genes. The relative abundance was higher for Bacillus and Candida in Braffedon, and Burkholderia and Neodeightonia in Yaokro. Commonly genera identified from rhizosphere included Bacillus, Burkholderia, Pseudomonas, Streptomyces, Cryptococcus, Penicillium, Purpureocillium and Trichoderma. The most abundant endophytes identified were Pantoea, Candida, Cryptococcus, Bacillus, Pseudomonas, Penicillium, Aspergillus and Rhodotorula. Genera limited to symptomless palms included Arthrininum, Chaetomium, Phialemonium, Fusarium, Klebsiella and Candida. Results indicate that the CILY phytoplasma may be a factor determining the level of diversity of a microbial community in a given location. Our research provides the basis to investigate the possible effect of endophytic and rhizosphere microbes against the CILY phytoplasma to further effectively improve the management of CILY in Grand-Lahou. Key words: Cote d’Ivoire lethal yellowing phytoplasma, endophytes, biocontrol, coconut.

Pedobacter lusitanus sp. nov., isolated from sludge of a deactivated uranium mine.

Strain NL19T is a Gram-stain-negative, aerobic bacterium that was isolated from sludge of a deactivated uranium mine in Portugal. 16S rRNA gene sequence analysis revealed that strain NL19T is a member of the genus Pedobacter and closely related to the strains Pedobacter himalayensis MTCC 6384T, Pedobacter cryoconitis DSM 14825T, Pedobacter westerhofensis DSM 19036T and Pedobacterhartonius DSM 19033T. It had a DNA G+C content of 40.8 mol%, which agreed with the genus description. The main fatty acids included C16 : 1ω7c, C14 : 1ω5c, C4 : 0, iso-C17 : 0, iso-C17 : 0 3-OH, C16 : 0, anteiso-C15 : 0 and iso-C15 : 0 3-OH. The main lipids present were phospholipids (60 %) and sphingolipids (35 %). The most abundant phospholipids included phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine. Menaquinone-7 (MK-7) was the only isoprenoid quinone detected. DNA-DNA hybridization similarities between strain NL19T and Pedobacter himalayensis MTCC 6384T, Pedobacter cryoconitis DSM 14825T, Pedobacter westerhofensis DSM 19036T and Pedobacter hartonius DSM 19033T were 15.3 , 16.2 , 11.5 and 16.0 %, respectively. Strain NL19T can also be distinguished from these four species based on gyrB and intergenic transcribed spacers (ITS) sequences and by some phenotypic traits such as NaCl tolerance, pH, growth temperature and carbon source utilization. Strain NL19Trepresents a novel species of the genus Pedobacter, for which the name Pedobacter lusitanus sp. nov. is proposed. The type strain is NL19T (=LMG 29220T=CECT 9028T). An amended description of Pedobacter himalayensis is also included.

Development of a Real‐time Fluorescence Loop‐mediated Isothermal Amplification Assay for Detection of Burkholderia gladioli pv. alliicola

AbstractBurkholderia gladioli pv. alliicola is a causal agent of rot on a wide range of hosts including onion and tulip. It is one of quarantine phytopathogenic bacteria in China. To reduce the economic losses associated with this pathogen, simple and rapid detection methods are needed. In this study, an efficient loop‐mediated isothermal amplification (LAMP) assay with a real‐time fluorometer was developed. The analysis of 16S‐23S rRNA intergenic transcribed spacer (ITS) sequences showed considerable variability between different Burkholderia species and B. gradioli pathovars. A set of LAMP primers was designed based on the ITS region. The sensitivity and specificity of the developed assay were evaluated at the optimal temperature of 65°C. The primers were specific for B. gladioli pv. alliicola and did not react to strains of others species and other pathovars in the species B. gladioli. The sensitivity of the real‐time LAMP assay was 1 fg DNA which was 100 times higher than that of conventional PCR. The method was verified by testing natural samples and inoculated onion seeds, and it showed effectiveness. The real‐time LAMP assay established in this study is an effective method for detection of B. gladioli pv. alliicola.

Rapid differentiation among Lactobacillus, Pediococcus and Weissella species from some Nigerian indigenous fermented foods

The taxonomic differentiation among lactic acid bacteria (LAB) using only phenotypic methods is inadequate. In the past, several phenotypic techniques have been applied in identification of different species of LAB from Nigerian indigenous fermented foods. In this study, two less-expensive and rapid molecular techniques were employed in the identification and differentiation of LAB species isolated from some Nigerian fermented foods: gari (cassava mash), fufu (cassava dough) and ogi (maize gruel) to avoid the common errors that are synonymous with the use of conventional methods. The size of the amplified products for the LAB species using universal primers fD1 and rD1 was about 1500 bp. The differentiation of the three predominant LAB, Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa to species level was performed by intergenic transcribed spacer (ITS-PCR) and amplified ribosomal DNA restriction analysis (ARDRA) using three restriction endonucleases: HinfI, HaeIII and RsaI. In summary, the study demonstrated that the ARDRA and ITS-PCR analysis are simple, fast and efficient methods that can be used for the routine identification and rapid differentiation of LAB species which are often regularly used as probiotics, starter culture and biopreservative in fermented foods consumed since they are generally regarded as safe (GRAS).

Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification.

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the β-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml-1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml-1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml-1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

Biogeography of sulfur-oxidizing Acidithiobacillus populations in extremely acidic cave biofilms.

Extremely acidic (pH 0–1.5) Acidithiobacillus-dominated biofilms known as snottites are found in sulfide-rich caves around the world. Given the extreme geochemistry and subsurface location of the biofilms, we hypothesized that snottite Acidithiobacillus populations would be genetically isolated. We therefore investigated biogeographic relationships among snottite Acidithiobacillus spp. separated by geographic distances ranging from meters to 1000s of kilometers. We determined genetic relationships among the populations using techniques with three levels of resolution: (i) 16S rRNA gene sequencing, (ii) 16S–23S intergenic transcribed spacer (ITS) region sequencing and (iii) multi-locus sequencing typing (MLST). We also used metagenomics to compare functional gene characteristics of select populations. Based on 16S rRNA genes, snottites in Italy and Mexico are dominated by different sulfur-oxidizing Acidithiobacillus spp. Based on ITS sequences, Acidithiobacillus thiooxidans strains from different cave systems in Italy are genetically distinct. Based on MLST of isolates from Italy, genetic distance is positively correlated with geographic distance both among and within caves. However, metagenomics revealed that At. thiooxidans populations from different cave systems in Italy have different sulfur oxidation pathways and potentially other significant differences in metabolic capabilities. In light of those genomic differences, we argue that the observed correlation between genetic and geographic distance among snottite Acidithiobacillus populations is partially explained by an evolutionary model in which separate cave systems were stochastically colonized by different ancestral surface populations, which then continued to diverge and adapt in situ.