A TaqMan Real-Time PCR Assay for Early and Accurate Detection of Hypomontagnella monticulosa, an Emerging Pathogen Causing White Leaf Spot on Pachira glabra Pasq.
Article information
Abstract
Hypomontagnella monticulosa is an emerging pathogen of Pachira glabra Pasq. causing white leaf spot, a damaging fungal disease of P. glabra in southern China. The early and proper detection and qualification is of fundamental for understanding epidemiology and developing preventive measures of this fungus. Using the second largest subunit of the RNA polymerase II gene as target, a quantitative TaqMan real-time polymerase chain reaction assay was developed for the detection and quantification of H. monticulosa in P. glabra leaves. This method could specifically recognize all tested H. monticulosa strains, while no cross-reaction was observed in closely related Hypoxylon species. Sensitivity of the assays was determined to be as low as 0.05 fg/μL (2, 300 copies/μL) of plasmid DNA, 0.5 pg/μL of mycelia genomic DNA, and 0.001% of target DNA mixed with leaf tissue DNA. The two-stage induction of H. monticulosa DNA was observed during the infection process, suggesting that this assay could be used to monitor the growth dynamics of this fungus in the whole disease process. Additionally, the assay could not only effectively detected H. monticulosa in naturally infected P. glabra trees in fields, but also accurately evaluate the differences in resistance among varieties of P. glabra. Therefore, the current study provides a rapid and accurate technology for monitoring and qualification of H. monticulosa infection in P. glabra, and will be applicable for prediction and control of the disease but also for the study of plant-H. monticulosa interaction.
Commonly known by its nonscientific names such as Money tree and Lucky tree, Pachira glabra Pasq., is a perennial flowering tree of the Malvaceae family (El-Din et al., 2021; Milagres et al., 2018). The oil extracted from its edible seeds is rich of mono- and polyunsaturated fatty acids associated with humans health improvement (Ayodele and Badejo, 2022.; Tebe et al., 2024) Traditionally, this species has been intensively used to treat stomach ulcers and headache (Abd El-Ghani, 2016; El-Din et al., 2021). A variety of different compounds with biological activities, including naphthoquinone derivatives, favonoids, sterols, and terpenoids, have been isolated and identified from stem barks, roots and leaves of P. glabra. Previous pharmacological studies have shown that P. glabra possesses anti-infammatory, anti-microbial and insecticidal activities (Lawal et al., 2014).
Being a native species of the Brazilian Atlantic Forest (Milagres et al., 2018), P. glabra is widely cultivated as an ornamental garden plant species in China and other countries. With the expansion of large-scale plantation of P. glabra, this species is repeatedly encountered by fungal diseases, leading to considerable losses of production and quality. For instance, P. glabra saplings with leaf anthracnose (Wu et al., 2023), leaf brown spot (Deng et al., 2024), and white leaf spot (Tian et al., 2023) were frequently observed during our periodic investigations of tree nurseries in Hunan Province, 2021 to 2022. In Brazil, a novel Diaporthe species, D. pachirae has been reported to cause leaf spot on P. glabra (Milagres et al., 2018). Among them, the white leaf spot disease eventually leads to early leaf death and abscission, thus seriously affecting the growth of P. glabra (Tian et al., 2023).
The diagnosis of white leaf spot on P. glabra can be easily accomplished by examining the characteristic symptoms and signs on the infected leaves. However, discernible damages often occur late in the interaction between hosts and pathogens, making it difficult to identify the taxa involved and monitor the taxa development during the initial stage of infection. Thus, the early and accurate monitoring of pathogens is critical for the prediction and management of diseases. Traditionally, the identification of phytopathogenic fungi is mainly based on morphological and observation and cultural examination (Khakimov et al., 2022). However, the delimitation of closely related fungi based on the observation of cultural characteristics is rather complicated and often unrealistic (Guglielmo et al., 2007). Moreover, culture-based identification needs fungal isolation and subsequent culture, which are time-consuming and tedious and, in some cases, impossible.
The causal agent of white leaf spot on P. glabra, Hypomontagnella monticulosa (Tian et al., 2023), belongs to the Hypomontagnella genus. This newly erected genus has been recently segregated from the Hypoxylon according to morphology and chemotaxonomic studies along with multi gene phylogeny, and now comprises only five species with H. monticulosa as type species (Lambert et al., 2019). H. monticulosa has been frequently reported as endophytes of some plant species including Zingiber griffithii, mangrove, and Indian sandalwood (Huang et al., 2023; Lutfia et al., 2021; Ouyang et al., 2025).
Species differentiation is challenging due to the immense morphological and phenotypic plasticity within the Hypoxylon and closely related genera like Hypomontagnella (Wendt et al., 2018). Currently, multi-locus phylogenetic analysis has extensively been applied to infer species limits within these genera (Kuhnert et al., 2014; Sir et al., 2016; Stadler, 2011). Different types of molecular markers are used for the molecular taxonomy, including the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA (rDNA), the large subunit (LSU) of the nuclear rDNA, alpha-actin (ACT), beta-tubulin (TUB2), and the second largest subunit of the RNA polymerase II (RPB2) (Suwannasai et al., 2013; Wendt et al., 2018). These molecular loci could be used for identification and qualification of given pathogenic species within the genera, if appropriate primers and/or probes are designed with cares.
PCR-based methods provide promising tools for rapid, sensitive, and specific routine diagnoses of plant diseases (Huang et al., 2023). Using either nuclear or mitochondrial ribosomal DNA regions, Guglielmo et al. (2007) developed and optimized multiplex polymerase chain reactions (PCRs) allowed for dependable identification of wood rotting fungal pathogens directly from standing trees, such as H. thouarsianum var. Thouarsianum (Ouyang et al., 2025). Real-time PCR offers a rapid, accurate, and culture-independent tool, and is extensively applied for detection of a wide range of phytopathogens (Venbrux et al., 2023). Compared with other real-time PCR assays, TaqMan probe-based PCR exhibits higher specificity, sensitivity, and accuracy because the probe only binds to the sequence region of target DNA between forward and reverse PCR primers and then produces specific fluorescent signals (Holland et al., 1991). Taking advantage of its outstanding properties, TaqMan probe real-time PCR assays have been employed to detect a diversity of plant pathogens (Gu et al., 2024; He et al., 2023; Wang et al., 2018).
The main objective of the present study is to develop a diagnostic TaqMan real-time PCR assay targeting RPB2 gene for early in planta detection and quantification of H. monticulosa, the causal agent of white leaf spot on P. glabra (Tian et al., 2023). The specificity, sensitivity, and effectiveness of this molecular approach were assessed using fungal culture, artificially inoculated and naturally infected plants.
Materials and Methods
Fungal isolates
Nineteen fungal strains including seven strains of H. monticulosa, 13 strains of Hypomontagnella closely-associated Hypoxylon species (Hypoxylon howeanum, H. jianfengense, H. medogense, H. perforatum, H. sublenormandii, H. investiens, and H. trugodes, were used in this study (Table 1). Fungal strains were revived from glycerol (20%) stocks and subsequently cultured on potato dextrose agar plates at 25°C under dark conditions for 5 to 7 days before use.
Strains and origins of Hypomontagnella monticulosa strains and closely related fungi, and the Ct values of the TaqMan real-time PCR assays targeting the second largest subunit of the RPB2 gene
Plant growth
P. glabra saplings were obtained from a local nursery, and transferred into a growth chamber for another 14 days before leaf inoculation. The growth conditions of plants were set as previously described (Gu et al., 2024).
DNA extraction
Fungal mycelia and plant materials were harvested and frozen in liquid nitrogen. These samples were ground to fine powder by a disposable tissue grinding pestle (Sangon Biotech, Shanghai, China). Genomic DNA (gDNA) of fungal mycelia and plant tissues were extracted by DNA extraction kit (BioFlux, Hangzhou, China) according to the manufacturer’s instructions. DNA samples were stored at −20°C for use. The quality and quantity of DNA extracts was measured using an ultra-micro UV spectrophotometer (NanoPHotometer-NP80; Implen GmbH, Munich, Germany).
Real-time primers and TaqMan probe design
RPB2 sequences of the target or non-target fungi were retrieved from National Center for Biotechnology Information (NCBI) database. The multiple sequence alignment of these sequences was performed with the ClustalW program in the MEGA11 software (Tamura et al., 2021). Primer premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) was used to design real-time PCR primers and TaqMan probe. 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). Synthesis of primers and probe were synthesized by Jieteng Biological Co. (Kunming, China) (Gu et al., 2024).
The specificity of primer pairs RPB2Hm-F (5′-CGGTTATGTTGCAGGAGATTACT-3′) and RPB2Hm-R (5′-CTTCTGGTCACCCCAGTTAC-3′) was initially evaluated by conventional PCR, using the DNA samples extracted from the fungal strains as listed in Table 1. The PCR amplification was performed on an SCI1000-G PCR gradient gene amplifier (SCILOGEX, Shanghai, China) following the procedures described in our previously published paper (Gu et al., 2024) except that the annealing temperature was set at 54°C in the present study. Amplicons were visualized on a gel containing 1% (w/v) of standard agarose after electrophoretic separation. PCR amplicon size were expected to be 146 bp in length.
The specificity of primers and probe were further verified by amplification-curve analysis of real-time PCR as described below using 50 ng of gDNA from target and non-target fungi as templates. The amplification curve was inspected with respect to background and curve shape. When primer specificity is high, the amplification curve shows a distinct exponential growth phase (log-linear phase) with a stable baseline and no nonspecific peaks.
Cloning of target sequences
The partial sequence of RPB2 gene in H. monticulosa strain TT422 (Tian et al., 2023) was amplified using primer set RPB2Hm-F and RPB2Hm-R, which was the same as that of real-time PCR assays as described in Section 2.4. The identity of amplified products was confirmed by DNA sequencing (Jieteng Biological Co.). The PCR amplicons were ligated into a cloning vector with pBM16A Toposmart Cloning Kit (Biomed, Beijing, China). The resulting pBM16A-T-RPB2 plasmid was transformed into Escherichia coli DH5α for plasmid DNA (pDNA) extraction. The pDNA concentration was determined with an ultra-micro spectrophotometer (NanoPHotometer-NP80; Implen GmbH) and converted to copy number according to the following formula:
TaqMan real-time PCR assay
The real-time PCR assay was performed on the CFX96 Real-Time System Thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) following the procedures described previously (Gu et al., 2024) except that the annealing temperature was set at 50.8°C, the concentrations of primers at 0.8 μmol/L, and the concentration of probe at 0.2 μmol/L in the present study. Signal threshold levels were set automatically by the system.
Sensitivity and stability of TaqMan real-time PCR assay
The sensitivity of TaqMan real-time PCR assay was tested by the standard curve method. DNA concentrations of pBM16A-T-RPB2 were subject to 10-fold serial dilutions ranging from 101 to 107 copies/μL. Linear regression curves were established between the logarithm of the pBM16A-T-RPB2 DNA concentrations and the Ct values. Each run contained a negative control using sterile ddH2O instead of DNA templates. The efficiency (E) of PCR amplification was calculated according to the formula (Ginzinger, 2002):
The effects of P. glabra leaf DNA extracts on the detection of the target fungus were assessed as described earlier by Gu et al. (2024). Leaf DNA extracts were prepared based on the experimental procedure described by He et al. (2023). Briefly, a series of leaf DNA extracts (0, 0.5, 1, 1.5, 2, and 2.5 μL) were mixed with 1 μL of 5 ng/μL fungal gDNA, and added to a total volume of 20 μL real-time PCR reaction mixture. The leaf DNA extracts and sterile ddH2O were used as negative controls, and gDNA of the target fungus without P. glabra leaf extracts were used as a positive control. Standard curves were also obtained by amplifying 10-fold serial dilutions of DNA extracts from the P. glabra leaves ranging from 50 to 0.005 ng/μL.
Validity of TaqMan real-time PCR on artificially inoculated plants
For artificial inoculation, healthy leaves were selected, slightly wounded, and sprayed with a conidial suspension (1 × 106 CFU/ml) of H. monticulosa strain TT422 as described by Tian et al. (2023). The inoculated plants were kept at a green house under the growth conditions as previously described (Gu et al., 2024). The disease symptoms of P. glabra leaves were observed and photographed at 6, 12, 24, 36, 48, 60, 84, and 108 hours post inoculation (hpi). To ascertain the inoculated pathogen responsible for the observed disease symptoms, pathogen strains were re-isolated from a piece of symptomatic leaf tissue using tissue isolation method (Tian et al., 2023), placed on oatmeal agar medium plate with lactic acid (3 mL/liter), and incubated at 28°C for 10 to 15 days for observation of cultural and morphological features (Lambert et al., 2019).
For evaluation of the validity of the real-time PCR assays on artificially infected P. glabra, DNA was extracted from the diseased leaves as described above. A total of 50 ng gDNA of leave tissues was used as a template to conduct real-time PCR. gDNA of H. monticulosa TT422 was used as positive controls. DNA of healthy P. glabra leaves and sterile ddH2O were used as negative controls.
Validity of TaqMan real-time PCR on naturally infected plants in fields
Healthy, asymptomatic but suspected to be infected, and symptomatic leaves were sampled, respectively, as described earlier by Gu et al. (2024). DNA samples were extracted from P. glabra leaves as described above, diluted to 50 ng/μL with sterile ddH2O for real-time PCR detection. DNA of healthy leaves was used as blank controls. The amount of H. monticulosa gDNA was calculated according to the gDNA standard curve. H. monticulosa in plant tissues was isolated and identified based on cultural and morphological characters as described above.
The standard of disease grades was defined as follows: 0 refers to no lesion; 1, 3, 5, and 7 refer to lesion area occupied <5%, ≥5% but <12%, ≥12% but <25%, ≥25% but <50% leaf area, respectively; 9 refer to lesion area occupied ≥50% leaf area or death of leaves.
Valuation of P. glabra resistance to white leaf spot using TaqMan real-time PCR
P. glabra cultivars ‘Jixiang’, ‘Bubugao’, and ‘Zhaocaimao’ with varying resistance levels were used. Saplings were sprayed with H. monticulosa conidial suspension (1 × 106 conidia/ml, containing 0.05% Tween 20) as described as Tian et al. (2023). The plants were enveloped in a transparent plastic bag for 2 days to maintain a humidity-saturated environment. The inoculated leaves were sampled at 0 and 7 days post-inoculation (dpi). Five leaves per sapling were collected each time for real-time PCR assays. Symptoms development were observed at 7 dpi and the disease index was calculated according to the standard of disease grades described above.
Statistical analysis
Statistical analysis was conducted using SPSS for Windows version 24.0 (IBM Co., Armonk, NY, USA). The data represent means ± standard deviation (SDs) calculated from three biological replicates. Differences between treatments were compared by the least significant difference test at at P < 0.05.
Results
Specificity of primer set and TaqMan probe
The obtained RPB2 sequences of nineteen fungi isolates (Table 1) were used for multiple sequence alignments. The RPB2 gene regions that are conserved for H. monticulosa strains but different from the non-target fungi were utilized to design specific primers and TaqMan probe. Based on the results of multiple sequence alignments (Fig. 1), the primer pairs RPB2Hm-F/RPB2Hm-R (5′-CGGTTATGTTGCAGGAGATTACT-3′ and 5′-CTTCTGGTCACCCCAGTTAC-3′, respectively) and TaqMan probe RPB2Hm-P (5′-CGCTAAGCCCGCCATCATCACCAAC-3′) were designed for molecular differentiation of these fungal species. Primer-BLAST analysis against NCBI database (https://www.ncbi.nlm.nih.gov/) showed that the primers RPB2Hm-F/RPB2Hm-R and the probe RPB2Hm-P were only specific to H. monticulosa, and no identical or highly similar sequence was observed in other published sequences of Hypomontagnella and closely associated Hypoxylon species in NCBI database.
Alignment of the second largest subunit of the RNA polymerase II (RPB2) gene sequences of Hypomontagnella monticulosa and closely related Hypoxylon species. The position of primer pairs RPB2Hm-F/RPB2Hm-R and probe RPB2Hm-P (line) designed for the detection of H. monticulosa by TaqMan Real-time PCR are highlighted with two arrows and one line, respectively. 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.
A conventional PCR was used to analyze the specificity of the primer set. The result showed that only the H. monticulosa strains were successfully detected, yielding a 146-bp PCR fragment, which was confirmed through sequencing. No electrophoretic bands were observed for the nontemplate control and the closely related Hypoxylon and Hypomontagnella species, including Hypoxylon howeanum, H. jianfengense, H. medogense, H. perforatum, H. sublenormandii, H. investiens, H. trugodes, and Hypomontagnella submonticulosum (Fig. 2), indicating the specificity of this PCR assay for white leaf spot pathogen.
Conventional PCR amplification of the second largest subunit of the RNA polymerase II (RPB2) gene sequences of Hypomontagnella monticulosa (lane [L] 2 to L8) and closely related Hypomontagnella and Hypoxylon species (L9 to L20) using species-specific primer pairs RPB2Hm-F/RPB2Hm-R. M, marker (100 to 2,000 bp); L1, nontemplate control; L2, TT 422; L3, HM-1; L4, HM-2; L5, HM-3; L6, HM-4; L7, HM-5; L8, HM-6; L9, XZ85 (H. howeanum); L10, XZ116 (H. howeanum); L11,J177 (H. jianfengense); L12, J177-1 (H. jianfengense); L13, XZ320 (H. medogense); L14, XZ61 (H. medogense); L15, AH183 (H. perforatum); L16, X2069 (H. sublenormandii); and L17, X2018 (H. sublenormandii); L18, CGMCC 3.15570 (H. submonticulosum); L19, Z129 (H. investiens); L20, XZ202 (H. trugodes).
To further verify the specificity of primers, the melting curves were established for real-time PCR using fungal gDNA as templates. Positive fluorescent signals were obtained only from the target fungi strains, but not from the non-target ones (Table 1, Fig. 3). The Ct values of the seven H. monticulosa strains were 20.6–22.7 cycles with an average of 22.0 cycles, while those of the eleven non-target Hypoxylon species were more than 35 cycles (Table 1), again confirming the specificity of the primer pairs and TaqMan probe for target fungal species.
Amplification curves of the genomic DNA of Hypomontagnella monticulosa strains (curves 1–7) using primer pairs RPB2Hm-F/RPB2Hm-R and probe TaqMan RPB2Hm-P. No obvious fluorescent signals were observed from the DNA samples extracted from the non-target fungi, including H. howeanum, H. jianfengense, H. medogense, H. perforatum, H. sublenormandii, H. submonticulosum, H. investiens, and H. trugodes (8–19). The data represent mean values ± standard deviations (SDs) calculated from three replicates. RFU, relative fluorescence unit.
Sensitivity and stability of TaqMan real-time PCR assay
A standard curve was plotted by tenfold serial dilution of cloned RPB2 DNA (pBM16A-T-RPB2, pDNA) of H. monticulosa TT-422 strain ranging from 100 to 107 copies/μL (Fig. 4A). The results showed that there was a robust linear correlation between the log 10 concentrations of pDNA and Ct values, with high correlation coefficient (R2 = 0.998) and high amplification efficiency (E = 110.1%). The lowest DNA concentration that could be detected was as low as 2,300 copies (0.05 fg, Fig. 4B). The sensitivity of the real-time PCR was 1,000 times higher than that of a conventional one (Supplementary Fig. 1).
TaqMan real-time PCR assays on Hypomontagnella monticulosa TT-422 pDNA (pBM16A-T-RPB2) (2.3 × 100 to 2.3 × 107 copies μL−1) and gDNA (0.005 to 50 ng μL-1) diluted with sterile ddH2O. (A) The standard curve for H. monticulosa TT-422 pDNA. (B) Amplification curves for H. monticulosa TT-422 pDNA. (C) Amplification curves for H. monticulosa TT-422 gDNA. (D) The standard curve for H. monticulosa TT-422 gDNA. The data represent mean values ± standard deviations calculated from three replicates. RFU, relative fluorescence unit.
gDNA from mycelia of H. monticulosa TT-422 was serially diluted with sterile double distilled water (ddH2O), ranging from 50 ng to 0.5 pg/μL. Real-time PCR was performed using the fungal gDNA to obtain standard curves and amplification curves. A linear relationship between gDNA quantity and the Ct values was observed with R2 = 0.996, a slope value of −3.12 and E = 109.2% (Fig. 4D). The lowest detection level was determined to be 0.5 pg when fungal gDNA was used. (Fig. 4C).
The potential effect of leaf DNA extract on quantitative results was evaluated using serial dilution of H. monticulosa TT-422 gDNA (5 ng/μL) with different volumes of P. glabra leaf extracts. The results showed no obvious differences between the Ct values of real-time PCR reactions with or without the addition of leaf extracts (P = 0.245 > 0.05) (Table 2). This observation indicates that the amplification stability of the real-time PCR assays was not significantly affected by the presence of the host background. As shown in Fig. 5B, the relationship between Ct values and H. monticulosa TT-422 gDNA was expressed by the equation y = −3.17x + 46.11 (R2 = 0.998; E = 106.8%). The lowest detection level with plant tissue DNA was determined to be 0.5 pg/μL (Fig. 5A).
The effect of Pachira glabra leaf DNA extracts on the Ct values of the TaqMan real-time PCR assays
TaqMan real-time PCR assays on Hypomontagnella monticulosa TT-422 gDNA (0.005 to 50 ng) diluted with the DNA extracted from Pachira glabra leaves. (A) Amplification curves. (B) The standard curve. The data represent mean values ± standard deviations calculated from three replicates. RFU, relative fluorescence unit.
Detection in artificially-inoculated P. glabra
P. glabra saplings inoculated with H. monticulosa TT-422 were sampled to evaluate the validity of the real-time PCR method. No symptoms were observed on the P. glabra leaves following inoculation up to 24 hours (Fig. 6A). However, the DNA amount of H. monticulosa could be detectable, reaching 3.54, 4.50, and 5.63 pg per 50 ng DNA of leave extracts at 6, 12, and 24 hpi, respectively (Fig. 6B). During the period of symptomatic infection, the DNA amount of H. monticulosa progressively increased over time in the inoculated leaves. Tiny white to pale yellow spots first occurred sporadically on the adaxial leaf surfaces at 36 hpi, when the DNA amount of H. monticulosa was 43.06 pg per 50 ng DNA of P. glabra leaves. By 60 hpi, white or pale yellow spots became denser and larger, and several began to merge with each other. Meanwhile, small light black to black spots emerged on the abaxial leaf surfaces. The DNA amount of H. monticulosa DNA reached 50.52 pg per 50 ng of total DNA at this time. Re-isolation of H. monticulosa isolates was consistently achieved from each diseased leaves using a tissue isolation technique. These findings suggest that the real-time PCR assays could monitor the proliferation progression of the fungus in the whole process of disease, and exhibit a potential application in fields.
Time-course infection with Hypomontagnella monticulosa on Pachira glabra leaves during 0 to 108 hours post-inoculation with 106 conidia suspensions (1 × 106 CFU/mL). (A) Photographs of infected leaves. (B) DNA amount of H. monticulosa was quantified by real-time PCR. The data represent means ± standard deviations calculated from three replicates.
Detection in naturally-infected P. glabra
H. monticulosa was detected for naturally infected leaves collected from P. glabra from gardens using this molecular method. Among the twenty-five asymptomatic but suspected to be infected leaves, obvious fluorescent signals were detected in nine leaves with average DNA amount of 3.41 pg per 50 ng DNA sample. This result indicates that white leaf spot symptoms could probably be developed in these asymptomatic leaves. There was no evident amplification observed for DNA extract from the other sixteen leaves without symptoms (Table 3). The nine asymptomatic leaves tested positive by the real-time PCR, were also detected by conventional PCRs. The results showed that only four asymptomatic leaves exhibited positive amplifications, demonstrating that the conventional PCR was less sensitive than the real-time PCR. No obvious amplification signals were observed when DNA from healthy leaves used. Moreover, there was a positive correlation between the disease severity and the amount of fungal DNA. Higher amounts of H. monticulosa DNA were detected in P. glabra leaves with severe symptoms than in leaves with only slight or mild symptoms. Again, H. monticulosa was re-isolated from each of the infected leaves using tissue-isolation method.
Detection of Hypomontagnella monticulosa in symptomless and infected leaves of Pachira glabra in fields using TaqMan real-time PCR and conventional PCR
Evaluation resistant levels of P. glabra cultivars
To validate the real-time PCR quantification technique of H. monticulosa in different P. glabra cultivars and get insight into their responses to H. monticulosa infection, pathogenicity tests were performed by spray inoculation in three cultivars, i.e., ‘Jixiang’, ‘Bubugao’, and ‘Zhaocaimao’. The resistance of the P. glabra cultivars to H. monticulosa was evaluated using visual symptoms assessment and real-time PCR assays. As showed in Fig. 7, ‘Zhaocaimao’ was more susceptibile than ‘Bubugao’ and ‘Jixiang’. The quantitative results for H. monticulosa DNA content demonstrated a statistically significant positive correlation with the disease severity index (Table 4), which was determined by measuring the leaf lesion area at 7 dpi.
Representative symptoms observed on cultivars of ‘Jixiang’ (A), ‘Bubugao’ (B), and ‘Zhaocaimao’ (C) exhibiting varying resistance levels to white leaf spot at 7 days post-inoculation. Leaves of saplings were sprayed with 106 conidia suspensions (1 × 106 CFU/mL), and wrapped in a transparent plastic bag for 2 days to maintain a humidity-saturated environment for symptom development.
Relationship between resistance of Pachira glabra cultivars against white leaf spot and Hypomontagnella monticulosa DNA amount determined by real-time PCR analysis
Discussion
Species within Hypoxylon and closely related genera are considerably diverse, mostly present as endophytes in a variety of living plants and are difficult to distinguish solely on a morphological basis (Sánchez-Ballesteros et al., 2000). Some species cause tree canker and wood rots in the necrotrophic phase, but mostly initiate latent infection as symptomless endophytes in living tissues (Castillo et al., 2023; Swiecki et al., 2005). Given that H. monticulosa has been frequently reported as an endophyte in plants (Ayodele and Badejo, 2022; Ouyang et al., 2025), it is highly likely that this fungal species can also be aggressive saprophytes or opportunistic pathogens when plants are encountered by varied internal and /or external environmental factors (Dutta et al., 2014). Recently, H. monticulosa has been identified as an emerging pathogen causing white leaf spot on P. glabra (Tian et al., 2023). To date, no preventive and therapeutic measures have been developed due to the lack of sufficient knowledge about the life cycle and epidemiological dynamics of this pathogen. A major challenge in managing this disease in P. glabra trees is early detection of H. monticulosa due to its survival for extended periods in quiescent stage in the host plant. Under favorable environmental conditions, the incidence and severity of the disease may be rapidly increased. However, effective diagnostic strategies for early-stage detection of white leaf spot in P. glabra remain underdeveloped. Therefore, establishing a specific, sensitive, and effective assay to detect and quantify latent infection by H. monticulosa is indispensable for early prediction of the disease to reduce the danger of outbreak on P. glabra.
Accurate and precise identification of phytopathogenic species within Hypoxylon and closely related genera based on morphology is often infeasible due to their limited or overlapping cultural and morphological characters, and high phenotypic variance under different cultural conditions (Wendt et al., 2018). Therefore, morphological and cultural characters are not easily used to differentiate species. PCR-based molecular methods are expected to supply an ideal solution for disease diagnosis (Khakimov et al., 2022; Venbrux et al., 2023). In the current study, the TaqMan real-time PCR assay targeting RPB2 marker allows a specific, sensitive, and effective tool for detection H. monticulosa causing white leaf spot on P. glabra. This new diagnostic assay is suitable for use in sensitive and rapid screening and controlling of white leaf spot on P. glabra, and will also facilitate the research of plant-H. monticulosa interaction as well as screening for resistant cultivars.
An effective PCR assays relies on molecular markers appropriate for design of highly selective primers that could specifically amplify the target sequences (Gu et al., 2024; Hayden et al., 2004). Gene markers, including ITS, LSU, ACT, TUB2, and RPB2, are routinely used in molecular differentiation of Hypoxylon and allies (Suwannasai et al., 2013; Tang et al., 2009; Wendt et al., 2018). Notably, the ITS of nuclear rDNA, which is commonly recognized by the community as standard barcode of fungi (Schoch et al., 2012), resulted in a limited resolution of the hypoxyloid taxa (Triebel et al., 2005). By contrast, the RPB2 gene is more effective in inferring phylogenetic relationships due to its slow evolution as a protein-coding gene (Hongsanan et al., 2017; Wendt et al., 2018). In fungal genome, RPB2 is a single-copy gene (Prencipe et al., 2023). As reported by Schena et al. (2013), real-time PCR assays based on single-copy genes enable a higher accuracy for quantitative detection. The occurrence of high level of fixed single nucleotide polymorphisms (SNPs) among different species can be used for the design of primers and probes. Real-time PCR methods are ideal for the detection of SNPs (Schena et al., 2004). A single-base change could distinguish sequences of the most closely related species within Ramularia eucalypti species complex (Schena et al., 2013). This study, therefore, aimed to design real-time PCR primers and probe specific to H. monticulosa based on the sequence variances in the RPB2 gene region. The TaqMan real-time PCR assays using the primer set RPB2Hm-F/RPB2Hm-R and probe RPB2Hm-P could accurately identified H. monticulosa using mycelia DNA from pure cultures, DNA mixtures of the target pathogen and leaf tissues, and DNA from the infected leaves of P. glabra. No cross-reaction was observed with the closely related Hypoxylon species (Figs. 2 and 3), indicating that these assays were species-specific. It is unknown that other Hypoxylon species could cause diseases on P. glabra. However, a number of Hypoxylon species live as as endophytes in a diverse of living plants (Sánchez-Ballesteros et al., 2000). If the closely related Hypoxylon species listed in Table 1 coexist with H. monticulosa in P. glabra tissues, the specificity of the primers used in this study can still be guaranteed to differentiate the target from its close allies.
Effectiveness and reliability of TaqMan real-time PCR techniques also depend on high sensitivity of primers and probe that could specifically bind to the target sequences (Gu et al., 2024). In this study, an optimized TaqMan real-time PCR assay was developed, and used to diagnose and quantify H. Monticulosa causing leaf white spot on P. glabra. The method could reliable detect as low as 0.05 fg/μL (2.3 × 103 copies/μL) of pDNA (Fig. 4B), 0.5 pg/μL of gDNA (Fig. 4C), and 0.001% of target DNA mixed with leaf tissue DNA (i.e., 0.5 pg target DNA/50 ng plant tissue DNA) (Fig. 5A). This detection threshold is 10 times higher than that of the RPB2-based real-time TaqMan PCR technology for detecting Puccinia striiformis sensu stricto in wheat, and comparable to that of the same method detecting P. graminis in the same host (Liu et al., 2015). Real-time PCR technique appears to be more sensitive than conventional PCR (Schena et al., 2004; Venbrux et al., 2023). The sensitivity of the real-time PCR was 1,000 times higher than the conventional PCR (Supplementary Fig. 1). Thus, the sensitivity and efficiency of the real-time PCR method is sufficient for accurate detection and quantification of H. monticulosa in P. glabra leaves.
It is necessary to assess the potential interference of plant tissues on in planta detection and quantification of fungal pathogen (Schena et al., 2004). Polysaccharides and phenolic compounds introduced from host plants during DNA extraction may interfere with PCR amplification by inhibiting the detection of target DNA (Schena et al., 2004; Schenk et al., 2023). In the present study, for determining the effects of plant background on quantitative results, H. monticulosa gDNA was serially diluted with DNA extract of P. glabra leaves. The results showed the dilution level could circumvent any bias due to PCR inhibitors derived from plant tissues. Accordingly, the presence of P. glabra leaf tissues exerted no significant effects on the sensitivity, efficiency, and reproducibility of the molecular assays.
Generally, no difference in visible signs can be observed between healthy and infected tissues during early stage of plant fungal diseases. Early detection and identify the fungal agents is critical for prediction and controlling the progression of diseases within and between host plants. In this study, the presence of H. monticulosa was sensitively and reliably detected by the molecular assays in artificially infected leaves at 6 hpi. However, the first visible white or pale yellow spots usually occurred at 36 hpi (Fig. 6A). These findings reveal that the real-time PCR method can be used to detect H. monticulosa in the latent phase in host leaves. In addition, the two-stage increase of H. monticulosa DNA was found during the infection process (Fig. 6B). This result was correlated well with the visual observation, confirming that this method is suitable for monitoring the infection dynamics of H. monticulosa in P. glabra trees during both latent and symptomatic stage.
The effectiveness and feasibility of this molecular assay were further validated for H. monticulosa detection in naturally infected P. glabra leaf samples collected from fields. The results showed that the DNA amount of H. monticulosa detected by the real-time assay correlated positively with the disease grades (Table 3). The infection was also confirmed by the tissue isolation method. Therefore, this newly developed assay represents a rapid and reliable diagnostic tool to detect white leaf spot of P. glabra at early stages of disease development in fields.
Finally, H. monticulosa biomass was quantified using the real-time PCR across P. glabra cultivars with varying resistance levels. As showed in Table 4 and Figure 7, the disease severity, as measured by leaf lesion area at 7 dpi, showed a statistically significant positive correlation with the DNA content of H. monticulosa. The findings demonstrate the robustness of the qPCR method across diverse genetic backgrounds and underscore its effectiveness in screening programs aimed at identifying disease-resistant cultivars.
Nevertheless, the present qPCR method has limitations. For example, DNA extraction is time-consuming and requires complex thermal cycling equipment, making it preferably performed in laboratory settings. If only used for qualitative purposes, i.e., presence or absence of specific microorganisms, isothermal amplification techniques like loop-mediated isothermal amplification (LAMP) can circumvent these limitations (Venbrux et al., 2023), and our team is currently developing LAMP method for H. monticulosa detection.
In the present study, an effective TaqMan real-time PCR method targeting the RPB2 gene has been developed for specific and accurate detection of H. monticulosa on the host P. glabra. This method represents a rapid and reliable diagnostic strategy to detect white leaf spot on P. glabra at the initial stages of infection, thus is suitable for application in early prediction and management of the disease to reduce the potential danger of prevalence.
Notes
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
The earlier batch of forestry reform and development project in 2022 funded by central government provided financial support.
Electronic Supplementary Material
Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).