A quantitative real-time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds


E-mail: robert.conner@agr.gc.ca


Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum lindemuthianum. Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real-time PCR-based diagnostic assay was developed for detecting C. lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA (rDNA) region consisting of part of the18S rDNA, 5.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C. lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C. lindemuthianum genomic DNA. To explore the correlation between the lesion area and the DNA amount of C. lindemuthianum in bean seed, seeds of the navy bean cultivar Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real-time PCR assays.


Bean anthracnose, caused by the fungus Colletotrichum lindemuthianum, is one of the most devastating diseases of dry bean (Phaseolus vulgaris) (Ishikawa et al., 2008). The anthracnose pathogen produces lesions on the leaves, stems and pods of beans. Early severe anthracnose infection can result in crop defoliation and large reductions in crop yield and seed quality (Tu, 1988). Pod infection usually leads to seed infection and discoloration (Schwartz et al., 2005). Seed infection has been shown to reduce germination and seedling vigour (Ravi et al., 1999). The use of pathogen-free seed is one of the key components in an integrated strategy for the control of this disease (Schwartz et al., 2005).

Infected seed and crop debris are the two most common sources of primary inoculum for anthracnose outbreaks (Schwartz et al., 2005). Crop rotation for 2–3 years has been recommended for the control of bean anthracnose. A recent field study demonstrated that seed infection alone at a level of 7% resulted in yield losses in the susceptible navy bean cultivar Navigator that ranged from 15 to 32% (Conner et al., 2004). Growth habit and row spacing appear to have little effect on anthracnose development in the field, but seed infection had a major impact on disease development and yield loss (Conner et al., 2006, 2009). It also has been demonstrated that transmission rates of the anthracnose fungus from infected seed to developing seedlings rose with an increase in size of the anthracnose lesions on the seed (Tu, 1983). The same study showed that symptomless seed infected by the anthracnose fungus could produce diseased seedlings. The efficacy of fungicide seed treatment on the control of anthracnose was strongly influenced by lesion size on bean seed (Tu, 1996). The application of fungicide seed treatment to bean seeds with large lesions (i.e. >2 mm) was ineffective at eliminating subsequent spread of the disease to emerging seedlings. Seed lots can be sorted to discard seeds with obvious lesions, but infected symptomless seed can be an important source of primary infection (Tu, 1983). However, it has long been recognized that the detection of pathogens in symptomless seeds is extremely difficult (Schaad & Frederick, 2002).

Traditionally, diagnosis of bean anthracnose infection was based on isolation of the pathogen from infected plant tissues onto nutrient agar. This method usually requires 10–14 days before the presence of the pathogen can be verified. However, a PCR-based system for rapid and sensitive detection of the anthracnose fungus in all types of bean tissue was developed. It permits the diagnosis of anthracnose within a 24 h period, providing qualitative data on the presence or absence of the bean anthracnose pathogen (Chen et al., 2007).

Real-time quantitative PCR was first developed to meet specific technical requirements, such as a high sensitivity and specificity, which were not easily achieved with other classical techniques. It is now becoming a routine tool because of its demonstrated reliability and rapidity (Gachon et al., 2004). Real-time PCR assays enable both pathogen detection and quantification simultaneously. They have the advantages of speed, accuracy and sensitivity over conventional PCR-based techniques (Schaad & Frederick, 2002). Real-time PCR has been used to quantify the extent of seed infection for a number of fungal pathogens of cereal, fruit and oilseed crops (Bates et al., 2001; Cullen et al., 2002; Guillemette et al., 2004; Debode et al., 2009; Cottyn et al., 2011). The amount of target DNA can be quantified by constructing a standard curve that relates the intensity of fluorescence to known amounts of template DNA. Moreover, real-time PCR assays have been used to detect and measure the presence of specific fungi, bacteria, viruses and viroids for research purposes (Kuan et al., 2011).

This study was undertaken to develop a real-time PCR assay to detect C. lindemuthianum and explore the relationship between anthracnose lesion size on navy bean seed and the amount of C. lindemuthianum genomic DNA in the seed.

Materials and methods

Source of materials

All of the microbial isolates used in this study are listed in Table 1. The fungal and bacterial cultures were started from stock cultures and grown on potato dextrose agar (PDA; Difco) at room temperature for 10–14 days. Then, an agar plug of each fungal or bacterial culture was transferred into liquid potato dextrose broth (PDB) and grown in shake culture at 100 rpm in the dark for 3–7 days (Chen et al., 2007). Seeds of the white navy bean cv. Navigator (Smith et al., 2009) were used for detection of C. lindemuthianum. All of the C. lindemuthianum-infected and symptomless seeds of cv. Navigator were collected from a field site at the Morden Research Station. Greenhouse-grown seed of an anthracnose-resistant bean differential line, G2333 (Young et al., 1998), was used as the pathogen-free control.

Table 1. Origin of fungal and bacterial isolates, and host plant samples used to develop and test a real-time PCR assay for amplification of the bean anthracnose pathogen, Colletotrichum lindemuthianum, using the ClF432/ClR533 primer set
  1. a

    AAFC, Agriculture and Agri-Food Canada; CCFC, Canadian Collection of Fungal Cultures; AAFRD, Alberta Agriculture, Food and Rural Development.

  2. b

    D, detected; ND, not detected.

1 Colletotrichum lindemuthianum Race 2 Phaseolus vulgaris Michigan State UniversityD
2 Colletotrichum lindemuthianum Race 17 Phaseolus vulgaris AAFC-HarrowD
3 Colletotrichum lindemuthianum Race 23 Phaseolus vulgaris AAFC-HarrowD
4 Colletotrichum lindemuthianum Race 31 Phaseolus vulgaris AAFC-HarrowD
5 Colletotrichum lindemuthianum Race 73 Phaseolus vulgaris AAFC-MordenD
6 Colletotrichum lindemuthianum Race 89 Phaseolus vulgaris AAFC-HarrowD
7 Colletotrichum lindemuthianum Race 105 Phaseolus vulgaris AAFC-MordenD
8 Colletotrichum lindemuthianum Race 1096 Phaseolus vulgaris AAFC-MordenD
9 Colletotrichum acutatum DAOM 214715UnknownCCFCND
10 Colletotrichum acutatum DAOM 214992UnknownCCFCND
11 Colletotrichum brassicae DAOM 116226UnknownCCFCND
12 Colletotrichum capsici DAOM 212661UnknownCCFCND
13 Colletotrichum circinans DAOM 151616UnknownCCFCND
14 Colletotrichum coccodes DAOM 60413UnknownCCFCND
15 Colletotrichum dematium DAOM 96293UnknownCCFCND
16 Colletotrichum destructivum DAOM 179749UnknownCCFCND
17 Colletotrichum destructivum DAOM 216020UnknownCCFCND
18 Colletotrichum fuscum DAOM 216112UnknownCCFCND
19 Colletotrichum gloeosporioides DAOM 183087UnknownCCFCND
20 Colletotrichum gloeosporioides DAOM 214710UnknownCCFCND
21 Colletotrichum gloeosporioides DAOM 225300UnknownCCFCND
22 Colletotrichum graminicola DAOM 231462UnknownCCFCND
23 Colletotrichum higginsianum DAOM 225478UnknownCCFCND
24 Colletotrichum lini DAOM 183091UnknownCCFCND
25 Colletotrichum musae DAOM 182828UnknownCCFCND
26 Colletotrichum pisi DAOM 196850UnknownCCFCND
27 Colletotrichum subliniolum DAOM 212374UnknownCCFCND
28 Colletotrichum trifolii DAOM 197037UnknownCCFCND
29 Colletotrichum truncatum DAOM 215174UnknownCCFCND
30 Uromyces phaseoli Mb05-70 Phaseolus vulgaris AAFC-MordenND
31 Sclerotinia sclerotiorum Phaseolus vulgaris AAFC-MordenND
32Fusarium solani f. sp. phaseoli Phaseolus vulgaris AAFC-MordenND
33Fusarium solani f. sp. pisi98Fus016-1 Pisum sativum AAFC-MordenND
34 Rhizoctonia solani Phaseolus vulgaris AAFC-MordenND
35Pythium spp.P-p3 Pisum sativum AAFRD-LacombeND
36 Botrytis cinerea B-3 Lupinus angustifolius AAFRD-LacombeND
37Xanthomonas campestris pv. phaseoliBXP-2 Phaseolus vulgaris AAFC-MordenND
38Pseudomonas syringae pv. phaseolicolaHB06-2 Phaseolus vulgaris AAFC-MordenND
39 Corynebacterium flaccumfaciens OSB-3 Phaseolus vulgaris AAFC-LethbridgeND
40 Phaeoisariopsis griseola MYA-2353 Phaseolus vulgaris University of GuelphND
41Phaseolus vulgaris (G2333) AAFC-MordenND
42DNA-grade water ND

Seed lesion area calculation

A total of 75 seeds with different sized lesions on only one side were harvested in 2006, 2007 and 2008 and were photographed on both sides with a Sony Cyber-Shot DSC-T7 digital camera (Sony Inc.). Squares of gridded paper (1 cm2) were photographed for use as the calibration file with the same camera settings. The area of each lesion was highlighted in green using photoshop cs2 software (Adobe Systems Inc.) and then assess image analysis software (L. Lamari, American Phytopathological Society) was used to quantify the lesion area of 75 C. lindemuthianum-infected bean seeds.

DNA extraction and ITS sequencing

Each of the infected and symptomless seeds was surface-sterilized using 70% ethanol and rinsed three times with sterilized water and air-dried. The seeds were individually ground into a fine powder with a sterilized mortar and pestle. A total of 50 mg of fine powder from each seed was used for DNA extraction with the DNeasy Plant Mini Kit (QIAGEN) according to the manufacturer's protocol. One hundred microlitres of elution buffer AE from the DNeasy Plant Mini Kit was used to elute the genomic DNA, and the eluate was used as the template for real-time PCR assays. Each reference genomic DNA was extracted from microbial cultures with the same kit. The concentration of pure DNA of C. lindemuthianum and the other microbial species was estimated using a NanoDrop-1000 spectrophotometer (Thermo Scientific) at 260 nm. DNA quality was evaluated according to the value of A260 nm/A280 nm (Sambrook et al., 1989). All DNA samples were stored at −20°C for later use.

The internal transcribed spacer (ITS) sequence of each microbial culture was amplified with the universal ITS4/ITS5 primer set (White et al., 1990). PCR products were purified with the MinElute PCR Purification Kit (QIAGEN) and then sent to the University Core DNA Services at the University of Calgary to be sequenced with both ITS4 and ITS5 primers. All sequences were clarified manually and aligned using clustalW embedded in the BioEdit sequence alignment editor (Hall, 1999). All sequences obtained in this study have been deposited into GenBank under accession numbers EU400129 to EU400156.

Real-time PCR primers and probe design

ITS fragments were sequenced from seven races of C. lindemuthianum, 21 other Colletotrichum spp. isolates and 9 other bean pathogens (Fig. S1). The ITS sequence of P. vulgaris was obtained from GenBank through the National Center for Biotechnology Information (NCBI) website. All above sequences were aligned to a sequence matrix which was used to design C. lindemuthianum-specific real-time PCR primers. Based on the ITS sequence of race 73 of C. lindemuthianum, 20 possible primer sets were designed under the parameters specified for real-time PCR using the online primer design program, PrimerQuest (http://www.idtdna.com/scitools/applications/primerquest/). Of the 20 primer sets, only one reverse primer showed potential specificity for C. lindemuthianum and only the last three bases of the reverse primer appeared to be specific to Clindemuthianum. This reverse primer, named C1R533, is located in both the ITS2 and 18S rDNA regions (Fig. S1).

The putative C. lindemuthianum-specific reverse primer information was programmed into primer express (v. 3.0; Applied Biosystems) to design forward primers as well as TaqMan MGB probes for the assay. The primer set was analysed for specificity using the blast program at the NCBI website (http://www.ncbi.nlm.nih.gov/blast). The primers were then synthesized by Invitrogen. The MGB probe was obtained from Applied Biosystems.

Real-time PCR amplifications

The real-time PCR assay and data analysis were performed with primer set ClF432/ClR533 on the StepOne (48-well) real-time thermal cycler (Applied Biosystems). All reactions were run in a total volume of 20 μL using 48-well microwell plates. For the SYBR Green dye assay, each PCR reaction contained 1× TaqMan Fast Universal PCR Master Mix, 500 nm of each primer ClF432 (5′-GGAGCCTCCTTTGCGTAGTAAC-3′) and ClR533 (5′-ACCTGATCCGAGGTCAACCTTGTT-3′), 0·75× SYBR Green I and 1 μL template DNA. The real-time PCR reactions were run at the default settings of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. For the TaqMan MGB probe assay, each PCR reaction contained 1× TaqMan Fast Universal PCR Master Mix, 500 nm of each primer ClF432 and ClR533, 100 nm internal TaqMan MGB probe ClP442 (6FAM-AGGGCACTCCTGCCGT-MGBNFQ) and 1 μL template DNA in a total volume of 20 μL. For the TaqMan MGB probe assay, all reactions were run at 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. For the SYBR Green dye assay, all reactions were run at 95°C for 10 min, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s, and a melt curve phase of 1 cycle at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s (ramp speed 0·3°C s−1) was programmed at the end of each run.

The specificity of the primer set used in this study was evaluated in two steps: first by finding the homologous DNA sequences by means of the blast program, and secondly by detecting the fluorescent signal of the target DNA using the real-time PCR assays. For the SYBR Green dye assay, the melt curve determined if the melting temperature of the PCR product using the genomic DNA of seed samples is the same as that of the PCR product of the genomic DNA of pure C. lindemuthianum. To quantify C. lindemuthianum genomic DNA, and to test the amplification efficiency and the detection limits of the real-time PCR assays, a 10-fold dilution series of C. lindemuthianum genomic DNA (5 × 100 to 5 × 106 fg) was used to create a standard curve for both the SYBR Green dye and the TaqMan MGB probe real-time PCR assays. Both standard curves were constructed by plotting the mean Ct value (= 3) versus logarithmic concentrations of C. lindemuthianum as measured with the primer set ClF432/ClR533. Triplicates were always used for each sample. Each run included two negative control samples, in which the DNA template was replaced by sterile water, and three standard samples which contained 500, 50 and 5 pg of genomic DNA from a pure culture of C. lindemuthianum race 73. The three standard samples were used to check the efficiency of the PCR amplification and calculate the amount of pathogen DNA in the seed samples. The samples were retested if the standard deviation of the Ct values for the three replicates was >0·5. Statistical correlations were determined between the ratio of lesion area and seed area of the Clindemuthianum-infected seeds to the log of C. lindemuthianum DNA quantity detected for both assays.


Specificity of primer set ClF432/ClR533

For the two primers, ClF432 and ClR533, only the reverse primer, ClR533, showed a high specificity for C. lindemuthianum, clearly distinguishing it from the other Colletotrichum spp. and the other bean pathogens. blast results of the reverse primer ClR533 did not show significant homology to other published sequences in the GenBank. Based on the sequence information of the ITS regions of the microbial strains evaluated, the specific reverse primer ClR533 was identified, which met the real-time PCR primer design criteria. This primer is located between the ITS2 region and 28S rDNA gene and the last three target-specific bases are located at the 3′ end of ITS2 region (Fig. S1). The specificity of the primer set was also verified by the results of the real-time PCR tests with both TaqMan MGB probe and SYBR Green dye assays. Positive amplification of DNA was observed only for the seven races of C. lindemuthianum, but not from the other Colletotrichum species, other bean pathogens or the host plant G2333 DNA (Table 1), indicating that the primer set ClF432/ClR533 was very specific to C. lindemuthianum. With the SYBR Green dye assay, only one melting temperature value was detected for the PCR products, which means only the target amplicon was amplified and no primer dimers or unexpected amplicons were produced (Fig. S2).

Standard curve and detect sensitivity

Strong linear relationships between the Ct values and the log of the amount of fungal DNA (fg) were shown by high correlation coefficients (i.e. R2 = 0·995 for the SYBR Green dye assay and R2 = 0·997 for the TaqMan MGB assay; Fig. S3). The quantitative detection limit of both assays was as low as 5 fg fungal DNA within the range of 5 × 100 to 5 × 106 fg of C. lindemuthianum genomic DNAs. The Ct value of 5 fg standard samples was 36·4 for the SYBR Green dye assay and 35·9 for the TaqMan MGB assay (Fig. S3).

Isolations from symptomless cv. Navigator seeds

The 30 symptomless cv. Navigator seeds collected each year (2006–2008) from the same field site as the other infected and the symptomless seeds for real-time PCR detection, were surface-sterilized then cultured for 7 days at room temperature on PDA. No fungal growth was observed on the medium from any of the symptomless seeds.

Real-time PCR tests on bean seeds

The mean Ct values of the 75 infected seeds with visible lesions ranged from 16·02 to 32·34 for the TaqMan MGB probe assay and 16·50 to 28·45 for the SYBR Green dye assay. The amount of C. lindemuthianum genomic DNA in the 75 infected seeds, which was estimated based on the known standard samples, ranged from 573·68 fg to 2·38 ng for the TaqMan MGB probe assay (Fig. 1) and 1·59 pg to 2·25 ng for the SYBR Green dye assay (Fig. 2). Statistical correlations between the ratio of lesion area and seed area of the C. lindemuthianum infected seeds to the log of detected C. lindemuthianum DNA per reaction were obtained for the TaqMan MGB probe assay (R2 = 0·52; Fig. 1d) and the SYBR Green dye assay (R2 = 0·55; Fig. 2d). Moreover, among the correlation values for seed samples of three years, the value of year 2006 was higher than the other two years (R2 = 0·74 for the TaqMan MGB probe assay, R2 = 0·72 for the SYBR Green dye assay). These results indicated that the size of lesions on the seed correlated with the amount of pathogen DNA in the seed.

Figure 1.

Correlation between the ratio of lesion area and seed area of the Colletotrichum lindemuthianum infected seeds to the log of detected C. lindemuthianum DNA per reaction with the TaqMan MGB Probe assay. Seed samples: (a) 2006; (b) 2007; (c) 2008; (d) 2006, 2007 and 2008.

Figure 2.

Correlation between the ratio of lesion area and seed area of the Colletotrichum lindemuthianum infected seeds to the log of detected C. lindemuthianum DNA per reaction with the SYBR Green assay. Seed samples: (a) 2006; (b) 2007; (c) 2008; (d) 2006, 2007 and 2008.

For the 90 symptomless navy bean seeds, weak fluorescent signals were detected in 89 seeds (SYBR Green assay) and 87 seeds (TaqMan assay) with mean amount of C. lindemuthianum DNA of the three replicates ranging from 4·33 to 438·83 fg (SYBR Green assay) and 1·14 to 130·54 fg (TaqMan assay), but no C. lindemuthianum DNA was detected in one symptomless seed 8S52 (SYBR Green assay) and three symptomless seeds 8S47, 8S51 and 8S52 (TaqMan assay). The mean detected amount of C. lindemuthianum DNA of symptomless seeds was 79·61 fg (SYBR Green assay) and 22·45 fg (TaqMan assay). This suggested that the symptomless seeds had a low amount of fungal DNA in the seeds, although the isolation test results of a parallel sample of 90 symptomless seeds from the same field site showed that the pathogen was not culturable.


This is the first report on the use of real-time PCR technology to quantify the extent of colonization of dry bean seed by the anthracnose pathogen C. lindemuthianum. This real-time PCR assay has a distinct advantage over previously developed PCR or conventional assays to detect anthracnose infection in bean seeds and other plant tissue (Chen et al., 2007; Wang et al., 2008) in that it quantifies the amount of infection in the seed. This study repeatedly showed a good correlation between lesion size and the amount of pathogen DNA detected by real-time PCR in individual bean seeds. The real-time PCR assay also has the advantage that it can be completed in half the time it takes to run a nested PCR assay (Cullen et al., 2002; Gachon et al., 2004). It has also been shown that real-time PCR assays are less laborious and more sensitive than nested PCR or duplex PCR assays (Gachon et al., 2004; Debode et al., 2009).

It was also evident that the real-time PCR assay was more sensitive than more traditional assays or other PCR procedures for detection of the anthracnose pathogen (Chen et al., 2007; Wang et al., 2008). Accuracy is another advantage of the real-time PCR assay for bean anthracnose. The use of a closed-tube PCR assay greatly reduces the risk of false-positive results due to cross-contamination compared to that of nested or semi-nested PCR assays (Tomlinson et al., 2005).

The probes designed for this study were highly specific for the detection of DNA of C. lindemuthianum. Tests with closely related species of C. lindemuthianum and other common pathogens of dry beans demonstrated the specificity of the real-time PCR assay. Positive amplification of DNA was observed only for the seven races of C. lindemuthianum, but did not occur with DNA from the other Colletotrichum species, other bean pathogens or the resistant race differential line G2333 (Young et al., 1998) that had been grown under disease-free conditions in a greenhouse.

The conventional PCR primers used to detect C. lindemuthianum in bean tissue (Chen et al., 2007) required some modification in order to be converted into a suitable probe for real-time PCR. The results demonstrated the versatility of this real-time PCR assay for use as a diagnostic tool to specifically detect the presence of anthracnose infection in seed. The results from the TaqMan MGB probe and SYBR Green dye assays were quite similar. This contrasts with the results reported by Debode et al. (2009) who observed that the ITS-TaqMan probe assay was 3·6 times more efficient in detecting DNA of C. acutatum from strawberries (Fragaria chiloense var. ananassa) than the ITS-SYBR Green assay. Demontis et al. (2008) compared TaqMan MGB probe and SYBR Green dye assays for the detection of Phoma tracheiphila in Citrus species and reported close agreement in the results of the two different real-time PCR assays. They also pointed out that the SYBR Green dye assay was less expensive and easier to set up than the TaqMan assay.

Seedborne infection is the most common means of spread for the anthracnose pathogen into new fields, or of new races of C. lindemuthianum into new regions (Tu, 1988). Lesions or discoloration of the seed caused by anthracnose are relatively easy to observe on navy beans and other bean classes that have a white seed coat. However, in bean classes with coloured seed, such as pinto beans and black beans, it can be difficult to observe anthracnose lesions if the disease symptoms are not severe. Differences in seed colour would not pose a challenge to detection of the anthracnose fungus for the real-time PCR assay.

It has been well established that not all bean seed that are infected with C. lindemuthianum produce lesions or any external symptoms (Tu, 1996). Latent infection can be detected by conventional procedures involving the plating of seed on nutrient agar, but such procedures are quite laborious, take a considerable amount of time for the fungus to grow and then require skilled personnel to identify the pathogen at the species level (McCartney et al., 2003; Guillemette et al., 2004). Studies with other pathogens have shown that real-time PCR provides the most sensitive method for detecting latent infections (Schaad & Frederick, 2002; Demontis et al., 2008; Debode et al., 2009).

In this study, image analysis technology was used to measure lesion size precisely and to determine the percentage of the seed surface that was covered by lesions by taking lesion size and seed size into account. A recent study by De Coninck et al. (2012) demonstrated that image analysis was more precise in measuring Cercospora leaf spot lesions on sugar beet (Beta vulgaris) leaves than were visual ratings. They also reported a strong correlation between real-time PCR quantification of DNA of Cercospora beticola and lesion size measurements on sugar beet leaves using digital image analysis, which enabled more efficient selection for disease resistance. The use of image analysis for disease assessments removes the lack of precision and subjectivity that is often associated with visual disease measurements.

A number of diseases and physiological conditions can produce discoloration and lesions on the bean seed that are difficult to distinguish visually from those of anthracnose. Similarly, the symptoms of anthracnose on the leaves and stems are sometimes confused with other diseases and disorders (Chen et al., 2007), so further research should be undertaken to explore the use of this real-time PCR assay in the diagnostics of foliar diseases of dry beans. Research should be conducted to evaluate the use of this real-time PCR assay in quantifying anthracnose in large seed samples of bean rather than just individual seeds. Often anthracnose seed infection occurs at very low frequencies which, if used for planting, can subsequently result in adverse effects on seed quality by the end of the growing season (Conner et al., 2009). Sampling based on assays of samples consisting of a composite of a large number of seeds might be possible with a testing procedure as sensitive as the real-time PCR assay developed in this study, which would greatly reduce the risk of planting seed lots with low frequencies of infection. This technique could be used to remove some of the ambiguities that are inherent in current certification schemes to ensure bean seed is pathogen-free, which are limited by small sample sizes and low sensitivity. A new real-time PCR based assay would be a useful tool in studies on the ecology and epidemiology of C. lindemuthianum. Similar procedures have been used in other host–pathogen research to examine factors influencing pathogen survival in soil, water and crop debris (Cullen et al., 2002; Cottyn et al., 2011; Kuan et al., 2011), disease development (Debode et al., 2009) and population dynamics and disease thresholds (Cullen et al., 2002). The availability of a highly sensitive and specific assay for bean anthracnose could assist in further research to determine the inoculum concentration thresholds that are required for transmission of the anthracnose fungus from infected seeds to their seedlings. This assay might also be used to examine anthracnose transmission on contaminated equipment (Tu, 1988) or materials in the field or in seed handling facilities.

Reports on the use of real-time PCR as a diagnostic tool for plant pathogens are becoming more numerous (McCartney et al., 2003; Demontis et al., 2008). The specificity and sensitivity of real-time PCR in pathogen detection should make its use in disease diagnosis and quantification more routine. Real-time PCR machines that can process DNA samples in 384-well plates, with a high throughput that can process plates continuously over a 24 h period, will further facilitate its use for rapid diagnosis of large numbers of seed or tissue samples (McCartney et al., 2003; Gachon et al., 2004; Guillemette et al., 2004). Initially the costs for a real-time PCR machine and the reagents required limited its use to research studies rather than for diagnostic activities (Cullen et al., 2002; McCartney et al., 2003), but these costs are expected to be reduced over time (Schaad & Frederick, 2002; Gachon et al., 2004). Real-time PCR assays for the detection and quantification of seedborne pathogens are starting to be offered as a commercial service (McCartney et al., 2003). It is expected that the real-time PCR protocol described in this study will be used in commercial disease diagnostic laboratories for the rapid detection of anthracnose in bean seed lots.


This research was financially supported by the Agricultural Adaptation Council, the Manitoba Pulse Growers Association, the Ontario White Bean Producers, Ontario Coloured Bean Growers, Syngenta Crop Protection and BASF Canada. The authors are very grateful to W. C. Penner and D. B. Stoesz of AAFC Morden, T. Henderson of AAFC Brandon and B. Luit of the University of Manitoba for their technical assistance on this study.