Methyl Red Test Research Articles

Biochemical And Physiological Characterization Of Actinomycetes Isolated From Rhizospheric Regions In The Soils Of Arachis Hypogea L. And Gossypium Herbaceum L. Near The Gir Wildlife Sanctuary

30 isolates of actinomycetes were collected from the soil samples of Arachis hypogea L. and Gossypium herbaceum L. near the fields located surrounding the Gir Wildlife Sanctuary area of Junagadh District,Gujarat State,India. All the isolates were subjected to serial dilution process. The present study aimed to identify and characterize these isolates using biochemical and physiological tests. From the morphological tests and gram staining procedures conducted prior to the biochemical and physiological tests, it was already observed that Actinomycetes are Gram positive , filamentous shaped bacteria. It was also observed that these bacteria contain hyphae, mycelium as well as sporangia which is able to release spores. It was able to produce yellow pigments, posses an earthy aroma and showed optimum growth under aerobic conditions at temperature about 28 ⁰C, pH 7 and 5% w/v NaCl Concentration. Therefore, it was considered as a mesophilic, basophilic and moderate Salt tolerant by nature. The isolates showed variations in carbon utilization but able to utilize almost all carbon sources. These bacteria were also able to give positive results in methyl red test, nitrate reduction tests, urea hydrolysis, citrate utilization tests etc. Altogether, the results indicated that these isolates i.e. Actinomycetes are able to utilize almost all nutrient sources available in the rhizosphere region of Arachis hypogea L. and Gossypium herbaceum L.

Antibiotic sensitivity profile of multidrug-resistant (MDR) Escherichia coli isolated from dairy cow's milk in Probolinggo, Indonesia

Abstract. Widodo A, Lamid M, Effendi MH, Khairullah AR, Riwu KHP, Yustinasari LR, Kurniawan SC, Ansori ANM, Silaen OSM, Dameanti FNAEP. 2022. Antibiotic sensitivity profile of multidrug-resistant (MDR) Escherichia coli isolated from dairy cow's milk in Probolinggo, Indonesia. Biodiversitas 23: 4971-4976. The presence of resistant bacteria in animal products such as milk can be a new threat because it is directly related to the human food chain. Resistant Escherichia coli has been widely studied and detected in farms in developing countries. The aim of present study was to determine the antibiotic resistance profile of E. coli bacteria from dairy cows taken during the milking process from several dairy farms in Probolinggo district, Indonesia. A total of 150 milk samples were obtained from farms and E. coli was isolated and identified on Eosin methylene blue (EMB) media and biochemical test, such as Triple Sugar Iron Agar (TSIA) and indole test, methyl red test, Voges-Proskauer test, and citrate test (IMViC) were also performed. The antibiotic sensitivity profile was screened using the Kirby-Bauer test and results were interpreted according to the CLSI standard. The results showed that 124/150 (82.67%) E. coli bacteria exhibited highest percentage of antibiotic resistance to tetracycline (13.71%), streptomycin (9.68%), trimethoprim (8.87%), chloramphenicol (0.87%), and aztreonam (1.61%). A total of 9/124 (7.26%) E. coli isolates were detected as multidrug-resistant (MDR) and 1/9 (0.81%) E. coli isolate was suspected as extended spectrum ?-lactamase (ESBL) bacteria which was resistant to aztreonam antibiotic. Thus, the threat of multidrug-resistant (MDR) E. coli can come from milk which can affect public health.

Molecular detection of Enterotoxigenic Escherichia coli Toxins and Colonization Factors from Diarrheic Children in Pediatric Teaching Hospital, Sulaymaniyah, Iraq

Enterotoxigenic Escherichia coli (ETEC) is one well-established causative agent of diarrhea in the developing countries among young children. This prospective study was performed at Laboratories of University of Sulaimani (in Sulaymaniyah City/Iraq) from September to October 2021which aimed to determine the prevalence of ETEC among children and the most prevalence colonization factor (CFA/I) among ETEC. One hundred and twenty-five fresh stool samples were collected from hospitalized – children with diarrhea at Dr. Jamal Ahmed Rashid’s Pediatric Teaching Hospital. The collected samples were cultured on MacConkey and eosin methylene blue agar as selective and differential media for Gram- negative bacteria. Colonies were identified through Gram staining and biochemical tests including: Indole, methyl red, and catalase reaction test. Vitek-2 machine was depended to test some obtained isolates. Most of isolates (60%) showed positive results for E. coli – out of this percentage, 14 (18.66%) were positive for ETEC using polymerase chain reaction assay identifying stable and labile toxins (LTs). It was noticed that all of the ETEC isolates were stable toxin producer isolates whereas LT producer isolates were not identified. Colonization factor 5 (CS5) has been detected among three ETEC isolates (21.42%), meanwhile, 11 isolates (78.57%) have not expressed colonization factors at all.

Biochemical Characterization and Antimicrobial Susceptibility Test of the Bacterial Strain Isolated from Sandwich in Rajshahi University, Bangladesh

This study was conducted to isolate and observe the morphological and biochemical characteristics of bacterial strains present in the sandwich. A single bacterial colony was isolated from a sandwich collected from different restaurants located in the area of the University of Rajshahi by plating from the diluted primary bacterial suspension of the liquid medium onto an agar solidified mineral salt medium after purifying through filter paper. The isolated bacterium was found to be Gram-positive, coccus, motile, lactose-non-fermenting, and could utilize different carbohydrates. Bacterial strain A showed a positive result for the Methyl Red test, the Catalase test, the Indole test, and the Simmons citrate agar test. The optimum culture condition of the isolate was pH 8 and the salt concentration was 0.1 gm/100 ml. The Minimum Inhibitory Concentration (MIC) value against Vancomycin was 50mg/ml and the viable cell count indicated 459×107 CFU/ml. The result showed that the isolated bacterial strain A was resistant to Vancomycin and amoxicillin, whereas it was susceptible to gentamycin, ciprofloxacin, and chloramphenicol. This bacterial strain A can grow to a harmful extent after a certain time of incubation, which may cause a health hazard.

Ignatzschineria rhizosphaerae sp. nov. Isolated from Rhizosphere Soil of the Halophyte Kalidium cuspidatum.

A Gram-staining negative, non-motile, aerobic, rod-shaped bacterium, designated strain HR5S32T, was isolated from rhizosphere soil of the halophyte Kalidium cuspidatum, in Tumd Right Banner, Inner Mongolia, northern China. Strain HR5S32T grew at 10-40°C (optimum 30°C), pH 6.0-10.0 (optimum pH 9.0), and 0-12% (w/v) NaCl (optimum 2%). It was positive for catalase, methyl red test, Voges-Proskauer test, and nitrate reduction, but negative to oxidase, urease and hydrolysis of Tween 80. The phylogenetic trees based on the 16S rRNA gene sequences and whole genome both showed that strain HR5S32T was most closely related to Ignatzschineria indica FFA1T (= KCTC 22643T). Ubiquinone-8 (Q-8) was the major respiratory quinone. Phosphatidylglycerol, phosphatidylethanolamine, and phospholipid were the major polar lipids. Its major fatty acids were Summed features 8 (C18:1 ω6c and/or C18:1 ω7c), C16:0, Summed features 3 (C16:1ω6c and/or C16:1 ω7c), and C14:0. The genome consisted of a 3,074,733bp circular chromosome, with a G + C content of 38.8%, predicting 2,763 coding sequence genes, 70 tRNA genes and 6 rRNA. The values of the average nucleotide identities (ANI) and digital DNA-DNA hybridization (dDDH) values of strain HR5S32T to I. indica FFA1T were 74.6% and 22.0%, respectively, hence significantly lower than the thresholds of 95% for ANI and 70% for DDH for species delineation. The results of phenotypic, physiological, genotypic, and phylogenetic tests allowed the differentiation of strain HR5S32T from its closely related species. Ignatzschineria rhizosphaerae sp. nov. is therefore proposed, and the type strain is HR5S32T (= CGMCC 1.19435T = KCTC 92093T).

Occurrence and Antimicrobial Susceptibility Profile of Escherichia Coli Isolates from Vended Milk in University of Abuja Community, Federal Capital Territory, Nigeria.

Purpose: This study was conducted to establish occurrence and antimicrobial susceptibility profile of Escherichia coli (E. coli) isolates from cow milk (Nono) sold within University of Abuja campuses.
 Methodology: Fifty (50) samples of “nono” milk were randomly collected from vendors in mini and main campus of University of Abuja from August to October 2021. All samples were analyzed using pour and surface plating methods. Presumptive E. coli isolates on Eosin methylene blue agar were characterized using preliminary and standard biochemical identification methods such as microbial plate count, Gram staining, oxidase, indole, methyl red, voges-proskauer, citrate and catalase tests. Antimicrobial susceptibility testing was conducted on positive isolates using the disc diffusion method.
 Findings: Out of the 50 samples of nono milk cultured, thirty (30) yielded growth for Escherichia coli which appeared as greenish metallic sheen on EMB agar plate and Gram-negative short rods on microscopic examination, representing an overall prevalence of 60%. The overall viable count of all samples was extremely high, with 8.7x10^7cfu/ml recorded as the highest colony forming unit per milliliter (CFU/ml) and 1.5×10^7cfu/ml recorded the lowest colony forming unit per milliliter (CFU/ml). All isolates were indole positive, methyl red positive, oxidase negative, citrate negative, catalase positive and voges-proskauer negative. E. coli isolates in this study were susceptible to sparfloxacin (66.7%), gentamicin (66.7%), ciprofloxacin (60%), streptomycin (60%), and tarivid (53.3%) and resistant to Septrin (73.3%), Augumentin (60%), Pefloxacin (66.7%), chloramphenicol (60%) and amoxicillin (53.3%). In conclusion, this study showed that the degree of contamination of E. coli in vended milk in the study area is higher than the maximum permissible bacteria count recommended by Codex Alimentarius standard for fermented milk products which is indicative of potential hazard to consumers.
 Recommendation: Public enlightenment on hygienic handling of milk during milking and especially vending by milk vendors is hereby recommended. Use of cool thermos for milk hawking is also advocated.

Coliform bacteria screening and evaluating chemical composition of raw milkfrom dairy farms of Sylhet Sadar, Bangladesh

The study aims screening of coliform bacteria and evaluates chemical constituents of farm supplied raw milk at Sylhet sadar, Bangladesh. A total of 50 milk samples (5 samples from each farm) were collected from randomly selected 10 different farms. Samples were diluted (1:1000000) and bacteria cultures were done in EMB (Eosin methylene blue) agar. Further, Indole test, Methyl red test, Voges-Proskauer were done to differentiate environmental and fecal coliform (Escherichia coli). Chemical analysis of milk was done using automatic milk-analyzer. Total coliform count (TCC) was found to be ranged between 0.6 × 106 to 7.8 × 106 colony forming unit/ml (cfu/ml) and the biochemical tests showed negative results for Indole and Methyl red test and positive for Voges-Proskauer test, respectively confirming the absence of fecal coliform (Escherichia coli) and presence of environmental coliforms. Mean values of Chemical constituents were found to be fat% (4.48%), Solid-Not-Fat% (8.55%), Protein% (3.20%), Lactose% (4.66%) and Mineral% (0.68%). Adulteration with added water in milk was also found with a mean of 0.89%. This study will help to generate an idea about coliform contamination scenario and quality of farm milk found at the particular study area.

Pathological And Cultural Characteristics Of Different Isolates Of Xanthomonas Axonopodis Pv. Malvacearum

Bacterial blight caused by Xanthomonas axonopodis pv. malvacearum infects nearly all crop stages and results in significant loss in cotton yield, seed index, oil percentage and ginning out turn. Bacterium pathogenic, cultural and biochemical variability were assessed in the laboratory, and the results revealed that the isolates Xam-2, Xam-5 and Xam-8 were the most virulent of the tested isolates. In terms of cultural and biochemical tests,the bacterium was gram negative, rod shaped and produced mucoid colonies that were slightly raised, circular, light to dark yellow colour, on NA medium. Showed positive results in KOH solubility, starch hydrolysis, catalase, gelatine liquefaction, tolerance to salt 1% test and negative to indole and methyl red test.

Isolation and characterisation of endophytic bacteria present in the leaves of Glycosmis pentaphylla (Retz.) Correa

Endophytes are an endosymbiotic group of microorganisms that dwell in plant tissues and are reservoirs of bioactive compounds. However, the researches on this area are limited. The present investigation was undertaken to isolate and characterise endophytic bacteria from the leaves of Glycosmis pentaphylla (Retz.) Correa. An effective surface sterilisation procedure was developed from the experiment to isolate endophytic bacteria from the leaves of the candidate species. There was no bacterial growth observed in the sterility test. A total of 3 endophytic bacteria were isolated from leaves of G. pentaphylla. Isolates showed distinct morphological and biochemical characteristics. Biochemical characterisation of the isolates was performed by following Bergey’s manual of systematic bacteriology. Two isolates were found gram-positive, and one was gram-negative. All three isolates were found positive for the catalase test and negative for the indole test; two isolates (GP-1, GP-3) were positive for the oxidase test; 2 isolates (GP-1, GP-2) were found positive for both citrate and methyl red test. Some plant growth-promoting activities of the isolates were also performed. All the 3 isolates (GP-1, GP-2, and GP-3) were found positive for the ammonia production test; isolate GP-2 was found positive for phosphate solubilisation test; isolate GP-3 was found positive for both IAA production and lipase activity test. The isolated endophytic bacteria survived in different salt concentrations.

Characterization of Bacteria Isolates from Surfaces of Corroded Zinc and Copper Coupons

This research characterized bacteria isolates from surfaces of corroded zinc and copper coupons. Corroded zinc and copper materials were collected carefully, from two different locations in Ken Saro-Wiwa Polytechnic Campus and were put in sterile McCartney bottles. Bacteriological analysis such as Gram staining, motility test, catalase test, indole test, oxidase test, methyl red test, Voges-Proskauer test, and citrate utilization test were carried out t o ascertain the bacteria responsible for the corrosion. Two bacteria were isolated; Enterococcus which was seen to be responsible for the corrosion of zinc and Escherichia coli; which was responsible for the corrosion of copper. It is recommended that materials that can inhibit biocorrosion should be used to control or treat metallic substances of corroded materials.

Assessment of Microbiological Quality of Raw Milk and Identification of Pathogenic Bacteria

Milk contains important nutrients such as minerals, vitamins, proteins and lipids and are consumed by all age group of humans around the world. It is impossible to avoid contamination of milk with micro-organisms because presence of nutrients therefore quality of milk can be determined by the microbial content in milk. Objective: To investigate the microbiological quality of raw milk. Methods: In the present study, there were 30 cow milk samples collected from different dairy farms of Lahore. Firstly, a surf field mastitis test was performed for detection of clinical and sub-clinical mastitis. The microbial isolation was performed by microbial culturing and biochemical tests and antibiotic sensitivity test was performed for isolated bacteria. These isolated bacterial DNA was extracted and amplified by 16S rRNA PCR. The precipitated amplicon was sequenced by 16S rRNA sequencing. The results were evaluated statistically to check the level of significance among them. Results: The Chi-square values of catalase test, oxidase test, indole test, methyl red test, Voges Proskauer test and triple sugar iron were 12.42, 13.77, 8.77, 9.02, 10.67 and 4.29 respectively and the p-values were 0.034, 0.031, 0.042, 0.039, 0.044 and 0.056 respectively on MacConkey Agar. The Chi-square values of catalase test, oxidase test, indole test, methyl red test, Voges Proskauer test and triple sugar iron were 12.44, 11.98, 9.38, 7.02, 14.22 and 10.43 respectively and p-values were 0.034, 0.045, 0.039, 0.012, 0.022 and 0.053 receptively on Mannitol salt Agar. The Chi-square and p-values of gram staining bacteria were 13.99 and 0.034 respectively and showed the significant relationship among them. Mastitis test were presented the value of Chi-square 17.86 and p-value 0.029. The ANOVA table on DNA isolation method were exposed the highly significant relationship among the variables. Conclusions: There was a significant association between different treatments. Different pathogens can grow in milk and milk products and produce toxic metabolites. Products that are contaminated by these toxic metabolites when consumed may results in food poisoning

Sensitivity Test of Bandotan Leaf Extract (Ageratum conyzoides) Against Pseudomonas aeruginosa Bacteria

The leaves of Bandotan (Ageratum conyzoides) are a plant thought to have antibacterial properties. This study aims to determine the sensitivity of Bandotan leaf extract in inhibiting the growth of the bacteria Pseudomonas aeruginosa. This study used a stock extract of Bandotan leaves from the Pharmacology Laboratory and a bacterial isolate of P. aeruginosa in the Microbiology Laboratory of the Faculty of Veterinary Medicine, Universitas Syiah Kuala, which was identified by Gram staining, indole test, Methyl Red test, and confectionery test. The research method was carried out by planting the re-identified bacterial isolates on Nutrient Broth (NB) media, incubated at 37ºC for 24 hours. Then the turbidity composition of the isolates was arranged to match the turbidity in 0.5 McFarland solution. Furthermore, the sensitivity test of the extract on Mueller Hinton Agar (MHA) media was carried out by levelling the bacterial isolates on the surface of the media and attaching a disc containing bandotan leaf extract with a concentration of 25%, 50%, 75% and gentamicin disk as a positive control and distilled water as a negative control. All treatments were incubated at 37ºC for 24 hours, and then the inhibition zone was measured using millimeters (mm) callipers. The results showed that concentrations of 25%, 50% and 75%, respectively, had an inhibition zone of 8.16 mm, 9.82 mm, and 16.08 mm, respectively. In contrast, the average inhibition zone for gentamicin was 25, 30 mm and 0 mm distilled water. Therefore, it can be concluded that the Bandotan leaf extract is sensitive to growth inhibition of P. aeruginosa bacteria.

Prevalence of Urinary Tract Infection during Pregnancy at Tertiary Care Hospital: A Cross-Sectional Study

Aim: the aim of this study was to determine the prevalence of urinary tract infection during pregnancy and the microorganism associated with it. Study design: A cross-sectional study Place and Duration: This study was conducted at CDF Hospital Hyderabad, Pakistan from June 2020 to June 2021 Methodology: A total of 150 women were included in this study. All the women were pregnant and aged between 18 to 50 years. Urine samples were collected from all the participants. From each sample, bacteria were isolated, and then the isolated bacteria were stained. Also, indole test, catalase test, voges proskauer test, urease test, citrate utilization test, methyl-red test, and coagulation test were performed for the identification of the bacteria. Results: The results of this study showed that almost 16 percent of pregnant women had urinary tract infections. Women aged from 20 years to 30 years showed a higher incidence of urinary tract infection. Urinary tract infection was more prevalent in unemployed participants than in employed participants. In the same way, participants belonging to rural areas had a higher urinary tract infection rate as compared to patients belonging to urban areas. Educated women showed less incidence of UTI as compared to uneducated or less educated women. The results also showed that the women in the third trimester of their pregnancy had the highest prevalence of UTI. Escherichia coli was the most common cause of UTI in our study. The least common cause of UTI was Staphylococcus epidermidis. Conclusion: This study concluded urinary tract infection is one of the most common complications during the gestational period. Keywords: urinary tract infection, bacteria, pregnancy, prevalence.

Complementation of Biochemical and Physiological Assays with Functional PGPR Based Assays to Screen Potential Isolates

The work deals with the biochemical characterization of rhizospheric isolates including Indole, Methyl Red, Voges Proskauer, Citrate test (IMViC test) based quantitative biochemical assay, PGPR properties like nitrogen assimilation (in microbes), phosphate solubilization etc. A special aspect of finding the correlation between the biochemical tests and PGPR properties were also studied. Methyl red test and tryptophanase assay (by indole test) were found to be positively and negatively correlated with phosphate solubilization. Vogues Proskeur and citrate utilization tests were negatively correlated with the phosphate solubilization. In future, these biochemical tests can be used as determining factors to identify phosphate solubilizing bacteria. Moreover, a group of bacteria was identified by the scatter plot analysis which shows low acid production with high phosphate solubilization. Lastly, we have given a new approach of screening rhizospheric bacteria based on motility on nitrogen deficient media.

Bacteriological quality of community well water and public health concerns in Enugu urban, Nigeria

Background: Water is a basic necessity used by humans for both domestic and industrial uses. Next to air, water is essential to life. It takes up about 71% of the earth’s surface. The objective of this study is to determine the bacteriological quality of well water in Enugu urban, Nigeria
 Methodology: A total of 60 domestic wells were selected from Abakpa, Obiagu and Achara layouts in Engu urban, Nigeria by stratified random sampling method, with 20 wells selected from each area based on location of well sites and construction parameters. Water samples were collected from each well using a sterile 200ml plastic bottle for bacteriological analysis to estimate total bacteria count in colony forming unit (cfu)/ml, total coliform count in most probable number (mpn)/100ml, and faecal coliform count in most probable number (mpn)/100ml. Bacterial isolates were identified using Gram reaction and conventional biochemical tests including catalase and coagulase for Gram positive bacteria, and oxidase, citrate utilization, hydrogen sulfide, indole, urease, methyl red, Voges Proskauer, and sugar fermentation tests for Gram negative bacteria. Antibiotic susceptibility testing (AST) of each isolate was performed by the disk diffusion method against selected antibiotics including penicillin G (10µg), ciprofloxacin (5µg), streptomycin (10µg), amoxicillin-clavulanic acid (20/10µg), and trimethoprim-sulfamethoxazole (25µg), and result interpreted using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) break points. Comparative statistics of the data was performed using analysis of variance (ANOVA) with p<0.05 considered statistically significant.
 Results: The well water in the three layouts were heavily contaminated as shown by comparatively high mean total bacteria counts of 0.8825±0.66x104 cfu/ml, 0.8435±0.6413x104 cfu/ml, and 0.8384±0.5948x104 cfu/ml for Abakpa, Obiagu and Achara layouts respectively (p=0.9714). The mean total coliform counts were 5.15±5.284, 5.45±4.31 and 5.05±4.763 mpn/100ml (p=0.8038), and the mean faecal coliform counts were 2.4±3.393, 2.65±2.796 and 2.05±2.35 mpn/100ml (p=0.9631) for Abakpa, Obiagu and Achara layouts respectively. A total of 50 pathogenic bacterial isolates were identified; Klebsiella pneumoniae 21 (43.8%), Escherichia coli 13 (30.0%), Proteus spp 6 (12.5%), Pseudomonas aeruginosa 6 (12.5%), and Staphylococcus aureus 2 (4.2%). The AST result shows that 75% of K. pneumoniae, E. coli, Proteus spp and S. aureus were resistant to all five antibiotics tested.
 Conclusion: These findings showed high faecal contamination of domestic well water sources, which poses a significant infection risk to the community. Proper water treatment measures and personal hygiene practices are recommended, and well sites should be located at a safe distance from septic tanks, pit latrines, flowing gutters and refuse dump sites.

Multi-drug Resistant Escherichia Coli from Poultry: A Potential Source of Antimicrobial Resistance Spread in Poultry Farm Environment

Background: Escherichia coli is known to inhabit the gastrointestinal tract of poultry and other animals. E. coli infection can cause major economic losses in poultry production. The development of resistance to the commonly used antimicrobials in the treatment of such infection can be a setback in the poultry sector. Objective: This study was carried out to determine the antibiotic resistance profile of E. coli isolated from chickens in Imo State, Nigeria. Materials and Methods: Twelve poultry farms were selected across the 3 senatorial zones of the state using purposive random sampling. A total of 120 cloacal samples were collected from chickens using sterile swabs. The samples were streaked onto MacConkey agar (MCA) and incubated at 37o C overnight. Pink colonies on the MCA were streaked onto eosin-methylene blue agar, incubated for 24 hours at 37o C, and confirmed by indole, methyl red, Voges-Proskauer, and citrate (IMVIC) tests. Antimicrobial susceptibility test was carried out using disc diffusion technique, and the inhibition zone diameter was measured and recorded as resistant or susceptible. Results: Twenty isolates out of the 120 samples were identified as E. coli. Twenty isolates were highly resistant to 5 antibiotics out of 10 antibiotics used. Antibiotic resistance patterns were as follows: amoxicillin (AMP) (95%), cephalothin (CEP) (90%), nalidixic acid (NA) (85%), trimethoprim (SXT) (85%), cefoperazone (PEF) (80%), ampicillin (AMP) (70%) ofloxacin (OFX) (15%), ciprofloxacin (CIP) (10%), gentamicin (CN) (10%), and streptomycin (S) (10%). Sixteen resistance patterns were recorded with AMC-SXT-AMP-CEP-NA-PEF being the most prevalent. Conclusion: This study shows that multi-drug resistant E. coli strains are present in poultry farms in Imo State.

First Report of Pantoea dispersa Causing Brown Blotch Disease in Flammulina filiformis in China.

Flammulina filiformis (previously known as F. velutipes) is one of the most frequently cultivated and consumed edible mushrooms in China. In October 2020, brown blotch disease was observed on the pileus of F. filiformis at a mushroom factory in Ganzhou (25.74°N; 114.95°E), Jiangxi, China, with a disease incidence of approximately 6%. Symptoms initially appeared as small, irregular spots on the infected pileus, with color ranging from pale yellow to light brown. Such spots were enlarged and pitted at high relative humidity within several days, and finally caused malformation of the caps and yield reduction. To isolate the causal agent, the blotches on F. filiformis caps were homogenized and diluted with sterilized distilled water, and the resulting suspension (100 μl) was spread onto LB agar plates. After incubation at 28°C for 48 h, three colonial types were obtained: (i) yellow, convex, and smooth colonies, (ii) light yellowish, irregular, and rough colonies, and (iii) milky white, glistening, and smooth colonies. The first colonial type was predominant. A single colony of each type was randomly selected and streaked on fresh LB agar plates to obtain pure cultures namely PF1, PF2 and PF3 respectively. To test pathogenicity of the three isolates, young F. filiformis fruiting bodies (8- or 10-day-old primordium) grown in culture bottles were inoculated by spraying a bacterial suspension (108 CFU/ml, 3 ml per bottle on average), and cultivated in a mushroom house at 15±2°C and 95% relative humidity. The fruiting bodies sprayed with sterilized distilled water served as controls. The pathogenicity tests repeated three times, and at least five culture bottles were included in each experiment. Among the three types of bacteria, only strain PF1 induced symptoms similar to the original disease. The brown spots were observed on treated pileus 10 days after inoculation, and a fresh weight reduction of 30.9% per culture bottle was observed. In contrast, those fruiting bodies treated with water remained asymptomatic. Same yellow colonies were also re-isolated from the infected pileus, and identified by subsequent methods. The strain PF1 was gram negative, motile, and short rods. Biochemical analysis showed that the strains belonged to genus Pantoea (positive for citric acid, inositol, mannitol, methyl red test, and Voges-Proskauer test but negative for lysine, ornithine, phenylalanine, H2S, urease, D-melibiose, sorbitol, adonitol, and raffinose). Further PCR amplification and sequencing of four genes, 16S rRNA gene with primer 27F/1492R, fusA gene with primer fusA3/fusA4, gyrB gene with primer gyrBf1/gyrBr1, and rpoB genes with primer Vic3/Vic2 (Delétoile et al. 2009; Palemon et al. 2021), were performed to identify the species. A BLASTn showed 100%, 100%, 99.51%, and 99.26% homology, respectively, with those of P. dispersa (MT072166, CP045216, CP076369, MH015168). The four gene sequences were deposited in GenBank (accession numbers: MZ373179, MZ393661, MZ393662, MZ393663). A phylogenetic analysis based on the four concatenated genes also showed that the strain PF1 well clustered with the type strain of P. dispersa. This species has been reported to cause leaf blight in rice (Toh et al. 2019), soft rot in Agave angustifolia (Palemon et al. 2021), and bulb decay in onion (Chang et al. 2018). To the best of our knowledge, this is the first report of P. dispersa causing brown blotch diseases on cultivated F. filiformis, which was previously known to be caused by Pseudomonas tolaasii (Lee et al. 2002). Our results also indicate P. dispersa could induce malformation of pileus and lead to a severe yield loss if not controlled effectively. Therefore, it should be considered in future disease management of F. filiformis cultivation.

Isolation, Biochemical Characterization and Antagonistic Study of Pseudomonas Fluorescens in Soil Collected from Different Locations in Kerala

Plant Growth Promoting Rhizobacteria (PGPR) is bacteria that populates roots and enhance the growth by a variety of mechanisms. Generally, PGPR promote plant growth directly by either increasing nutrient acquisition for example nitrogen, phosphorus, potassium and essential minerals, or modulating plant hormone levels, or by indirectly decreasing the inhibitory effects of pathogens on plant growth and development as biocontrol agents. In this study, an attempt was made to collect rhizospheric soil samples with native Pseudomonas population from different locations of Kerala state and to identify the native Pseudomonas fluorescens strains, a potent biocontrol agent in controlling pest, insects, nematodes and various pathogenic microbes as well as PGPR in the rhizosphere under UV light and further characterize them morphologically and culturally. Utmost P. fluorescens strains showed positive PGPR exertion. The study showed that Pseudomonas is an effective factory growth promoting bacterium. In the present study, soil samples were collected from different locales in Kerala (Trivandrum, Kollam and Kottayam) in order to insulate the soil microbes and also checking its negative parcels with other microbes. The observed colonies were identified as Pseudomonas fluorescens by checking the green fluorescence emitted by the colonies when exposed to UV light. These fluorescent colonies were selected and confirmed as Pseudomonas fluorescens using biochemical tests like MR test, Citrate utilisation test and Catalase test. The Methyl red (MR) test was used to identify the glucose utilizing property of Pseudomonas fluorescens. A change from yellow to cherry red colour indicates positive result. The Citrate Utilisation test was used to identify the ability of Pseudomonas fluorescens to utilize citrate as its sole carbon and energy source thereby confirming that the isolated colony is of the particular species. Catalase test was used to identify the ability of catalase containing Pseudomonas fluorescens to convert Hydrogen peroxide into Water and Oxygen. Presence of thick effervescence was noted confirming the catalase utilising ability of Pseudomonas fluorescens. Coagulase test is used in order to differentiate Staphylococcus aureus that is positive which produce the enzyme coagulase, from S. epidermis and S. saprophyticus negative one which do not produce coagulase.

Evaluation of Antibiotic Resistant and Metal Tolerances Capability of Petroleum (hydrocarbon) Degrading Bacteria

Diesel oil is considered one of the important products of crude oil which constitute a vital source of pollution in the environment. Biodegradation of complex hydrocarbon requires cooperation of more than a single species. This is true in case of pollutants made up of crude oil or petroleum. Soil contains varieties of microorganism that can be established in any natural environment. The bacterial isolates were characterized biochemically by indole test, methyl red test, citrate utilization Test, voges proskauer test, starch hydrolysis, catalase production test, nitrate reduction test, antagonism test, oligodynamic susceptibility and antibiotic sensitivity tests.

Isolation and Characterization of Bio-surfactant Producing Bacteria

Oil spillages are so common in many fields which lead to contaminated soil. Soil polluted land filling contains bacteria that produce a broad spectrum of bio-surfactants. Bio-surfactant as an efficient biological surfaceactive agent may provide an alternative solution for the removal of heavy metals from industrial wastes. The biosurfactant has more potential efficiency in the process of remedying natural resources. Surfactin, Iturin and Fengycin are widely studied bio-surfactants. The present study was aimed to isolate and characterize the biosurfactant producing bacteria from the soil samples collected from the petrol bunk. Two isolates obtained were used for the further analysis. Catalase, Lactose fermentation, Citrate utilization tests were positive for isolate 1 and identified as Staphylococcus sp. Starch hydrolysis, Methyl red test, Catalase, Lactose fermentation, Citrate utilization tests and Nitrate reduction were positive for isolate 2 and identified as Bacillus sp. The samples were anlayzed for the Bio-surfactant property using Haemolytic activity, oil drop assay, oil collapse activity and emulsification test. The functional group was detected using the FTIR analysis. Bacillus sp. strains have more potent biosurfactant property than Staphylococcus sp. and they act as bio-surfactants in removing the stains. Biochemical characterization of isolated samples, determination of bio-surfactant producing organism by oil spreading activity, drop collapse assay, oil spreading activity and biofilm formation were confirmed by Bacillus species bacteria