*Corresponding author Introduction
The emergence of resistant Colletotrichum spp. isolates against Quinone outside Inhibitors (QoI; FRAC 11) fungicides is a current global threat to horticultural cultivation (Cortaga et al., 2023). Resistance towards QoI fungicide, especially Pyraclostrobin, has been attributed to mutation from glycine to alanine at codon 143 (G143A) in cytochrome-b gene (cyt b) of C. siamense in strawberry (Fragaria × ananassa) (Hu et al., 2023). Additionally, Forcelini et al. (2016) revealed that C. acutatum in strawberry had developed mutations at G143A and F129L (Phenylalanine to Leucine at codon 129) following repeated exposure to Pyraclostrobin. Similar mutations have been reported with resistance to other QoI fungicides, including Azoxystrobin, which has been associated with G143A mutation in C. siamense in blueberry (Vaccinium sect. Cyanococcus) (Hu et al., 2015), G143 in C. siamense on apple (Malus domestica) (Chechi et al., 2019), both G143A and F129L in C. nymphaeae on strawberry (Luo et al., 2021), as well as G143A in C. truncatum on soybean (Glycine max) (Rogério et al., 2024).
Numerous Colletotrichum spp. involve in the anthracnose and dieback epidemics in mango (Mangifera indica), causing significant challenge to production worldwide (Li et al., 2020; Nurlaelita et al., 2024; Widiastuti et al., 2023, 2024; Wu et al., 2020). The most prevalent one, Colletotrichum asianum, has been widely documented across multiple countries, including Sri Lanka (Krishnapillai and Wijeratnam, 2014), Brazil (Vieira et al., 2014), Ghana (Honger et al., 2014), Malaysia (Zakaria et al., 2015), South Africa (Sharma et al., 2015), Ethiopia (Abera et al., 2016), China (Mo et al., 2018), Australia (Giblin et al., 2018), Vietnam (Li et al., 2020), Philippines (Alvarez et al., 2020), Mexico (Tovar-Pedraza et al., 2020), Thailand (Rattanakreetakul et al., 2023), Taiwan (Lin et al., 2023), Peru (Vilcarromero-Ramos et al., 2023), and Fiji (Lomavatu et al., 2024). In Indonesia, C. asianum was first officially reported in mango cultivated in Indramayu region, West Java (Benatar et al., 2021).
Currently, the disease management largely relies on chemical-based fungicides, particularly Strobilurin class (QoI fungicides), with Azoxystrobin first introduced by Syngenta in 1996. Notably, the first resistance to this fungicide, involving a G143A mutation, appeared in 1998 in cereal crops (Bartlett et al., 2002). The allele-specific and restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR) method for detecting mutations in the cytochrome b gene based on cDNA, as published by Isa and Kim (2022), has been shown to successfully identify the G143A mutation in C. gloeosporioides and C. acutatum in response to Pyraclostrobin. Those two species belong to distinct well-characterized species complexes, though they share the same genus (Pardo‐De la Hoz et al., 2016). It has been known that allele-specific and RFLP-PCR detection methods are widely favored for detecting resistance to QoI fungicides considering their efficiency, reliability, and rapid results (Dodhia et al., 2021; Finger et al., 2014; Kiniec et al., 2022; Miles et al., 2021; Obuya and Franc, 2016; Rosenzweig et al., 2015).
Given the diversity within Colletotrichum species and their species groups, it is essential to verify that molecular approaches for detecting gene mutations perform consistently and are practical when implemented in different species. In this study, we further investigate whether the resistance profile of C. asianum, which is part of the Cg species complex, can be effectively detected using the allele-specific and RFLP-PCR method developed by Isa and Kim (2022). Moreover, no case of Pyraclostrobin resistance in C. asianum associated with the cytochrome b gene had been reported in mango to date.
Materials and Methods
Validation outline.
The molecular detection method for G143A mutation in the Colletotrichum species complex, as outlined by Isa and Kim (2022), was evaluated for its applicability to be applied across different species within the same species complex. C. asianum was selected as the test subject for this validation. This study employed two approaches: a bioassay (conidial germination and germ tube elongation) and a PCR-based test, including conventional, allele-specific, and RFLP-PCR methods, which are further detailed below. A slight modification in each test did not significantly affect the validity of the result.
Isolates and fungicides.
Four mango-isolated C. asianum (HM_BDIY, MLG_TJT, HM_IJB, and 4A-3) (Nurlaelita et al., 2024) and four citrus-isolated C. gloeosporioides (CR3, CR5, CA6, and CR11) (Shidiq et al., 2024) maintained in the Phytopathology Laboratory, Universitas Gadjah Mada were used in this study as shown in Table 1. The isolates were regularly grown on potato dextrose agar (PDA; HiMedia Laboratories Pvt. Ltd., Thane, India) at room temperature during the study. The QoI fungicide Pyraclostrobin (cabrio 250 g/l a.i., emulsifiable concentrate [EC]; BASF, Jakarta, Indonesia) was selected to evaluate the control efficacy in vitro.
Table 1.
List of isolates in Colletotrichum gloeosporioides species complex used in this study
Pyraclostrobin fungicide sensitivity evaluation.
Pyraclostrobin-amended PDA was used as food poisoning technique for all isolates of C. asianum and C. gloeosporioides. The medium was prepared by mixing PDA powder with double-distillated water (3.9 g/100 ml) in an Erlenmeyer. Fungicide stock solution was prepared by passing through a sterile syringe filter (Ø0.22 µm). PDA medium was autoclaved at 120°C and then cooled to 50-55°C. Afterward, the fungicide stock solution was added to the melted PDA and mixed gently to form concentrations of 0.1, 0.5, 1, 5, 10, and 100 ppm, respectively. As for control, the PDA medium was made without any amendment. For inoculation, mycelium discs were taken using a cork borer (Ø0.5 cm) at each fungal isolate's active growing part of the mycelia from 7 days old PDA culture. The discs were transferred to the Pyraclostrobin-amended PDA in Petri dish plates under sterile condition of laminar airflow. After inoculation, Petri dish was sealed and stored in a plas-tic box at room temperature (28°C) under dark conditions. Each fungicide concentration treatment was repeated thrice. Mycelial growth was observed 9 days after inoculation to determine the effective concentration to inhibit 50% of the population (EC50 value). The inhibition rate was measured by comparing unamended control growth and fungicide-amended treatments minus 0.5 cm, according to the method used by Isa and Kim (2022), with slight modification.
Germination rate and germ tube length inhibition.
A germination test was performed by preparing different concentrations of Pyraclostrobin (0.1, 0.5, 1, 5, 10, and 100 ppm). Initially, the stock solution (10, 100, and 1,000 ppm) was prepared by filtering using a sterile syringe filter (Ø0.22 µm). Then, 7-10 days old conidium of C. gloeosporioides and C. asianum were collected by diluting them with sterile dd-water (5-10 ml) using a glass rod spreader. The concentration was adjusted to 105 conidia/ml in a 1.5 ml microtube mixed with fungicide. Forty microliters of potato dextrose broth solution (50% [v/v]) was added to previously mentioned mixture of conidia and fungicide to accelerate the fungal growth. After immersion for about 1 hr, each 50 µl of final conidial suspension was placed in four different positions on a single glass slide using a micropipette as a replication. Then, the samples were kept in a storage box that had been modified to preserve humidity at room temperature under dark conditions. The percentage of germinated conidia and germ tube length were examined under 5 µl of Lactophenol cotton blue after 18 hr of incubation using an Olympus CX31 microscope (Olympus, Tokyo, Japan). The number of germinated conidia per 100 conidia was presented as percentage germination test result. On the other hand, germ tube length was measured by counting the length of 15 germinated conidia in every respective concentration. Then, the conidial and germ tube inhibition rates (%) were calculated by comparing the difference between control and treatment, divided by control. Each treatment was done using four replications.
Extraction of RNA and cDNA synthesis.
Approximately 100 mg of mycelia from each isolate of C. asianum and C. gloeosporioides were scraped off from a 7-day-old PDA culture and ground using liquid nitrogen in a 2 ml microtube. The RNA extraction was carried out using Total RNA Mini Kit (plant) (Geneaid, Taipei, Taiwan) according to the manufacturer's protocol. The extracted RNA was then quantified for quality control using a Nanodrop machine (BioDrop Duo+, Biocrom Ltd., Leicestershir, England, UK). Subsequently, cDNA was synthesized according to ReverTra Ace TM qPCR RT Kit (Toyobo, Co., Ltd., Osaka, Japan) using 10 µl of mixture: 2 µl of 5× RT Buffer, 0.5 µl of RT Enzyme Mix, 0.5 µl of Primer Mix, RNA (final concentration of 50 µg/ml), and nuclease-free water. The incubation was then carried out using a thermal cycler (Bio-Rad T-100; Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 1 hr at 37°C and continued at 98°C for 5 min. Then, cDNA was stored at −20°C until needed.
A mplification and sequencing of the cytochrome b gene.
The partial cytochrome b gene region was sequenced from cDNA according to the method by Isa and Kim (2022), using the forward primer Cacytb-P2 (5’-CAT AGT AAY ACA GCT TCT G-3’) and reverse primer Cacytb-R (5’-GGA ATA GAT CTT AAT ATA GC-3’). For allele-specific PCR. A 50 µl PCR mixture containing 25 µl MyTaq HS Red Mix (Bioline, London, UK), 2 µl forward and reverse primer each, 2 µl cDNA, and 19 µl ddH20. For amplification, the PCR program was run using the protocol: initial denaturation at 94°C for 4 min, 30 cycles of denaturation at 98°C for 10 sec, annealing at 37°C for 30 sec, and extension at 72°C for 1 min, followed by a final extension step at 72°C for 7 min. The target band was then visualized under a UV transilluminator on 1.5% agarose gel amended with FloroSafe DNA Stain (Axil Scientific Pte, Ltd., Singapore) after separated using electrophoresis at 100 V for 40 min. Thirty μl of successfully amplified PCR products were then sent for Sanger sequencing. The sequence data was analyzed using MEGA11 software version 11.0.13 (The Pennsylvania State University, University Park, PA, USA).
Allele-specific PCR evaluation.
Applying the method developed by Isa and Kim (2022), a pair of forward primer CacytbF (5’-GGG TAT AGG TTT CCT GGG TTA TG-3’) and reverse primer S-mmc (5’ATA AGG TTA GTA ATA ACT GTT GCC C-3’) for sensitive isolate, and R-mmc (5’-ATA AGG TTA GTA ATA ACT GTT GCC G-3’) for resistant isolate was used to detect possible G143A point mutation in C. asianum and C. gloeosporioides. A 10 µl PCR mixture containing 5 µl MyTaq HS Red Mix, 1 µl of each forward and reverse primer, and 3 µl cDNA was prepared and subjected to PCR using protocol: initial denaturation at 95°C for 4 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 1 min 20 sec, followed by a final extension step at 72°C for 5 min. The amplified target bands were then visualized under a UV transilluminator on 1.5% agarose gel amended with FloroSafe DNA Stain after separated using electrophoresis at 50 V for 50 min.
RFLP-PCR.
The PCR product from amplification was used to perform RFLP-PCR following Isa and Kim (2022) procedure with slight modification on the incubation time. In 30 µl of the final reaction mixture, 10 µl PCR product from previous cytochrome b amplification, 2 µl 10x buffer (G buffer), 1 µl Fnu4HI enzyme (10 U/µl), and 17 µl ddH2 O were mixed and incubated at 37°C for 3 hr followed by deactivation at 65°C for 20 min. Then, electrophoresis at 50 V for 50 min on 1.5% agarose gel amended with FloroSafe DNA Stain was performed, and the result was viewed under a UV transilluminator.
Data analysis.
Probit analysis using a general linear model was conducted using JMP Pro 13 (SAS Institute, Inc., Cary, NC, USA) to determine the EC50 value. Germination rate and germ tube length inhibition were analyzed using a non-parametric test (Kruskal-Wallis's test, at P<0.05) and followed by Dunn's Test. Furthermore, Spearman's range correlation test was conducted to determine the relationship between two quantitative variables on the bioassay test. All obtained data were analyzed and confirmed against the reference method.
Results
Mycelial inhibition rate and EC50 value of Pyraclostrobin.
Seven different concentrations of Pyraclostrobin were evaluated against the C. asianum and C. gloeosporioides on their mycelial growth, and the results were shown in Fig. 1. Based on the reference method (Isa and Kim, 2022), a resistant isolate was determined by an inhibition rate lower than 40% at concentration of 10 ppm, implies that there was no resistance isolates detected (Fig. 1A). However, notable growth was observed on the HM_IJB isolate (Fig. 1B) which compared to Fig. 1A. Using probit analysis, EC50s of Pyraclostrobin were varied from 0.01 to 1.37 ppm with the highest one was in HM_IJB isolate (Table 2).
Fig. 1.
Comparison of Colletotrichum asianum (HM_BDIY, MLG_TJT, HM_IJB, and 4A-3) and C. gloeosporioides (CR3, CR5, CA6, and CR11) isolates’ growth response to Pyraclostrobin fungicide by food poisoning technique. (A) Relative growth (%) to control and (B) front view of the culture. Five different fungicide concentrations were used by amending it to potato dextrose agar (PDA) media. The response was recorded at 9 days post inoculation at 28°C. Each concentration treatment was repeated thrice. Red arrows: lowest inhibition rate.
Table 2.
Toxicity and effective concentration (EC50) profile of Pyraclostrobin on C. asianum and C. gloeosporioides isolates
| Isolate | N | EC50 (ppm) | 95% FL | Log (concentration)±SE | X2 | df |
|---|---|---|---|---|---|---|
| HM_BDIY | 21 | 0.12 | 0.0690-0.1850bc | 0.41±0.04 | 02.36 | 4 |
| MLG_TJT | 21 | 0.08 | 0.0348-0.1401ab | 0.32±0.04 | 13.06 | 4 |
| HM_IJB | 21 | 1.37 | 0.9273-1.967c | 0.30±0.03 | 16.62 | 4 |
| 4A-3 | 21 | 0.14 | 0.0649-0.2434bc | 0.27±0.03 | 13.86 | 4 |
| CR3 | 21 | 1.35 | 0.9445-1.8938c | 0.33±0.03 | 08.99 | 4 |
| CR5 | 21 | 0.01 | 0.0003-0.049a | 0.13±0.03 | 12.49 | 4 |
| CA6 | 21 | 0.15 | 0.0503-0.301bc | 0.19±0.03 | 03.84 | 4 |
| CR11 | 21 | 0.59 | 0.3475-0.931c | 0.26±0.03 | 09.46 | 4 |
Effect of Pyraclostrobin on conidial germination and germ tube elongation.
In both conidial germination (Fig. 2A) and germ tube elongation (Fig. 2B), HM_IJB showed the lowest inhibition rate of Pyraclostrobin compared to other isolates. Furthermore, there were statistically significant differences in HM_IJB compared to some isolates. Concentration of 1 ppm of Pyraclostrobin was significantly enough to inhibit the germination and germ tube elongation of both C. asianum and C. gloeosporioides compared to the control. The isolates were completely inhibited at concentrations ranging from 5 to 100 ppm. Moreover, the Spearman correlation values of 0.93 and 0.94 for conidial and germ tube inhibition, respectively, indicated a very strong positive correlation between the concentrations and the inhibition.
Fig. 2.
Inhibition rate (%) of (A) conidial germination and (B) germ tube length of Colletotrichum asianum (HM_BDIY, MLG_TJT, HM_IJB, and 4A-3) and Colletotrichum gloeosporioides (CR3, CR5, CA6, and CR11) after being treated with five different concentrations of Pyraclostrobin using the fungicide-immersing method. The inhibition rate was counted 18 hr after treatment. A non-parametric test (Kruskal-Wallis's test, at P<0.05) followed by Dunn's Test was done as statistical analysis. Spearman's range correlation test indicates the pattern of two variables. The arrows (red) indicate an isolate which shows lower inhibition rate or possesses resistancy (isolate of IJB_HM).
PCR-based test for resistance detection.
Three PCR-based methods were conducted to detect and validate the resistance profile of all of the isolates. Direct sequencing of the cytochrome b region using conventional PCR was achieved using a reference primer set. Gel electrophoresis visualization of the cytochrome b region presented an identical 617 bp length in all isolates (Fig. 3). Nonetheless, sequencing result, listed in NCBI GenBank with accession no. PV690112-PV690119, showed a shift in codon at 129 from Phenylalanine (wild type) to Leucine (mutated type) of C. asianum isolate HM_IJB as shown in Fig. 4. On the other hand, allele-specific PCR confirmed that there was no resistant isolate due to G143A mutation detected since all DNA bands were visible using the sensitive primer sets (Fig. 5). In addition to that, RFLP-PCR targeting specific site of mutation (G143A) showed no cut region using Fnu4HI restriction enzyme (Fig. 6).
Fig. 3.
Cytochrome b amplification using primer CacytbP2/CacytbR used in the reference method of Colletotrichum asianum (HM_BDIY, MLG_TJT, HM_IJB, and 4A-3) and Colletotrichum gloeosporioides (CR3, CR5, CA6, and CR11). M: DNA Marker (100 bp plus ladder).
Fig. 4.
Substitution of nucleotide from TTC to TTA resulted in phenylalanine (F) to leucine (L) change at codon 129 of Colletotrichum asianum (HM_IJB) while no glycine (G) to alanine (A) mutation was observed at codon 143. (A) Sequencing result of cytochrome b region showing mutation nucleotide; and (B) illustrative figure indicating region of cytochrome b's gene shifting. Isolates of C. asianum were HM_BDIY, MLG_TJT, HM_IJB, and 4A-3 and isolates of C. gloeosporioides were CR3, CR5, CA6, and CR11.
Fig. 5.
Allele-specific polymerase chain reaction using primer CacytbF/R-mmc (A) or S-mmc (B) used in the reference method to confirm the lack of G143A mutation on the tested isolates of Colletotrichum asianum (HM_BDIY, MLG_TJT, HM_IJB, and 4A-3) and Colletotrichum gloeosporioides (CR3, CR5, CA6, and CR11). M: DNA Marker (100 bp plus ladder).
Fig. 6.
Restricted fragment result using Fnu4HI enzyme as used in the reference method to detect specific mutation of Colletotrichum asianum (HM_BDIY, MLG_TJT, HM_IJB, and 4A-3) and Colletotrichum gloeosporioides (CR3, CR5, CA6, and CR11). (A) Gel electrophoresis visualization; and (B) cleavage region by Fnu4HI enzyme. M: DNA Marker (100 bp plus ladder).
Discussion
In this paper, validation of molecular tests (Isa and Kim, 2022) revealed a sensitive profile to Pyraclostrobin on C. asianum and C. gloeosporioides with no G143A mutation. Thus, this study confirmed the repeatability of the reference method, particularly when cDNAs which successfully amplified the cytochrome b region of C. gloeosporioides and C. asianum were used as templates. The slight adjustment to the bioassay test concentration was determined to not influence the result accuracy. Furthermore, allele-specific and RFLP-PCRs was found to be likely sufficient to determine resistance associated with G143A mutation in the Cg species complex.
Other important finding is that the direct sequencing inadvertently identified a substitution at codon 129 from Phenylalanine to Leucine (F129L) in HM_IJB (Fig. 4). This mutation was not detectable by RFLP-PCR because the restriction enzyme (Fnu4HI) specifically targets G143A site. Therefore, conventional PCR followed by direct sequencing offers a robust advantage in detecting single nucleotide polymorphisms (SNPs) within the cytochrome b gene region. The F129L mutation, while conferring moderate resistance towards Pyraclostrobin, is considered less significant than that of the G143A mutation, which exhibits high levels of resistance (Wei et al., 2021).
Interestingly, the F129L mutation identified in this study was encoded by the TTA codon, a variant not previously reported in Colletotrichum asianum from mango but previously observed in Colletotrichum truncatum from soybean in association with Azoxystrobin resistance (Cortaga et al., 2023; Poti et al., 2023). This TTA SNP, arising from TTC (wild type), could be analogous to mutations seen in Alternaria solani genotype II, where mutations are likely propagated via conidial dispersal rather than single evolutionary events (Nottensteiner et al., 2019). If that pattern holds true, it suggests the mutation may have developed in a nearby mango orchard where C. asianum was isolated. Consequently, the PCR-based approach from the reference study may be not suitable for detecting other sites of mutation in Colletotrichum species, and a further enhanced method is warranted.
A thorough analysis of bioassay involving mycelial growth, conidial germination, and germ tube elongation tests on Colletotrichum isolates demonstrated consistency with molecular testing results. Mycelial growth inhibition results indicated that no isolate displayed high resistance; all remained sensitive to Pyraclostrobin based on the reference standards. Regarding the EC50 values, two isolates (CR3 and HM_IJB) exhibited elevated values (1.35 and 1.37 ppm, respectively) compared to others, suggesting that higher concentrations may be increasingly crucial to control them. Although mycelial growth inhibition generally indicated sensitivity, the EC50 range of 0.11 to 10 ppm for Pyraclostrobin was actually classified as resistant (Forcelini et al., 2016).
This study confirmed that the method developed by Isa and Kim (2022) is effective not only for C. gloeosporioide s and C. acutatum but also for C. asianum and thus potentially to other members of Cg species complex. The focus of this method on a single mutation site may, however, limit its ability to detect other potential SNPs. Sequencing the target region may still provide a reliable approach for identifying gene shifts. To our knowledge, this is the first report identified F129L mutation in C. asianum infecting mango which presents a not observed before TTA SNP. Further research is needed to develop resistance assays specifically for the F129L mutation, and an extensive sample study is recommended to determine the significance of this mutation for the future use of QoI fungicides.
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