Rapid Detection of Colletotrichum siamense from Infected Tea Plants Using Filter-Disc DNA Extraction and Loop-Mediated Isothermal Amplification.

The pathogen Colletotrichum siamense causes tea anthracnose, resulting in economic losses to the Chinese tea industry. To effectively diagnose this pathogen in the field, we developed a loop-mediated isothermal amplification (LAMP) method using highly specific primers with a sensitivity of 1 pg/µl designed for amplifying the CAL gene, which was 10 times higher than that of conventional PCR. Additionally, to improve the method for obtaining DNA samples required for on-site diagnosis, we used the filter-disc DNA extraction method, which does not require special instruments and can be completed in a few minutes, and found that it effectively meets the requirements for the LAMP reaction. Finally, we combined LAMP with a filter-disc DNA extraction method (FDE-LAMP) to diagnose different degrees of disease in inoculated samples and 20 samples from the field. The results showed that the procedure had sufficient sensitivity for pathogen detection. Therefore, the FDE-LAMP procedure could greatly contribute to managing and preventing tea anthracnose in the field.

A LAMP-Based Toolbox Developed for Detecting the Major Pathogens Affecting the Production and Quality of the Chinese Medicinal Crop Aconitum carmichaelii.

Aconitum carmichaelii Debeaux is a traditional Chinese medicinal herb that has been utilized for approximately 2,000 years. However, as cultivation has increased, there have been more reports of A. carmichaelii infections caused by four major pathogenic fungal species, Fusarium oxysporum, F. solani, Mucor circinelloides, and Sclerotium rolfsii, resulting in increased disease incidences and limited production and quality. To detect these infections, we developed a LAMP-based toolbox in this study. The cytochrome c oxidase subunit 1 (cox1) gene, translation elongation factor-1α (EF-1α), internal transcribed spacer (ITS) regions of rDNA, and alcohol dehydrogenase 1 (ADH1) gene, respectively, were used to design species-specific LAMP primer sets for F. oxysporum, F. solani, S. rolfsii, and M. circinelloides. The results showed that the LAMP-based toolbox was effective at detecting pathogens in soil and plant materials. We also used this toolbox to investigate pathogen infection in the main planting regions of A. carmichaelii. Before harvesting, F. oxysporum, M. circinelloides, and S. rolfsii were commonly found in the planting fields and in infected A. carmichaelii plants. Therefore, the toolbox we developed will be useful for tracking these infections, as well as for disease control in A. carmichaelii.

Development of Culture Medium for the Isolation of Flavobacterium and Chryseobacterium from Rhizosphere Soil.

An effective medium designated phosphate separately autoclaved Reasoner's 2A supplemented with cycloheximide and tobramycin (PSR2A-C/T) has been developed for the isolation of Flavobacterium and Chryseobacterium strains from the plant rhizosphere. It consists of Reasoner's 2A agar (R2A) prepared by autoclaving phosphate and agar separately and supplementing with 50 mg L(-1) cycloheximide and 1 mg L(-1) tobramycin. A comparison was made among the following nine media: PSR2A-C/T, PSR2A-C/T supplemented with NaCl, R2A agar, R2A agar supplemented with cycloheximide and tobramycin, 1/4-strength tryptic soy agar (TSA), 1/10-strength TSA, soil-extract agar, Schaedler anaerobe agar (SAA), and SAA supplemented with gramicidin, for the recovery of Flavobacterium and Chryseobacterium strains from the Welsh onion rhizosphere. Flavobacterium strains were only isolated on PSR2A-C/T, and the recovery rate of Chryseobacterium strains was higher from PSR2A-C/T than from the eight other media. In order to confirm the effectiveness of PSR2A-C/T, bacteria were isolated from onion rhizosphere soil with this medium. Flavobacterium and Chryseobacterium strains were successfully isolated from this sample at a similar rate to that from the Welsh onion rhizosphere.

A species-specific method for detecting pathogenic Streptomyces species from soil and potato tubers in Argentina / A species-specific method for detecting pathogenic Streptomyces species from soil and potato tubers in Argentina.

Potato common scab is caused by several soil-inhabiting pathogenic Streptomyces species. In the present study, a species-specific PCR method was used to detect Streptomyces species in potato tuber lesions and soils. Total genomic DNA from soil samples from six locations and tuber samples from four potato cultivars (Spunta, Shepody, Innovator and Russet Burbank) were assessed. Streptomyces scabies, Streptomyces acidiscabies, and Streptomyces turgidiscabies were detected in soybean, tobacco and potato soils and in all potato varieties except Russet Burbank. The phylogenetic analysis of the sequences obtained confirmed the identification. The method proposed proved to be time-saving and cost effective for the rapid detection of Streptomyces species. This is the first report of the detection of S. acidiscabies and S. turgidiscabies in soils and potato tubers from Argentina.

A species-specific method for detecting pathogenic Streptomyces species from soil and potato tubers in Argentina.

Potato common scab is caused by several soil-inhabiting pathogenic Streptomyces species. In the present study, a species-specific PCR method was used to detect Streptomyces species in potato tuber lesions and soils. Total genomic DNA from soil samples from six locations and tuber samples from four potato cultivars (Spunta, Shepody, Innovator and Russet Burbank) were assessed. Streptomyces scabies, Streptomyces acidiscabies, and Streptomyces turgidiscabies were detected in soybean, tobacco and potato soils and in all potato varieties except Russet Burbank. The phylogenetic analysis of the sequences obtained confirmed the identification. The method proposed proved to be time-saving and cost effective for the rapid detection of Streptomyces species. This is the first report of the detection of S. acidiscabies and S. turgidiscabies in soils and potato tubers from Argentina.

A species-specific method for detecting pathogenic Streptomyces species from soil and potato tubers in Argentina. / A species-specific method for detecting pathogenic Streptomyces species from soil and potato tubers in Argentina.

Potato common scab is caused by several soil-inhabiting pathogenic Streptomyces species. In the present study, a species-specific PCR method was used to detect Streptomyces species in potato tuber lesions and soils. Total genomic DNA from soil samples from six locations and tuber samples from four potato cultivars (Spunta, Shepody, Innovator and Russet Burbank) were assessed. Streptomyces scabies, Streptomyces acidiscabies, and Streptomyces turgidiscabies were detected in soybean, tobacco and potato soils and in all potato varieties except Russet Burbank. The phylogenetic analysis of the sequences obtained confirmed the identification. The method proposed proved to be time-saving and cost effective for the rapid detection of Streptomyces species. This is the first report of the detection of S. acidiscabies and S. turgidiscabies in soils and potato tubers from Argentina.