Molecular Identification
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2023 ◽  
Vol 83 ◽  
S. P. M. Cotta ◽  
M. S. Marins ◽  
I.E. Marriel ◽  
U.G.P Lana ◽  
E.A. Gomes ◽  

Abstract Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

2021 ◽  
Vol In Press (In Press) ◽  
Folasade Muibat Adeyemi ◽  
Nana-Aishat Yusuf ◽  
Rashidat Ronke Adeboye ◽  
Omotayo Opemipo Oyedara

Background: The virulence factors of enterococci play a major role in the pathogenicity of enterococcal strains. Objectives: This study aimed to evaluate virulence factors and detect selected virulence and resistance genes in vancomycin-resistant Enterococcus (VRE) from clinical samples from southwest Nigeria. Methods: The VRE isolates (n = 85) recovered from clinical samples were characterized using conventional microbiology techniques, and molecular identification was made with ddlE primers. Phenotypic screening for five virulence determinants and detection of virulence and resistance genes using a polymerase chain reaction were carried out. Results: Phenotypic identification revealed 61 Enterococcus faecium and 24 Enterococcus faecalis. All the isolates hydrolyzed bile. Moreover, 88.2% of the isolates produced biofilm; however, 72.9% of the isolates produced gelatinase enzyme. Altogether, six isolates (7%) produced all five virulence factors. The least virulence factor expressed by the two species E. faecium and E. faecalis was DNase at 21.3% and 29.2% followed by cytolysin at 27.9% and 41.7%, respectively. Only 25 isolates (29.4%), including 23 E. faecium (37.7%) and only 2 (8.3%) E. faecalis isolates, revealed bands with molecular identification. Additionally, VRE isolates showed bands for asa1 (16%); only 1 (4%) isolate had the hyl gene and vanB gene, respectively. Conclusions: The absence of vanA and low detection of vanB resistance genes suggest the possible presence of other van types and emphasizes the need for further investigations on the incidence of other van genes using molecular screening methods in enterococci isolates in Nigeria for surveillance purposes. Moreover, the low occurrence of virulence genes implies that there might be other mediators of pathogenicity involved in Enterococcus virulence traits.

Yuwadee Somsap ◽  
Patcharaporn Boonroumkaew ◽  
Attawit Somsap ◽  
Rutchanee Rodpai ◽  
Lakkhana Sadaow ◽  

A rare ocular dirofilariasis case along with the clinical characteristics, treatment, and outcome is reported. A whitish roundworm (10.6 cm long and 0.5 mm width) emerged from the pterygium, a triangular tissue growth on the cornea of the eye, of a male patient. The worm had a rounded anterior part, mouth without lips, smooth cuticular surface, and short rounded posterior tail with spicules: these features suggested that it was a male Dirofilaria sp. Molecular identification confirmed that the worm belonged to Dirofilaria immitis. This is the first molecular confirmation that D. immitis is a causative agent of ocular dirofilariasis in Thailand: dirofilariasis is a newly emerging zoonotic disease. Physicians should be alert to zoonotic filarial worms and knowledgeable about treatment of this disease.

Plant Disease ◽  
2021 ◽  
Jiahao Lai ◽  
Guihong Xiong ◽  
Bing Liu ◽  
Weigang Kuang ◽  
Shuilin Song

Blueberry (Vaccinium virgatum), an economically important small fruit crop, is characterized by its highly nutritive compounds and high content and wide diversity of bioactive compounds (Miller et al. 2019). In September 2020, an unknown leaf blight disease was observed on Rabbiteye blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E). Disease surveys were conducted at that time, the results showed that disease incidence was 90% from a sampled population of 100 plants in the field, and this disease had not been found at other cultivation fields in Nanchang. Leaf blight disease on blueberry caused the leaves to shrivel and curl, or even fall off, which hindered floral bud development and subsequent yield potential. Symptoms of the disease initially appeared as irregular brown spots (1 to 7 mm in diameter) on the leaves, subsequently coalescing to form large irregular taupe lesions (4 to 15 mm in diameter) which became curly. As the disease progressed, irregular grey-brown and blighted lesion ran throughout the leaf lamina from leaf tip to entire leaf sheath and finally caused dieback and even shoot blight. To identify the causal agent, 15 small pieces (5 mm2) of symptomatic leaves were excised from the junction of diseased and healthy tissue, surface-sterilized in 75% ethanol solution for 30 sec and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, and then incubated on potato dextrose agar (PDA) at 28°C for 5-7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were white with sparse creeping hyphae. Pycnidia were spherical, light brown, and produced numerous conidia. Conidia were 10.60 to 20.12 × 1.98 to 3.11 µm (average 15.27 × 2.52 µm, n = 100), fusiform, sickle-shaped, light brown, without septa. Based on morphological characteristics, the fungal isolates were suspected to be Coniella castaneicola (Cui 2015). To further confirm the identity of this putative pathogen, two representative isolates LGZ2 and LGZ3 were selected for molecular identification. The internal transcribed spacer region (ITS) and large subunit (LSU) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004) and LROR/LR7 (Castlebury and Rossman 2002). The sequences of ITS region (GenBank accession nos. MW672530 and MW856809) showed 100% identity with accessions numbers KF564280 (576/576 bp), MW208111 (544/544 bp), MW208112 (544/544 bp) of C. castaneicola. LSU gene sequences (GenBank accession nos. MW856810 to 11) was 99.85% (1324/1326 bp, 1329/1331 bp) identical to the sequences of C. castaneicola (KY473971, KR232683 to 84). Pathogenicity was tested on three blueberry varieties (‘Rabbiteye’, ‘Double Peak’ and ‘Pink Lemonade’), and four healthy young leaves of a potted blueberry of each variety with and without injury were inoculated with 20 μl suspension of prepared spores (106 conidia/mL) derived from 7-day-old cultures of LGZ2, respectively. In addition, four leaves of each variety with and without injury were sprayed with sterile distilled water as a control, respectively. The experiment was repeated three times, and all plants were incubated in a growth chamber (a 12h light and 12h dark period, 25°C, RH greater than 80%). After 4 days, all the inoculated leaves started showing disease symptoms (large irregular grey-brown lesions) as those observed in the field and there was no difference in severity recorded between the blueberry varieties, whereas the control leaves showed no symptoms. The fungus was reisolated from the inoculated leaves and confirmed as C. castaneicola by morphological and molecular identification, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. castaneicola causing leaf blight on blueberries in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing effective control strategies, reducing economic losses in blueberry production, and promoting the development of the blueberry industry.

Chukwunonso Francis Obi ◽  
Ikenna Onyema Ezeh ◽  
Michael Ikenna Okpala ◽  
Onyinye Agina ◽  
Paschal Ugochukwu Umeakuana ◽  

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2025
Aaron J. Simkovich ◽  
Yinzi Li ◽  
Susanne E. Kohalmi ◽  
Jonathan S. Griffiths ◽  
Aiming Wang

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Zahra Arab‐Mazar ◽  
Maryam Niyyati ◽  
Ehsan Javanmard ◽  
Monireh Kamali ◽  
Zohreh Lasjerdi ◽  

2021 ◽  
Vol 17 (1) ◽  
pp. 35
Wartono Wartono

<p>Chili (Capsicum annuum L.) is a vegetable commodity with high economic value which is widely cultivated by farmers in Indonesia. One of the obstacles faced in chili cultivation is stem rot disease. This study aimed to identify the pathogens that caused stem rot in chili plants obtained from one location in Sindangjaya Village, Cipanas District, Cianjur Regency, West Java Province based on morphological and molecular analyses. Pathogen identification was performed with morphological and molecular approaches. The morphological characters observed included colony shape, sporangium diameter, and mating type. The pathogenicity of the isolates was assayed by inoculating chili stems aged 40 days. Molecular identification was carried out using two pairs of primers for ITS regions and TEF-1 gene. Based on the results of morphological and molecular identification, as well as pathogenicity tests, it was confirmed that Phytophthora capsici pathogen was the causal agent of stem rot in chili plants collected from Sindangjaya Village. Further study is needed to determine the spread of the disease, damage, and yield loss caused by stem rot disease, as well as how to prevent and control the disease.</p>

2021 ◽  
Vol 8 ◽  
Ahmed Gareh ◽  
Amira A. Saleh ◽  
Samar M. Moustafa ◽  
Amin Tahoun ◽  
Roua S. Baty ◽  

Cystic echinococcosis has been considered one of the major parasitic zoonoses which is associated with severe economic losses. The present study was undertaken to investigate the occurrence, organ distribution, cyst fertility, and viability of cystic echinococcosis in slaughtered camels and cattle from various abattoirs in Assiut Governorate, Egypt. The work also involved morphological, morphometric, and molecular identification of the parasite. The occurrence of hydatid cysts was investigated in total number of 100 lungs of camels and 574 liver and lungs of cattle admitted to three slaughterhouses at Assiut Governorate, Egypt. Moreover, several individual variable factors, including organ involvement, age, sex, and hydatid cyst characteristics, were studied to identify their possible association with the occurrence of the disease. Genomic DNA was extracted from the hydatid cysts, followed by molecular identification of the parasite through amplification of ribosomal DNA internal transcribed spacer (ITS) regions. Hydatid cysts were found in 6 camels (6%) out of 100 inspected camels, while 5 hydatid cysts (0.87%) were detected in a total number of 574 cattle examined. The parasite was detected exclusively in lungs of camels, while lungs were the main organ infected by the parasite in cattle and one hydatid cyst was found in the liver (0.17%). In camel, 66.7, 16.65, and 16.65%of detected cysts were fertile, sterile, and calcified, respectively, while in cattle, these percentages were 60, 20, and 20%, respectively. None of the studied variable factors were significantly associated with the occurrence of the disease in camels, with the exception that all cysts were found in the lung. Conversely, we found a significant association (P &lt; 0.05) between the age and sex of the slaughtered cattle and the occurrence of hydatid cysts. In this respect, the rate of infection was higher in female cattle and those cattle more than 5 years (P &lt; 0.05). The morphological, morphometric, and molecular studies confirmed the presence of the parasite. Taken together, our results concluded that camels and cattle play a potential role in maintaining the transmission cycle of this zoonotic parasite.

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