Agar Medium Research Articles

IN VITRO PROPAGATION COLLA AROMATIC ROXB

This experiment was tested the propagation of Colla aromatica Roxb with tissue culture, the study objective to study of propagation possibility of Colla aromatica Roxb (Homalomena aromatica Schott. Araceae), this study found the possibility of disinfecting Colla aromatica Roxb using sodium hypochlorite solution in contains of 15 and 20 for 5 and 10 minutes, bacteria contamination was 0%, fungal contamination was 14.29%, the survival rate was 85.71% At the same time without Chemicals stimulate growth of plant, can't cause callus form callus in the case . After rearing young shoots of for 30 days, feeding Benzyl Adenine Pure (BAP) at a contains of 3 mg/l combined with Naphthalene Acetic Acid (NAA) at a contain of 1 mg/l, there was a high number of shoots 1.88 shoots, but at the same time, giving NAA only was found to be statistically different. after culture the shoots in Agar media for 60 days, it was found that the result of giving NAA combined with BAP for the elongation of the shoot is not statistically different, from the observation that giving NAA combined with BAP with a high contains, will have the characteristics of the shoot elongation that is better than giving NAA combined with low contains

Pyricularia oryzae enhances Streptomyces griseus growth via non-volatile alkaline metabolites.

Chemical compounds that affect microbial interactions have attracted wide interest. In this study, Streptomyces griseus showed enhanced growth when cocultured with the rice blast fungus Pyricularia oryzae on potato dextrose agar (PDA) medium. An improvement in S. griseus growth was observed before contact with P. oryzae, and no growth-promoting effect was observed when the growth medium between the two microorganisms was separated. These results suggested that the chemicals produced by P. oryzae diffused through the medium and were not volatile. A PDA plate supplemented with phenol red showed that the pH of the area surrounding P. oryzae increased. The area with increased pH promoted S. griseus growth, suggesting that the alkaline compounds produced by P. oryzae were involved in this growth stimulation. In contrast, coculture with the soilborne plant pathogen Fusarium oxysporum and entomopathogenic fungus Cordyceps tenuipes did not promote S. griseus growth. Furthermore, DL-α-Difluoromethylornithine, a polyamine biosynthesis inhibitor, prevented the increase in pH and growth promotion of S. griseus by P. oryzae. These results indicated that P. oryzae increased pH by producing a polyamine.

Isolation and Molecular Characterisation of Chlorogonium sp. from Industrial Wastewater

Microalgae are photosynthetic, unicellular microorganisms also known as phytoplankton. They are small plant-like entities. In this research, the sample were collected from cement factory in a sterilised 20L container wrapped with foil paper and were transported down to Federal University, Wukari where it was kept in refrigerator at biochemistry laboratory. Blue-Green media (BG-11) was prepared. Wastewater containing Microalgae obtained from cement wastewater pond were cultivated in BG-11 medium to determine the growth of the organism. BG-11 medium contained macronutrients, trace metals and some vitamins which aid the growth of the organism. The wastewater sample containing with suspected microalgae was inoculated (inoculum ratio = 25%) and incubated under atmospheric CO2 at room temperature (30±2°C) in our laboratory for two to three weeks during the incubation period. In order to purify the isolates, the upper growth layer was first decanted into a freshly produced medium and then plated on BG-11 media that had been solidified with 1% agar-agar. For several of the cultures, growth on the agar plates continued for around three weeks. Following repeated sub-culturing, the emerging greenish colonies were re-emerged into a sterile BG-11 agar medium. In isolation of organism from the industrial cement waste water, the isolate was identified by morphological and molecular identification by extracting the DNA, run the electrophoretic analysis and PCR using primers for 18S rRNA eukaryotic microalgal and then run the sequence analysis. The results of this study obtained, indicated that, the electrophoretic result show the band has 1800-2000base pair and the organism isolated from the industrial cement waste water were chlorogonium sp. with a percent similarity of 78.65% and accession number of OR886595 based on data Gene Bank blast results.

Characteristics of Candida albicans Derived From HIV-Positive Individuals With Oral Candidiasis: Genotyping, Phenotypic Variation, Antifungal Susceptibility, and Biofilm Formation.

Oral candidiasis (OC) is one of the most common mucosal infections in those afflicted with HIV/AIDS. This study aimed to provide detailed information on the phenotype, genotype, antifungal susceptibility, and biofilm formation ability of oral Candida albicans isolated from HIV-infected patients with OC. A total of 25 C. albicans isolates were collected from oral lesions of HIV-infected patients referred to Behavioral Diseases Counseling Center affiliated with Ahvaz Jundishapur University of Medical Sciences, Iran. The antifungal susceptibility testing was done according to CLSI M27 guideline (fourth edition). The crystal violet method was used to evaluate the biofilm formation ability of isolates. Different phenotypes were identified on yeast extract-peptone-dextrose agar medium supplemented with phloxine B. Genotyping analysis of the isolates was performed using high-resolution melting (HRM) assays and ABC genotyping. The highest and lowest susceptibility of the C. albicans isolates was found for fluconazole 24 (96%) and ITC 18 (72%), respectively. Forty-eight percent of the isolates had high biofilm formation ability and exhibited gray cell type. The most common genotype was genotype B (52%). HRM analysis of HIS3, EF3, and CDC3 markers showed three, four, and five different groups, respectively. Investigating the phenotype, antifungal susceptibility and biofilm formation ability of the C. albicans isolates obtained from oral lesions of HIV-infected patients revealed that the dominant genotypes in the current research could cause more serious infections from the oral source. We recommend further research with a larger sample size to determine the molecular epidemiology of C. albicans among HIV patients in Iran.

Clear zone formation in microdroplets for high-throughput screening for lactic acid bacteria.

Droplet microfluidic-based technology is a powerful tool for biotechnology, and it is also expected that it will be applied to culturing and screening methods. Using this technology, a new high-throughput screening method for lactic acid bacteria was developed. In this study, the conventional culture of lactic acid bacteria that form clear zones on an agar medium was reproduced in water-in-oil droplets, and only the droplets in which lactic acid bacteria grew were collected one by one. Using this method, the specific recovery of Lactiplantibacillus plantarum from a mixture of L. plantarum and Escherichia coli and the acquirement of lactic acid bacteria from an environmental sample were successful. This method could be applied to various conventional screening methods using the clear zone as a microbial growth indicator. This has expanded the possibilities of applying droplet microfluidic-based technology to microbial cultivations.

Cellulase gene expression in the thermophilic Thermomyces lanuginosus isolated from compost

Thermophilic fungi are superlative microorganisms for enzyme production, especially cellulase, and their using in biotechnological applications is due to their stability at utmost temperatures. In the current investigation, we isolated six genera encompassing six fungal species and one species variety from 30 samples of compost at 45 °C and 55 °C. Thermomyces lanuginosus was the most rampant species. The colony diameter of T. lanuginosus ranged from 2.8 to 4.3 cm at 45 °C on yeast-starch agar (YpSs) medium with white or greyish-brown mycelia. Fifteen isolates of T. lanuginosus were cellulase producers with variable competencies with a C/Z range of 1.09–1.38 cm. Fascinatingly, the clear zone diameter was much bigger when using Iodine than those obtained using Congo red. T. lanuginosus isolate no. 33 produced substantial amounts of cellulase on the four used media: Corncob (CC), Corncob treated with NaOH (C-NA), Yeast Peptone Dextrose (YPD), and CarboxyMethyl Cellulose (CMC) with the highest activity on CC; 143.9 μg/min, also cellulase gene expression levels of cel6Aq, cel7Aq, and bgl3Aq genes exhibited higher fold changes in the CC condition (7.26-fold, 11.51-fold, and 3.39-fold, respectively). X-ray fluorescence (XRF) analysis revealed the presence of 11 minerals with higher concentrations in CC than in C-NA. Supplementation of corncob medium with rosemary essential oil (CR) completely inhibited cellulase production. It adversely affected the growth, and changes in conidia, which were depicted using a scanning electron microscope (SEM). Interestingly, the conidia appeared much bigger than other media, and the large conidia diameter was 10.2–12.1 μm.

First report of Rhizoctonia blight caused by Rhizoctonia solani on Chinese cabbage (Brassica rapa subsp. chinensis) in India.

During January and February 2021, foliar blight symptoms were observed on the leaves of Chinese cabbage (Pak choi) at Lembucherra research farm, College of Agriculture, Tripura, India. The incidence of disease symptoms ranged from 5 to 10% of the plants observed in the field. The symptomatic leaves showed grayish colored water-soaked lesions with an irreguar shape and size. A total of 10 symptomatic leaves (1 leaf per plant) from Chinese cabbage infected plant were sampled, surface decontaminated with 1% NaOCl, washed twice in sterile water, plated on 2% water agar, and incubated at 25 ± 2°C. Hyphal tips from mycelium of 7-day old culture (2 isolates from two different plants) with right-angled branching were transferred to potato dextrose agar (PDA) media (SRL, India). Cream or light brown hyphae that branched at right angles, with septa near the point of the origin of hyphae, and a slight constriction at the base of the branch) were visible under a microscope. Olive-brown sclerotia were observed after 5 days of incubation. Multiple nuclei per cell were visible after staining with 4', 6-diamidino-2-phenylindole (Estandarte et al. 2016). Based on morphological characteristics (Parmeter et al. 1970) the isolates TP36 and TP37 were identified as Rhizoctonia solani. The internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were amplified with ITS1& ITS4 (White et al. 1990) and (GAPDH F-5'- CAAGGAGAACCCAGGTGTTAAG-3' and GAPDH R- 5'-GGCGTCGAAGATAGAAGAGTGT-3') respectively for both isolates and sequenced (accession #. PP458158, PP458159, PP425343, PP425344). BLASTn analysis showed 99.26%( 668/673 nt) to 99.46% (659/664 nt) identity with R. solani sequences (GenBank MG397062.1 and KX674524.1) for ITS and 98.42% (552/562 nt) to 100% 540/540 nt)identity with R. solani sequences (GenBank HQ425709.1 and CP102644.1) for GAPDH. Isolates TP36 and TP37 were deposited in the Indian Type Culture Collection (ITCC), New Delhi as R. solani (nos. 9154 and 9319, respectively). Both isolates were amplified using (anastomosis group) AG1 subgroup specific primers (Matsumoto 2002; Prashantha et al. 2021) to identify their AG. The presence of a 265 bp amplicon for both isolates suggested that they belong to AG1-IA. A multilocus analysis of R. solani isolates from different host plants with concatenated sequences ITS and GAPH showed that TP36 and TP37 are closely related to rice isolate RS107. A pathogenicity test on five plants per treatment was conducted and repeated twice on one month old Chinese cabbage plants (hybrid, TOKITA, India) grown under glasshouse conditions in a sterilized mixture of soil and sand (3:1) at 27-28oC during January 2024 at ICAR-IARI, New Delhi. R. solani isolates TP36 and TP37 were grown on PDA and plants were inoculated by placing single sclerotia of 10-day old colony on different plant parts and covering it with moist cotton. After 7 day, typical lesions of R. solani infection were visible. No symptoms were observed on the control plants. The fungus was reisolated from the inoculated plants and identified as R. solani based on morphology. R. solani has previously been reported to cause disease on some members of Brassicaceae in different countries (Budge et al. 2009; Hua et al. 2014). Based on literature available this is the first report of R. solani infecting Chinese cabbage in India.

Mycobolome of Phialomyces macrosporus across OSMAC-based assorted culture media.

The fungus Phialomyces macrosporus was cultured using the One Strain Many Compounds (OSMAC) strategies to evaluate its metabolome. Variations in the nutrient culture media, culture regime, and cultivation parameters can significantly influence fungal extract quantity and chemical diversity. This study aimed to explore the mycobolome of P. macrosporus in five different culture media and two different cultivation conditions using NMR-based metabolomics. Principal component analysis (PCA) of 1H NMR spectra revealed clear differentiation between these samples, highlighting the rice dextrose agar medium (RDA) and potato dextrose broth (PDB) as standard complex media for conducting a fungal metabolite screening program.

In vitro Evaluation on Efficacy of Bioagents and Plant Extracts against Early Blight of Tomato (Alternaria alternata)

AIMS: In vitro evaluation on efficacy of Bioagents and plant extracts against Early blight of tomato (Alternaria alternata) Study Design: CRD (Completely Randomized Design). Place and Duration of Study: A trial was conducted in plant pathology laboratory at HRS Venkataramannagudem, Dr. Y.S.R. Horticultural University, Venkataramannagudem, during 2022. Methodology: The efficacy of bioagents were tested against isolates for radial growth inhibition on suitable media (Potato dextrose media and Nutrient agar media) using dual culture technique under in vitro conditions. The poisoned food technique was followed to evaluate theefficacy of botanicals in inhibiting the mycelial growth of test pathogen. Results: In the present investigation total nine bioagents and four plants extracts were tested against Alternariaalternata under in vitro. Results revealed that among the nine antagonists., Trichoderma harzianum, Tichoderma viride, Tichoderma longibraciatum (TCT4), Trichoderma reesi (TCT10), A10 (Tichoderma asperellum), A28 (Trichoderma harzianum), Trichoderma virens and two bacterial bioagents Pesudomonas fluorescens and Baciilus subtillis tested against A. alternata, maximum reduction in colony growth of A. alternata was observed in A10 (T. asperellum) (69.50%) and significantly superior over all other bioagents tested which was followed by A28 (T. harzianum) (62.96%) and the next best was T. virens (60.28%). Least inhibition was noticed in Bacillus subtillis (46.69%). Total four plant extracts neem leaves (Azadirachta indica), onion bulb (Allium cepa), garlic bulb (Allium sativum), and turmeric rhizome (Curcuma longa) tested at three concentrations (5, 7.5, 10 %) with suitable control by poisoned food technique against A. alternata, onion bulb extract (44.07%) which was found superior to all other tested botanicals. Least mean per cent inhibition was recorded with turmeric (21.34%).

First Report of Klebsiella variicola Causing Tomato Pith Necrosis in Yunnan Province, China.

Tomato (Solanum lycopersicum L.) is a key vegetable crop in China. In August 2023, an outbreak of bacterial pith necrosis in tomato occurred in Lufeng County, Yunnan Province, China, affecting over 40% of the tomato plants in a greenhouse. The stems of infected plants developed a waterlogged soft rot and the disease progressed, the lower leaves and lateral branches of infected plants gradually wilted and died. A longitudinal cut of the stem revealed hollow pith with brown vascular tissue. To isolate the pathogen, the plant surface was disinfested with 75% ethanol. Then, a piece of infected tissue from the base of the stem was excised and immersed in sterile water for 2 min. A small amount of liquid was streaked onto TTC (2,3,5-triphenyltetrazolium chloride) agar medium using an inoculation loop, and plates were incubated at 28℃ for 24 h. Colonies on the TTC plate were white, indicating that the pathogen was not Ralstonia solanacearum. Colonies grown on LB (Luria-Bertani) agar medium were randomly selected and subjected to preliminary pathogenicity tests. Based on the results, a colony named Kv4 was selected and purified through six subcultures in LB agar medium. Biochemical tests showed the strain utilized D-sorbitol, raffinose and citrate but not adonitol, and was positive for methyl red, D-glucose (acid), urea hydrolysis, lysine decarboxylase, and motility, and negative for phenylalanine deaminase, H2S production, indole production, and ornithine decarboxylase. These characteristics align with Klebsiella species (Garrity et al. 2007). To determine the species of strain Kv4, partial sequences of the 16S rDNA, phoE, leuS, and rpoB genes were amplified (Barrios-Camacho et al. 2019) and sequenced. Through BLASTn analysis, strain Kv4 sequences of 16S rDNA (OR888750) had 99.47% identity (1488/1496 bp), phoE (OR899599) had 98.69% (605/613 bp) identity, leuS (OR899598) had 99.07% identity (959/968 bp), and rpoB (OR899597) had 97.69% (633/648 bp) identity with Klebsiella variicola strain FF0907. Using the ClustalW algorithm in MEGA11 for nucleotide sequence alignment, phylogenetic trees were constructed with 16S and phoE, leuS, and rpoB via the neighbor-joining method, confirming strain Kv4 as K. variicola. To test pathogenicity, the roots of 25 'Moneymaker' tomato plants with four to five true leaves were wounded, then each plant inoculated with a 15 mL bacterial suspension (OD600=0.6) of strain Kv4, while the control plants received sterile water. Plants were incubated at 28℃ with a 16 h photoperiod. Experiments were done twice. At 15 days after inoculation (DAI), all plants inoculated with Kv4 showed yellowing, unevenly distributed small black necrotic spots on the leaf surface, and purple-brown soft rot at the stem base. By 18 DAI, there was a gradual transformation of the stem bases from green to purplish brown. At 21 DAI, 60% of the inoculated plants displayed brownish soft rot at the stem base. In contrast, the control plants remained symptom-free. The pathogen was re-isolated from the stem and identified as K. variicola via sequence analysis of 16S, phoE, leuS, and rpoB. In recent years, several new bacterial pith necrosis diseases were reported in tomato (Guo et al. 2023; Ivić et al. 2023). This is the first study documenting K. variicola causing bacterial pith necrosis in tomato. Once considered a benign plant endophyte, Sun et al. (2023) reported K. variicola causing banana sheath rot in Guangdong and Guangxi Provinces, China. Malik et al. (2023) reported that K. variicola caused leaf streak in sorghum in India. This report of bacterial pith necrosis in tomato caused by K. variicola strain Kv4 underscores the escalating threat posed by emerging pathogens to agricultural production. The emergence of K. variicola as a tomato pathogen complicates plant disease management strategies.

First report of basal rot of yellow dragon fruit (Selenicereus megalanthus) caused by Fusarium oxysporum in Peru.

Cultivation of yellow dragon fruit (Selenicereus megalanthus) in Peru has recently expanded (Verona-Ruiz et al. 2020). In August 2021, approximately 170 of 1,110 dragon fruit cuttings (15.3%) in the university's nursery (6°26'10'' S; 77°31'25'' W) showed basal rot symptoms. Initial symptoms included small brown spots on the base of stems, expanding towards the top that became soft and watery. All symptomatic plants eventually died, i.e., a severity of 100%. The disease was more prevalent on cuttings during the rooting phase than on well-established cuttings. We collected five symptomatic cuttings from throughout the nursery. Four sections of 1 × 1 cm2 of tissue adjacent to the diseased area were excised from each cutting, immersed for 1 min in 2% NaClO, rinsed twice with sterile distilled water, placed on potato dextrose agar (PDA) medium (four sections per Petri plate, five plates), and incubated at 25°C for 7 days. Morphologically similar mycelia grew from all sections, and five monosporic isolates were obtained, one per plate. Colonies grew fast, reaching 60 to 64 mm in 7 days, and produced violet-white cottony aerial mycelia with orange sporodochia on PDA, and abundant macro- and microconidia on synthetic nutrient-poor agar. Macroconidia were straight to slightly curved, typically with 2 to 3 septa, 16.6 to 23.3 × 1.7 to 3.7 µm (n = 30); microconidia were oval or kidney-shaped, and commonly hyaline, 6.7 to 16.4 × 2.5 to 4.7 µm (n = 40). Genomic DNA was extracted from isolate AFHP-100, then the ITS region and the TEF1 and RPB2 partial genes were amplified and sequenced (Accession numbers PP977433, OR437358, PP537149) following Gardes and Bruns (1993) and O'Donnell et al. (1998). We conducted a BLASTn search of ITS sequence against the NCBI "nr" database and local 'megablast' searches of TEF1 and RPB2 sequences against FUSARIUM-ID v.3.0 (Torres-Cruz et al. 2022). We found 100%, 98.19 to 99.84%, and 98.81 to 99.76% identities in ITS, TEF1, and RPB2 sequences, respectively, to the ex-epitype and other reference strains of Fusarium oxysporum (CBS 144134, NRRL26406, among others). A maximum likelihood phylogenetic analysis with a TEF1-RPB2 concatenated dataset with FUSARIUM-ID sequences also showed isolate AFHP-100 was F. oxysporum. A pathogenicity test was carried out by inoculating wounded healthy roots of three cuttings with submersion in a 5 × 106 conidia/ml suspension for 25 min. Then, the inoculated plants were planted in sterile soil. One cutting with wounded roots submerged in sterile water served as a control. In parallel, sterile soil was inoculated with 20 mL of the conidial suspension, and another three healthy cuttings were planted. A cutting planted in noninoculated soil also served as a control. Basal rot symptoms developed in all inoculated plants after 25 days. After re-isolation, the same fungus, corroborated based on micromorphology and TEF1 sequence (PP335689), was recovered, fulfilling Koch's postulates. The isolate was deposited in the KUELAP Herbarium (voucher KUELAP-3214), located and administered by the National University Toribio Rodriguez de Mendoza de Amazonas, in Chachapoyas, Peru. Fusarium oxysporum has been reported to cause basal stem rot in Bangladesh and Argentina (Mahmud et al. 2021; Wright et al. 2007), and stem blight in Malaysia (Mohd Hafifi et al. 2019) on dragon fruit. This is the first report of F. oxysporum causing basal rot in S. megalanthus in Peru. This fungus is among the most destructive plant pathogens, and the rapid expansion of the crop in Peru requires a comprehensive knowledge of the biotic factors influencing production. Therefore, this report is foundational to implementing proper control strategies.

Exploring the Potential of Turmeric as a Natural Antibacterial Agent: In Vitro Evaluation of Crude Paste and Aqueous Extract

Background: There is growing concern in the use of natural antimicrobial compounds due to the emergence of antibiotic resistance. One such contender is turmeric (Curcuma longa), an herb known for its usage in medicine for centuries; nowadays, however, its prospects in pharmacology are being actively discussed. Since access and utilization of modern health care is scarce in Bangladesh, such natural cures are worth researching. Objective: To assess the in vitro antibacterial activity of turmeric’s crude paste and aqueous extract against Staphylococcus aureus and Escherichia coli. Methods: The study was carried out in the Department of Pharmacology with the collaboration of Department of Microbiology, Mymensingh Medical College, Bangladesh, over a period of one year from July 2010 to June 2011, and it consisted of three experiments: Effects of crude turmeric paste incorporated in nutrient agar media, Minimum Inhibitory Concentration of Aqueous Turmeric Extract and Amikacin by broth dilution method, and Subculture method to confirm bactericidal effect. Results: CTP demonstrated effective growth of S. aureus at 10% concentration and E. coli at 30% concentration. The MIC of ATE was found to be 800 μg/ml for S. aureus and 2000 μg/ml for E. coli. The MIC for amikacin was 10 μg/ml for both organisms. These effects have been further substantiated in subculture studies regarding their bactericidal properties. Conclusion: From the findings of the current study, it is evident that both turmeric and its crude paste had profound antibacterial effects against S. aureus and E. coli, though the effect of crude paste was observed to be stronger than that of the aqueous extract. Although not as strong as Amikacin, its organic nature and possibility of side effects make it worth pursuing. Hence, this work adds to the literature on the fact that turmeric has health benefits as highlighted by traditional medical practice and points to the need to find new natural sources of .............

The yeast Dothiora sorbi IOJ-3 naturally produced various filamentous sectors with distinct abilities by undergoing DNA demethylation

Some fungi have demonstrated the ability to adapt rapidly to changing environments by exhibiting morphological plasticity, a trait influenced by species and environmental factors. Here, an anamorphic yeast strain IOJ-3 exhibited unique sectorization characteristics, naturally producing diverse filamentous sectors when cultivated on potato dextrose agar (PDA) medium or natural culture medium for durations exceeding 13 days. The strain IOJ-3 and its filamentous sectors were identified as Dothiora sorbi. The morphology of the sectors was consistent and heritable. The life cycle of strain IOJ-3 was investigated through microscopic observation, emphasizing the development of conidiogenous cells as a crucial stage, from which filamentous sectors originate. Some physiological characteristics of IOJ-3 and filamentous sectors are compared, and strain IOJ-3 has a higher antibiotic tolerance than two filamentous sectors, IOJ-3a expands faster on the culture medium, and IOJ-3b can penetrate cellophane. A transcriptomic analysis was conducted to investigate the differentially expressed genes between the yeast form IOJ-3 and its two filamentous sectors, revealing a total of 594 genes that exhibited consistent differential expression relative to IOJ-3, including 44 silencing genes in IOJ-3 that were activated. Gene Ontology analysis indicated that these differentially expressed genes were primarily associated with the cellular component category. Furthermore, adding 5-Azacytidine accelerated filamentous sectorization and increased the proportion of filamentous cells of strain IOJ-3 in PD liquid media, suggesting that the filamentous sectorization observed in strain IOJ-3 is linked to processes of DNA demethylation. In conclusion, this study sheds light on the biological characteristics of D. sorbi regarding morphological transitions and provides substantial direction for exploring genes related to fungal filamentous development.

Assessing management strategies for mitigating Rhizoctonia damping-off in sugar beet cultivation

Rhizoctonia solani is recognized as one of the most economically significant pathogens that can cause both crown root rot (CRR) and damping-off (DO) in the sugar beet (SB) plant. This report details the first investigative study on R. solani as a fungus causing DO of SB in Morocco. The identification of the fungal pathogen was done both based on morphological features as well as on molecular tools utilizing internal transcribed spacer region of ribosomal DNA (ITS rDNA) and specific primers. In vitro evaluation of the antifungal effect of four commonly used fungicides, tebuconazole, azoxystrobin, trifloxystrobin, and difenoconazole, revealed their effectiveness in inhibiting the growth of pathogenic strains on potato dextrose agar (PDA) medium. Bacillus subtilis Y1336, available in the market, was tested as a potential biocontrol agent (BCA) against R. solani-induced DO. The effectiveness of the BCA was confirmed through double culture tests on PDA, in vitro seed inoculation with both B. subtilis and R. solani, and by employing the antagonist agent as a seed coat in greenhouse settings. This marks the first instance of the effectiveness of B. subtilis Y1336 against R. solani-induced DO in SB. B. subtilis Y1336 should be integrated into SB damping-off control strategies, in conjunction with the fungicides that have been shown to be more effective.

Genetic structure and pyrimethanil resistance of Botrytis spp. causing gray mold on strawberry from greenhouses in Zhejiang, China

Gray mold caused by Botrytis spp. is a common disease on various hosts, including strawberries. In this study, we obtained 59 Botrytis isolates from strawberries from greenhouses in Zhejiang Province, China. Identification of the sampled isolates at species level was performed by a duplex PCR assay method, the result showed that, in Zhejiang, gray mold on strawberry fruits is caused by a complex of Botrytis groups including B. cinerea group N (47.5 %) and B. cinerea group S (52.5 %). The sensitivities of all Botrytis isolates to pyrimethanil were determined based on discriminatory dose method on L-asparagine-based agar medium plate. Our results showed that the isolates obtained from the greenhouses with continuous use of pyrimethanil developed severe resistance to pyrimethanil, and the resistance frequencies of B. cinerea group N and group S isolates were 89.3 % and 41.9 %, respectively. By sequencing, four different resistance-related point mutations were identified in 38 Botrytis isolates: Bcpos5L412F (16 isolates, 42.1 %), Bcpos5L412V (14 isolates, 36.8 %), Bcmdl1E407K (2 isolates, 5.3 %), and Bcpos5L412S⸱Bcmdl1E407K (1 isolate, 2.6 %). The exogenous addition of methionine could not completely alter the resistance of Botrytis isolates to pyrimethanil. In this study, the pyrimethanil resistance in Botrytis isolates was steadily inherited, and compared to the pyrimethanil-sensitive isolates, the resistant mutants exhibited good fitness in sporulation capacity, spore germination rate, and virulence on strawberries. In conclusion, our results provided a description of the genetic structure of Botrytis groups complex on strawberry fruits and reminded growers to focus on the stable pyrimethanil resistance in Botrytis isolates, caused by point mutations in BcPos5 and BcMdl1.

First report of Neopestalotiopsis clavispora Causing guava scab in China.

Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province, China. Guavascab was corky with ovoid or round lesions on the surfaces of green fruits. Gradually the lesions sunk. Disease incidence was estimated as 85% in 500 investigated plants in about 50 ha. Twenty diseased fruits were collected from twenty trees in the field. From each fruit the margin of the diseased tissues was cut into 2 mm × 2 mm pieces; surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, successively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Thirty-four isolates were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover pure cultures of three isolates (PGNC-1, PGNC-2, and PGNC-3) . The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, these colonies form black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 15.8 to 21.2 µm × 4.5 to 6.5 µm (n = 40). The three median cells were versicolored, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (4.5 to 5.5 µm long; n = 40) and three apical appendages (19.2 to 24.5 µm long; n = 40). The morphological characteristics of the isolates were consistent with the description of Neopestalotiopsisclavispora (Maharachchikumbura et al. 2012). Molecular identification was performed using PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). Sequences were generated from the isolates using primers for the rDNA ITS (ITS1/ITS4), TEF1-α (EF1-728F/EF1-986R), and β-tubulin (T1/βt2b) loci (Maharachchikumbura et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, OQ996557 to OQ996559; TEF, OR101037 to OR101039; β-tubulin, OR100971 to OR100973). The sequences of the isolates were 100% identical to the type strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis. The isolates clustered with N. clavispora (MFLUCC12-0280 and MFLUCC12-0281). N. clavispora and Pestalotiopsis clavispora are synonyms. The pathogenicity was tested in vivo. Plants (cv. Zhenzhu) were grown ( 3 years old) in a quarantine orchard at 25℃ to 32℃ with 60 to 80% relative humidity in May 2022. Disease-free green fruits were inoculated. Sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) and sterile distilled water (control) for about 15 s before they were fixed on the wounded fruits with transparent tape. Five fruits on one plant per isolate were inoculated. Five fruits on one plant severed as control. The test was performed thrice. Disease symptoms were found on the inoculated fruits after 20 days, whereas the controls remained healthy. The pathogen was re-isolated from infected fruits and was phenotypically identical to the original isolates thus fulfilling Koch's postulates. Neopestalotiopsis or Pestalotiopsis spp. were reported to be the causal agents of guava scab in Colombia and in Hawaii (Keith et al. 2006; Solarte et al. 2018). N. clavispora has been reported to cause disease in a broad range of hosts (Ge et al. 2009; Chen et al. 2018), but not in guava. This is the first report of N. clavispora causing guava scab in China. There would be no harvest if this disease is left unmanaged.

Biochemical and Molecular Identification of Azospirillum brasilense Bacteria and Evaluation of Their Efficiency in Producing Hormones, Dissolving Phosphorus, and Fixing Nitrogen

The study aimed to isolate A.brasilense bacteria from the soil of the rhizosphere of different plants and different locations in Al-Diwaniyah Governorate. They were identified in two ways. The first was the routine method, which included studying the microscopic and cultural characteristics and biochemical tests of the isolates. The second method was molecular, using polymerase chain reaction (PCR) technology and using primers. It also included testing the efficiency of these isolates in dissolving tricalcium phosphate (TCP) on Pikovskaya agar medium, fixing nitrogen in the liquid nutrient medium (N.B), and measuring the amount of hormone production using an HPLC device. The results of isolation and regular and molecular identification the presence of ten isolates of bacteria bearing the characteristics of A. brasilense bacteria, out of fifteen local bacterial isolates, took the following symbols and sequences (Az2, Az3, Az5, Az6, Az7, Az9, Az11, Az12, Az13, Az14), as the results showed confirmation of the identification of the bacterial isolates identified by biochemical tests. Using a specialized primer to amplify the 462bp fragment of the 16S ribosomal RNA gene, the results of testing the efficiency of the bacteria in dissolving phosphate (TCP) showed that the isolate (Az13) outperformed the highest value in its effectiveness in dissolving metallic phosphorus through the diameter of the clear zone around the colony, which was effective in dissolving phosphate of up to 3.89 mm. As for the nitrogen fixation efficiency test, the isolate (Az3) excelled in the amount of fixed nitrogen reaching 12.44 mg.L-1. As for the amount of its production of growth regulators for Auxins, Gibberellins, and Cytokinins, the isolate (Az3) recorded the highest value in the amount of Auxins production ( Indol-3-acetic acid) its concentration reached 34.4 µg.ml-1, and as for the production of the hormone Gibberellins (GA3), the isolate (Az3) recorded the highest value amounting to 34.7 µg.ml-1, as for the production of the hormone Cytokinins (CK), The isolate (Az11) recorded the highest value, amounting to 28.8 µg.ml-1.

Evaluation of the contribution of media derived from various animal livers on the production of Lucilia sericata

The effects of liver from different animals and agar- media on the production of Lucilia sericata (Meigen 1826) larvae were investigated to determine the best medium for producing larvae for wound therapy. The research was conducted in two phases. The best liver for generating L. sericata larvae was determined in the first phase, using media with beef, porcine, lamb, and chicken livers gelled with agar. In the first phase of the research, it was established that chicken liver was acceptable since the number of flies emerging from puparia was the highest at 80.75%. The preparation and content of the best medium for developing L. sericata larvae were determined in the second phase using chicken liver, raw, cooked, agar, and agar+salt. The number of flies emerging from puparia on the medium with chicken liver + salt + agar was 95.7% in the second phase, followed by 95% of flies coming out of the pupa in the medium prepared with chicken liver and agar. Finally, as the number of flies developing in these two mediums was not significantly different, we believe that the chicken liver and agar medium are most suitable for developing larvae.

Luteipulveratus flavus sp. nov. isolated from two lichen species.

Two novel strains, YIM 133132T and YIM 133296, were isolated from lichen samples collected from Yunnan Province, Southwest PR China. YIM 133132T and YIM 133296 are aerobic, Gram-staining-positive, non-motile actinomycetes. They are also catalase-positive and oxidase-negative, and YIM 133132T formed flat yellowish colonies that were relatively dry on YIM38 agar medium. Flat yellowish colonies of YIM 133296 were also observed on YIM38 agar medium. YIM 133132T grew at 25-35 °C (optimum 25-30 °C), pH 6.0-9.0 (optimum pH 7.0) and in the presence of 0-8% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains YIM 133132T and YIM 133296 represented members of the genus Luteipulveratus and exhibited high sequence similarity (96.93%) with Luteipulveratus halotolerans C296001T. The genomic DNA G+C content of both strains was 71.8%. The DNA-DNA hybridisation (dDDH) values between YIM 133132T and YIM 133296 were 85.1%, and the DNA-DNA hybridisation value between YIM 133132T and YIM 133296 and L. halotolerans C296001T was 23.4%. On the basis of the draft genome sequences, the average nucleotide identity (ANI) between strains YIM 133132T and YIM 133296 and L. halotolerans C296001T was 80.8%. The major menaquinones that were identified were MK-8(H4), MK-9 and MK-8(H2). The polar lipids were diphosphatidylglycerol and phosphatidylinositol. On the basis of the morphological, physiological, biochemical, genomic, phylogenetic and chemotaxonomic characteristics, strains YIM 133132T and YIM 133296 can be clearly distinguished from L. halotolerans C296001T, and the two strains represent a novel species for which the name L. flavus sp. nov. is proposed. The type strain is YIM 133132T (CGMCC= 1.61357T and KCTC= 49824T).

Alarming Prevalence of Nosocomial Pseudomonas aeruginosa among Patients with Infected Burn Wounds

Background: Recently, the prevalence of nosocomial Pseudomonas aeruginosa has become a concern due to its involvement in increased morbidity and mortality, especially those highly virulent strains associated with burn wounds in burn centers. Therefore, this cross-sectional study aimed to isolate and determine the prevalence of nosocomial Pseudomonas aeruginosa among burned inpatients. This study was carried out between September 2023 and March 2024. A total of 355 pus, purulent fluid and necrotic tissue swabs were obtained from burn victims with burns at the Burn Centers in Najaf City, Iraq. 85/355 (23.9%) of isolates under study were detected as Pseudomonas aeruginosa based on laboratory methods (microscopic, biochemical and culture). All test isolates appeared to grow optimally on cetrimide and chromium agar media, and colonies appeared green on these media when examined by the naked eye and greenish fluorescence under ultraviolet light. However, colonies on blood agar were bright and metallic, and the most common type of lysis was β-hemolysin followed by α-hemolysin. On Nutrient agar, most of the isolates were characterized by positive multi-pigment production 57/85 (67.1%), while 28/85 (32.9%) of the isolates were negative pigment production. Conclusion: The rate of virulent P. aeruginosa in the current study raises the degree of alarm, especially among burn patients because they do not have the means of defense that qualify them to resist this virulent pathogen. Also, the older age groups were more affected by it, which increases the levels of risk because elderly patients have weak immunity in addition to most of them being infected with other diseases such as diabetes, which increases the complications associated with burn infections. The impact of Pseudomonal infections on females in most age groups was one of the results of utmost importance and the real reasons behind it must be known. According to numerous studies and reports, the possession of this dangerous pathogen of multiple virulence factors such as its ability to form biofilms delays or prevents the patient's recovery and increases the likelihood of death in burn inpatients. Also, the possession of these virulent bacteria of a large genome makes them exceptionally adaptable and resistant to unfavorable environments.