Abstract
A real-time quantitative PCR assay using a species-specific primer pair was developed to rapidly and accurately quantify Valsa mali, the causative pathogen of apple Valsa canker (AVC), in crabapple seeds, crabapple seedlings, apple twigs and apple seeds. Surveys were conducted in different regions, and crabapple or apple seeds were collected for V. mali detection by qPCR assay. Our results showed that 12.87% to 49.01% of crabapple seeds collected from different regions were positive for V. mali. The exopleura and endopleura were the two major areas of V. mali infection in crabapple seeds. The presence of V. mali infection in crabapple seeds was also confirmed by a high-throughput sequencing approach. With the growth of crabapple seedlings, the concentration of V. mali gDNA in crabapple seedlings gradually increased until eight or more leaf blades emerged. One-year-old twigs from an apple scion nursery were infected with V. mali, and only apple seeds from infected apple trees showing evident Valsa canker symptoms carried V. mali. In conclusion, this study reports that crabapple seeds and apple seeds carried V. mali as latent inoculum sources. V. mali infected not only apple tissues but also crabapple seedlings, which are the rootstocks of apple trees. This study indicated that the inoculum sources for AVC vary. Application of a novel qPCR assay can potentially improve the accuracy of early diagnosis, and is helpful to reveal the epidemic regularity of AVC.
Highlights
Valsa mali Miyabe & Ymada, the pathogen that causes apple Valsa canker (AVC), is widely spread in eastern Asia[1,2,3] and causes serious losses to apple production, especially in China[4]
In addition to the typical symptoms of AVC, in some cases, latent infections by the pathogens can exist in apple tissues[8], which could be a potential risk of canker disease in apple trees, especially in newly established apple orchards
Ct values of a 10-fold DNA dilution series of a V. mali strain as the ordinate were plotted against the logarithm of the amount of sample copy number as the abscissa, which was calculated by the DNA concentration
Summary
Valsa mali Miyabe & Ymada (anamorph Cytospora sacculus), the pathogen that causes apple Valsa canker (AVC), is widely spread in eastern Asia[1,2,3] and causes serious losses to apple production, especially in China[4]. China is the largest producer of apples; the average incidence of AVC is approximately 52.7%, which severely limits the development of the apple industry[5] Control of this disease is difficult because the pathogen can infect the phloem and xylem of the host tissue. The infected grafted twigs and rootstocks without typical symptoms might be the primary source of infection in newly planted orchards This hypothesis is still difficult to confirm because of the difficulty of detecting V. mali in symptomless tissues. A nested PCR assay was developed to detect V. mali on symptomless and symptomatic apple tissues, and the accuracy of nested PCR for detection of the pathogen from the symptomless samples was relatively high[11] This method could not be used to quantify the amount of pathogen in apple plants
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