Development of a TaqMan Real-Time PCR for Early and Accurate Detection of Anthracnose Pathogen Colletotrichum siamense in Pachira glabra

Anthracnose, caused by Colletotrichum siamense, is a destructive disease of Pachira glabra in southern China. Early and proper monitoring and quantification of C. siamense is of importance for disease control. A calmodulin (CAL) gene-based TaqMan real-time PCR assay was developed for efficient detection and quantification of C. siamense, which reliably detected as low as 5 pg of genomic DNA and 12.8 fg (5800 copies) of target DNA. This method could specifically recognize all tested C. siamense isolates, while no amplification was observed in other closely related Colletotrichum species. The assay could still detect C. siamense in plant mixes, of which only 0.01% of the tissue was infected. A dynamic change in the amount of C. siamense population was observed during infection, suggesting that this real-time PCR assay can be used to monitor the fungal growth progression in the whole disease process. Moreover, the method enabled the detection of C. siamense in naturally infected and symptomless leaves of P. glabra trees in fields. Taken together, this specific TaqMan real-time PCR provides a rapid and accurate method for detection and quantification of C. siamense colonization in P. glabra, and will be useful for prediction of the disease to reduce the epidemic risk.


Introduction
Pachira glabra Pasq., well known as the money tree or the lucky tree, is a medium-sized perennial evergreen tree belonging to the Malvaceae family.Being a native species of the Brazilian Atlantic Forest, it was introduced to China for landscaping use due to its ornamental value.The tree grows fast, and has a light and soft wood, which can be applied to manufacture light objects such as boxes, toys, and stationery stuff [1].Its mature seeds, young leaves, and flowers are edible [2].Moreover, the leaves of Pachira glabra, known to contain phenolic acids, quercetin flavonols, and glucoside derivatives, have been a traditional source for local people for some time, and used as a traditional remedy for stomach problems, ulcers, and headache [3].With the continuous increase of plantation area in southern China, P. glabra is frequently decimated by fungal diseases, causing enormous economic losses.In our previous studies, white leaf spot and anthracnose, caused by Hypomontagnella monticulosa and Colletotrichum siamense, respectively, were frequently observed in P. glabra trees [4,5].Recently, leaf brown spot, caused by Diaporthe phoenicicola, has been observed on P. glabra by our lab [6].An anthracnose was observed with the incidence of up to 30% on P. glabra leaves.P. glabra anthracnose develops into sunken, The specificity of the primers CALCs-F and CALCs-R was first analyzed by a conventional PCR.The primers set amplified a 183-bp PCR product, confirmed through sequencing, with DNA extracted from C. siamense cultures.No amplification was observed for other closely related Colletotrichum species, including C. aenigma, C. alienum, C. aotearoa, C. asianum, C. boninese, C. conoides, C. fructicola, C. gloeosporioide, C. grevilleae, C. grossum, C. musae, C. theobromicola, Diaporthe phoenicicola (the causal agent of P. glabra leaf brown spot), and the nontemplate control (Figure 2).Then, the specificity of the primer set and TaqMan probe was checked using a TaqMan real-time PCR.The results showed that positive signals were obtained only from the target fungi species, but not from the nontarget ones (Table 1, Figure 3), indicating the primer set CALCs-F/CALCs-R and TaqMan probe CALCs-P were specific to C. siamense.The Ct values of the eight C. siamense isolates were 22-28 cycles, while those of the thirteen nontarget Colletotrichum species were more than Plants 2024, 13, 1149 4 of 14 39 cycles (Table 1).The Ct values of the target and nontarget fungi were well separated into two different groups.These observations confirmed the specificity of the primer set and TaqMan probe for target species.
multiple sequence alignments.The results showed 7 to 24 nucleotide differences when compared with the closely related Colletotrichum species (Figure 1).Thus, based on the nucleotide differences of the CAL regions, the primer set CALCs-F/CALCs-R (5′-GTG-GACATGCGGAATCCT-3′ and 5′-TCAAAGACCTATTCAGAGTCAACATAT-3′, respectively) and TaqMan probe CALCs-P (5′-AATACAGGCCCACTGACTGGTCTTC-3′) were designed for molecular determination of these species.BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=Blast-Search&LINK_LOC=blasthome, accessed on 3 July 2022) analysis of the designed primers and probe against the NCBI database showed that the primers CALCs-F/CALCs-R were specific to C. siamense and there were not any identical or highly similar sequences in any other sequenced organisms.2).Then, the specificity of the primer set and TaqMan probe was checked using a TaqMan real-time PCR.The results showed that positive signals were obtained only from the target fungi species, but not from the nontarget ones (Table 1, Figure 3), indicating the primer set CALCs-F/CALCs-R and TaqMan probe CALCs-P were specific to C. siamense.The Ct values of the eight C. siamense isolates were 22-28 cycles, while those of the thirteen nontarget Colletotrichum species were more than 39 cycles (Table 1).The Ct values of the target and nontarget fungi were well separated into two different groups.These observations confirmed the specificity of the primer set and TaqMan probe for target species.(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).The data represent mean values ± SE calculated from three replicates.

Sensitivity and Stability of the TaqMan Real-Time PCR Assay
A standard curve was plotted by tenfold serial dilution of cloned target DNA (pDNA) ranging from 10 0 to 10 7 copies/µL (Figure 4a).The standard curve showed that there was a strong linear correlation between the logarithm of the pDNA concentrations and Ct values, with high correlation coefficient (R 2 = 0.998) and high amplification efficiency (E = 99.3%).The amplification curves of pDNA showed a reliable detection limit down to 1, 000 copies (Figure 4b), the sensitivity of which was 100 times higher than that of a conventional PCR (Figure S1).

Sensitivity and Stability of the TaqMan Real-Time PCR Assay
A standard curve was plotted by tenfold serial dilution of cloned target DNA (pDNA) ranging from 10 0 to 10 7 copies/µL (Figure 4a).The standard curve showed that there was a strong linear correlation between the logarithm of the pDNA concentrations and Ct values, with high correlation coefficient (R 2 = 0.998) and high amplification efficiency (E = 99.3%).The amplification curves of pDNA showed a reliable detection limit down to 1, 000 copies (Figure 4b), the sensitivity of which was 100 times higher than that of a conventional PCR (Figure S1).

Sensitivity and Stability of the TaqMan Real-Time PCR Assay
A standard curve was plotted by tenfold serial dilution of cloned target DNA (pDNA) ranging from 10 0 to 10 7 copies/µL (Figure 4a).The standard curve showed that there was a strong linear correlation between the logarithm of the pDNA concentrations and Ct values, with high correlation coefficient (R 2 = 0.998) and high amplification efficiency (E = 99.3%).The amplification curves of pDNA showed a reliable detection limit down to 1, 000 copies (Figure 4b), the sensitivity of which was 100 times higher than that of a conventional PCR (Figure S1).Five tenfold serially diluted gDNA of C. siamense with ddH2O, ranging from 20 ng to 5 pg, were also evaluated using this target gene.The regression curve generated with a slope value of −3.53 (E= 91.9%, R 2 = 0.937).The lowest detection level with fungal gDNA was determined to be 5 pg (Figure 4c,d).
To evaluate the potential effect of leaf extract on quantitative results, 1 µL of C. siamense CS-1 gDNA (5 ng/µL) was serially diluted with P. glabra leaf extract (0, 0.5, 1, 1.5, 2, 2.5 µL).Statistical analysis showed that there were no statistically significant amplification results regardless of the presence or absence of host leaf extract in the C. siamense isolate CS-1 gDNA (p = 0.785 > 0.05) (Table 2).Therefore, the TaqMan real-time PCR was not significantly affected by plant background using primer set CALCs-F/CALCs-R and TaqMan probe CALCs-P.A standard curve was generated by serial dilution of 50 ng gDNA of fungal mycelia with DNA of healthy leaves, resulting in a slope of −3.43 and R 2 value of 0.977 with an E value of 95.7% (Figure 5).The lowest detection level with infected plant DNA tissue was determined to be 5 pg/µL.Five tenfold serially diluted gDNA of C. siamense with ddH 2 O, ranging from 20 ng to 5 pg, were also evaluated using this target gene.The regression curve generated with a slope value of −3.53 (E = 91.9%,R 2 = 0.937).The lowest detection level with fungal gDNA was determined to be 5 pg (Figure 4c,d).
To evaluate the potential effect of leaf extract on quantitative results, 1 µL of C. siamense CS-1 gDNA (5 ng/µL) was serially diluted with P. glabra leaf extract (0, 0.5, 1, 1.5, 2, 2.5 µL).Statistical analysis showed that there were no statistically significant amplification results regardless of the presence or absence of host leaf extract in the C. siamense isolate CS-1 gDNA (p = 0.785 > 0.05) (Table 2).Therefore, the TaqMan real-time PCR was not significantly affected by plant background using primer set CALCs-F/CALCs-R and TaqMan probe CALCs-P.A standard curve was generated by serial dilution of 50 ng gDNA of fungal mycelia with DNA of healthy leaves, resulting in a slope of −3.43 and R 2 value of 0.977 with an E value of 95.7% (Figure 5).The lowest detection level with infected plant DNA tissue was determined to be 5 pg/µL.The standard curve.The data represent mean values ± SE calculated from three replicates.

Detection in Artificially Inoculated P. glabra
The disease progression of C. siamense in P. glabra leaves after artificial inoculation was monitored with visual observations and real-time PCR analyses.During the first 24 h post inoculation (hpi), P. glabra leaves remained symptomless (Figure 6a).However, the amount of C. siamense DNA steadily increased by 28.5 to 85.7 pg per 50 ng DNA of leaves over this period (Figure 6b).These findings suggest that the real-time PCR method allows the monitoring of growth progression during the latent phase.During the symptomatic stage of the infection, the amount of C. siamense DNA largely increased over time in the inoculated leaves.Small black spots first occurred sporadically at 36 hpi, when the C. siamense DNA was 1159.2 pg per 50 ng DNA of P. glabra leaves.By 60 h, leaf spots became denser and larger, and several began to coalesce.The amount of C. siamense DNA reached 1578.52 pg per 50 ng of total DNA at this time.These results showed that the TaqMan realtime PCR method could monitor the growth progression of C. siamense in the whole process of disease.

Detection in Artificially Inoculated P. glabra
The disease progression of C. siamense in P. glabra leaves after artificial inoculation was monitored with visual observations and real-time PCR analyses.During the first 24 h post inoculation (hpi), P. glabra leaves remained symptomless (Figure 6a).However, the amount of C. siamense DNA steadily increased by 28.5 to 85.7 pg per 50 ng DNA of leaves over this period (Figure 6b).These findings suggest that the real-time PCR method allows the monitoring of growth progression during the latent phase.During the symptomatic stage of the infection, the amount of C. siamense DNA largely increased over time in the inoculated leaves.Small black spots first occurred sporadically at 36 hpi, when the C. siamense DNA was 1159.2 pg per 50 ng DNA of P. glabra leaves.By 60 h, leaf spots became denser and larger, and several began to coalesce.The amount of C. siamense DNA reached 1578.52 pg per 50 ng of total DNA at this time.These results showed that the TaqMan real-time PCR method could monitor the growth progression of C. siamense in the whole process of disease.
2.4.Detection in Naturally Infected P. glabra C. siamense was detected for naturally infected leaves in fields by real-time PCR.For the 25 asymptomatic but suspected to be infected leaves, fluorescent signals were detected in seven leaves with an average DNA content of 85.11 pg per 50 ng DNA sample, suggesting that anthracnose symptoms would probably be developed in these seven asymptomatic leaves.No amplification was observed for DNA extracted from the other 18 asymptomatic leaves (Table 3).The seven asymptomatic leaves were also tested by conventional PCR.Only two leaves exhibited positive amplifications, indicating the conventional PCR method was less sensitive than the real-time PCR.Fluorescent signals occurred with DNA extracted from naturally infected leaves.There was a positive correlation between disease severity and amount of fungal pathogen DNA.C. siamense was detectable in these samples by tissue isolation.3).The seven asymptomatic leaves were also tested by conventional PCR.Only two leaves exhibited positive amplifications, indicating the conventional PCR method was less sensitive than the real-time PCR.Fluorescent signals occurred with DNA extracted from naturally infected leaves.There was a positive correlation between disease severity and amount of fungal pathogen DNA.C. siamense was detectable in these samples by tissue isolation.

Discussion
Species within the CGSC cause destructive anthracnose diseases on a wide variety of economically valuable crops [7,11,12].There are obstacles to differentiate and detect CGSC species in diseased plant materials, including limited morphological differentiation among species, variation in pathogenicity and cultural morphology, and common latency or quiescence in host cells [15,26].Thus, the molecular method is expected to supply an ideal solution for disease diagnosis.It is essential to accurately and rapidly identify CGSC species that cause anthracnose disease so as to develop effective control strategies.In this study, the TaqMan probe-based real-time PCR assay using CAL marker provides a specific, sensitive, and effective method for detecting C. siamense, the causal agent of P. glabra anthracnose; thus, it is suitable for application in early prediction of P. glabra anthracnose to reduce the epidemic risk.
Plants 2024, 13, 1149 9 of 14 Successful detection of pathogens by PCR-based methods needs primers to satisfy the criteria, including uniqueness of the amplification sequence to the target pathogen of interest and conservation across populations of the target pathogen [27].Several molecular protocols based on real-time PCR amplification have been previously developed for detection and quantification of Colletotrichum species, targeting different molecular markers such as ITS [18,21], GAPDH [22], CAL [17], ACT [20], cutinase (Cuti) [19], and ApMat [25].In this study, real-time PCR primers and a probe specific to C. siamense were designed based on the sequence differences in the CAL gene region.The TaqMan real-time PCR method accurately identified and quantified C. siamense using DNA from pure cultures, a DNA mixture of the target fungus and plant tissues, and DNA from the infected P. glabra leaves.This assay is species specific, as indicated by lack of amplification of nontarget species (Figures 2 and 3).Systematic analyses of phylogenetic informativeness profiling and Bayesian concordance using various molecular markers reveal that APN2/MAT-IGS, APN2, the intergenic spacer between GAPDH, and a hypothetical protein (GAP2-IGS), and the longest TUB2 are the most suitable gene markers for reliably discriminating species within the CGSC, while CAL, along with GAPDH and GS, has intermediate informativeness values [14,28].However, the CAL gene does contain unique sequence motifs that can be used to successfully differentiate C. siamense from other closely related species in this study.A similar study was conducted by Kaur et al., who developed a diagnostic TaqMan real-time PCR assay for accurately detecting and quantifying C. theobromicola in boxwood using the CAL marker [17].These assays suggest that molecular targets with relatively intermediate resolution for large genera like Colletotrichum could still be used for identification and qualification of a given species within the genera, if appropriate primers and probes are designed with special care.
The effectiveness of TaqMan real-time PCR assays relies on not only high specificity, but also on high sensitivity of primers and a probe that could specifically amplify the target sequences.In the present study, an optimized protocol of a TaqMan real-time PCR assay was proposed and used to diagnose and quantify C. siamense, the causal agent of P. glabra anthracnose, at very low concentrations of fungal DNA.The assay could reliably detect as low as 12.8 fg/µL (5.8 × 103 copies/µL) of target DNA, 5.0 pg/µL of gDNA, and C. siamense in plant mixes, of which 0.01% of the total tissue DNA was pathogen DNA.This detection threshold is comparable to that of the CAL-based real-time TaqMan PCR technology for detection of C. theobromicola in boxwood [17], and slightly lower than that of the CAL-targeted loop-mediated isothermal amplification (LAMP) for detection of C. siamense in infected tea plants [29].However, LAMP could not provide quantitative data of target DNA, so it is not suitable for monitoring the dynamics of the pathogen [30].The detection limit of the present assay targeting the CAL gene was higher than that of the assay targeting the ApMat gene in Chinese fir (1 ng/µL of gDNA) and lower than the assay targeting the multi-copy ITS in rubber trees (100 fg/µL of gDNA) reported previously [18,25].This might be attributed to the different copies of these gene markers in the fungal genomes.The ITS region has been suggested to represent the universal barcode for fungi [31] but with several concerns [32].However, the utility of this region is limited for systematic identification and delimitation in Colletotrichum [14].Bhunjun et al. evaluated the phylogenetic significance of five molecular markers for species delineation within each species complex of Colletotrichum, and found that the ITS region was the least parsimony informative locus [33].We attempted to design the primers and probe of TaqMan realtime PCR based on the ITS to differentiate and quantify C. siamense from the other closely related Colletotrichum species listed in Table 1, and no satisfactory amplification signals were obtained.The efficiency of the present assay using both fungal DNA in water and the same DNA spiked with DNA from P. glabra leaves was within the acceptable limits from 91.9% to 99.3% (−3.53 > slope > −3.34) (Figures 4d and 5b).Similar efficiencies were displayed in the real-time assays for detection of C. theobromicola in boxwood and C. siamense in rubber trees [17,18].Accordingly, the sensitivity and PCR efficiency of the present method are sufficient for accurate detection and quantification of C. siamense in P. glabra trees.Some compounds introduced from host plants during DNA extraction, such as polysaccharides and phenolic substances, are potential PCR inhibitors [31].A prerequisite for in planta DNA quantification is to avoid the potential interference of plant tissues in detection and quantification of pathogens.As suggested by Du et al. [18], DNA extracts of plant material were diluted to 50 ng/µL in this study.The results showed the dilution level could preclude any bias due to PCR inhibitors originating from plant tissues.Accordingly, the presence of P. glabra leaf samples did not affect the efficiency, sensitivity, and reproducibility of the real-time PCR assays.
C. siamense is a hemibiotrophic fungus that can remain in a latent state in host tissues [15].At the latent stage, no visible difference can be observed between healthy and infected tissues.Therefore, early and accurate detection is critical for controlling this fungus known to cause delayed discernable symptoms in host plants.In the present study, the presence of C. siamense was successfully detected by using the real-time PCR assay in artificially inoculated leaves at 2 hpi, while the first visible lesion usually occurred at 36 hpi (Figure 6a).The results indicate that this assay can be used to detect C. siamense in the latent phase in host leaves.Moreover, the two-stage induction of C. siamense DNA was observed during the infection process (Figure 6b), which was consistent with visual observation, confirming that this method is useful for monitoring the dynamics of C. siamense in P. glabra trees.
Finally, the effectiveness and potential application of the real-time assay were validated for C. siamense detection in naturally infected P. glabra leaves collected from fields.The infection was also confirmed by the tissue isolation method.The amount of C. siamense DNA detected by the real-time assay correlated positively with the disease severity (Table 3).Therefore, the TaqMan PCR technique presented here is an efficient detection strategy for predicting and monitoring the epidemics of C. siamense in fields.

Fungal Isolates
In total, eight isolates of C. siamense from P. glabra and fourteen isolates of nontarget species from various hosts and geographical regions were used in this study (Table 1).The C. siamense isolates used in this study were obtained from the infected P. glabra plants collected from three provinces in China, including Hunan [5], Guangxi, and Guangdong.The nontarget isolates included 13 other closely associated Colletotrichum species and one isolate of Diaporthe phoenicicola gpg2023-1 [6], which causes leaf brown spots on P. glabra as mentioned above.The fungal isolates were revived on potato dextrose agar (PDA) plates at 28 • C under dark conditions for 5 to 7 days.Then, mycelial plugs taken from the edge of fungal colonies were transferred to new PDA plates under the same conditions.

Plant Growth
About one-year-old P. glabra plants purchased from a local nursery were planted in peaty soil, and cultivated in a greenhouse at 25 • C under natural sunlight.For leaf inoculation experiments, healthy individuals were moved into a growth chamber at 25 • C for another 14 days before inoculation.The parameters were set as follows: 12-h photoperiod per day, photo flux density 125 µmol m −2 s −1 , relative humidity 80 to 85%.

DNA Extraction
Genomic DNA (gDNA) of 5-to-7-day-old fungal mycelia and 0.5 g of plant material were extracted according to the instructions of the Biospin Fungal Genomic DNA Extraction Kit (BioFlux, Hangzhou, China).DNA samples were stored at −20°C for use.The quality and quantity of DNA samples were determined by ultra-micro spectrophotometer (NanoPHotometer-NP80; Implen GmbH, Munich, Germany) at the ratio of absorption at 260 and 280 nm.

Real-Time Primers and TaqMan Probe Design
Known sequences of the CAL gene region were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/).The CAL sequences of C. siamense from the isolates CS-2 to CS-8 and D. phoenicicola gpg2023-1 were amplified as described above.These sequences were aligned and compared using MEGA11 with default parameters, and the regions of nucleotide differences in the CAL sequences between target species and nontarget ones were used to design the real-time PCR primers and TaqMan probes using Primer premier 5.0 (PREMIER Biosoft International, San Francisco, CA, USA).The probe was labeled at the 5' end with 6-carboxyfluorescein (FAM) as a reporter dye, and modified at the 3' end with the quencher dye tetramethylcarboxyrhodamine (TAMRA).The primers and probe were synthesized by Jieteng Biological Co. (Kunming, China).
The specificity of primer set was evaluated by a conventional PCR as described above, and further verified by amplification curve analysis of a real-time PCR as described below.The specificity of the TaqMan probe was assessed by a real-time PCR.Both the conventional PCR and real-time PCR were conducted with 50 ng gDNA of the target and nontarget species as templates.

TaqMan Real-Time PCR Assay
The real-time PCR assays were performed in a total volume of 20 µL containing 10 µL of 2× TaqMan Mix (BioFlux, Hangzhou, China), 0.4 µL of each forward and reverse primer, and 1.8 µL of the TaqMan probe, 1 µL of DNA templates (50 ng/µL), 0.2 µL of Taq polymerase (5 U/µL) (BioFlux, Hangzhou, China), and 6.2 µL of sterile ddH 2 O.The CFX96 Real-Time System Thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) was used for amplification and fluorescence measurement.The thermocycling conditions were as follows: initial denaturation at 95 • C for 2 min, followed by 40 cycles of denaturation at 95 • C for 10 s, then annealing and extension at 63.9 • C for 30 s. Signal threshold levels were set automatically by the system.Triplicate reactions were performed in each assay and each assay was repeated at least twice.

Sensitivity and Stability of the TaqMan Real-Time PCR Assay
To evaluate the sensitivity of the TaqMan real-time PCR assay, standard curves were generated by amplifying 10-fold serial dilutions of pDNA (pBM16A-T-CAL) ranging from 10 0 to 10 7 copies/µL.Sterilized ddH 2 O instead of template DNA was served as negative control.Triplicate reactions were performed in each assay, and each assay was repeated at least twice.Each run contained a negative control using sterile ddH 2 O instead of DNA templates.The samples with a stable Ct value and amplification curve ratio were selected to establish the linear regression curves between the logarithm of the template concentration and the Ct values.The PCR amplification efficiency (E) was calculated by the formula [34]: E = (10 1/-slope − 1) × 100. ( To assess whether leaf extracts of P. glabra could interfere with the detection of C. siamense, a series of leaf extracts (0, 0.5, 1, 1.5, 2, and 2.5 µL) were mixed with 1 µL of 5 ng/µL gDNA and added into a total real-time PCR reaction volume of 20 µL.Leaf extracts were prepared according to the method described by He et al. [25].The leaf extracts and sterile ddH 2 O were included in each real-time PCR run as negative controls.In the positive control, gDNA of C. siamense isolate CS-1 culture without P. glabra leaf extracts was used as a template.Triplicate reactions were performed in each assay and each assay was repeated at least twice.Standard curves were also generated by amplifying 10-fold serial dilutions of DNA extracted from the P. glabra leaves infected by C. siamense isolate CS-1 (50 to 0.005 ng/µL).

Validity of the TaqMan Real-Time PCR on Artificially Inoculated Plants
Conidial suspension of C. siamense isolate CS-1 were prepared from PDA cultures.The conidia were counted by a blood cell counting chamber.Conidial suspension (containing 0.7% glucose and 0.05% Tween 20) was sprayed on the leaves of healthy P. glabra plants until runoff.The inoculated plants were maintained at a growth chamber under the growth conditions described above.The disease symptoms of plants were observed and recorded at 2, 6, 12, 24, 30, 36, 48, and 60 hpi.Three leaves were randomly picked from the inoculated plants during each time, surface-sterilized through sequential immersion in 0.5% sodium hypochlorite then 75% ethanol, and thoroughly washed with sterilized water three times.Then, the leaves were dried with filter paper and frozen quickly with liquid nitrogen for DNA extraction and real-time PCR assays.4.9.Validity of the TaqMan Real-Time PCR on Naturally Infected Plants in Fields P. glabra leaves were sampled as described by Du et al. [18] with minor modifications.Briefly, 25 leaves without lesions but suspected to be infected with C. siamense, 3 naturally infected leaves for each disease grade, and healthy leaves were collected, and placed in sealed bags.Leaf DNA was extracted as described above.DNA samples were diluted to 50 ng/µL with sterile ddH 2 O for real-time PCR detection using DNA of healthy leaves as blank controls.The amount of C. siamense gDNA was estimated by comparing Ct values to the gDNA standard curve.C. siamense in plants was detected using a tissue separation method as described previously [5] and identified based on cultural and morphological characteristics [11].
Disease grades were determined based on six symptom grades as follows: 0 refers to no lesion, 1 refers to lesion area occupied <5% leaf area, 2 refers to lesion area occupied ≥5% but <10% leaf area, 3 refers to lesion area occupied ≥10% but <20% leaf area, 4 refers to lesion area occupied ≥20% but <40% leaf area, and 5 refers to lesion area occupied ≥40% leaf area or death of leaves.

Statistical Analysis
Data represent mean values ± standard errors (SEs) calculated from three replicates.Analysis of variance (one-way ANOVA) and multiple comparisons of differences between assays (Duncan's multiple range test, p < 0.05) were performed using SPSS for Windows version 24.0 (SPSS Inc., Chicago, IL, USA).

Conclusions
In the current work, an efficient TaqMan real-time PCR assay has been developed for specific and accurate detection of C. siamense on the host P. glabra tree by targeting the CAL gene.This assay enables a rapid and reliable diagnostic method to detect P. glabra anthracnose at early stages of the disease, thus is proper for application in early prediction of the disease to reduce the risk of epidemics.This method can be used in a high-throughput selection of anthracnose-resistant P. glabra cultivars during germplasm identification, and also valuable for official phytosanitary purposes.The qPCR assay is about to enter into a contract with a private sector user.

Figure 1 .
Figure 1.Alignment of calmodulin (CAL) sequences of Colletotrichum siamense, other closely related Colletotrichum species, and Diaporthe phoenicicola, highlighting the position of the primers CALCs-F/CALCs-R (arrows) and probe CALCs-P (line) designed for detecting C. siamense by TaqMan realtime PCR.Gaps are marked as dashes in sequences, and rose red, blue, and green boxes represent 100%, ≥75%, and ≥50% of nucleotide identity among the aligned sequences, respectively.

Figure 1 .
Figure 1.Alignment of calmodulin (CAL) sequences of Colletotrichum siamense, other closely related Colletotrichum species, and Diaporthe phoenicicola, highlighting the position of the primers CALCs-F/CALCs-R (arrows) and probe CALCs-P (line) designed for detecting C. siamense by TaqMan real-time PCR.Gaps are marked as dashes in sequences, and rose red, blue, and green boxes represent 100%, ≥75%, and ≥50% of nucleotide identity among the aligned sequences, respectively.

Figure 5 .
Figure 5.The TaqMan real-time PCR assays on Colletotrichum siamense CS-1 gDNA (ranging from 0.005 to 50 ng) diluted with the DNA extract of Pachira glabra leaves.(a) Amplification curves.(b) The standard curve.The data represent mean values ± SE calculated from three replicates.

Figure 5 .
Figure 5.The TaqMan real-time PCR assays on Colletotrichum siamense CS-1 gDNA (ranging from 0.005 to 50 ng) diluted with the DNA extract of Pachira glabra leaves.(a) Amplification curves.(b) The standard curve.The data represent mean values ± SE calculated from three replicates.

Figure 6 .
Figure 6.Colletotrichum siamense time-course infection on Pachira glabra leaves during 0 to 60 h post inoculation with 10 6 conidia/mL.(a) Photographs of leaves infected.The white arrows indicate disease spots at the onset times.(b) C. siamense DNA quantification.The data represent mean values ± SE calculated from three replicates as quantified by TaqMan real-time PCR.

2. 4 .
Detection in Naturally Infected P. glabra C. siamense was detected for naturally infected leaves in fields by real-time PCR.For the 25 asymptomatic but suspected to be infected leaves, fluorescent signals were detected in seven leaves with an average DNA content of 85.11 pg per 50 ng DNA sample, suggesting that anthracnose symptoms would probably be developed in these seven asymptomatic leaves.No amplification was observed for DNA extracted from the other 18 asymptomatic leaves (Table

Figure 6 .
Figure 6.Colletotrichum siamense time-course infection on Pachira glabra leaves during 0 to 60 h post inoculation with 10 6 conidia/mL.(a) Photographs of leaves infected.The white arrows indicate disease spots at the onset times.(b) C. siamense DNA quantification.The data represent mean values ± SE calculated from three replicates as quantified by TaqMan real-time PCR.

Table 1 .
Strains and origins of Colletotrichum siamense and other fungi species, and specificity of the TaqMan real-time PCR assays targeting calmodulin gene.

Table 2 .
The effect of Pachira glabra leaf extract on the Ct value of the TaqMan real-time PCR assays.

Table 2 .
The effect of Pachira glabra leaf extract on the Ct value of the TaqMan real-time PCR assays.

Table 3 .
Detection of Colletotrichum siamense in symptomless and infected leaves of Pachira glabra in the field using the TaqMan real-time PCR and conventional PCR.

Table 3 .
Detection of Colletotrichum siamense in symptomless and infected leaves of Pachira glabra in the field using the TaqMan real-time PCR and conventional PCR.

DNA Amount [log (pg per 50 ng of Sample DNA)] Detection with Conventional PCR 1 Detection with the Tissue Isolation Method 1
1 D, detected; ND, not detected.The data represent mean values ± SE calculated from three replicates.