Meanwhile, the specificity of our probe was again determined by searching the nucleotide databases. 2145 0 obj
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0000000016 00000 n
0000012477 00000 n
Entry into the bud meristem occurred between 6 and 36h after the teliospore deposition [5]. In order to detect the dynamic range of the assay on infected sugarcane, pathogen-free plantlets of variety FN40 were artificially inoculated with the smut pathogen and genomic DNA extracted from samples at 12h, 24h, 48h, 120h, 168h, and 336h. The sample collected at 0h was used as the mock control. A slow phase of smut pathogen growth was observed from 0h to 12h; then a logarithmic growth phase appeared from 12h to 60h, followed by a stationary phase and declining phase. For growth curve observation, smut spores were eluted to 5 106 spores/mL with 300mL sterile PDA liquid medium (containing 75g/mL streptomycin) and cultured at 140rpm at 28C [10]. ) times more sensitivity than conventional PCR. 0000015335 00000 n
0000008958 00000 n
Although visible in detecting the presence of the pathogen, conventional PCR was insufficient to quantify the pathogen in nature. 0000028547 00000 n
0000012696 00000 n
Each run of TaqMan real-time PCR contained three replicates, as well as three smut-DNA-template positive controls, mock controls (pathogen-free plantlets syringe-inoculated with sterile ddH2O), and blank controls (without template DNA). Single colonies were transferred onto the new PDA medium and cultured at 28C for 5d. Fungal mycelial DNA was extracted using the SDS method [10] and eluted with sterile water containing 100g/mL RNase A. ) and the highest at 24h ( In addition, the sensitive and accurate quantification of the smut pathogen by TaqMan PCR assay is beneficial in giving insight into the mechanisms of sugarcane-smut pathogen interaction. 0000022666 00000 n
0000014009 00000 n
By germination assay, teliospores from smut whip were transferred onto the PDA liquid medium containing 75g/mL streptomycin and cultured at 140rpm at 28C. Smut resistance is an important agronomic trait due to the serious loss in sugarcane stalk yield caused by smut pathogen [6, 31, 32]. Conventional PCR amplified product with a length of 459bp was detected on 1.5% agarose gel. 0000013568 00000 n
0000012099 00000 n
Furthermore, quantification of pathogen was verified in pathogen-challenged buds in different sugarcane genotypes, which suggested its feasibility for evaluation of smut resistance in different sugarcane genotypes. 0000020937 00000 n
"pF[[k%Hw.&(S% TGo4H8 I 2/ Correlation coefficient ( Therefore, the primers bEQ-F/bEQ-R and probe bEQ-P were selected for further experimentation. 0000015241 00000 n
(Table 1). These DNA samples were subjected to the TaqMan assay. 0000024335 00000 n
The standard curve was generated using tenfold serial dilutions with ddH2O from This method is more convenient than serological technique and hybridization assays using DNA probes, which are time consuming; insensitive and additional technical skills are required. 0000009197 00000 n
The observation of teliospore germination process by microscopy is shown in Figure 4(a). Two-bud sets of the sugarcane genotypes of Yacheng05-179 (resistant) and ROC22 (susceptible) were inoculated with 0.5L smut suspension as described above. 0000016010 00000 n
The This assay was capable of detecting the smut pathogen at the initial stage (12h) of infection and suitable for inspection of sugarcane pathogen-free seed cane and seedlings. copies/L) of +1 leaf gDNA of ROC22 infected with smut pathogen, which was 125 times more sensitive than that of conventional PCR (Table 1 and Figure 3). 0000011375 00000 n
0000019498 00000 n
In generated standard curves, linearity between the TaqMan real-time PCR Ct values and target concentration was observed over eight orders of magnitude in ten-fold serial dilutions in triplicate (Figure 1). 0000006971 00000 n
Then dilutions were detected by TaqMan real-time PCR as described above. 0000015445 00000 n
The sensitivity for the detection and quantification limits of the primers was investigated based on the plasmid with a known copy number of the bE insert. Sugarcane cultivar FN40 (a widely grown cultivar in China) was provided by the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture (Fuzhou, China). Figure 6 showed that the results of the smut pathogen quantification and the difference of copy numbers between resistant and susceptible varieties were significant. The TaqMan probe (bEQ-P) and the primers (bEQ-F/bEQ-R) were designed according to the sequence of smut bE gene using the Primer Express software version 2.0 (ABI Applied Biosystems, USA). 0000018097 00000 n
0000026574 00000 n
This could be applied to (i) sugarcane seeds or stalks importation and export inspection for smut pathogen, (ii) evaluation of smut resistance between or within several or a batch of sugarcane genotypes by quantifying the copy number of smut pathogen in asymptomatic smut-infected sugarcane, and (iii) supervision and management of the pathogen-free seed cane of sugarcane. 0000007949 00000 n
0000020592 00000 n
0000010046 00000 n
0000006176 00000 n
to 0000006803 00000 n
0000038083 00000 n
0000013746 00000 n
to 0000018444 00000 n
The authors especially thank Andrew C. Allan in The New Zealand Institute for Plant & Food Research Ltd. (Plant and Food Research), Mt Albert Research Centre, Auckland, New Zealand, for his critical revision and valuable comments on this paper. 0000009926 00000 n
to One positive clone was referred to as pbE. 0000013300 00000 n
The detection of smut pathogens is important at the early stages of sugarcane colonization [20] since it is difficult to differentiate it from other fungi based on mycelial morphology. 0000005815 00000 n
to Due to a huge amount of spores released by smut whips, it is difficult to stop the infection or reinfection of sugarcane including pathogen-free seedlings or plantlets. 0000003605 00000 n
The specificity of PCR-based TaqMan assay was performed using 100ng DNA of S. scitamineum, Phoma sp., Fusarium moniliforme, Pestalotia ginkgo, Fusarium oxysporum, and Helminthosporium sacchari. copies) of pbE DNA and 10 fg ( 0000015148 00000 n
0000017202 00000 n
Primers bEQ-F (5-TGAAAGTTCTCATGCAAGCC-3) and bEQ-R (5-TGAGAGGTCGATTGAGGTTG-3) were designed to yield a 123bp fragment of the bE gene. For the evaluation of TaqMan assay efficiency and the calculation of the copy number of the smut pathogen, a standard curve was generated using pbE DNA in each TaqMan assay. 0000014259 00000 n
The promycelia were found and began to detach at 12h. There were many free promycelia at 24h, and these promycelia lengthened as the culturing continued. was the slope and 0000010132 00000 n
0000005559 00000 n
The copy numbers decreased by 74.2% at 336h compared to those at 168h, but still higher than those of 12h. Sugarcane genotypes Yacheng05-179 (smut resistant) and ROC22 (smut susceptible) were artificially inoculated in the buds and used for investigation of the correlation between copy number of S. scitamineum and smut resistance. However, one of the limitations of the conventional PCR method is that they do not allow quantifying the amount of S. scitamineum in the sugarcane, and there is no report on sensitive detection and quantification assay for this pathogen. 660 dalton/bp [11]. 0000014956 00000 n
This was used for gradient dilution in sterile water and plated onto potato dextrose agar (PDA) containing 75g/mL streptomycin (Shenggong, China). 0000009559 00000 n
Thus, it is useful in the production and supervision of pathogen-free sugarcane seed cane in the programme of pathogen-free seed cane in mainland China. to In the present study, a set of TaqMan real-time PCR primers and a probe, which were reliable and specific, were designed according to the sequences of smut bE gene and used for the detection and quantification of smut in sugarcane. Entry into the meristem of the buds occurs between 6h and 36h after the teliospore deposition [5]. Conventional diagnostic approaches involve the application of morphological identification, which is time consuming and thus not conducive to control the spread of disease for the three months of disease period [3], or the isolation of the pathogen followed by biochemical identification and pathogenicity tests (requires more than one week) [7], or serological testing which requires high quality antibodies [8], or electron microscopy which needs expensive equipment [8]. This technique, including TaqMan real-time PCR, has been widely used for the diagnosis of pathogens such as planta botrytis cinerea [24], sugarcane yellow leaf virus [19], maize chlorotic mottle virus [8], and cucumber vein yellowing virus [25]. 0000006027 00000 n
With the development of the PCR technique, the required detection sensitivity could be achieved. 0000018908 00000 n
at 168h. In contrast, copies in susceptible variety varied from 0000012901 00000 n
), slope (S), and efficiency ( The end-point dilution of these two assays in which a positive result was recorded was compared. 0000019692 00000 n
copies/L). The TaqMan assay R2 value was 0.998; Ct values ranged from 0000011830 00000 n
Due to its high specificity, sensitivity, accuracy, and speed, real-time PCR is a suitable detection technique [23]. At 48h, a large number of spores had detached, which lead to many microspores and resulted in turbidity of the liquid medium. The standard curve was generated using pbE DNA with the R2 value in the linear regression being 0.994. Schenck [22] found that conventional PCR assays were significantly more sensitive and efficient than microscopy for smut pathogen detection. 0000024886 00000 n
Standard curve analysis was based on threshold cycles values (Ct) and serial dilutions of pbE DNA (103 to 1010). The above results demonstrated that the application of TaqMan real-time PCR assay in relatively accurate quantification of the target DNA was possible, which also showed a wider dynamic range of nearly 1000 ( At each time point of 0h, 12h, 24h, 48h, 120h, 168h, and 336h after inoculation treatment, three culms were sampled for DNA extraction by CTAB-based protocol as reported [14]. 0000037399 00000 n
Similarly, Singh et al. Sporisorium scitamineum is a fungal smut pathogen epidemic in sugarcane producing areas. This assay showed excellent results with regard to sensitivity, compared to conventional PCR. A linear regression analysis ( 0000018662 00000 n
The PCR conditions for this standard curve were adopted to perform the further reactions to estimate the copy number of smut pathogen by TaqMan real-time PCR assay. to Quantification of smut pathogen in the buds was accomplished by calculating the target amplicon copy number based on TaqMan real-time PCR. ), which revealed a linear relationship between the quantities of templates and Ct values, was performed with To further validate the sensitivity of this TaqMan method, +1 leaf gDNA of ROC22 infected with smut was serially diluted five fold and measured by TaqMan real-time PCR and conventional PCR. 0000019048 00000 n
0000007844 00000 n
0000016524 00000 n
The sequence of the smut bE gene was chosen as the PCR target gene for confirmation of the presence of smut, as reported by Albert and Schenck [9]. paratuberculosis [27]. , with the lowest at 12h ( 2013, Article ID 942682, 9 pages, 2013. https://doi.org/10.1155/2013/942682, 1Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture/Fujian Agriculture and Forestry University, Fuzhou 350002, China. The infected buds may either produce symptoms or exist as a latent infection which may germinate and produce black whips in the following season [4]. Smut-infected whips were collected from sugarcane cultivar ROC22 cultivated in the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture (Fuzhou, China). A BLAST comparison of the 123bp region of the smut bE gene, for which the primers were designed, showed no similarity with other sequences but had a 100% sequence identity with the target bE gene in GenBank. The (Figure 5). 0000037279 00000 n
The growth curve could be divided into slow phase (012h), logarithmic growth phase (1260h), and stationary phase (60h-). When the concentration of the +1 leaf gDNA was reduced to 100ng/L, no apparent amplification was observed on agarose gel (Figure 3). Based on the TaqMan PCR assay, the copy numbers of the pathogen at 12168h in smut resistant variety Yacheng05-179 challenged by S. scitamineum were much lower (ranging from Pathogen detection is a crucial procedure in the import and export of sugarcane stalk during germplasm exchange and in the supervision and management of pathogen-free cane or plantlets from tissue culture. The pathogen-free FN40 four-month-old plantlets were syringe-inoculated from the basal portion up to 2cm length with 0.5L of the smut suspension containing 5 106 spores/mL in 0.01% (v/v) Tween-20 [7, 13]. , with much higher conidial densities than those of the resistant variety especially at first sampling point ( The TaqMan real-time PCR assay established here can shorten testing time and be used as a tool for the detection and quantification of this pathogen in sugarcane. Early detection and proper identification of the smut are an essential requirement in its management practice. 0000018198 00000 n
Here, for the first time, we report the development of a TaqMan real-time PCR assay for the smut, which is compared with conventional PCR, is much more sensitive. In summary, the present study confirmed that these designed primer sets and probe are highly specific and sensitive for smut detection. If the Ct value exceeds 35, the sample should be detected again, from which no Ct value means negative, otherwise positive. 0000016786 00000 n
0000033636 00000 n
The amplified bE fragment (459bp) was purified from a 1.5% agarose gel and cloned into Escherichia coli DH5 cell, using pMD18-T vector (TaKaRa, China). copies (Figure 2). Some promycelia outgrew basidiospores at 36h, and a small amount of them began to detach to form microspores. The development and severity of the smut disease depend on environmental conditions, the resistance of sugarcane genotypes, and the interaction between sugarcane, S. sporisorium, and environment. Initial quantities of smut pbE DNA templates were 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag, and 10ag, approximately 1 This work was funded by National Natural Science Foundation of China (no. 0000025428 00000 n
The TaqMan probed bEQ-P (5-TGCTCGACGCCAATTCGGAG-3) contained 6-carboxy-fluorescein (FAM) reporter dye at the 5 end and 6-carboxytetramethylrhodamine (TAMRA) fluorescent quencher at the 3 end. 0000008252 00000 n
0000018544 00000 n
Song, L. P. Xu, and R. K. Chen, Differential gene expression in sugarcane in response to challenge by fungal pathogen, Y. Que, L. Xu, J. Lin, M. Ruan, M. Zhang, and R. Chen, Differential protein expression in sugarcane during sugarcane- sporisorium scitamineum interaction revealed by 2-DE and MALDI-TOF-TOF/MS,, D. M. Gong and R. K. Chen, Advances in the study on resistance mechanism and heritance to sugarcane smut caused by, Y. X. Que, L. P. Xu, J. W. Lin, T. S. Chen, R. K. Chen, and Y. L. Li, Establishment of evaluation system of smut resistance for sugarcane varieties,, L. P. Xu, Y. Q. Lin, and H. Y. Fu, Evaluation of smut resistance in sugarcane and identification of resistance in sugarcane varieties,. 0000019790 00000 n
HVmp;I%;&06P&chgdLM)s:iI~499]!IC"! 1 !OKwX-; h{'`F W%W m QB
G;:]I&j~?_roleE]%K[Z9H;]Xtu:x}G)?TfBxx xf %Yl->U5FV]G~"|'{
'|_~i2`8ol{?xs^?]:
ozg7:~Yz'D2p^jk(%ck:Rp.e1Fc^Y,i%]p2v UX/HEiv"NU^]1D__s),p/Eh8)(^*SSh@aBE53@S~CSQ:i6h@7Xdn`jr"#NU^Px>UYQqUd;5-,qRTT4AgX/Qdp*_RP yPdNJrkOZFqlJ*. Teliospores were mixed and sealed in plastic bags and then stored at 4C. The sequences used to design testing primers and probe were compared to other organisms in National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) to confirm the specificity and were commercially synthesized by TaKaRa Biotechnology (Dalian, China). In this study, the TaqMan assay was successfully applied to quantify the smut pathogen in tissue cultured plants (FN40) challenged by the pathogen (Figure 5), and the results indicated that this assay was capable to detect the pathogen at the early stage (12h) of the challenge and at the limit of copies/L of smut pathogen was detected, and the corresponding fluorescent levels continued to increase from 24h ( After that, five buds from both varieties were excised at 0h, 12h, 24h, 48h, and 168h after inoculation, respectively. A TaqMan Real-Time PCR Assay for Detection and Quantification of, Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture/Fujian Agriculture and Forestry University, Fuzhou 350002, China. Ma = mean value of three technical replicates; P. Padmanaban, K. C. Alexander, and N. Shanmugan, Effect of smut on growth and yield parameters of sugarcane,, Y. X. Que, L. P. Xu, J. W. Lin, R. K. Chen, and M. P. Grisham, Molecular variation of Sporisorium scitamineum in Mainland China revealed by RAPD and SRAP markers,, A. R. Sundar, E. L. Barnabas, P. Malathi, and R. Viswanathan, A mini-review on smut disease of sugarcane caused by Sporisorium scitamineum, in, K. C. Alexander and K. Ramakrishnan, Infection of the bud, establishment in the host and production of whips in sugarcane smut (, L. P. Xu, R. K. Chen, and P. H. Chen, Analysis on infection index of smut caused by, N. Singh, B. M. Somai, and D. Pillay, Smut disease assessment by PCR and microscopy in inoculated tissue cultured sugarcane cultivars,, Y. Zhang, W. Zhao, M. Li, H. Chen, S. Zhu, and Z. 0000010650 00000 n
In this study, was a fixed value as 1.987 1011 for the dynamic range of smut which was obtained by the copy number for each serial dilution (100pg 10 ag).