Journal of Microbiology

Research Support, Non-U.S. Gov'ts

NOTE] Anaerobic Cellulolytic Rumen Fungal Populations in Goats Fed with and without Leucaena leucocephala Hybrid, as Determined by Real-Time PCR: Ching Mun Kok , Chin Chin Sieo , Hui Yin Tan , Wan Zuhainis Saad , Juan Boo Liang , Yin Wan Ho; J. Microbiol. 2013;51(5):700-703. Published online October 31, 2013; DOI: https://doi.org/10.1007/s12275-013-2540-z

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Abstract: The effect of Leucaena leucocephala hybrid-Bahru (LLB), which contains a high concentration of condensed tannins, on cellulolytic rumen fungal population in goats was investigated using real-time PCR. The fungal population in goats fed LLB was inhibited during the first 10 days of feeding, but after 15 days of feeding, there was a tremendous increase of fungal population (157.0 μg/ml), which was about fourfold more than that in control goats (39.7 μg/ml). However, after this period, the fungal population decreased continuously, and at 30 days of feeding, the fungal population (50.6 μg/ml) was not significantly different from that in control goats (55.4 μg/ml).

Simultaneous Detection of Waterborne Viruses by Multiplex Real-Time PCR: Lae-Hyung Kang , Se-hwan Oh , Jeong-Woong Park , Yu-Jung Won , Sangryeol Ryu , Soon-Young Paik; J. Microbiol. 2013;51(5):671-675. Published online September 14, 2013; DOI: https://doi.org/10.1007/s12275-013-3199-1

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Abstract: Norovirus, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus are all common causes of gastroenteritis. Conventional diagnoses of these causative agents are based on antigen detection and electron microscopy. To improve the diagnostic potential for viral gastroenteritis, internally controlled multiplex real-time polymerase chain reaction (PCR) methods have been recently developed. In this study, individual real-time PCRs were developed and optimized for specific detections of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus group B1. Subsequently, individual PCRs were combined with multiplex PCR reactions. In general, multiplex real-time PCR assays showed comparable sensitivities and specificities with individual assays. A retrospective clinical evaluation showed increased pathogen detection in 29% of samples using conventional PCR methods. Prospective clinical evaluations were detected in 123 of the 227 (54%) total samples used in the multiplex realtime PCR analysis. The Norovirus genogroup II was found most frequently (23%), followed by Rotavirus (20%), the Hepatitis A virus (4.5%), Coxsackievirus (3.5%), and Norovirus genogroup I (2.6%). Internally controlled multiplex real-time PCR assays for the simultaneous detection of Rotavirus, Coxsackievirus group B, the Hepatitis A virus, and Norovirus genogroups I and II showed significant improvement in the diagnosis of viral gastroenteritis.

NOTE] Evaluation of Fusarium Head Blight in Barley Infected by Fusarium graminearum: Woo-Ri Kang , Duk-Ju Hwang , Shin-Chul Bae , Theresa Lee , Soonok Kim , Il-Pyung Ahn; J. Microbiol. 2013;51(4):540-543. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3338-8

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Abstract: Fusarium head blight, which is primarily caused by Fusarium graminearum, is a devastating disease in the barley field. A real-time PCR protocol was developed to evaluate the growth of this pathogen in the host plant tissues. All four strains harbored the gene encoding ATP-BINDING CASSETTE TRANSPORTER (FgABC; FGSG_00541) as a single copy within their genomes. Our Southern blot result was identical with the genomic data for F. graminearum strain PH-1. Based on the crossing point (CP) values obtained in our TaqMan real-time PCR analysis, two standard curves describing the relationship among the CP value, FgABC copy number, and amount of fungal DNA were constructed. Chronological enumeration of fungal growth was coincided with the symptom development.

Quantification of Rice Sheath Blight Progression Caused by Rhizoctonia solani: Mukhamad Su’udi , Jong-Mi Park , Woo-Ri Kang , Duk-Ju Hwang , Soonok Kim , Il-Pyung Ahn; J. Microbiol. 2013;51(3):380-388. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-3274-7

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Abstract: Rhizoctonia solani has a wide host range, including almost all cultivated crops and its subgroup anastomosis group (AG)-1 IA causes sheath blight in rice. An accurate measurement of pathogen’s biomass is a convincing tool for enumeration of this disease. Mycological characteristics and molecular diagnosis simultaneously supported that all six strains in this study were R. solani AG-1 IA. Heterokaryons between strains Rs40104, Rs40105, and Rs45811 were stable and viable, whereas Rs40103 and Rs40106 did not form viable fused cells, except for the combination of Rs40106 and Rs40104. A primer pair was highly specific to RsAROM gene of R. solani strains and the amplified fragment exists as double copies within fungal genome. The relationship between crossing point (CP) values and the amount of fungal DNA was reliable (R2>0.99). Based on these results, we determined R. solani’s proliferation within infected stems through real time PCR using a primer pair and a Taqman probe specific to the RsAROM gene. The amount of fungal DNA within the 250 ng of tissue DNA from rice cv. Dongjin infected with Rs40104, Rs40105, and Rs45811 were 7.436, 5.830, and 5.085 ng, respectively. In contrast, the fungal DNAs within the stems inoculated with Rs40103 and Rs40106 were 0.091 and 0.842 ng. The sheath blight symptom progression approximately coincided with the amount of fungal DNA within the symptoms. In summary, our quantitative evaluation method provided reliable and objective results reflecting the amount of fungal biomass within the infected tissues and would be useful for evaluation of resistance germplasm or fungicides and estimation of inoculum potential.

Quantification of Rice Brown Leaf Spot through Taqman Real-Time PCR Specific to the Unigene Encoding Cochliobolus miyabeanus SCYTALONE DEHYDRATASE1 Involved in Fungal Melanin Biosynthesis: Mukhamad Su’udi , Jong-Mi Park , Woo-Ri Kang , Sang-Ryeol Park , Duk-Ju Hwang , Il-Pyung Ahn; J. Microbiol. 2012;50(6):947-954. Published online December 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2538-y

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Abstract: Rice brown leaf spot is a major disease in the rice paddy field. The causal agent Cochliobolus miyabeanus is an ascomycete fungus and a representative necrotrophic pathogen in the investigation of rice-microbe interactions. The aims of this research were to identify a quantitative evaluation method to determine the amount of C. miyabeanus proliferation in planta and determine the method’s sensitivity. Real-time polymerase chain reaction (PCR) was employed in combination with the primer pair and Taqman probe specific to CmSCD1, a C. miyabeanus unigene encoding SCYTALONE DEHYDRATASE, which is involved in fungal melanin biosynthesis. Comparative analysis of the nucleotide sequences of CmSCD1 from Korean strains with those from the Japanese and Taiwanese strains revealed some sequence differences. Based on the crossing point (CP) values from Taqman realtime PCR containing a series of increasing concentrations of cloned amplicon or fungal genomic DNA, linear regressions with a high level of reliability (R2>0.997) were constructed. This system was able to estimate fungal genomic DNA at the picogram level. The reliability of this equation was further confirmed using DNA samples from both resistant and susceptible cultivars infected with C. miyabeanus. In summary, our quantitative system is a powerful alternative in brown leaf spot forecasting and in the consistent evaluation of disease progression.

Journal Article

Possible Translocation of Periodontal Pathogens into the Lymph Nodes Draining the Oral Cavity: G. Amodini Rajakaruna , Makoto Umeda , Keisuke Uchida , Asuka Furukawa , Bae Yuan , Yoshimi Suzuki , Ebe Noriko , Yuichi Izumi , Yoshinobu Eishi; J. Microbiol. 2012;50(5):827-836. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2030-8

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Abstract: Numerous publications have reported the presence of periodontopathogenic bacteria in peripheral and central vascular lesions. However, it is unclear how this bacterial translocation occurs. The objective of this study was to investigate whether periodontopathic bacteria are translocated to lymph nodes proximal to the oral cavity. Obtaining lymph node samples is not ethically feasible unless they are excised as part of the surgical management of patients with cancer. This study analyzed formalin-fixed and paraffin-embedded lymph nodes, histologically negative for cancer cell invasion, that were excised from 66 patients with histories of head and neck cancer. Real-time PCR was performed to amplify the 16S ribosomal DNA fragments from Porphyromonas gingivalis, Treponema denticola, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Prevotella intermedia. The relationship between bacterial detection and cancer severity, gender, and the use of anti-cancer therapy was examined by Fisher’s exact test. P. gingivalis, T. forsythia, and P. intermedia were present in 17%, 8%, and 8% of the samples of submandibular and submental lymph nodes, respectively. There were no significant relationships between bacterial detection and the cancer disease status, patient gender or use of anticancer therapy. According to these data, it appears that the translocation of periodontopathic bacteria may occur via lymphatic drainage, irrespective of the cancer disease status, gender or anticancer therapy.

Research Support, Non-U.S. Gov'ts

Cloning and Expression Analysis of a Chitinase Gene Crchi1 from the Mycoparasitic Fungus Clonostachys rosea (syn. Gliocladium roseum): Zhongwei Gan , Jinkui Yang , Nan Tao , Zefen Yu , Ke-Qin Zhang; J. Microbiol. 2007;45(5):422-430.; DOI: https://doi.org/2594 [pii]

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Abstract: Clonostachys rosea (syn. Gliocladium roseum) is a well-known biocontrol agent and widely distributed around the world. In this study, an endochitinase gene Crchi1 was isolated from the mycoparasitic fungus C. rosea using the DNA walking strategy. The Crchi1 ORF is 1,746 bp long and interrupted by three introns. The cloned gene Crchi1 encodes 426 amino acid residues and shares a high degree of similarity with other chitinases from entomopathogenic and mycoparasitic fungi. Several putative binding sites for transcriptional regulation of Crchi1 in response to carbon (5''-SYGGRG-3'') and nitrogen (5''-GATA-3'') were identified in the upstream of Crchi1. Expression of Crchi1 gene in different carbon sources was analyzed using real-time PCR (RT-PCR). We found that the Crchi1 expression was suppressed by glucose but strongly stimulated by chitin or solubilized components of the cell wall from Rhizoctonia solani. Phylogenetic analysis of chitinases from entomopathogenic and mycoparasitic fungi suggests that these chitinases have probably evolved from a common ancestor.

Molecular Survey of Latent Pseudorabies Virus Infection in Nervous Tissues of Slaughtered Pigs by Nested and Real-time PCR: Hyun A Yoon , Seong Kug Eo , Abi George Aleyas , Seong Ok Park , John Hwa Lee , Joon Seok Chae , Jeong Gon Cho , Hee Jong Song; J. Microbiol. 2005;43(5):430-436.; DOI: https://doi.org/2279 [pii]

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Abstract: In this study, the prevalence and quantity of a latent pseudorabies virus (PrV) infection in the nervous tissues of randomly selected pigs was determined via nested and real-time PCR. The nervous tissues, including the trigeminal ganglion (TG), olfactory bulb (OB), and brain stem (BS), were collected from the heads of 40 randomly selected pigs. The majority of the nervous tissues from the selected pigs evidenced a positively amplified band on nested PCR. In particular, nested PCR targeted to the PrV glycoprotein B (gB) gene yielded positive results in all of the BS samples. Nested PCR for either the gE or gG gene produced positive bands in a less number of nervous tissues (57.5% and 42.5%, respectively). Real-time PCR revealed that the examined tissues harbored large copy numbers of latent PrV DNA, ranging between 100.1 and 107.2 (1-1.58x107) copies per 1 g of genomic DNA. Real-time PCR targeted to the PrV gE gene exhibited an accumulated fluorescence of reporter dye at levels above threshold, thereby indicating a higher prevalence than was observed on the nested PCR (100% for BS, 92% for OB, and 85% for TG). These results indicate that a large number of farm-grown pigs are latently infected with a field PrV strain with a variety of copy numbers. This result is similar to what was found in association with the human herpes virus.

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