Yellow Colonies Research Articles

Detection of Cholera Toxin-Producing Vibrio cholerae in Phytoplankton from Santubong and Samariang Estuaries

Many cholera outbreaks worldwide were associated with cholera toxin-producing Vibrio cholerae. The bacteria are ubiquitous in aquatic environment, whilst phytoplankton is associated with adaptation of the Vibrio species. This study was conducted to detect cholera toxin-producing Vibrio cholerae, and to determine association of the selected water physicochemical parameters with the number of the bacteria. In this study, a total of ten phytoplankton samples were collected at Santubong and Samariang Estuaries in Kuching, Sarawak. Water physicochemical parameters (temperature, pH and salinity) were recorded. Vibrio bacteria were cultivated on thiosulfate citrate bile-salts sucrose selective agar and analysed for cholera toxin-producing Vibrio cholerae using polymerase chain reaction by targeting ctxA gene that encodes for virulence cholera enterotoxin subunit A. The result revealed that a range of 1.0 × 107 – 8.0 × 107 CFU/ml of yellow colonies growing on the thiosulfate citrate bile-salts sucrose agars. Inversely, no samples were positive with cholera toxin-producing Vibrio cholerae. The physicochemical parameters at Samariang Estuary were more associated with the number of bacteria in the samples compared to Santubong Estuary.

Flavivirga rizhaonensis sp. nov., a marine bacterium isolated from intertidal sand.

A bacterial strain designated RZ03T was isolated from an intertidal sand sample from the Yellow Sea in China and characterised using a polyphasic taxonomic approach. Cells of strain RZ03T were observed to be Gram-stain negative, aerobic, and oxidase and catalase positive rods showing gliding motility and forming yellow colonies. Growth was found to occur at 7-30°C (optimum, 25°C), at pH 5.5-9.5 (optimum, pH 6.5-7.0) and with 0.5-5% NaCl (optimum, 1.5-2%). Phylogenetic analysis based on 16S rRNA gene sequences indicates that strain RZ03T clusters within members of the genus Flavivirga of the family Flavobacteriaceae and is closely related to the type strains Flavivirga amylovorans JCM 17112T and Flavivirga jejuensis JCM 17113T (97.9% and 97.5% similarity, respectively). The predominant cellular fatty acids are iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and iso-C15:0 3-OH and the major respiratory quinone is MK-6. Polar lipids include phosphatidylethanolamine, three unidentified aminolipids, an unidentified phospholipid and four unidentified lipids. The genome of strain RZ03T is 4.88 Mbp with a G+C content of 32.2mol%. A total of 4152 genes are predicted, with 4052 protein-coding genes, 51 RNA genes and 49 pseudogenes. This polyphasic study suggests that strain RZ03T represents a novel species in the genus Flavivirga, for which the name Flavivirga rizhaonensis is proposed. The type strain is RZ03T(= KCTC 62833T = MCCC 1K03615T).

Vibrio parahaemolyticus and Vibrio harveyi causing Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei (Boone, 1931) isolated from Malaysian shrimp ponds

Acute hepatopancreatic necrosis disease (AHPND) first emerged as a new shrimp disease in 2009 that heavily affected shrimp industry leading to global economic losses. The etiological agent was previously identified as Vibrio parahaemolyticus that carries a plasmid containing toxins (PirA and PirB). However, recent researches revealed that V. parahaemolyticus is not the only bacterial species capable of causing AHPND, thus this study screened on bacterial strains with AHPND toxins from Penaeus vannamei shrimps in Malaysia. Out of the 86 isolated total strains, 12 AHPND positive strains were arbitrarily selected and were evaluated in in vivo assay using Artemia franciscana as a model organism. All the 12 AHPND positive strains with PirA and PirB genes demonstrated significant mortalities (P < 0.05) of A. franciscana compared to the negative control. The 12 AHPND positive strains were identified using molecular methods of 16S rRNA, RctB and RpoD region amplifications belonged to the Harveyi clade and were closely related to V. parahaemolyticus and Vibrio harveyi. Further test showed that the yellow colony V. harveyi strain BpShHep24 was found to be more virulent than the green colony V. parahaemolyticus strain BpShHep31 in shrimp P. vannamei challenge test. Histological examination of shrimp hepatopancreas challenged with yellow colony V. harveyi strain BpShHep24 showed massive sloughing of hepatopancreas tubules of epithelial cells into the lumen, haemocyte infiltrations, proximal-to-distal lesion of hepatopancreas and collapsed tubule epithelia within 24 h.

Mutants defective in the production of encapsulin show a tan-phase-locked phenotype in Myxococcus xanthus.

Myxococcus xanthus, a myxobacterium, displays phase variation between yellow phase and tan phase. We found that deletion of the encA gene encoding encapsulin and the encF gene encoding a metalloprotease causes formation of tan colonies that never transform into yellow colonies. The encA and encF mutants were defective in the production of DK-xanthene and myxovirescin. They did not produce extracellular polysaccharides; hence, the cells did not aggregate in liquid and showed reduced swarming on agar plates. The mutants had defective sporulation, but were rescued extracellularly by wild type cells. All these traits indicate that the encA and encF mutants are likely to be tan-phase-locked, and encapsulin has a close relationship with phase variation in M. xanthus. The encA and encF genes are localized in the same gene cluster, encBAEFG (MXAN_3557~MXAN_3553). Unlike the encA and encF genes, deletion of other genes in the cluster did not show tan-phase-locked phenotype.

Sphingomonas panacisoli sp. nov., bacterium isolated from soil in South Korea.

A newly isolated bacterial strain designated as HKS19 was isolated from a ginseng cultivation soil sample collected in South Korea. Cells of the strain HKS19 were Gram-stain-negative, rod, oval-shaped and they formed yellow colonies when grown on R2A agar at 30 °C. HKS19 showed the highest 16S rRNA gene sequence similarity (98.6%) with Sphingomonas asaccharolytica NBRC 15499T. Its growth was observed at 10-37 °C (optimum 30 °C), pH 6-9 (optimum pH 7), and in the presence of 0-1% NaCl (optimum 0%). The genome size of HKS19 was 3.4 Mb and the G+C content was 65.1 mol%. The main polar lipid of strain HKS19 was diphosphatidylglycerol (DPG), the predominant respiratory quinone was Q-10 and the major fatty acids were a summed feature 8 (C18 : 1 ω6c / C18 : 1 ω7c) and C16 : 0. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic analysis, strain HKS19 represents a newly isolated species of the genus Sphingomonas, for which the name Sphingomonas panacisoli is proposed. The type strain is HKS19T (=KACC 18881T=LMG 29564 T).

Flavobacterium stagni sp. nov., isolated from a freshwater reservoir.

Strain WWJ-16T was isolated from a freshwater reservoir in Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set (92 protein clusters) indicated that strain WWJ-16T is affiliated with the species in the genus Flavobacterium. Strain WWJ-16T was most closely related to Flavobacterium fontis MIC3010T (97.9 % 16S rRNA gene sequence identity) and Flavobacterium squillarum CMJ-5T (97.6 %). Strain WWJ-16T showed 77.4 % average nucleotide identity and 20.6 % digital DNA-DNA hybridization identity with F. fontis MIC3010T. Cells of strain WWJ-16T were Gram-stain-negative, strictly aerobic, non-motile, rod-shaped and formed yellow colonies. Optimal growth occurred at 25 °C, pH 7 and in 0.5-1 % NaCl (w/v). Strain WWJ-16T contained iso-C15 : 1 G and iso-C15 : 0 as predominant fatty acids. The major hydroxyl fatty acids were iso-C15 : 0 3-OH, iso-C17 : 0 3-OH and iso-C16 : 0 3-OH. The polar lipid profile consisted of phosphatidylethanolamine, four uncharacterized aminophospholipids and four uncharacterized phospholipids. The major polyamine was homospermidine. The major isoprenoid quinone was MK-6. The genomic DNA G+C content of strain WWJ-16T was 39.4 mol%, as determined by genome sequencing. The genotypic and phenotypic characteristics indicate that strain WWJ-16T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium stagni sp. nov. is proposed. The type strain is WWJ-16T (=BCRC 81125T =LMG 30600T=KCTC 62515T).

First Report of Fusarium oxysporum and Fusarium redolens Causing Wilting and Yellowing of Wild Rocket (Diplotaxis tenuifolia) in the United Kingdom

In summer 2015, a UK grower reported crop losses of 20% (>£170k) in glasshouse-grown wild rocket (Diplotaxis tenuifolia, cvs. Voyager and Napoli) with disease symptoms initially seen as chlorosis of leaf veins followed by wilting, yellowing and plant death. Diseased plants were collected from three areas of a glasshouse with further samples taken from two areas of another glasshouse in 2017. Roots were washed, surface sterilized in 70% ethanol for 10s, rinsed twice in sterile water and 5mm sections placed on potato dextrose agar (PDA) containing 20 µg/ml chlorotetracycline. Following incubation at 20°C for 4 days, fungal colonies were sub-cultured onto PDA and examined after 14 days. All colonies resembled Fusarium with four morphotypes identified. Morphotype 1: flat, white colonies, abundant oval to cylindrical microconidia (9.4 x 4.6 µm), no macroconidia. Morphotype 2: pale yellow colonies which darkened with age to orange/brown, no microconidia, abundant macroconidia (33.3 x 5.7 µm), 3-7 septate, dorsiventral curvature, hooked basal cell and papillate apical cell. Morphotype 3: white colonies, cotton-like aerial mycelium with pink pigmentation on the underside. Abundant cylindrical (occasionally oval) microconidia (8.9 x 3.6 µm), sparse macroconidia (15.2 x 4.1 µm), slightly curved and slender, 3 septate with hooked basal cell and pointed apical cell. Morphotype 4: white colonies, cotton like aerial mycelium with orange/red pigmentation on the underside, abundant cylindrical microconidia (8.2 x 3.3 µm), macroconidia extremely sparse (15.4 x 3.9 µm), slightly curved and slender, 3-4 septate with hooked basal cell and pointed apical cell. Morphotype 1 was most frequently isolated from diseased plants. DNA was extracted from cultures of each morphotype (19 x type 1, 4 x type 2, 1 x type 3 and 4) and PCR carried out to amplify and sequence part of the translation elongation factor 1-α (EF1-α) gene (Taylor et al., 2016). Following BLAST searches and phylogenetic reconstruction (Fig. S2), isolates were identified as Fusarium redolens (morphotype 1, GenBank accession no. MK172061), Fusarium equiseti (morphotype 2, MK172060) and Fusarium oxysporum (morphotype 3, MK172059 and type 4, MK172058). F. oxysporum isolates were only identified in 2015. The pathogenicity of each Fusarium sp. was determined on six wild rocket cultivars. Roots of five week-old plants (eight plants per isolate/cultivar combination) were trimmed by 2-3mm and soaked in 100 ml of conidial suspension (1 x 106 cfu/ml) for 10 minutes before transplanting into compost in 9 cm pots. Control plants were dipped in sterile water. Pots were placed in a glasshouse (27°C day, 20°C night) and disease symptoms recorded twice weekly. Typical symptoms of wilting and yellowing (particularly in leaf veins) were observed 12 days after inoculation with F. oxysporum and F. redolens, while uninoculated control plants and those inoculated with F. equiseti remained healthy. Fusarium was consistently re-isolated from symptomatic plants, and following DNA extraction, PCR and sequencing of the EF1-α gene, sequences matched the identity of the inoculated isolate, thus fulfilling Koch’s postulates. This is the first report of Fusarium wilt of rocket in the UK and to our knowledge the first ever report of F. redolens causing wilt of wild rocket. F. oxysporum has been reported to cause wilt of wild rocket in Italy (Garibaldi et al. 2003) whilst F. redolens is known to cause wilt of chickpea (Jimenez-Fernandez et al. 2011).

Flavobacterium amnicola sp. nov., isolated from a sub-tropical stream.

Strain LLJ-11T, isolated from water sampled from a freshwater stream in Taiwan, was characterized using a polyphasic taxonomy approach. Cells of strain LLJ-11T were Gram-stain-negative, strictly aerobic, motile by gliding, rod-shaped and formed translucent yellow colonies. Optimal growth occurred at 25 °C, pH 7 and 0 % NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain LLJ-11T is affiliated with species in the genus Flavobacterium. Strain LLJ-11T was most closely related to Flavobacterium amniphilum KYPY10T with 98.0 % 16S rRNA gene sequence identity. The DNA-DNA relatedness of strain LLJ-11T with respect to Flavobacterium species was less than 35 %. Average nucleotide identity and digital DNA-DNA hybridization values between strain LLJ-11T and the type strains of other closely related Flavobacterium species were 70.0-76.3 % and 21.1-23.9 %, respectively. Strain LLJ-11T contained iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, iso-C15 : 0 3-OH, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and summed feature 9 (iso-C17 : 1ω9c and/or 10-methyl C16 : 0) as the predominant fatty acids. The major isoprenoid quinone was MK-6. The polar lipid profile consisted of phosphatidylethanolamine, three uncharacterized aminophospholipids and two uncharacterized phospholipids. The major polyamine was homospermidine. The DNA G+C content was 34.8 mol%. On the basis of the phylogenetic inference and phenotypic data, strain LLJ-11T is considered a representative of a novel species within the genus Flavobacterium. The name Flavobacterium amnicola sp. nov. is proposed, with strain LLJ-11T (=BCRC 81124T=LMG 30599T=KCTC 62514T) as the type strain.

Molecular identification and antibiogram profiles of respiratory bacterial agents isolated from cattle reared in some selected areas of Mymensingh division, Bangladesh

Respiratory bacterial infections in cattle are very common all over Bangladesh causing high economic loss. This research was performed with a view to proper control of respiratory bacterial infections of cattle in Bangladesh. A total of 100 nasal samples were collected on the basis of clinical signs. From the collected samples isolation, identification and characterization of the bacterial agents was done using cultural, biochemical and molecular techniques. Antibiogram profiles of the isolated agents were studied by disc diffusion method. Pasteurella multocida, Staphylococcus aureus and Escherichia coli were successfully isolated and identified from the collected samples. The isolated Pasteurella multocida produced small, round, opaque colonies on blood agar; Staphylococcus aureus produced golden yellow colony in mannitol salt agar; E. coli produced black color colonies with metallic sheen on EMB agar. Pasteurella multocida showed Gram negative, bipolar rods. Staphylococcus aureus showed Gram positive, cocci shaped and E. coli showed Gram negative, small rod shaped. Among 100 nasal samples 16 were found to be positive for Pasteurella multocida, 21 for Staphylococcus aureus and 13 for E. coli on the basis of cultural and biochemical characteristics. The antibiogram study reflected that ciprofloxacin, tetracycline and chloramphenicol should be first choice of treatment of respiratory bacterial infections caused by the isolated 3 bacteria. Pasteurella multocida was further characterized by PCR where 16 isolates showed positive band at 460 bp and Pasteurella multocida type A at 1044 bp. The present research work covering antibiogram study is a preliminary report in the context of Bangladesh.&#x0D; Asian Australas. J. Biosci. Biotechnol. 2019, 4 (1), 60-66

Flavobacterium sufflavum sp. nov., isolated from a freshwater lake.

A bacterial strain, BBQ-12T, was isolated from a freshwater lake in Taiwan. The strain was Gram-stain-negative, strictly aerobic, motile by gliding, rod-shaped and formed yellow colonies. Optimal growth occurred at 25 °C, pH 6 and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain BBQ-12T formed a phylogenetic lineage in the genus Flavobacterium. Strain BBQ-12T was most closely related to Flavobacterium fluminis 3R17T with 98.1 % 16S rRNA gene sequence similarity. Strain BBQ-12T showed 74.4-83.1 % average nucleotide identity and 16.0-21.8 % digital DNA-DNA hybridization identity with the type strains of other closely related Flavobacterium species. Strain BBQ-12T contained iso-C15 : 0, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and anteiso-C15 : 0 as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, four uncharacterized aminophospholipids and three uncharacterized phospholipids. The major polyamine was homospermidine. The major isoprenoid quinone was MK-6. The DNA G+C content of the genomic DNA was 34.2 mol%. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain BBQ-12T should be classified as a novel species of the genus Flavobacterium, for which the name Flavobacteriumsufflavum sp. nov. is proposed. The type strain is BBQ-12T (=BCRC 81049T=LMG 30051T=KCTC 52809T).

Advanced diagnosis of vibriosis among some marine fishes in lake Temsah, Egypt

Vibriosis is a serious disease affecting marine water fishes. The present study was applied on 320 marine fishes of four equal different species (Sparus auratus, Mugil cephalus, Mugil seheli and Tilapia zillii) each 80. Fishes were collected seasonally and randomly from Lake Temsah in Ismailia governorate from September2015 to August 2016. The signs and lesions of severe septicemia, hemorrhages and ulcerations were observed among the examined fishes. Isolated bacteria were observed as yellow and green pigmented colonies on TCBS media, and as creamy colored colonies on TSA media with 2.5% NaCl concentration. The causative agent was identified as vibrio sp. Concerning gene expression, most isolates were molecularly identified using the pvsA gene primers giving a product size of 338-bp size and 348-bp for V. alginolyticus and V. parahemolyticus respectively. The highest prevalence of vibriosis was recorded in T. zillii (63.75%), M.seheli (37.5%) then M.cephalus and Sparus auratus (28.75%) while the total prevalence was (39.69%). The highest seasonal prevalence was recorded in summer (81.25%) followed by spring (35%) then autumn (23.75%) and winter (18.75%). The highest prevalence of organ specifity was in liver, kidneys then spleen and gills. The histopathological studies showed activation of melanomacrophage centers, degeneration, necrosis and congestion in liver, kidney, spleen, and gills.

First report of Rhizopus stolonifer causing Rhizopus bunch rot on grapes in Pakistan

During June 2016, a postharvest survey of table grapes was carried out in the main fruit markets of Attock (33°46'07.9"N 72°21'43.0"E) and Jehlum (32°56'22.3"N 73°43'31.4"E) districts of Punjab Province. At the time of sampling, two cultivars (King’s Ruby and Perlette) were sampled at five different locations of both districts. Disease incidence % of bunches averaged 3 to 4 %. Some infected fruit appeared water-soaked, light brown and covered by fluffy mycelium consisting of erect sporangiophores with black sporangia on the top (Figure 1). Symptomatic tissue pieces were surface-sterilized with 0.1% NaOCl for 30 sec, rinsed three times with sterile distilled water, dried on filter paper for 45 sec and incubated on potato dextrose agar (PDA) at 25°C. After 1 day, mycelium on PDA was transferred to a fresh PDA plate and incubated at 25°C with a 12-h photoperiod. Within 3 days, white to yellow colonies with black aerial sporangia were formed (Figure 2). A total of 64 isolates were examined morphologically. Sporangiophores were erect, light brown and 623 to 3800 µm long. One to three rhizoids were observed opposite to each sporangiophore. Sporangia were black, globose to sub-globose, 91 to 124 μm in diameter. Columellae were conical to cylindrical and 86 to 187 μm long × 72 to 205 μm wide. Sporangiospores were hyaline to light dark grey, globose, ellipsoidal in shape and 6.8 to 12.4 × 3.6 to 12.5 μm (Figure 3). These features were identical to the description of Rhizopus stolonifer (Ehrenb.) Vuill (Liou et al., 2007). For molecular identification, the ITS1-5.8S-ITS2 region of two representative isolates (Rizo 05 and Rizo 07) was amplified with primers ITS1/ITS4 (White et al., 1990). Sequence comparison of two isolates Rizo05 and Rizo 07 (Accession no. MH348205 and MH356272) revealed 100% identity with previously reported isolates of Rhizopus stolonifer (Accession no. MG865992, KU729185, HM051076, and MF374842). To complete Koch’s postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of Rizo 05 and Rizo 07 were pipetted onto three non-wounded and four wounded asymptomatic grape berries (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries to serve as a negative control. Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice (Ghuffar et al., 2018). Black to light brown, fluffy mycelium with the original symptoms was observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control (Figure 4). The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Previously, Rhizopus stolonifer has been reported as a pathogen on grapes in Chile (Latorre et al., 2002). To our knowledge, this is the first report of Rhizopus stolonifer causing bunch rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against Rhizopus rot of grapes in Pakistan.

A putative novel Methylobacter member (KRF1) from the globally important Methylobacter clade 2: cultivation and salient draft genome features.

Methane oxidation by methanotrophs is a very important environmental process in the mitigation of methane. Methylobacter (Mtb.) clade 2 members have been reported as dominant methane oxidisers in soils and sediments worldwide. We enriched and purified a methanotroph from a tropical rice field soil sample from India. The highly enriched culture showed the presence of motile, long and thick rods (3-5µm × 0.9-1.2µm) and minor presence of short, thin rods. The culture was purified on agarose medium and formed yellow colonies which showed the presence of only thick and long rods, henceforth termed as strain KRF1. Based on 16S rRNA gene sequence analysis, strain KRF1 shows close phylogenetic affiliation to Methylobacter tundripaludum SV96T (98.6% similarity). Due to the taxonomic novelty, and being the first member of Mtb. related to Mtb. tundripaludum from the tropics, the draft genome was sequenced. From the blastx analysis of the contigs, it was clear that the culture still had contamination of another organism, a Methylophilus species. The data binned in two clear bins: Mtb. related contigs and Methylophilus-related contigs. The binned draft genome of KRF1 shows features including the typical pathways for methane metabolism, denitrification and the presence of molybdenum iron and vanadium-iron nitrogenase genes. KRF1 is phylogenetically distinct from the five strains of Mtb. tundripaludum including SV96T, Lake Washington strains and OWC strains, showing ~ 26% DDH and ~ 81% ANIb values and a unique position in a phylogenomic tree. Subsequently, KRF1 has been completely purified from its methylotrophic partner and a pure culture has been established and maintained in a WDCM approved culture collection, the MACS Collection of Microorganisms (as MCM 1471). KRF1 is thus the first cultured member of a putative novel species of Methylobacter clade 2 isolated from the tropics.

First Report of Bacterial Leaf Spot of Crown-of-Thorns (Euphorbia milii) Caused by Xanthomonas axonopodis pv. poinsettiicola in Taiwan

Crown-of-thorns (Euphorbia milii; Euphorbiaceae) is a popular plant widely grown as a potted flower or hedge in many countries (Marys and Romano 2011). In 2017, leaf spot symptoms were observed on potted E. milii in a plant market located in Nantun District, Taichung, Taiwan. Over 90% of the plants onsite were infected. Leaf lesions were round, had necrotic centers, and had margins that appeared yellow when viewed from the upper leaf surface and water-soaked when observed from underneath. Symptomatic tissues were cut and examined under a light microscope. Samples with bacterial streaming were streaked onto nutrient agar, and yellow, round colonies were recovered. Strains Eu1, Eu2, and Eu3 were obtained from three different plants. They produced yellow mucoid colonies on yeast dextrose carbonate (YDC) agar, grew at 35°C or on SX medium (a semiselective medium), and could degrade starch, esculin, casein, and lipid, similar to many known xanthomonads (Schaad et al. 2001). The isolates also induced hypersensitive reaction on tobacco (cv. Taiyen 5) 48 h postinfiltration. Identification of Eu1 to Eu3 using the Biolog microbial identification system (Biolog, Hayward, CA) showed that they are Xanthomonas campestris pv. poinsettiicola (X. axonopodis pv. poinsettiicola) with probabilities of 84 to 98%. Multilocus sequence analysis targeting partial sequences of the lacF, lepA, fusA, gapA, gltA, and gyrB genes (Almeida et al. 2010) were conducted, and Eu1 to Eu3 had identical sequences for every fragment (accession nos. MH068853 to MH068858). Comparing their concatenated sequences (2,744 bp) with those from reference/type strains of other xanthomonads able to produce symptoms on spurges (Rockey et al. 2015) revealed that Eu1 to Eu3 share 98.65% sequence identity with X. axonopodis pv. poinsettiicola LMG849ᴾᵀ and have lower sequence similarity with X. hortorum pv. pelargonii LMG7314ᴾᵀ (94.06%), X. codiaei LMG8678ᵀ (93.55%), and X. campestris pv. campestris LMG568ᴾᵀ (93.51%). Koch’s postulates were fulfilled by spraying bacterial suspensions (OD₆₀₀ = 0.3; on average 7 × 10⁸ CFU/ml; in 0.02% Silwet L-77) onto all aboveground parts of cutting-propagated E. milii (approximately 10 cm tall; var. Olympus). The test was conducted with every isolate, each inoculated onto three plants. As controls, three plants were sprayed with bacteria-free solutions (0.02% Silwet L-77). Inoculated E. milii were placed separately in plastic bags for 48 h to maintain humidity. Within 1 month, all inoculated plants developed leaf spots similar to those found in the plant market, and the controls did not. Bacteria reisolated from inoculation-infected plants had the same colony morphologies as Eu1 to Eu3 on YDC agar. Colony polymerase chain reaction and sequencing of gapA of the reisolates showed that their sequences were identical to those of Eu1 to Eu3. A representative strain, Eu1, was spray-inoculated onto four poinsettia plants (approximately 8 cm in height) using the same inoculum density as mentioned above. Leaf spots appeared on all of the treated plants but not on four plants sprayed with bacteria-free solution as mentioned above. Natural infection of poinsettia and E. milii by X. axonopodis pv. poinsettiicola has been reported in the United States (Rockey et al. 2015). In Taiwan, however, only bacterial leaf spot of poinsettia has been recorded (Lee et al. 2006). This study is the first report of X. axonopodis pv. poinsettiicola naturally infecting E. milii in Taiwan. Because many nurseries and plant markets in Taiwan often grow E. milii and other spurges in close proximity and at high density, care should be taken to prevent the spread of the disease within and across host species.

Previous diet affects the amount but not the type of bait consumed by an invasive ant.

Recent research on multiple invasive ant species has revealed the importance of carbohydrates for achieving high activity levels and outcompeting native ants. However, comparatively little is known about the role of diet and macronutrient preferences for uptake of insecticidal baits used to control invasive ants. We tested whether diet affected yellow crazy ant (Anoplolepis gracilipes Fr Smith) survival and behavior, and whether bait preference would be complementary to past diet. We found that colonies fed only crickets for 28 days had fewer live workers and queens, and less brood per live queen than colonies fed crickets + honeydew but did not differ significantly from colonies fed only honeydew. Colonies that had been fed only crickets were more active (as assessed by interaction with a novel object), retrieved 16-17 times more bait per worker overall, and consumed more of the six bait types than cricket + honeydew and honeydew-only fed colonies. However, prior diet did not affect bait choice. The two highest sugar bait formulations combined accounted for most of the bait consumed across all treatments (cricket-only 74.8% ± 28.1; cricket + honeydew 69.2% ± 12.4; honeydew-only 62.5% ± 30.4). Yellow crazy ant colonies fare better without protein than without carbohydrates. Yellow crazy ants ate the most bait when fed only crickets but did not choose baits complementary to their previous diet. Baits in a sugar-rich carrier may be most effective for the control of yellow crazy ants, regardless of the relative availability of macronutrients. © 2019 Society of Chemical Industry.

The Frequency of Staphylococcus aureus Classical Enterotoxin Genes in Raw Milk Samples in Zanjan, Iran

Background: Staphylococcus aureus is one of the major causes of food poisoning. Since milk is a nutritious source of proteins and vitamins, it could provide the optimal conditions for the growth of several bacterial pathogens, such as S. aureus. The present study aimed to assess the frequency of Staphylococcus aureus classical enterotoxin genes in raw milk samples in Zanjan, Iran. Methods: In total, 82 bovine, unpasteurized milk samples were collected from the dairy farms in various rural areas in Zanjan, Iran. The isolation and identification of S. aureus were performed using the Baird-Parker agar, routine biochemical tests, and polymerase chain reaction (PCR) targeting the S. aureus-specific femA gene. In addition, staphylococcal enterotoxin genes (e.g., sea, seb, sec, sed, and see) were assessed using PCR. Results: Following the appearance of yellow colonies with yellow zones on Mannitol salt agar, 21 S. aureus isolates (25.6%) were detected. In total, 80.9% of the isolates were positive for the presence of SE genes, and the most frequent SE gene was sea (88.2%), followed by see (58.8%), and seb (52.9%). Furthermore, 76.5% of the isolates had two or more SE genes simultaneously. Conclusion: According to the results, the presence of enterotoxigenic S. aureus in the studied raw milk samples confirmed the possible risk posed on the public health. Therefore, it is recommended that the quality of dairy product quality programs be optimized in order to intensify the sanitary inspection of these products.

Altererythrobacter amylolyticus sp. nov., isolated from lake sediment.

An aerobic, motile, Gram-stain-negative bacterium, designated strain NS1T, was isolated from interfacial sediment from Taihu Lake, China. The strain formed yellow colonies on R2A medium. Cells were ovoid to rod-shaped and non-spore-forming. Growth occurred at 15-40 °C (optimum, 28 °C), at pH 5.0-10.5 (optimum, 6.5-7.5) and in the presence of 0-1 % (w/v) NaCl (optimum, 0 %). Phylogenetic trees based on 16S rRNA gene sequences showed that strain NS1T represented a member of the genus Altererythrobacter and had the highest sequence similarity to Altererythrobacter troitsensis CCTCC AB 2015180T (97.1 %). The average nucleotide identity value between strain NS1T and the closest related strain based on their genomes was 78.6 %. The predominant ubiquinone was Q-10. The major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, an unidentified phospholipid, an unidentified glycolipid and six unidentified lipids. The genomic DNA G+C content was 66.6 mol%. On the basis of phenotypic and genotypic characteristics, strain NS1T represents a novel species of the genus Altererythrobacter, for which the name Altererythrobacter amylolyticus sp. nov. is proposed. The type strain is NS1T (=CGMCC 1.13679T=NBRC 113553T).

Preliminary and Biochemical Characterization of Bacterial Strains Isolated from Ramie Cane Soils

For the present study a total of 18 bacterial isolates were isolated from Ramie canes soils. Preliminary characteristics like colony characteristics and substrate concentrations were studied for the microorganisms. Physicochemical factors affecting the bacterial growth. The three seasons winter, summer and rainy seasons were also affected the growth of the bacteria. Various substrates like starch peptone, xylan peptone, Glycerol, Aspargine, glucose nitrate agar, carboxymethyl cellulose agar and pectin peptone agar media were studied. Maximum colony growth was recorded in pectin peptone agar media. Cream white, orange, red, yellow, lemon yellow and watery white colonies were recorded. The size of the bacterial strains was recorded in 1-55 mm in diameter. All the strains were entire lobed, convex having smooth and rough surface. of the 18 strains 11 were gram positive rods and the remaining strains were negative rods. Among the 18 strains, we were selected six strains for further studies like effect of seasons on growth and amino acid infilteration of bacteria. Maximum growth was recorded in winter season with 85.0 colony count in the presence of CCLS strain. There are 10 amino acids were introduced into the bacteria, with maximum growth was WCES bacterial strain. The results of this study reveal that the soil microflora associated with the ramie cane soils. These bacterial strains were used in the process of decortication and degumming.

Antibacterial activities of Physalis angulata herb extract on white feces diseases (WFD) in Litopenaeus shrimp vannamei

Litopenaeus vannamei shrimp is one of the leading export commodities in fisheries from Indonesia, however White Feces Diseases (WFD) cause deaths up to 30% and decrease their growth and production. WFD is caused by the accumulation of several Vibrio bacteria in shrimp water and intestines. One of the local herbs that has the potential as antiproliferation, antidiabetic, anticytotoxic commonly used in the treatment of humans diseases is Physalis angulata known as ciplukan. The aim of this study was to obtain ciplukan extract which has an antibacterial activity of Vibrio sp., and to analyze the anti-bacterial effect of ciplukan extract on Vibrio bacteria from vannamei infected with WFD. This study used descriptive and experimental methods using a randomized block design. Data collection methods were carried out through direct observation of the object under study. Observation data were analyzed by F- test followed by HSD. The results showed that the extract of ciplukan plants contained antibacterial active ingredients. Chloroform solvent extracts had the best antibacterial activity with 13 mm inhibition zone on green bacterial colonies and 16.5 mm in yellow bacterial colonies derived from shrimp intestinal infected with WFD. The best concentration that provides the highest inhibition zone as obtained by 10% chloroform extract.

Aspergillus flavus (Link) toxins reduces the fitness of dengue vector Aedes aegypti (Linn.) and their non-target toxicity against aquatic predator

Mosquito that accountable for dispersal of dengue fever is Aedes aegypti Linn. and considered to be a chief vector for dengue especially in South Asian countries. Aspergillus flavus is considered to be wild growing green yellow colonies and synthesis highly regulating aflatoxins (B1, B2, G1 and G2) as a secondary metabolite. Mycotoxins of A. flavus showed its efficacy against III and IV instars of Ae. aegypti with more than 90% mortality at the prominent dosage of 2 × 108 conidia/ml. The proximate lethal concentrations (LC50 and LC90) of mycotoxins against third and fourth instars was 2 × 105 and 2 × 107 respectively. Correspondingly, sub-lethal dosage of mycotoxin A. flavus significantly inhibited the level of α- β-carboxylesterase and SOD activity and upregulated the level of major detoxifying enzymes GST and CYP450. Moreover, sub-lethal dosage also showed higher deterrent and fecundity effects. Gut-histological examination reveals that the A. flavus considerably affected the gut epithelial cells along with the inner gut lumen as compared to the control. The non-target screening of A. flavus against two aquatic predators (A. bouvieri and Tx. splendens) display more than 80% of mortality rate against both the species at the dosage of 2 × 1016 (two-fold-higher dosage used in larval assays). Thus the biosafety assessment suggests that A. flavus display higher toxicity against the non-targets and it is not-recommended to apply it directly to the aquatic habitat of dengue mosquito which shares their living space with other beneficial insects.