Detection of Alternaria solani with high accuracy and sensitivity during the latent period of potato early blight

Abstract

Early blight (EB) disease, caused mainly by Alternaria solani, is an economic threat to potato and tomato production worldwide. Thus, accurate and sensitive detection of the fungal pathogen of this disease in plants at the early infection stage is important for forecasting EB epidemics. In this study, we developed an RNA-based method that enables highly accurate and sensitive A. solani detection in a whole potato leaf at a single spore level based on quantitative real-time polymerase chain reaction (qPCR). We discovered jg1677, a highly expressed gene whose full-length coding sequence is very specific for A. solani, by analyzing A. solani transcripts isolated from enhanced high throughput transcriptome of infected potato leaves by A. solani and using the National Center for Biotechnology Information's basic local alignment search tool. The specificity of the primers derived from jg1677 was determined using 22 isolates of common potato pathogens, including seven Alternaria isolates. Detecting jg1677 transcripts with qPCR is 1,295 times more sensitive than detecting genomic DNA. In addition, the expression pattern of jg1677 at different infection stages was determined by qPCR. What is more, jg1677 was expressed relatively stable between 15 and 35°C in infected leaves, and its expression was virtually unaffected in isolated leaves left at room temperature for 24 h. Our work provides a much more sensitive and accurate method compared to conditional DNA-based ones, permitting a very early diagnosis of EB and lowering the risk of EB epidemics.

Keywords: Alternaria solani; RNA-based; detection – plant pathogens; early blight disease; potato.

FIGURE 1

Acquisition workflow of A. solani-specific candidate genes. Obtaining total RNA from spray-inoculated potato leaves by A. solani, and separating A. solani transcripts from total reads. Sorting transcripts from highest to lowest according to their abundances, and BLAST them in NCBI. Finally, verified candidate transcripts.

FIGURE 2

Detection of the specificity of jg1677 primer. (A) Agar gel electrophoresis of the PCR products of each primer pair showed no bands for CK and jg1677 and bands for jg9669 and gene 023260. StActin7 as the loading control. (B) The sequence comparison showed that the specific primers q-jg1677-F and q-jg1677-R could not be matched to Bipolaris maydis ATCC 48331. (C) Detection of jg1677 is A. solani-specific. Lane M, DL2000-bp DNA marker; Lane 1, potato cDNA, the negative control; Lane 2–23, the loading order is the same as in Table 2.

FIGURE 3

Comparison of the sensitivity of RNA-based and DNA-based detection methods. (A) qPCR using DNA and RNA to detect A. solani jg1677 expression levels. Data are shown as means ± SD. Error bars represent SD. (B) Standard curves for qPCR detection of A. solani using single spore cDNA samples diluted fivefold, 20-fold, and 40-fold.

FIGURE 4

Expression of jg1677 at different temperatures. qPCR detection of jg1677 expression at different temperatures. Data are shown as the expression of jg1677 compared to that at 25°C. Error bars represent SD.

FIGURE 5

Expression levels of jg1677 before and post spore germination. qPCR detection of jg1677 expression at different germination periods. Data are shown as the expression of jg1677 compared to conidia. Error bars represent SD.

FIGURE 6

Expression levels of jg1677 of samples collected in plastic bags for different times at room temperature. qPCR detection of jg1677 expression for different sealing times. Data are shown as the expression of jg1677 compared to control. Error bars represent SD.