Rotary Shaker Research Articles

INVESTIGATING THE BIODEGRADATION OF LOW-DENSITY POLYETHYLENE BY PROTEUS AND SERRATIA SPP.

Plastics are gradually replacing ceramics, glasses and other materials in their day to day use, their short term application and release to the environment has made them a waste burden on cities and municipalities, where they cause environmental pollution and esthetic damage. There is therefore, a need to develop an eco-friendly protocol to manage plastic wastes. The role of microbes in bioremediation has been established, although, there is paucity of information on the use of Proteus and Serratia spp. to degrade plastics. Hence, the ability of Proteus and Serratia spp. to degrade low-density polyethylene was investigated. The microorganisms were isolated from waste dumpsites in Owerri Municipality and its immediate environs. 100ml enrichment cultures were dispensed into 10 labeled conical flasks, 1ml aliquot of 10-1 microbial dilutions of each organism were seeded each into 5 sets of the conical flasks and 0.5g of polyethylene powder was added as the sole source of carbon to each set of flasks, the set-up was incubated in a rotary shaker at 300c and 150rpm for 60 days. The optical density (OD600), temperature and PH changes were measured at 10 days interval, while enumeration of viable colonies in (cfu/ml) during incubation period was also carried out. Results obtained showed progressive but gradual increase in optical density, temperature and PH values and appearance of air bubbles in the test media indicating microbial activities and ability of the organisms to utilize the substrate as energy source. The viability count of the isolates during and at the end of the incubation period also showed gradual increase in viable colonies (cfu/ml). It is concluded that both Proteus and Serratia spp. were able to utilize the low-density polyethylene as sole carbon source, hence suggesting their ability to degrade the polymer.

Temporary immersion bioreactors as a useful tool for obtaining high productivity of phenolic compounds with strong antioxidant properties from Pontechium maculatum

Pontechium maculatum (Russian bugloss) is a medical plant belonging to the family Boraginaceae. Although this species is known as a medical plant rich in biologically active secondary metabolites, biotechnological studies about this valuable plant is still missing. The scientific objectives of this study were to investigate the biomass production, synthesis, and productivity of various phenolic acids, flavonoids, and shikonin in P. maculatum cultivated in various breeding systems. Additionally, the antioxidant activity of plant-derived extracts was evaluated. Plants were cultivated in a traditional agar-solidified medium, a liquid medium with rotary shaking, and a temporary immersion bioreactors Plantform™ (TIB), as well as cultivated in soil (ex vitro conditions). Analyses of the growth index and dry weight accumulation were performed on the collected material. In the extracts obtained from examined plants, total phenolic content was estimated, and qualitative and quantitative analysis of phenolic derivatives using DAD-HPLC was conducted, simultaneously with an analysis of antioxidant capacity. TIB stimulated the highest synthesis of all examined phenolic acids and shikonin. In TIB-cultivated shoots level of rosmarinic acid obtained a concentration of 3160.76 mg × 100 g− 1 dry weight (DW), and shikonin obtained a concentration of 77.26 mg × 100 g− 1 DW. Furthermore, plants from TIB were characterized by the highest productivity of all studied phenolic derivatives, what makes it very effective platform for the synthesis of biologically active secondary metabolites in Russian bugloss. Moreover, this article shows that P. maculatum is a rich source of various phenolic derivatives with high antioxidant potential.

Preparation of Biofertilizer from Azospirillum brasilense Bacteria and test its Impact on Growth and Yield of Wheat Triticum aestivum L. in Gypsiferous Soil

After confirm the bacterial strain that it is belong to Azospirillum brasilens and test the efficiency on nitrogen fixation , Indolic Acetic Acid (IAA) Hormone production , this strain was grown in slants and transferred to liquid broth in the rotary shaker to prepare mother (starter) culture which contain 5 x 109 CFU.ml-1 and then mixed starter culture with two types of carriers ( powder of date palm nucleus and powder of olive nucleus).
 After that, field experiment was conducted in gypsifores soil ( Typiccalcigypsids) to study the efficiency of this biofertilizer on promoting the wheat growth and yield at three levels of nitrogen (0 , 50% , 100%) of fertilizer recommendation .
 Results showed that the biofertilizer which was added alone either with date palm nucleus powder or with olive nucleus caused significant increases in plant high , dry weight of vegetative part , roots dry weight after 60 days from germination and the dry weight of straw and grain yield compare with control treatment with percentage increases of 22.68 , 92.84 , 171.01 , 75.5% for biofertilizer which held on date palm nucleus powder and 21.2 , 59.46 , 134.7 , 40.01 and 37.11% respectively . Also the results refered to not significant differences between two carriers. The result also appear that the beneficial effect of biofertilization continuous with increase of nitrogen levels , the treatment ( biofertilize with date palm nucleus powder + 100% nitrogen fertilizers) gave highest values in straw and grain yield where is ( 5.41 and 4.30 ) ton. ha-1 in compare with the same treatment which take half of fertilizers recommendation which gave (5.33 and 3.86) ton.ha-1. Also the result showed significant effect of bio-fertilization on the number of Azospirillum bacteria , ( 32 x 104 and 30 x 104 )CFU . gm-1soil for bio-fertilizer which was carried on date palm nucleus powder and olive nucleus powder, respectively in compare with control which gave 13.33 x 104 CFU. gm-1soil.

Genome Sequence Resource of Ustilago crameri, a Fungal Pathogen Causing Millet Smut Disease of Foxtail Millet.

Genome Sequence Resource of Ustilago crameri, a Fungal Pathogen Causing Millet Smut Disease of Foxtail Millet.

First Report of Anthracnose on Juglans regia Caused by Colletotrichum siamense in Korea.

Walnut (Juglans regia) is one of the main tree crops cultivated for nut production in South Korea with an estimated production of about 1,189 tons per year (Korea Forest Service 2020). In August 2021, anthracnose symptoms, including dark, depressed, irregularly shaped lesions on fruits and leaves of walnut cv. Sinlyeong, were observed at three orchards in Nonsan (36°10'22.5"N 127°06'14.8"E) and Suwon (37°16'04.7"N 126°55'22.3"E and 37°15'10.6"N 126°57'35.6"E). This led to severe yield loss of walnut fruit with a disease incidence of approximately 70 to 80% in each orchard. Three samples, including infected fruits and leaves, were randomly collected per site. Fungal isolates were isolated either from acervuli filled with conidial masses on infected walnut tissues or from plant tissues that were surface-disinfested, followed by plating onto 2% PDA. Colonies were initially white, later became pale brownish to light gray with concentric rings of salmon sporodochia. White to gray aerial mycelia, reaching 65 mm diameter in 5 days, were abundantly produced on PDA at 25 °C. Appressoria were brown, ovoid, and in some cases, clavate, 5.1-8.7 μm in length, and 3.2-5.1 μm in width (n = 50). Conidia were single celled, hyaline, cylindrical with rounded ends and smooth walls, guttulate, 13.6-18.8 µm in length, and 4.4-6.3 µm in width (n = 50). Setae were absent. Three isolates, i.e., one per orchard, were retained and deposited in the culture collection (CDH) of National Institute of Forest Science, Korea (Accession No. CDH052-054). The internal transcribed spacer (ITS) region of rDNA, beta-tubulin (TUB2) and a partial sequence of the actin (ACT) genes were amplified and sequenced for each of the isolates using the pair of primers, ITS1F/ITS4 (Gardes and Bruns 1993; White et al. 1990), T1/Bt2b (ODonnell and Cigelnik 1997; Glass and Donaldson 1995) and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. A BLAST search in GenBank revealed that the sequences of ITS (OK631731-733), TUB2 (OK665927-929) and ACT (OK665930-932) showed sequence identities of 98.6 to 99.6% to Colletotrichum siamense sequences (FJ972613, FJ907423, FJ907438). A maximum likelihood tree, based on a combined dataset of ITS, ACT and TUB2 gene sequences for Colletotrichum spp., revealed that the three isolates were clustered with type specimens of C. siamense. To prepare larger quantities of inoculum for the pathogenicity, mycelial plugs bearing acervuli taken from 2% PDA were incubated in a conical flask containing 200 ml of 2% potato dextrose broth at 25°C on a rotary shaker at 150 rpm for two weeks. Spore concentration was adjusted to 1.0 × 104 ml-1 conidia of C. siamense (CDH054). A 10 to 15 ml of spore suspension was then sprayed on each leaf of 12 seedlings of 'Sinlyeong' walnut (three-year-old), while 7 seedlings were treated with sterile distilled water as a control. Each treated seedling was covered by a plastic bag to maintain moisture for one day. Inoculation trials were repeated twice, in August and September 2021. Symptoms identical to those observed in the field developed four to five days after the inoculations from which the inoculated pathogen was successfully re-isolated, fulfilling Koch's postulates. However, no symptoms were observed in the control. To our knowledge, this is the first report of anthracnose on J. regia caused by C. siamense in Korea. This indicates that disease occurrences must be further rigorously surveyed at the nation-wide scale to effectively control the disease in the country.

Free Radical Scavenging Activity of Clove and Cumin Formulation Mediated Selenium Nanoparticl

Background: Selenium nanoparticles (SeNPs) have the potential to be used for various applications.The green SeNPs (selenium nanoparticles) are formed by simple mixing Withania somnifera (W. somnifera) leaves extract and selenious acid (H2SeO3) solution. Nanoparticles have revolutionized for targeted drug delivery by minimizing the toxicity and side effects that commercially available drugs may have on patients. In this study we have chosen the clove and cumin because it protects from cholesterol, inflammatory damage and it has antioxidant property Aim: The aim of the study is to analyze the antioxidant activity of clove and cumin formulation mediated selenium nanoparticles. Materials & Methods: Cumin and Clove were powdered and stored in an air-sealed container. 1g of cumin and clove formulation was mixed with 100 ml of distilled water and boiled for 15 minutes and the formulation was filtered with Whatmann No.1 filter paper. 30mM sodium selenite was prepared in 50 ml of distilled water. 50 ml of sodium selenite solution and 50 ml of clove and cumin formulation was mixed and kept in a rotary shaker for 48 hours. UV spectroscopy reading based on color change was observed. Results: The antioxidant effect of selenium nanoparticles showed a higher percentage of inhibition at 50µg/ml in DPPH Assay and H2O2 Assay. The clove and cumin extract showed an increased percentage of inhibition with increasing concentration. Conclusion: The clove and cumin formulation mediated selenium nanoparticles acts as potent antioxidant agents. It also has the least side effects compared to commercial drugs.

Gelatinase Microbial Morphology from Leather Defect

Microbes attach in pickle leather, wet blue leather, and upper shoeleather have been identified to have positive profile to produce extracellular protease enzymes with pH values of acidic to basic on 15% gelatin and 2% commercial skimmed milk as substrates. Defective tanned leather was added to a microbial growth solution enriched with 2% gelatin substrate and incubated for 7 days at room temperature on an open rotary shaker at 120 rpm and then stored at room temperature while waiting for gelatinase testing. There were 5 morphological colonies of microbial gelatinase from defective tanned skin, all of which were white.

FORMULATION AND EVALUATION OF IMPLANTABLE DRUG DELIVERY OF DISULFIRAM

Disulfiram is widely prescribed to discourage alcoholics from drinking alcohol. The effectiveness of oral disulfiram as a treatment for alcoholism is severely limited due to its poor bioavailability and poor patient compliance. To minimize the failure of the orally administered drug, efforts have been made to prepare alternative dosage form of subcutaneously implantable disulfiram pellets or tablets. In the present study an attempt has been made to design and evaluate a disulfiram implant using plain drug. The disulfiram implants have been formulated by direct compression and the effect of parameters like pH of site of administration (6.0 and 7.4) and sterilization were studied on in-vitro release of implantable disulfiram pellets. In- vitro release has been studied by vial and rotary flask shaker methods. The release kinetic mechanism from all the formulation was found to be zero order. The in vitro release data of all the formulations was compared with that of marketed disulfiram implant (Esperal). In contrast to other formulation in vitro release from marketed formulation appeared to be independent of hydrodynamics of diffusion (volume of dissolution media and agitation speed). The present study had revealed that, in-vitro drug release from implants is unaffected by pH of site of administration and gamma ray sterilization. Both the methods (Vial Method & Rotating Flask Method) of in vitro dissolution testing are found significantly different for formulations prepared in laboratory but not for marketed formulation, indicating the different mechanism of release of marketed formulation.

Enhanced Isolation of Streptomyces from Different Soil Habitats in Calamba City, Laguna, Philippines using a Modified Integrated Approach.

Streptomyces species are considered to be the most prolific sources of various bioactive secondary metabolites that are important for antibiotic production. Here, we describe a modified integrated approach to isolate Streptomyces species from diverse soil habitats, such as dumpsite, garden, forest, grassland, and riverside in Calamba City, Laguna, Philippines. A total of 25 soil samples were collected from a depth of 0-20 cm using systematic random soil sampling. All soil samples were air-dried, crushed, pretreated with calcium carbonate, and incubated on a rotary shaker. Isolation of Streptomyces in soil samples was then performed using the standard serial dilution plate technique on starch casein agar supplemented with nystatin (50 μg/ml) and ampicillin (5 μg/ml). Identification of the Streptomyces isolates was done using a polyphasic method that includes morphological and biochemical characterization. A total of 103 morphologically and biochemically distinct Streptomyces were isolated from diverse soil habitats. The number of Streptomyces isolates varied in each collection site, with the highest number collected from dumpsite soil and the least from forest soil. Most of the hydrogen sulfide producers were noted to be isolated from dumpsite samples. Moreover, more Streptomyces were isolated in soil habitats at higher altitudes with a slightly acidic to alkaline pH and a temperature ranging from 29 to 33°C. Employing the modified integrated approach, we have isolated up to 10 times more Streptomyces compared to early studies. These Streptomyces isolates can be valuable for future drug discovery and development research.

MOLECULAR CHARACTERIZATION AND ANTIBACTERIAL ACTIVITY OF BIOSURFACTANT PRODUCING ISOLATE STREPTOMYCES CELLULOSAE VAR. PD66

In the present study, the actinomycetes isolate named as PD66 (Streptomyces cellulosae var.) was screened and identified based on 16S rRNA nucleotide sequence and phylogenetic evolutionary relationship with other strains by National Collection of Industrial Microorganisms (NCIM), Pune. Biosurfactant production by the isolate PD66 was carried out using Kim’s medium with 3% olive oil as a carbon source and incubated at 27ºC for 7 days on rotary shaker at 150rpm. After 7 days, the contents of the flask were centrifuged and collected the cell free supernatant for performing the preliminary tests like oil displacement, para film-m test for confirmation of biosurfactant production, these two tests were given positive results for biosurfactant production obtained from the selected isolate PD66. Lipase activity was also checked and confirmed the extra cellular lipase enzyme release during the growth of isolate PD66 using tributyrine agar medium. Surface tension and emulsification index were also measured for evaluation of biosurfactant present in cell free supernatant broth using SLS (Sodium lauryl sulphate) as a standard. Determination of extracellular antibacterial activity of biosurfactant samples was determined for every 24hrs upto 10days using cell free supernatant by agar diffusion method. Selected isolate PD66 has showed antibacterial activity against E.coil and Staphylococcus aureus. After extraction of cell free supernatant with ethylacetate, the resultant crude extract was tested for identification of bioactive metabolite by TLC method and Bioautography technique.

Preparation of Al‐Dy core‐shell particles by electron beam treatment of Al powder with consequent magnetron deposition of Dy film

• A new method was proposed for coating a commercial aluminum powder by Dy. • Low-energy high-current electron-beam treatment and magnetron sputtering were used. • Surface smoothing and cleaning were detected after electron-beam refinement. • The average size of powder particles increases with the number of e-beam pulses. • Rolling and shaking of powder during the treatment causes uniformity of deposition. The paper presents a new method for coating commercial metal powders with rare‐earth elements (REE). This method consists of a two‐step treatment of a metal powder in a single vacuum cycle, namely the pulsed irradiation above the melting point by a low‐energy high‐current electron beam with subsequent magnetron sputtering of REE. This research reports the preparation of Al‐Dy core‐shell powder with an average diameter of particles of 40 µm. A powder holder system, providing rotary agitation and shaking of powder, was used to guarantee the uniform processing of each particle. It was established that electron beam irradiation leads to smoothing and cleaning the Al particles from surface oxides. With an increase in the number of pulses, the uniformity of processing increases; however, an increase in the average particle size occurs. Subsequent magnetron sputtering allows completely coating the surface of processed powder with dysprosium. The proposed method can be suitable for fabricating different metal‐REE core‐shell powders with necessary changes in processing parameters.

Successful application of microspore culture in pakchoi (Brassica campestris L. ssp. chinensis Makino var. communis Tsen et Lee) for hybrid breeding.

Microspore embryogenesis is an effective method of obtaining double haploid (DH) lines in only 1year. However, the microspore embryogenesis protocol was not efficient in pakchoi. This study aimed to establish an effective microspore culture protocol in pakchoi for hybrid breeding. The embryos were obtained from three genotypes (18SY01, 18SY02, 18SY03), but the frequency of microspore embryogenesis was significantly different. Globular embryos from three genotypes were placed into a rotary shaker (50 r/min, 25 ℃) for further culture to improve microspore embryogenesis and plantlet regeneration without callus development. Shake culture not only increased the frequency of cotyledonary embryos but also accelerated microspore embryogenesis in the NLN-13 liquid medium. Moreover, the doubled haploid rates of regenerated plants for the three genotypes were above 50%. The morphological characters and plot yield of DH lines were identified, and there were significant differences between them. According to the measurement of the self-compatibility index, all the DH lines were self-incompatible. Furthermore, the hybrid combination was prepared with the selected DH lines and the pakchoi genic male sterile line GMS010 to develop excellent hybrids. This work contributes to accelerating the application of microspore embryogenesis and supplying the DH lines in pakchoi hybrid breeding.

Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal)

Different parts of Shorea robusta (Sal) are being used in ancestral and ayurvedic medicines and are known to cure health ailments. The different phytochemicals present in S. robusta is known to possess antimicrobial property. The different botanical parts of this plant have been used in ayurvedic medicines to cure certain infectious diseases. The main aim of this study was to screen phytochemicals and antimicrobial activity of bark extract of S. robusta. Literatures were collected through books, journals and further additional information were collected from residents and traditional ayurvedic practitioners. The ethanolic bark extract of S. robusta was obtained through 70% ethanol in rotatory shaker for 72 hours at 37 ℃ and then the crude extract was dried, preserved and analyzed for phytochemical analysis and antimicrobial activity. The phytochemical screening of ethanolic extract of bark of S. robusta indicated presence of phytochemicals like, alkaloids, flavonoids, tannins, steroids, anthraquinone and absence of phlobatannins, terpenoids, starch and proteins. The extract of S. robusta on Staphylococcus aureus exhibited clear zone of inhibition of 21mm at minimum inhibitory concentration (MIC) of 2 mg/mL while on Escherichia coli exhibited clear zone of inhibition of 9 mm at MIC of 4 mg/mL. The antimicrobial activity may be conferred due to the presence of plant phytochemicals. S. robusta bark extract exhibiting significant minimum inhibitory concentration and antimicrobial activity indicates the efficacy of this plant to be considered for discovering and extracting new antimicrobial products against the pathogens. These findings need further support for appropriate formulation of the drug and its therapeutic use in clinical settings.

Photobacterium arenosum WH24, Isolated from the Gill of Pacific Oyster Crassostrea gigas from the North Sea of Germany: Co-cultivation and Prediction of Virulence

Cream colored bacteria from marine agar, strain WH24, WH77, and WH80 were isolated from the gill of the Crassostrea gigas a Pacific oyster with a filter-feeding habit that compels accompanying bacteria to demonstrate a high metabolic capacity, has proven able to colonize locations with changing circumstances. Based on the 16S rRNA gene sequence, all strains had high similarity to Photobacterium arenosum CAU 1568T (99.72%). This study involved phenotypic traits, phylogenetic analysis, antimicrobial activity evaluation, genome mining, Co-cultivation experiments, and chemical studies of crude extracts using HPLC and LC-HRESIMS. Photobacterium arenosum WH24 and Zooshikella harenae WH53Twere co-cultivated for 3 days in a rotary shaker at 160 rpm at 30 °C, and LC-MS monitored the chemical profiles of the co-cultures on the third day. The UV chromatograms of the extracts of the co-cultivation experiments show that Zooshikella harenae WH53T could be inhibited by strain WH24. The high virulence of Photobacterium arenosum WH24 was confirmed by genome analysis. Gene groups with high virulence potential were detected: tssA (ImpA), tssB (ImpB/vipA), tssC (ImpC/vipB), tssE, tssF (ImpG/vasA), tssG (ImpH/vasB), tssM (IcmF/vasK), tssJ (vasD), tssK (ImpJ/vasE), tssL (ImpK/vasF), clpV (tssH), vasH, hcp, lapP, plpD, and tpsB family.

Genome Resource of Colletotrichum spaethianum, the Causal Agent of Leaf Anthracnose in Polygonatum falcatum

Genome Resource of <i>Colletotrichum spaethianum</i>, the Causal Agent of Leaf Anthracnose in <i>Polygonatum falcatum</i>

Multiple Methods Synergistically Promote the Synchronization of Somatic Embryogenesis Through Suspension Culture in the New Hybrid Between Pinus elliottii and Pinus caribaea.

Pinus elliottii × Pinus caribaea is an interspecific pine hybrid of major economic importance. Somatic embryogenesis and plant regeneration in P. elliottii × P. caribaea on solid medium have been reported previously; however, a current limitation is the lack of a stable and effective method for its commercial use. The objective of this study was to establish a suspension culture system and evaluate the effect of multiple methods synergistically on the synchronization of embryo development in P. elliottii × P. caribaea. For the former, a protocol to initiate and establish a suspension culture system of P. elliottii × P. caribaea was presented. Based on biomass growth, the growth of embryogenic calli (EC) followed an S-shape curve in suspensions grown for a 15-day period, and the exponential phase of cell suspensions was reached between days 3 and 6. The initial packed cell volume (PCV) and revolutions per minute (rpm) have a significant effect on the proliferation of EC, and the highest proliferation multiple reached 6.86 (±0.06) at the initial density of 5 ml PCV under a 9–10 days transfer interval in the dark on a rotary shaker at 70 rpm. For the latter, the influence of abscisic acid (ABA), ammonium (NH4+), nitrate (NO3–), low temperature, and polyethylene glycol (PEG) on somatic embryogenesis was very significant. When EC were suspended in the medium at a presence of 37.84 μM/L ABA, as many as 274 mature cotyledonary embryos/ml PCV of cells were thereafter formed in the mature medium, and 266 somatic embryos were obtained on mature medium after suspension culture in liquid medium containing 10 mmol/L NH4+ and 30 mmol/L NO3–. Furthermore, reducing the concentration of 2,4-dichlorophenoxyacetic acid gradually and at 4°C incubation for 12 h in the initial exponential phase could promote the synchronization of somatic embryogenesis, which resulted in 260 mature cotyledonary embryos. This suspension culture system and method of synchronic control can be used in the large-scale production of P. elliottii × P. caribaea seedlings.

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

Pyrite Biooxidation by Acidophilic Archaea AcidiplasmaSp. MBA-1 Under Varied Conditions

The goal of this research was to study pyrite (FeS2 ) bioleaching by a strain of the genus Acidiplasma under different conditions (temperature, pH) to evaluate the potential role of Acidiplasma representatives in biooxidation of this sulfide mineral. To compare the role of Acidiplasma archaea in pyrite biooxidation with other acidophilic microorganisms, the experiments were also performed with representatives of othergroups of microorganisms predominant in biohydrometallurgical processes.Pure and mixed cultures of moderately thermophilic acidophilic microorganisms, including strains Acidithiobacillus caldus MBC-1, Sulfobacillusthermosulfidooxidans VKMV 1269T and Acidiplasmasp. MBA-1, were used. The experiments were carried out in flasks with 100 mL of mineral nutrient medium supplemented with 0.02% yeast extract and 1 g of pyrite on a rotary shaker for 20 days. Bioleaching was performed at 45, 55, and 60∘С. The results demonstrated that the representatives of the genus Acidiplasmaprovided a comparatively higher rate of pyrite bioleaching at high temperatures (55 and 60∘C) and low pH of the medium (1.0). Thus, according to the results, strains of thegenus Acidiplasma may provide a high rate of pyrite bioleaching at low levels ofpH. Therefore, the results suggest that archaea of the genus Acidiplasma may be promising microorganisms to improve bioleaching processes with an increase in the operational temperature, which is usually maintained at 40–45∘C in industrial-scale reactors.&#x0D; Keywords: biomining, bioleaching, acidophilic microorganisms, sulfide minerals, pyrite

First report of Sclerotinia sclerotiorum on watercress (Nasturtium officinale) in aquaponic system in Hungary.

Watercress (Nasturtium officinale) is an aquatic dicotyledonous vegetable belonging to Brassicaceae (Aiton 1812). Watercress was grown in an aquaponic system on fired clay ball medium at the Aquaponic Research Station of the University of Debrecen, in the city of Debrecen (Hungary). During January 2020, 3-month-old plants showed symptoms in aquaponic cultivation. A visual survey showed 30% of plants with symptoms. Leaves and stems withered and showed white cotton-like mycelium. Mycelia from infected plants were placed on potato dextrose agar (PDA) and incubated at 25°C for seven days. Single hyphal tips were transferred to produce a pure culture. All ten fungal isolates showed similar morphological characteristics on PDA. Colonies consisted of white mycelia after three days and globoid to irregular and black 2.5 to 7 (average, 3) mm (n = 100 from ten plates) sclerotia formed ten days later, which are the typical morphological features of Sclerotinia sclerotiorum (Mordue et al. 1976). Molecular identification was performed with one of the ten isolates (Scl_B). Mycelia were grown in 250 ml of potato dextrose broth in a rotary shaker at 175 rpm at 24°C for six days. DNA was extracted from mycelium using a Nucleospin plant II (Macherey-Nagel, Germany) according to the manufacturer's protocol. PCR amplification (Kim et al. 2014) was performed with primers ITS1/ITS4 for the internal transcribed spacer region (White et al. 1990) on a Primus 96 thermal cycler (MWG Biotech, Germany). Specific polymerase chain reaction was performed with primers SSasprF/SSasprR (Abd-Elmagid et al. 2013). PCR products were sequenced by Microsynth Austria GmbH. NCBI BLAST analysis of the 440-bp ITS sequence (Genbank MW012403.1) showed 100% identity with the sequence of S. sclerotiorum (MT177267.1, etc.). The 170-bp specific gene sequence (Genbank MW959042.1) had a 100% similarity to hypothetical proteins (Genbank MK028159.1), with a 99.4% similarity to a portion of the S. sclerotiorum aspartyl protease gene (AF271387.1). Pathogenicity tests were carried out by inoculating surface-disinfested, 30-day-old watercress plants in plastic pots (15x15x12 cm). In three repeated experiments 90 watercress plants were measured. 15 plants (one plant per pot) were planted into the five-times autoclaved substrate (Biorgmix: pH 6.1±0.5%, N:1.5%, P2O5:0.7%, K2O:0.5%, organic matter content:50%) and inoculated by ten wheat kernels that were colonized by S. sclerotiorum (Scl_B) (Garibaldi et al. 2019). 15 plants were planted into the substrate with ten non-inoculated kernels as a control. Plants were kept in an MLR-352 climatic test chamber (PHCbi, Japan) at 21 ± 1°C for 12 hr light:dark cycle. On the first day of the experiment complex nutrient solution (Tek-Land: N:5%, P2O5:5%, K2O:5%, B:0.01%, Cu:0,01%, Mn:0.02%, Mo:0.002%, Zn:0.016%) was used, then autoclaved water daily. Eight days later white mycelium appeared on every inoculated plant and five days later dark sclerotia formed on the stems. Based on the morphological characteristics the re-isolated pathogen was S. sclerotiorum. Similar results were detected in three repeated experiments with white mold fungus being reisolated from all 45 infected watercress plants. The 45 non-inoculated plants did not show any symptoms and any diseases. This pathogen has already been reported on watercress in the field (Farr et al. 1989; Boland and Hall 1994; Garibaldi et al. 2019). This is the first reported case of white mold on watercress in aquaponic system in Hungary.

IN VITRO COMPATIBILITY OF DIFFERENT HERBICIDES WITH ENTOMOPATHOGENIC FUNGUS BEAUVERIA BASSIANA

The in vitro effect of three herbicides commonly used in wheat or corn plant protection programs in Romania on mycelial growth of entomopathogenic Beauveria bassiana (BbTd1) were investigated. The formulations with Isoxaflutole 225 g/L + Thiencarbazone methyl 90 g/L + cyprosulfamide (safener) 150 g/L (Adengo), Nicosulfuron 40g/L (Nicogan) and dimethylamine salt 600g/L (Dicopur) were tested at three different concentrations (field recommendation- FR, half and twice the FR) both on solid and liquid media. On the solid media, the radial growth of the fungus was measured for 11 days. On the liquid media mycelial biomass was weighted after 7 days of incubation on a rotary shaker. Dicopur significantly inhibited the vegetative growth of B. bassiana BbTd1 in both culture media at tested concentrations. In liquid media, mycelial growth was stimulated by Adengo and Nicogan at different concentrations. No significant differences caused by Adengo and Nicogan compared to control of radial growth was detected. The results showed that Adengo and Nicogan could be used simultaneously with this entomopathogen in integrated control programs. Further in vivo studies are necessary to establish if Dicopur formulation can cause damage to field populations of entomopathogenic fungi.