Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR (IC-real-time RT-PCR) assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:10¹, 1:10², 1:10³, 1:10⁴, 1:10⁵ and 1:10⁶ (w/v, g/mL) were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:10² and 1:10³ dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:10⁶ dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:10⁶ dilution, DAS-ELISA could detect at least 1:10³ dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control.

In the fall of 2013 and 2014, peanut (Arachis hypogaea) pod rot specimens were collected from Laixi District, Qingdao City, Shandong Province, China. The peanut pods were black and rotted, and some were covered with white mycelia. Symptomatic pods were cut into 0.3–0.5 cm sections, surface disinfected with 70% ethanol solution (v/v) for 30 s, and soaked in 0.1% mercuric chloride solution for 60 s. The surface-sterilized samples were rinsed with sterile distilled water three times, dried, and placed on Czapek's Dox agar supplemented with 100 μg/ml of chloramphenicol. The plates were incubated in the dark at 28°C for 3 to 5 days. The fungal colonies were initially white and radial. The hyphae were septate, 4–10 μm wide, and had almost right-angled branching with a basal constriction, and were white to brown. As the hyphae grew, they produced sclerotia, which were white at first, and then turned dark brown. The sclerotia were ball shaped, elliptic, or irregular, 3.5–6.5 mm in diameter, had a rough surface, a...

SUMMARY: Deploying resistant cultivars is an economical and essential management method in controlling viral diseases, and there are several mutational resources for tobacco. In the present study, the inoculation of tobacco plants with tobacco viruses was performed in a greenhouse from 2011 to 2014 to identify mutants resistant to tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). The high-throughput screening included seeding uniformly, transplanting in seedbeds, inoculating by cloth brushes and reporting symptoms based on disease indices. A total of 4000 second generation segregating (M₂) mutants of tobacco cultivar Zhongyan100 were screened. Seeds from highly resistant mutant M₂plants were selected and planted separately. The M₃were grown and mutational stability was measured. For TMV, ten highly resistant plants were selected in the M₂generation and the mutation rate was 0·012%. In the M₃generation, there were seven mutants with hereditary high resistance and, according to the results of real-time polymerase chain reaction, theNgene was detected in all seven M₃. Two hereditary immune M₄mutants, one of which was a male sterile line, were identified and evaluated in the glasshouse and in the field. For CMV, seven highly resistant plants were selected from the M₂generation and the mutation rate was 0·009%. In the M₃generation, there was one mutant with hereditary high resistance. The results indicate that hereditary mutants may be identified in the M₄generation and back-crossed to wild-type Zhongyan100 to identify anti-viral genes.