Nutrient Agar Medium Research Articles

Micrococcus luteus: A Potential Keratinolytic Bacteria Isolated from Poultry Waste Dumping Site at Namakkal District, Tamil Nadu, India

: In this study, the experimental soil samples were collected from poultry waste dumping site at Namakkal district, Tamil Nadu, India. From the five soil samples, 15 bacterial colonies were isolated based on Keratinolytic activity with source of agar nutrient medium, nitrogen, carbon and feather meal powder. After the incubation period only 6 isolates showed clear zone of hydrolysis around colony in feather meal agar medium incubated at 37 C for 72 h. The Bacterial species were identified by the morphological and biochemical tests compared with Bergey’s manual of systematic bacteriology IV edition. The obtained bacterial genera from poultry waste dumping sites were Escherichia coli, Enterococcus faecalis, Micrococcus luteus, Klebsiella pneumoniae, Proteus sp., Staphylococcus aureus, Salmonella sp. and Pseudomonas aeruginosa. During the Keratinolytic activity the maximum zone of inhibition was noticed in the isolate No.3, and it was identified as Micrococcus luteus. The identified potential keratinolytic bacterial species culture was optimized with different temperature, pH, nitrogen and carbon sources for bulk production of enzyme. The bulk production of enzyme was precipitated with 20% of ammonium sulphate and it was purified with dialysis bag and subjected to assay procedure for determination of enzyme activity. The antibiotic sensitivity was tested on the isolated bacterial isolates and the highest antibiotic resistance was observed in Staphylococcus aureus (75%) and second most Salmonella (70%) and followed by Klebsiella pneumoniae (60%), Enterococcus faecalis (50%) and Escherichia coli (45%) .

Exploring Citrus Nematodes and its Antagonists in Citrus Growing Areas of Punjab, Pakistan

Citrus nematode (Tylenchulus semipenetrans) is a major threat to the citrus industry in Pakistan. During 2019-20, a survey was carried out for citrus nematode and its antagonists in citrus orchards (Both healthy and declining trees were samples) of Punjab, Pakistan. Bhalwal, Shahpur, Sillanwali, Kotmomin, Sargodha, Quaidabad, Jhang, and Rahim Yar Khan were surveyed for disease incidence, prevalence, and association of T. semipenetrans. Maximum disease prevalence (100%) was recorded in Rahim Yar Khan while minimum (20%) in Bhalwal and Kotmomin. Maximum slow decline with 80% field incidence was observed in Rahim Yar Khan while a minimum of 20% in Quaidabad. Soil and root samples were used for nematode extraction using Baerman's funnel method and the Whitehead Hemming tray method. A maximum number of J2s/100ml of soil were observed in soil samples taken from Rahim Yar Khan (1674) followed by Shahpur (1534), Qaidabad (1432), Sargodha (1347), and Bhalwal (1172). The highest number of nematode females per gram of root were recorded in Rahim Yar Khan (652) followed by Quaidabad (611), Bhalwal (490), Kotmomin (421), Sillanwali (387). Isolation of different fungal and bacterial antagonist isolates was also done by soil dilution plate technique using nutrient agar media and potato dextrose agar media. Identification of fungal antagonists was made on colony growth, color, and spore structure. Bacterial identification was also done on morphological characters and gram tests. Trichoderma harzianum, Trichoderma viride, Trichoderma koningii Trichoderma atroviride, Pseudomonas fluorescens, Pseudomonas putida, Bacillus cereus and Bacillus subtilis were found associated.

DETECTION OF NITROGENASE PRODUCING BACTERIA FROM THE SOIL OF LIWA BOTANICAL GARDEN

Liwa Botanical Gardens is an ex-situ conservation area for various types of plants. Each plant produces organic matter that will provide nutrients for the growth of nitrogen-fixing bacteria. This indicates the existence of an environment that supports the growth of nitrogen-fixing bacteria. Nitrogen is one of the nutrients needed by plants for their growth. However, the abundance of nitrogen in the atmosphere cannot be utilized directly by plants but needs to transform into ammonium and nitrate first. This transformation can be done by nitrogen-fixing bacteria through an enzymatic process. This research aims to obtain bacterial isolates that can fix nitrogen. Nitrogen-fixing bacteria were isolated using Nutrient Agar (NA) medium and furthered by nitrogenase activity detection test with semi-solid Nitrogen Free Bromothymol Blue (NFB). Nitrogen-fixing bacteria are characterized by color changes in the medium. The results obtained 22 isolates with 3 isolates detected capable of producing nitrogenase enzymes, namely TBP B3, TB1 B2, and TMA2 B2.

Occurrence of Crown Gall Disease on Japanese Spindle (Euonymus japonicas var. Green Rocket) Caused by Agrobacterium rosae in Iran.

Crown gall disease caused by diverse Agrobacterium species is one of the main biotic constraints in the ornamental plants industry in Iran (Mafakheri et al. 2017). In August 2019, Japanese spindle (Euonymus japonicus var. Green Rocket) plants showing crown gall symptoms were observed in a commercial greenhouse in Tehran, Iran. Infected plants were characterized by a visible overgrowth on their stems and crown. Bacterial isolation from the gall tissues was performed on nutrient agar (NA) and 1A media as described by Moore et al. (2001). The six resulted bacterial strains (A.E1 to A.E6) were evaluated using PCR primer pair F8360/F8361 amplifying a 453 bp DNA fragment in recA gene and confirmed as Agrobacterium sp. (Shams et al. 2013). Pathogenicity of the strains was evaluated in two independent assays on Japanese spindle plantlets as well as 10-15 day old tomato (Solanum lycopersicum cv. Sunseed 6189) and sunflower (Helianthus annuus cv. Armavirski) plants in greenhouse conditions using the needle prick method as described previously (Mafakheri et al. 2019). The reference strain A. radiobacter ICMP 5856 and sterile distilled water were used as positive and negative controls, respectively. Crown gall symptoms appeared 20-25 days post inoculation on the Japanese spindle plantlets as well as tomato and sunflower plants inoculated with the strains isolated in this study, while the negative control plants remained asymptomatic. Koch's postulates were accomplished by re-isolating on NA medium and PCR-based identification of the inoculated strains from the symptomatic plants. The representative strain A.E1 was subjected to multilocus sequence analysis (MLSA) using the sequences of four housekeeping genes (i.e. atpD, gyrB, recA, and rpoB) as described previously (Mafakheri et al. 2019). MLSA results revealed that the strain A.E1 is phylogenetically closely related to A. rosae. The sequences were deposited into GenBank under the accession numbers MT007962 to MT007965 for atpD, gyrB, recA, and rpoB, respectively. Further, the strain A.E1 was subjected to whole genome sequencing using Illumina HiSeq X platform. DNA extraction was performed using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany), DNA libraries were obtained with Nextera XT DNA Library Prep Kit (Illumina, USA), and de novo sequence assembly was performed using SPAdes genome assembler. The resulting whole genome sequence was deposited into the GenBank database under the accession number JAFJZW000000000. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were calculated among all the type strains of Agrobacterium species/genomospecies using standard criteria as detailed previously (Osdaghi et al. 2020; Chen et al. 2021). The strain A.E1 had 97% ANI and 72% dDDH values with A. rosae strain NCPPB 1650, suggesting that the bacterial strains isolated from Japanese spindle in Iran belong to A. rosae. This is the first report of A. rosae causing crown gall disease on Japanese spindle in Iran. The new crown gall disease could negatively affect the ornamental shrub production industry in central Iran unless strict sanitary measures are taken into the account in the nurseries in these areas. Further nationwide surveys and samplings are warranted to elucidate the economic impact of the pathogen on ornamental plant industry in the country.

Frateuria flava sp. nov., isolated from soil.

A Gram-stain-negative, aerobic, rod-shaped and non-motile novel bacterial strain, designated MAH-13T, was isolated from a soil sample. The colonies were observed to be yellow-coloured, smooth, spherical and 1.8-3.0 mm in diameter when grown on nutrient agar medium for 2 days. Strain MAH-13T was found to be able to grow at 20-40 °C, at pH 5.0-10.0 and with 0-1.0% NaCl (w/v). Cell growth occurred on tryptone soya agar, Luria-Bertani agar, nutrient agar and Reasoner's 2A agar. The strain was found to be positive for both oxidase and catalase tests. The strain was positive for hydrolysis of casein, starch, DNA and l-tyrosine. According to 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Frateuria and to be closely related to Frateuria terrea DSM 26515T (98.2% similarity), Dyella thiooxydans ATSB10T (98.2 %), Frateuria defendens HyOGT (97.9 %), Rhodanobacter glycinis MO64T (97.8 %) and Frateuria aurantia DSM 6220T (97.8 %). The novel strain MAH-13T has a draft genome size of 3 682 848 bp (40 contigs), annotated with 3172 protein-coding genes, 49 tRNA genes and three rRNA genes. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain MAH-13T and five closely related type strains were in the range of 73.7-85.5 % and 20.7-30.1%, respectively. The genomic DNA G+C content was determined to be 68.0 mol%. The predominant isoprenoid quinone was ubiquinone 8. The major fatty acids were identified as iso-C15:0, iso-C16:0 and summed feature 9 (iso-C17 : 1 ω9c and/or C16:0 10-methyl). On the basis of dDDH and ANI values, genotypic analysis, and chemotaxonomic and physiological data, strain MAH-13T represents a novel species within the genus Frateuria, for which the name Frateuria flava sp. nov. is proposed, with MAH-13T (=KACC 19743T=CGMCC 1.13655T) as the type strain.

Modification of nutrient agar medium to culture yet-unculturable bacteria living in unsanitary landfills

The Ceylon Journal of Science is a peer-reviewed journal published quarterly by the University of Peradeniya, Sri Lanka in March, June, September and December. It is aimed at publishing high quality research articles on topics related to different disciplines in Science. The journal accepts original research articles, book reviews, reviews and mini-reviews, short communications, opinions, research notes, and commentaries and notes. The journal strictly adheres to publication ethics as emphasized by the Committee on Publication Ethics (COPE). The Journal has its own website.The Ceylon Journal of Science is indexed in Sri Lanka Journals Online (SLJOL), Directory of Open Access Journals (DOAJ), Google Scholar and Zoological Records. According to the Google Scholar; H5-Index: 12H5-Median: 15According to the Exaly (1970 – 2021); Impact Factor: 0.6 (top 19%)Extended IF: 0.6 (top 19%) H-Index: 8 (top 28%)Citations/paper: 1.42

OBTAINING OF PLANT TISSUE CULTURE Scutellaria baicalensis GEORGI. AND ITS BIOCHEMICAL ANALYSIS

Aim. To obtain a plant tissue culture of S. baicalensis as a possible source of biologically active compounds (BAC) with a wide range of pharmacological action. Methods. Plant tissue culture, photocolorimetric method, reversed-phase high performance liquid chromatography (HPLC) method. Results. Two stably productive plant tissue culture strains (16SB3 and 20SB4) of S. baicalensis were obtained from fragments of roots seedling on a specially developed agar nutrient medium 5С01. The yield of dry biomass from 1 liter of this medium per passage (21st day of growth) for strain 16SB3 is 25–30 g, for strain 20SB4 – 30–40 g. The total content of flavonoids in dry biomass was in terms of routine for strains 16SB3 and 20SB4 – 0.6–0.9 and 0.7–0.9 mg/g, respectively, and the yield of flavonoids – 18–27 and 21–36 mg/l of nutrient medium, respectively. BAC typical for plants in nature, in particular, flavonoids vogonin, baikalein, neobaikalein, skulkapfavon and their derivatives, were found in the studied biomass of both strains. Conclusions. It was found that the biomass of the two strains of S. baicalensis plant tissue culture accumulated the same BAC, in particular, flavonoids, as do plants in natural conditions. The resulting plant tissue culture is promising as a possible source of Baikal skullcap BAC.

Differentiation of valuable for breeding sunflower lines by resistance to Phoma rot

The purpose of the research is differentiation of valuable for breeding sunflower lines by resistance to Phoma rot. The promising constant sunflower lines were estimated by their resistance to Phoma rot using methods of artificial inoculation in the greenhouse and laboratory conditions. In greenhouse conditions, there were used two methods of artificial inoculation with a fungus Plenodomus lindquistii in the bottom of leaf petiole of a sunflower plant at a stage of the first true leaf pair. Application of the pieces of agar nutrient medium with mycelium, pycnidia and picnospores (unbroken) allowed to identify resistant and mid-resistant sunflower lines (VK 900, L 86 and L 107, L 132). At inoculation with 0.2 ml of a fungus spore suspension (concentration of 1 × 106 spores/ml), five susceptible and one very susceptible sunflower lines (L 2385, L 103, L 136, L 131, L 128, and L 82) were determined. In laboratory conditions, the quick test for estimation of susceptibility to Phoma rot (dose of infection – 12 g) allowed to reveal more than 50 % of plants with healthy root system and two constant sunflower lines L 116 and L 136 with the affection degree of one point. Estimation of susceptibility to Phoma rot infection of the different organs (stem and root system) at artificial inoculation showed a possibility to differentiate sunflower lines by resistance of roots and stems and to select the most promising for the further work in breeding.

ISOLATION, MOLECULAR CHARACTERIZATION OF DIAZOTROPIC BACTERIA FROM VIRGIN SOIL FROM ARAKU VALLEY AND ASSESSMENT OF OF AMMONIA ACCUMULATION AT SALINE CONDITIONS

Nitrogen-fixing bacteria are widely distributed in nature where they reduce atmospheric nitrogen in soil or in association with plant. They have been found in a wide variety of terrestrial and aquatic habitats in both temperate and tropical regions of the word. Nitrogen-fixing bacteria are found in symbiotic associations with plants free living in soil. The objective of the present research was to isolate free living Nitrogen fixing bacteria from virgin soil samples in araku valley and assessment of their ammonia accumulation at saline conditions. 10 soil samples were collected in different place from virgin areas. For isolation of the free living nitrogen fixing bacteria, Nitrogen free media like Jensens Medium and Azotobacter Agar were used. Serially diluted soil samples were spread on the agar media and incubated for 48 hours. Eleven morphologically different bacteria were separated on made pure colonies on nutrient agar media. All bacteria were under go biochemical characterization which reveals that all these bacteria related to Azospirillum, Azotobacter and Clostridium. High ammonia liberating isolate MGN-10 was molecular characterizes as Azotobacter chroococcum and this soil application increase the plant growth in terms of growth parameters.

Synthesis, Characterization and Biological Evaluation of some Novel Substituted Indole-Coumarin Derivatives as Potential Antibacterial and Antifungal Agents

Aim: In the present work, we have designed and synthesized indole-fused coumarin derivatives as potential antibacterial and antifungal agents.
 Place and Duration of Study: The present work was carried out at PEA's Modern college of Pharmacy, Sector 21, Yamunanagar, Nigdi- 411044 between the duration of January-2021 to May-2021.
 Methodology: The in vitro antibacterial and antifungal activity was performed by disc diffusion method. The antibacterial activities were tested using agar nutrient medium against Bacillus subtilis, Bacillus pumilus, Escherichia coli, and Pseudomonas aeruginosa that are representative types of gram-positive and gram-negative organisms respectively. The anti-fungal activity of all the compounds were determined on potato dextrose agar medium against Aspergillus niger and Candida albicans. Clotrimazole 100 μg/ml was used as a standard and DMF was used as control.
 Results: It was observed that all the compounds were sensitive to the gram +ve bacteria. Compound 2c, 2e, and 2f were sensitive to gram –ve bacteria. Compounds 2a, 2b, and 2f were sensitive to both the fungal strains while all the derivatives were sensitive to Candida albicans.
 Conclusion: From present investigation it has been observed that compound 2f was sensitive against gram +ve, gram –ve, and both the strains of fungal strains. Structurally, compound 2f possess aldehyde functional group at indole nucleus. Therefore, from present study we have concluded that compound 2f is a lead molecule for the further development of potential antibacterial and antifungal agents.

Antioxidant and inhibitory activity of Roselle Extract (Hibiscus sabdariffa L.) against Methicillin-Resistant Staphylococcus aureus (MRSA)

Background: Hibiscus sabdariffa known as roselle, which belongs to the family Malvaceae, grows in sub-tropical and tropical region including Indonesia. Several studies have reported the benefits of roselle for health. Roselle extract has been shown to have antibacterial effect as a support for antibacterial therapy, especially for case of antibiotic resistance, and antioxidant effect which can neutralize free radicals. Purpose: The aim of this study was obtaining the minimum inhibitory concentration (MIC) and inhibition concentration (IC50) of roselle extract. Methods: Antibacterial effect of roselle extract against Methicillin-Resistant Staphylococcus aureus (MRSA) was tested in nutrient agar media using diffusion method. Antioxidant activity of roselle extract was performed by DPPH. The violet color of DPPH solutions that was reduced by roselle extract were measured using visible spectrophotometer at the wavelength of 516 nm. Result: The result of this study obtained the (MIC) and IC50 of roselle extract were 2,5% and 1251±202,32 ppm, respectively. Conclusion: It can be concluded that roselle extract have antibacterial effect against MRSA and antioxidant effect.

Anti-bacterial activity of Rosela Flower Extract (Hibiscus sabdariffa L.) against Extended-Spectrum Beta-Lactamase (ESBL) Eschericia coli

Roselle (Hibiscus sabdariffa L.) contains cyanidin-3-rutinoside, delphinidin, delphinidin-3-monoglucoside, cyanidin-3-monoglucoside, cyanidin-3-sambubioside, cyanidin-3,5-diglucoside may inhibit the growth of Extended-Spectrum-Beta-Lactamase (ESBL) Escherichia coli, but there is no research reported the determination of MIC of Roselle (Hibiscus sabdariffa L.) extract powder against ESBL E.coli ATCC 6110 and ATCC 5949 and their potency ratio compared to meropenem. This study aimed to determine the MIC of Roselle flower extract powder on the growth of ESBL E. coli ATCC 6110 and ATCC 5949 and determine the potency ratio compared to meropenem. This study used two methods in determining MIC, namely the agar diffusion method and the dilution method with Nutrient Agar media which was incubated at 37 ±1°C for 24 hours, while the determination of the potency ratio was carried out by diffusion method with the same media which was incubated at 37 ±1°C for 24 hours. The results obtained were diameter of inhibition zone (mm) which were then observed and analyzed to calculate the potency ratio. The results showed that the MIC of Roselle flower extract powder was obtained at a concentration of 12,500 ppm by diffusion method and at a concentration of 3,125 ppm by dilution method with 24 hours incubation at 37 ± 1o C and the potency ratio of Roselle flower extract powder compared to meropenem was 89.7% and 97.97% against ESBL E.coli ATCC 6110 and ESBL E.coli ATCC 5949 respectively.

Characterization of phosphate solubilizing plant growth promoting rhizobacterium Lysinibacillus pakistanensis strain PCPSMR15 isolated from Oryza sativa.

The rhizosphere soil is a source for a diversity of microorganisms which play a vital role in the enhancement of plant health through the mechanism of symbiotic interaction thereby influencing the plant growth. The present study aimed at isolating potential phosphate solubilizing rhizobacteria from rice (Oryza sativa) crop for which, four different rhizosphere soil samples were collected from different locations of Tiruvallur district, India. Isolates were cultured on nutrient agar medium followed by serial dilutions and different colonies with morphological variations were isolated from each dilution. A total of 52 bacteria were isolated and maintained as pure cultures. Out of the 52 isolates, 16 strains showed phosphate solubilizing ability and amongst them, 4 were highly potential which were subjected to morphological and biochemical characterization. Phosphate solubilizing bacterial strains when assessed for their possible effect of their inoculation on the growth and development of mung bean seeds significantly enhanced the growth of the plants. Furthermore, the potential bacteria were analysed for Indole Acetic Acid (IAA) production, which was found to be directly proportional to the plant growth promotion. Upon the comparative analysis of the four potential isolates, PCPSMR15 exhibited remarkable plant growth promoting traits. A detailed biochemical and molecular analysis identified the promising strain PCPSMR15 as Lysinibacillus pakistanensis. The present study, thus signifies the strain, PCPSMR15 for exploration as an inoculant for improving soil fertility, enhancing phosphorus availability to plants and improved crop production and sustainability.

Insights Into Manganese Solubilizing Bacillus spp. for Improving Plant Growth and Manganese Uptake in Maize.

Manganese (Mn) is an essential micronutrient for plant growth that is involved in the structure of photosynthetic proteins and enzymes. Mn deficiency is widespread mainly in dry, calcareous, and sandy soil, which leads to a significant decrease in crop yield. Mn-reducing bacteria promote the solubilization of Mn minerals, thus increasing Mn availability in soil. The present study aimed to assess the Mn solubilizing ability and plant growth-promoting potential of Bacillus spp. strains for maize plants with insoluble Mn compounds. Several Mn-solubilizing bacterial (MSB) strains were isolated from the maize rhizosphere using nutrient agar media amended with 50 mM MnO2. These strains were screened based on qualitative and quantitative solubilization of Mn, phosphorus, potassium, and zinc and production of ammonia. The majority of MSB strains were positive for catalase, protease, amylase, and oxidase activity, while more than 60% of tested strains were positive for lipase activity, and the production of indole-3-acetic acid and siderophores. Forty-five percent of the tested strains also showed solubilization of potassium. All the MSB strains were evaluated for their ability to promote plant growth and Mn uptake in the presence of MnO2 under axenic sand culture conditions. The results revealed that inoculation with MSB strains under sand culture significantly improved the growth of maize seedlings except for strains ASH7, ASH10, and ASH12. Comparatively, strains ASH6, ASH11, ASH19, ASH20, and ASH22 demonstrated a better increase in plant growth, fresh and dry biomass, and Mn uptake in roots and shoots than the other strains tested. All of these strains were identified as Bacillus spp. through 16S rRNA partial gene sequencing. Maize inoculation with these selected identified MSB strains also resulted in an increase in maize growth and nutrient uptake in maize roots and shoots under soil culture conditions in the presence of native soil Mn. The current study highlights the importance of MSB strain inoculation which could be a potential bioinoculants to promote plant growth under Mn deficiency.

Isolation And Characterization Of Biosurfactant Producing Bacteria From Different Environmental Soil Samples

Isolation And Characterization Of Biosurfactant Producing Bacteria From Different Environmental Soil Samples

First report of pearl millet bacterial leaf blight caused by Pantoea stewartii subspecies indologenes in India.

Pearl millet (Cenchrus americanus L.) field-grown plants of cv. 7042S shown unusual water-soaked lesions on leaf tips spreading towards the leaf base from Manasagangothri region (12.31°N 76.61°E), Karnataka, a southern Indian state during March 2020. Later those infected plants showed extensive necrosis and typical leaf blight symptoms with 70% disease incidence and 59% severity. Surface sterilized (3 x 3 mm) infected leaf tissues were crushed in 1mL sterile distilled water and streaked onto nutrient agar media. Bright-yellowish, circular, mucoid single bacterial colonies (PPi-M1) with regular margin were recovered after 24 hours of incubation at 28oC, and the same bacterial colonies were used for further biochemical and molecular characterization. The isolate, PPi-M1 found as gram-negative rods, gelatin, starch hydrolysis negative, and catalase, indole production positive. The partial sequence of 16S rRNA gene (primers: 27F/1492R) of the isolate PPi-M1 was amplified, sequenced, and curated sequence submitted to NCBI GenBank (accession number: MN808555). In nucleotide BLAST search for homologous sequences, 99.5% nucleotide matching similarity (1410bp) was observed with other Pantoea stewartii subspecies indologenes strains (MF163274; NR_104928) at NCBI database indicating that our isolate PPi-M1 belongs to this species. In Phylogenetic analysis using the Maximum Likelihood method and Tamura Nei model (1993), PPi-M1 formed a distinct cluster with other Pantoea stewartii strains with bootstrap value >95 and it was distant from P. allii, P. ananatis, P. agglomerans, and P. dispera. Besides, the subspecies-specific PCR assay and subsequent sequencing of galE and recA genes (primers: 3614galE/3614galEc; 3614recA/3614recAc; 372 and 223 bp) also confirmed the identity of the isolate as Pantoea stewartii subspecies indologenes. Further, the pathogenicity test was performed in-planta on 21 days old seedlings of pearl millet cv. CO-10. The bacterial suspension of isolate PPi-M1 (1x108 CFU/ml) was used for inoculation by leaf clipping method (Ke et al. 2017). All the inoculated plants (n=4 leaves per plant; 15 plants) maintained under greenhouse conditions (Temp: 27-29oC; RH: 80-85%) except mock (sterile water inoculation) shown similar water-soaked lesions from the cut end of the leaf, with a definite spreading margin and a typical leaf blight symptom in 8 dpi, as observed in the field. Re-isolated bacterial colonies from infected leaves shared similar morphological characters and molecular identity with inoculated culture, thus proving Koch's postulates. This pearl millet leaf blight causing bacterial strain PPi-M1 was deposited in the National Agriculturally Important Microbial Culture Collection, Mau, India (accession no.: NAIMCC-B-02508). Previously, P. stewartii was reported to cause leaf blight and rot diseases on rice and maize (Kini et al. 2016; Roper et al. 2011), also the international seed federation has instigated the phytosanitary measures highlighting its true seed transmission ability (Pataky et al. 2003). This study will supplement future pearl millet breeding programs, and to our knowledge, this is the first report of P. s. subsp. indologenes inciting pearl millet leaf blight disease in India.

First Report of Collar Rot in Purple Passion Fruit (Passiflora edulis) Caused by Neocosmospora solani in Yunnan province, China.

Purple passion fruit (Passiflora edulis Sims) is a perennial climbing vine native to South America that is grown worldwide as an edible tropical fruit with excellent nutritional value and high economic value (Zibadi et al. 2007). With the increasing expansion of the plantation area in China, considerable economic loss caused by collar rot has attracted wide attention. From 2018-2020, collar rot resulted in the death of many plants of P. edulis 'Mantianxing', a commercial cultivar in China, in southwest China's Yunnan province. The disease spread quickly, and field incidence reached more than 50%. Stem rot symptoms were observed at the base of the stem, about 5-10 cm from the ground, resulting in wilting, defoliation, and death of plants. Representative symptomatic samples were collected from the base of five plants, surface disinfested for 30 seconds with 75% ethanol and 15 min with 10% hypochlorite, washed three times with sterile distilled water, then transferred to potato dextrose agar (PDA) dishes. After 2 days in the dark at 28℃, emerging fungal colonies were purified on new PDA dishes cultured at 28℃ for 7 days. The mycelia were flocculent. The color of the surface and the reverse colony was white and cream, respectively. On synthetic nutrient agar (SNA) medium, microconidia were oval, ellipsoidal or reniform, 0- or 1-septate, and 6.7-23.1 μm in length (n>30); macroconidia were straight to slightly curved, 3- or 5-septate, and 30.8-53.9 μm in length (n>30). Genomic DNA, extracted from six isolates, was amplified with three pairs of primers, ITS1 and ITS4 (White et al. 1990) , EF1-728F and EF1-986R (Carbone and Kohn 1999), and fRPB2-5F and fRPB2-7cR (Liu et al. 1999). The amplicons from all six isolates were sequenced and identical sequences obtained. The sequence of one representative isolate was uploaded to NCBI (National Center for Biotechnology Information) and analyzed with BLASTn in the Fusarium MLST database (https://fusarium.mycobank.org). The sequence of the internal transcribed spacer 1 (ITS1) region (GenBank MN944550) showed 99.1% (449/453 bp) identity to Fusarium solani strain NRRL 53667 (syn: Neocosmospora solani, GenBank MH582405). The sequence of the translation elongation factor-1 (EF-1) gene (GenBank MN938933) showed 97.8% identity (263/269 bp) to F. solani strain NRRL 32828 (GenBank DQ247135). The sequence of the second largest subunit of RNA polymerase Ⅱ (RPB2) gene (GenBank MW002686) showed 98.7% identity (810/821 bp) to F. solani strain NRRL 43441 (GenBank MH582407). Based on a multilocus phylogenetic analysis of the ITS1, EF-1 and RPB2 sequences, coupled with the morphological characteristics, the isolate (designated as NsPed1) was considered to be Neocosmospora solani (syn: Fusarium solani) (Crespo et al. 2019). Subsequently, three-month-old healthy seedlings and 45-day-old cuttings of P. edulis 'Mantianxing' plants were inoculated with the isolate NsPed1 to test its pathogenicity. Stems were wounded, approximately 1-2 mm deep, in the collar region of plants at 2 cm above the soil. A disk (9 mm in diameter) of NsPed1-colonized PDA was placed on the wound. Sterile PDA served as controls. All plants were kept in a growth chamber with 28-30°C, 60% relative humidity, and 16/8-h light/dark photoperiod. Fifteen plants were used for each treatment and replicated three times. Two weeks after inoculation, the stems of the inoculated plants turned brown with a lesion, 2-5 cm in length, and the leaves wilted. These symptoms were similar to those of the diseased plants in the field. The control plants were asymptomatic. N. solani NsPed1 was re-isolated from the infected plants, satisfying Koch's postulates. Taken together, N. solani NsPed1 was identified as the causal pathogen of collar rot in P. edulis 'Mantianxing'. Knowledge of the causal organism of collar rot in purple passion fruit will lead to improved measures to prevent and control the disease in China and other countries.

Pigeonpea (Cajanus cajan L.) and Cowpea (Vigna unguiculata L.) Water as Alternative Growth Medium for Escherichia coli and Staphylococcus aureus in Laboratory with Minimum Infrastructure

The availability of non-synthetic media from natural ingredients is needed to answer the needs in laboratories where the price of nutrient media is quite expensive and there are limited supplies of material ware houses. Cowpea (Vigna unguiculata L.) and pigeonpea (Cajanus cajan L.) are the local foods of people of NTT (East Nusa Tenggara) which have a high enough nutritional content which has the potential to be developed into cheap, easy and simple non-synthetic media in making. The purpose of this study was to determine whether the agar media contained nutrient from cowpea and pigeonpea water can be used as a alternative for nutrient agar for the growth of Escherchia coli and Staphylococcus aureus bacteria. This research is a true experiment with posttest-only control design. The growth rate of S. aureus bacteria on pigeonpea medium, cowpea medium, nutrient agar medium, were 164 CFU/mL (SD=3,13), 161 CFU/mL (SD=3,02) and 164 CFU/mL (SD=3,21), respectively. The average growth of E. coli on cowpea medium, pigeonpea medium, and nutrient agar control medium were 163 CFU/mL (SD=2,79), 167 CFU/mL (SD-2,63) and 164 CFU/mL (SD=2,75) respectively. Test results ANOVA between pigeonpea medium, cowpea medium and nutrient medium in order to obtain p value = 0.145 (p> 0.05) for the growth of E. coli bacteria and p value = 0.393 (p> 0.05) for growth S. aureus. It was concluded that there was no difference between the number of bacterial colonies of E. coli and S. aureus on three medium. The pigeonpea medium and cowpea can be used to grow and alternative nutrient agar in order to grow bacteria E. coli and S. aureus.

Uji Daya Hambat Ekstrak Etanol Daun Inai (Lawsonia inermis L.) terhadap Pertumbuhan Staphylococcus aureus

Henna leaves are often used as traditional medicine, including as an antibacterial. Henna leaves contain several active components including phenols and flavonoids which act as antibacterial. The purpose of this study was to determine antibacterial activity of ethanol extract of henna leaves (Lowsonia inermis L.) against Staphyloccocus aureus. This research was conducted using descriptive method. In this study, ethanol extract of henna leaf was made by maceration method. Ethanol extract of henna leaves was tested against Staphylococcus aureus on Nutrient Agar (NA) medium using well diffusion method with 3 extract concentrations, namely 40%, 50% and 90%, tetracycline as positive control and aquadest as negative control with 3 repetitions each. The results of this study showed that inhibition zone diameter of henna leaves extract at a concentration of 40% was 21.29 mm, at a concentration of 50% was 23.1 mm and at a concentration of 90% was 24.4 mm, positive control was 27.93 mm and negative control did not show inhibition zone. The inhibition zone for all concentrations belongs to strong inhibition category.

Дослідження мікроцистинів як інгибиторів розвитку Phytophthora infestans

Information from foreign literary sources regarding microcystins of blue-green algae is presented. The current state of the problem of reservoirs «blooming» and the significance of this phenomenon for humans are reflected. The research was carried out in two stages: laboratory and full-scale. A pure culture of Phytophthora infestans was isolated, on which further studies were carried out. Isolation of isolates was carried out on agar nutrient medium. Potassium permanganate and ethanol were selected from the available antiseptics. Field experiments were carried out on experimental lines of Solanum lycopersicum by diagnosing signs of late blight disease. The determination of the potential negative effect of tomato plants treated with a suspension of cyanobacteria was carried out by the method of biotesting using Achatina fulica as a test object. The effect of blue-green algae toxins – microcystins – on colonies Ph. infestans in vitro is described. Photometric observation of the decrease in the number of colonies with a time interval of three days is presented. The phytophtorostatic effect of microcystins has been established. Degradation of Ph. infestans the next day were fixed after treatment of the colony with a suspension of cyanobacteria. An inhibitory effect was revealed, almost to the complete disappearance of symptoms in plants partially affected by late blight and grown in vivo. It has been determined that plants treated with microcystin can be considered safe for further consumption; the death of model organisms has not been registered.