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Article

New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple

by
Diana J. McHenry
and
Srđan G. Aćimović
*
Plant Pathology Laboratory, Alson H. Smith Jr. Agricultural Research and Extension Center, School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, Winchester, VA 22602, USA
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(5), 878; https://doi.org/10.3390/microorganisms12050878
Submission received: 28 February 2024 / Revised: 17 April 2024 / Accepted: 23 April 2024 / Published: 27 April 2024
(This article belongs to the Special Issue Colletotrichum Pathogens in Plants)
Versions Notes

Abstract

:
Bitter rot of apple is an economically important worldwide disease caused by different Colletotrichum species, depending on many factors such as climate, geography, other hosts, and crop management practices. Culture, morphology, and single-locus sequencing-based methods for identifying the Colletotrichum species are severely limited in effectiveness, while the multilocus sequence typing methods available for delineating species are costly, time-intensive, and require high expertise. We developed species-specific hydrolysis probe real-time PCR assays for the following nine Colletotrichum species causing bitter rot in the Mid-Atlantic U.S.A.: C. fructicola, C. chrysophilum, C. noveboracense, C. gloeosporioides s.s., C. henanense, C. siamense and C. theobromicola from the C. gloeosporioides species complex, and C. fioriniae and C. nymphaeae from the C. acutatum species complex. After searching 14 gene regions, we designed primers and probes in 5 of them for the nine target species. Four primer–probe set pairs were able to be duplexed. Sensitivity tests showed as little as 0.5 pg DNA were detectable. These real-time PCR assays will provide rapid and reliable identification of these key Colletotrichum species and will be critically important for studies aiming to elucidate their biology, epidemiology, and management on apples as the number one produced and consumed tree fruit in the U.S.A.

1. Introduction

Apple bitter rot is a severe disease leading to direct fruit losses ranging from 2 to 100% [1,2,3,4,5]. The economic impacts of bitter rot in the U.S.A. are estimated to be between $300 and $400 million annually. Wet and warm weather conditions favor bitter rot infections during the late spring and summer. Typical brown circular and flat to sunken lesions on apple fruit can occur both in the orchard and postharvest in storage [6,7,8].
This complex disease is caused by multiple fungal species in the genus Colletotrichum. There are three species complexes within Colletotrichum with pathogens infecting apple and pear fruits as follows: (1) acutatum species complex (CASC), (2) gloeosporioides species complex (CGSC), and (3) boninense species complex [9,10,11]. Over the last 8 years, efforts in the Mid-Atlantic U.S.A. have led to identifying the following nine species as causal agents of apple bitter rot: C. chrysophilum, C. fructicola, C. noveboracense, C. siamense, C. theobromicola, C. henanense and C. gloeosporioides sensu stricto (s.s.) from CGSC, and C. fioriniae and C. nymphaeae from CASC. C. chrysophilum is also the primary cause of the leaf form of this disease on apples called Glomerella leaf spot which, in Southeastern U.S.A. and several South American countries, can rapidly defoliate apple trees [12,13,14,15]. In grapes, often grown close to apples, Colletotrichum causes ripe rot disease. Up to 20 Colletotrichum species worldwide have been reported to be infecting grape berries, causing losses [16,17].
The Colletotrichum genus, encompassing over 200 known species, presents a challenge in taxonomy due to its high genetic variability. Initial attempts at classification relied on morphological characteristics, but issues arose from the lack of standardized culturing and ambiguous traits that were insufficient for quick differentiation. Various approaches, such as secondary metabolite profiling, pathogenicity testing, cross-mating, physiological studies, carbon source utilization, and molecular phylogeny, were employed to characterize the Colletotrichum species. However, a singular conserved DNA barcode proved elusive, with markers like GAPDH, ACT, CHS, HIS3, and TUB2 initially considered [18]. Subsequent studies revealed the limitations of a single DNA barcode marker for all Colletotrichum spp., prompting a multilocus approach. Vieira et al. [19] reported that a concatenated phylogeny with additional intergenic markers like APN2/MAT-IGS, GAP2-IGS, and APN2 differentiated the C. gloeosporioides complex, while HIS3, GAPDH, and TUB2 distinguished the C. acutatum species complex. This and other approaches uncovered novel species on apples like C. noveboracense [10] and C. orientalis [20] and identified a previously described species on bananas, C. chrysophilum [21], causing bitter rot on apples [10,22]. Ongoing efforts utilize whole genome sequencing and various descriptive genomics facets to refine the Colletotrichum spp. taxonomy [23]. Nevertheless, all these differentiation efforts require high expertise and are an obstacle for rapid and cheap pathogen identification for the facilitation of species-specific field or storage sample investigations and treatment, particularly for apple diseases caused by the Colletotrichum species.
Accurate and rapid identification of the Colletotrichum species causing apple bitter rot is vital for Malus resistance breeding [23,24]. It is also essential for the development of effective control strategies while minimizing risks for single-site fungicides resistance in these pathogens [5,11,25,26]. Furthermore, fast detection of the Colletotrichum spp. in early, untypical spots on flowers and leaves or rot symptoms on apple fruit would lead to more timely decisions in implementing effective management options. Finally, in North Carolina, Villani et al. [27] found that symptoms of apple bitter rot, predominantly caused by the species in the CGSC, are indistinguishable from rots caused by other fungal pathogens, e.g., Botryosphaeria obtusa, B. dothidea, Botrytis cinerea, and others. Furthermore, late fruit infections by Colletotrichum, just before apple harvest, lead to indistinguishable rot symptoms from the ones caused by other postharvest pathogens, expressing when fruit are prepared for or placed in cold storages. This necessitates rapid diagnostic assays to identify the Colletotrichum species as the primary cause of rot and distinguish it from other less invasive rots.
Molecular detection assays have been developed for many Colletotrichum species using various genes [28,29,30,31,32,33,34,35,36,37,38,39]. In several cases, only a few non-target Colletotrichum species were used in the specificity testing of the assay; often, only species found on the same host plant in the same geographical region were included [29,37,39]. This is a straight-forward, appropriate strategy for those small host–pathogen-geography systems, but it can lead to non-specific amplification or false positives when the assay is used outside that system. A PCR primer set could be species-specific for species A when tested among only species A, B, and C; the same primer set may also unintentionally amplify species Y and Z. In addition, given the uncertainty around past species delineation within the genus, and how closely related many Colletotrichum species are, it is important to include as many related species and as many isolates within each species as is feasible when testing new molecular detection assays.
In recent studies of the Mid-Atlantic Colletotrichum on apples, the most common species were C. fioriniae and C. chrysophilum [10,11,22]. Other species occurring on apple were C. fructicola, C. noveboracense, C. siamense, C. henanense, C. nymphaeae, C. gloeosporioides s.s., and C. theobromicola [10,11,22]. In the northern Mid-Atlantic, C. fioriniae and C. chrysophilum were most common, while C. fructicola was dominant in the south.
The aim of this study was to develop species-specific hydrolysis probe real-time PCRs for molecular detection and identification of the following nine causal agents of bitter rot on apple in the Mid-Atlantic U.S.A.: C. chrysophilum, C. fioriniae, C. fructicola, C. gloeosporioides s.s., C. henanense, C. noveboracense, C. nymphaeae, C. siamense, and C. theobromicola.

2. Materials and Methods

2.1. Fungal Isolates, Strains, Culture Media, and DNA Extraction

A total of 88 Colletotrichum isolates in 16 species, plus 10 other fungal species, apples, and grapes were used in this study (Table 1). Isolates were grown on PDA at 25 °C for DNA extraction, which was performed on mycelia with a DNeasy Plant Mini Kit (QIAGEN, Germantown, MD, USA). DNA quality was determined via gel electrophoresis. Isolates were previously identified to the species level [3,10,22,23,40,41,42,43,44,45,46,47,48,49].

2.2. Primer and Probe Design, Specificity Testing, and RT-PCR Optimization

The Colletotrichum GenBank accessions were downloaded from as many species and genes as possible, using multiple search strategies. In Geneious 2022.2.2 (Biomatters, Inc., Boston, MA, USA), the accessions were aligned, and duplicate sequences within species were removed, such that only unique accessions remained (Table S1). Accessions from the following 14 gene regions were visually examined for areas of high DNA polymorphism among species: ACT, ApMat, APN2, CAL, CHS, CYTB, GADPH, GS, HIS, ITS, ladA, rps3, SOD2, and TUB2. Primers and probes were then designed by eye within these areas. Primer and probe sequences were also assessed using a Nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 October 2022, 10 July 2023, and 5 October 2023).
Following the initial traditional PCR testing of primer sets, the primer–probe sets were tested and optimized, using annealing temperature and primer and probe concentrations, for hydrolysis probe real-time PCR on a Bio-Rad CFX96 Connect Real-Time System. The final real-time PCR volumes were 10 µL, using the SensiFAST Probe No-ROX (Bioline, London, UK), final primer (IDT, Coralville, IA, USA) and TaqMan probe (Applied Biosystems, Waltham, MA, USA) concentrations as listed in Table 2, and 1 µL DNA (1–50 ng/µL). Cycling conditions were an initial denaturation of 95 °C for 3 minutes, followed by 40 cycles of 95 °C for 5 seconds and the optimized annealing temperature (see Table 2) for 50 seconds. Multiplex PCRs were evaluated.
Specificity was validated using 88 isolates (Table 1), including fungi from other genera, as well as apples and grapes. Hydrolysis probe real-time PCR assays were performed during three independent experiments, with three technical replicates and no-template negative controls. To assess sensitivity, standard curves for each primer–probe set were constructed, and limits of detection (LoD) were determined in 8-step dilutions (1, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, and 0.0001 ng/µL) with 3 technical replicates, and each assay was performed three times; LoD was the lowest DNA concentration detectable across all three replicates in all three assays. Selectivity was examined by adding both Colletotrichum DNA (at concentrations 1, 0.1, 0.05, 0.01, 0.005, 0.001, and 0.0005 ng/µL) and apple DNA (1 ng/µL) for each assay. A two-tailed, paired Student’s t-test was used to compare Cq values, without apple DNA and with apple DNA, with significance at p < 0.05.
All templates that did not amplify for one of the newly designed primer–probe sets were tested with ITS1-F/ITS4 (Colletotrichum, Neonectria [53,54], Dc_09_F/Dc_09_R (Marssonina [55]) or COX-F/COX-R primers (plants [30]) to confirm that the DNA had no PCR inhibitors.

3. Results

Alignments of 1487 Colletotrichum GenBank accessions across 14 gene regions were visually assessed for high DNA polymorphism. Primer sets with good matches were discerned and tested for the following nine species in seven genes: C. chrysophilum (APN2 and ladA), C. fioriniae (CAL and GAPDH), C. fructicola (APN2 and ladA), C. gloeosporioides s.s. (GAPDH), C. henanense (ApMat), C. noveboracense (ApMat and ladA), C. nymphaeae (ACT and GAPDH), C. siamense (ApMat), and C. theobromicola (TUB2) (Table 2). After initial PCR testing, probes were designed for nine species in five genes (Table 2).
Primer sets were initially tested at 60 °C and 65 °C with traditional PCR. Sets for C. chrysophilum (CHLAD), C. fructicola (FRLAD), and C. siamense (SIAP) were also tested at 70 °C; additionally, CHLAD and FRLAD were tested at 74 °C. Results from these PCRs showed that an annealing temperature of at least 65 °C would be required for species-specific amplification. Therefore, for real-time PCR, testing began with an annealing temperature of 65 °C and was increased as needed (see Table S2 for the highest annealing temperatures at which the non-target species were amplified). Primer–probe concentrations were tested at final concentrations of 300 nM primers and 100 nM probe, and 600 nM primers and 200 nM probe.
Standard curve and LoD results are shown in Table 3. The real-time PCRs had high efficiencies and an LoD at 0.5 pg, except the primer–probe sets for C. noveboracense (NOLAD, 1 pg), and for C. fructicola and C. theobromicola (FRLAD and THTUB, 5 pg) (Table 3). The following four primer–probe set pairs were able to be duplexed: C. fioriniae (FICAL) and the C. nymphaeae (NYMG) primer–probe set, FRLAD and the C. siamense (SIAP) set, the C. gloeosporioides s.s. (GLG) set and NOLAD, and the C. henanense (HEAP) primer–probe set and THTUB (Table 2). Addition of apple DNA to each assay had no significant effect (Table S3).
NYMG was mostly species-specific, where C. lupini and a few C. fioriniae were amplified (Table 4). NOLAD also amplified a couple of C. nymphaeae (Table 4). The C. chrysophilum primer–probe set (CHLAD) amplified a few C. fioriniae, about a third of the C. fructicola isolates, and C. theobromicola (Table 4). FRLAD amplified a third of C. chrysophilum. Although non-specific amplifications did occur, their quantification (Cq) values were high and relative fluorescence units (RFUs) were low, as follows: Cq > 34 and RFU < 170 for CHLAD, and Cq > 37 and RFU < 100 for FRLAD, NOLAD, and NYMG (Figure S1). The primer–probe sets FICAL, GLG, HEAP, SIAP, and THTUB were species-specific, amplifying only the target species (Table 4). None amplified other fungi, apple, or grape.
In silico screening indicated that the following primer–probe sets may amplify other non-target species: C. aenigma, C. camelliae, and C. viniferum with FRLAD; C. nupharicola with NOLAD; C. scovillei with NYMG; C. aeschynomenes and C. salsolae with SIAP; and C. grevilleae and C. grossum with THTUB (Figure S2).

4. Discussion

Here, we present hydrolysis probe real-time PCR assays for the detection and identification of the following nine Colletotrichum species responsible for bitter rot of apple in the Mid-Atlantic U.S.A.: C. chrysophilum, C. fioriniae, C. fructicola, C. gloeosporioides s.s., C. henanense, C. noveboracense, C. nymphaeae, C. siamense, and C. theobromicola. After visually assessing 14 gene regions, we designed primers and probes in the following 5 gene regions for these nine species: ApMAT (C. henanense, C. siamense), CAL (C. fioriniae), GAPDH (C. gloeosporioides s.s., C. nymphaeae), ladA (C. chrysophilum, C. fructicola, C. noveboracense), and TUB2 (C. theobromicola). All were detectable from as low as 5 pg DNA, with most as low as 0.5 pg. The following four pairs of assays can be duplexed, which allows for quicker results if the whole panel is run: C. fioriniae with C. nymphaeae (both in CASC), C. fructicola with C. siamense, C. gloeosporioides with C. noveboracense, and C. henanese with C. theobromicola (in CGSC). These assays will provide faster identification of species than MLST, which is currently the most reliable molecular assay for species identification [22,56,57]. This is the first report of species-specific assays for C. chrysophilum, C. fioriniae, C. henanense, and C. noveboracense.
Many Colletotrichum species are very closely related, making any type of species delineation or identification challenging. Culture-based methods are time-intensive, require expertise, and are not always reliable [58]. MLST often requires 5–8 genes and high expertise to reliably resolve phylogenetic relationships [9,56,58,59,60]. Our primer–probe sets required as many as 20 mismatches among both the primers and probe (Figure S2) and annealing temperatures that were mainly > 68 °C in order to eliminate non-specific amplification (Table 2), underscoring the necessity of our manual, meticulous, wide-ranging search for polymorphic areas in genes from as many GenBank Accessions as we could find (Table S1).
However, the real-time PCR assays that amplified non-target species are not worrisome for us because the Cq values were high and RFUs were low for the non-target amplifications. Moreover, for most, we had another real-time PCR to confirm species identity (e.g., for any C. fructicola individuals that weakly amplify for CHLAD, it will strongly amplify for FRLAD).
The utility of species-specific quantitative detection assays for the Colletotrichum species infecting apples are numerous and far-reaching. More studies quantifying the seasonal spore release of different Colletotrichum spp. are needed to elucidate the key differences in the biology, ecology, epidemiology, and management of these pathogens. Colletotrichum management starts with cultural practices such as good orchard sanitation, as follows: removal of infection sources like diseased fruit mummies, cankered branches, and alternate hosts, and good tree canopy management for faster drying and better fungicide coverage [61,62,63]. However, quantification of propagules for different Colletotrichum species in various infection sources, pointing to their relative importance during the growing season, has not been explored. For example, apple buds have been largely overlooked as infection sources. The few existing reports showed that C. acutatum was isolated from 1.3% of apple buds in Norway [64], and 30 to 80% of apple buds in New Zealand [65], although these studies were likely dealing with several Colletotrichum spp. In Japan, Nekoduka et al. [61] reported fruit scars as the key overwintering sources for Colletotrichum. Buds are also sites for inoculum overwintering in plants such as sweet and sour cherry [66,67] and blueberry [68,69]. Even at low infection incidence, buds could play a large role as overwintering sites for Colletotrichum spp. [70]. Therefore, real-time PCR assays for Colletotrichum species will help reveal how these species survive in multiple locations in tree canopy, not being limited to cankers and mummies.
Sensitive detection assays could be used to determine the time of the first biotrophic infections in the season on apple fruit surfaces, which is the single most important event for apple producers. Knowing the time of first infections allows fungicide application against bitter rot before or during such an event, and this will mark the beginning of an effective spray program that must last until harvest. So far, the tree fruit pathologists in the main apple-growing regions of the East Coast U.S.A. have relied on observation and accumulated years of experience to recommend fungicides before the start of heavy bitter rot infection pressure. For example, in New York, the use of effective fungicides for bitter rot must not be delayed beyond 10 July, while in Pennsylvania, this cut-off period is mid to late June, and in Virginia, it is the end of May, early June. Even with the latest advances in our understanding of the ecology, epidemiology, and management of C. fioriniae [42,71], the more exact times of the year for the first Colletotrichum infections on fruit for each apple region remain undetermined.
The Colletotrichum species differ in their susceptibility to fungicides [10,43,44]. Our assays for rapid detection and identification of Colletotrichum species are critical to apple producers for refining the selection of fungicides in their spray programs. In addition to controlling bitter rot effectively, this also helps reduce the risk of Colletotrichum developing resistance to the single-site fungicides that growers currently rely heavily on (e.g., quinone outside inhibitors). Our detection assays can assist growers in improving fungicide programs during the growing season and in cold storage by strategically alternating classes of fungicides with different modes of action for higher control efficacy and fungicide resistance risk reduction. Once the Colletotrichum species is/are identified as apple rot cause, the current year spray programs can be actively improved, storage fungicides can be selected to mitigate rot spread in bins or packing lines, or fungicide choices and application strategies can be modified to prevent losses in current and the following season(s), respectively. In addition, our rapid detection and identification assays for Colletotrichum spp. will allow for the evaluation of different ways to improve the efficacy of existing control options for bitter rot and assist in the development of new ones.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms12050878/s1, Figure S1: Amplification plots showing high Cq values and low RFU values of non-specific amplifications; Figure S2: Alignments of available Colletotrichum accessions at primer and probe sites for each primer–probe set; Table S1: List of GenBank accessions used to assess areas of DNA polymorphism among Colletotrichum spp. (n = 1487); Table S2: Highest annealing temperature (°C) at which target and non-target species amplified for each primer–probe set; Table S3: Real-time PCR standard curve Cq values and effect of apple DNA on Cq (NA = no amplification during assay with apple DNA, nt = not tested because it was below LoD).

Author Contributions

Conceptualization, S.G.A. and D.J.M.; methodology D.J.M.; validation, D.J.M.; formal analysis D.J.M.; resources, S.G.A.; data curation, D.J.M.; writing—original draft preparation, D.J.M. and S.G.A.; writing—review and editing, S.G.A. and D.J.M.; visualization, D.J.M.; supervision, S.G.A.; project administration, S.G.A.; funding acquisition, S.G.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded in 2022 by the Virginia Agricultural Council (VAC), grant award number 800 to S.G.A. through the Virginia Department of Agriculture and Consumer Services; by the National Institute of Food and Agriculture through the New York State Specialty Crop Block Grant Program 2019–2021, project award number SCG 19 006/NYFVI 89379 to S.G.A.; by the New York State Department of Agriculture and Markets (NYSDAM) through the Apple Research and Development Program (ARDP) in 2020, project award number NYSDAM 136376 ARDP 6258793 to S.G.A; and by the S.G.A.’s unrestricted research funds.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors thank Mizuho Nita at Virginia Tech for providing access to isolates of Colletotrichum species and other fungi from grapes in his collection. We also acknowledge Fatemeh Khodadadi at the University of California, Riverside, for her contributions to writing the project proposals with S.G.A. to the Virginia Agricultural Council, while at Virginia Tech, and to the New York State Specialty Crop Block Grant Program, while at Cornell University. We also acknowledge Ricardo Delgado Santander at Washington State University for his contributions to writing the project proposal with S.G.A. to the New York State Specialty Crop Block Grant Program while at Cornell University.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Isolates used to test primer–probe sets.
Table 1. Isolates used to test primer–probe sets.
TaxonSample IDIsolateHostLocality
Colletotrichum acutatum species complex
C. acutatum s.s.VT1108PJ51Lycopersicon esculentum tomatoAuckland, New Zealand [41]
C. fioriniaeVA-16VA-16Ginger Gold appleFrederick Co. VA [22]
VA-44VA-44Honeycrisp appleMadison Co. VA [22]
VA-53VA-53Honeycrisp appleMadison Co. VA [22]
VA-1-6VA-1-6Wolf River appleBerkeley Co. WV [22]
VA-1-16VA-1-16pear[22]
VA-1-20VA-1-20Ginger Gold appleFrederick Co. VA [22]
VA-1-66VA-1-66Ambrosia appleRappahannock Co. VA [22]
VA-1-99VA-1-99Honeycrisp appleRappahannock Co. VA [22]
VA-2-9VA-2-9Gold Rush appleFrederick Co. VA [22]
VA-3-59VA-3-59Smokehouse or Rambo appleFauquier Co. VA [22]
VA-3-75VA-3-75Yellow York appleBedford Co. VA [22]
VA-3-96VA-3-96Golden Delicious appleBedford Co. VA [22]
VA-4-32VA-4-32York appleBedford Co. VA [22]
VA-4-99VA-4-99Golden Delicious appleRappahannock Co. VA [22]
VT0787VT0787 [22]
C. godetiaeVT1111JA8Prunus dulcis almondCA [40]
VT1112S1Rhododendron sp.Helsingborg, Sweden [48]
C. johnstoniiVT1114PJ49Citrus sp.Clifton, New Zealand [41]
VT1115PJ50Citrus sp.Clifton, New Zealand [41]
C. lupiniVT1118PJ62Lupinus mutabilisFrance [47]
VT1119PJ64Lupinus albaCanada [45]
C. nymphaeaeVA-1-22VA-1-22Ginger Gold appleFrederick Co. VA [22]
VA-1-24VA-1-24Ginger Gold appleFrederick Co. VA [22]
VT1124FREC138Robinia pseudoacaciaAdams Co. PA [43]
VT1125HC646Honeycrisp appleBourbon Co. KY [44]
VT1126Rdl96Empire appleBerks Co. PA [42]
C. pyricolaVT1127PJ12 New Zealand [46]
C. salicisVT1128FREC145Salix nigra black willowAdams Co. PA [42]
VT1129FREC146Salix nigra black willowAdams Co. PA [42]
Colletotrichum gloeosporioides species complex
C. chrysophilumVA-77VA-77Granny Smith appleMadison Co. VA [22]
VA-1-83VA-1-83Idared appleFrederick Co VA [22]
VA-2-25VA-2-25 Rappahannock Co. VA [22]
VA-2-32VA-2-32 Rappahannock Co. VA [22]
VA-2-37VA-2-37Golden Delicious appleAlbemarle Co. VA [22]
VA-2-67VA-2-67Law Rome appleAlbemarle Co. VA [22]
VA-2-85VA-2-85 Rappahannock Co. VA [22]
VA-2-100VA-2-100 Rappahannock Co. VA [22]
VA-3-4VA-3-4Golden Delicious appleFrederick Co. VA [22]
VA-3-33VA-3-33 Frederick Co. VA [22]
VA-4-86VA-4-86Greening appleFauquier Co. VA [22]
VA-5-13VA-5-13Granny Smith appleFauquier Co. VA [22]
VA-6-19VA-6-19Winter Banana appleFrederick Co. VA [22]
C. fructicolaVA-1-32VA-1-32Red Delicious appleAlbemarle Co. VA [22]
VA-1-44VA-1-44Golden Delicious appleAlbemarle Co. VA [22]
VA-1-49VA-1-49Granny Smith appleNelson Co. VA [22]
VA-1-58VA-1-58Golden Delicious appleNelson Co. VA [22]
VA-1-68VA-1-68Red Delicious appleAlbemarle Co. VA [22]
VA-1-71VA-1-71Golden Delicious appleNelson Co. VA [22]
VA-1-78VA-1-78Granny Smith appleNelson Co. VA [22]
VA-1-79VA-1-79Golden Delicious appleAlbemarle Co. VA [22]
VA-1-90VA-1-90Honeycrisp appleNelson Co. VA [22]
VA-1-91VA-1-91Granny Smith appleNelson Co. VA [22]
VA-2-21VA-2-21Golden Delicious appleAlbemarle Co. VA [22]
VA-2-35VA-2-35Golden Delicious appleAlbemarle Co. VA [22]
VA-2-54VA-2-54 Rappahannock Co. VA [22]
VA-3-39VA-3-39Harrison appleAlbemarle Co. VA [22]
VA-3-44VA-3-44Gala Supreme appleFrederick Co. VA [22]
VA-3-52VA-3-52Yates appleAlbemarle Co. VA [22]
VA-3-54VA-3-54Winter White Pearmain appleAlbemarle Co. VA [22]
VA-3-73VA-3-73Gala Supreme appleFrederick Co. VA [22]
VA-3-87VA-3-87Bramtot appleAlbemarle Co. VA [22]
VA-4-12VA-4-12Golden Delicious appleFauquier Co. VA [22]
VA-4-41VA-4-41GoldRush appleBedford Co. VA [22]
VA-4-53VA-4-53Winesap appleAlbemarle Co. VA [22]
VA-5-86VA-5-86Red Delicious appleFauquier Co. VA [22]
VA-5-88VA-5-88Royal Gala appleFrederick Co. VA [22]
VA-6-15VA-6-15Royal Gala appleFrederick Co. VA [22]
VA-6-16VA-6-16Buckeye Gala appleBotetourt Co. VA [22]
VA-6-28VA-6-28Pink Lady appleAlbemarle Co. VA [22]
VA-6-56VA-6-56Buckeye Gala appleBotetourt Co. VA [22]
VA-6-59VA-6-59Buckeye Gala appleBotetourt Co. VA [22]
VT1109HC540Honeycrisp appleBourbon Co. KY [44]
C. gloeosporioides s.s.VT1104DLC8appleFrederick Co. MD [43]
C. henanenseVT1105SHB6appleWestmoreland Co. PA [45]
VT1113SHB5aappleWestmoreland Co. PA [43]
C. kahawae cladeVT1116HC278Malus pumilaKY [3]
VT1117HC292Malus pumilaKY [3]
C. noveboracenseVT1106AFKH109Idared appleColumbia Co. NY [44]
VT1120PMBrms-1appleAdams Co. PA [44]
VT1121PMCMS-6751appleLehigh Co. PA [44]
VT1122Coll940Juglans nigraCherokee Co. OK [44]
VT1123PMEssl-10aappleLycoming Co. PA [44]
C. siamenseVA-6-10VA-6-10Granny Smith appleAmherst Co. VA [22]
VT1130DLC6aappleFrederick Co. MD [43]
VT1131KY146appleClinton Co. KY [44]
VT1132KY8appleHarlan Co. KY [44]
C. theobromicolaVA-41VA-41Granny Smith appleNelson Co. VA [22]
Other fungi
Botryosphaeria dothideaVT0745VT0745grapeFrederick Co. VA
Diaporthe sp.VT0748VT0748grapeFrederick Co. VA
Diplocarpon coronariaeVT1136BMO8appleAdams Co. PA [50]
VT1137BMO9appleAdams Co. PA [50]
VT1138Vtech4appleFrederick Co. VA [50]
VT1139Vtech5appleFrederick Co. VA [50]
Erysiphe necatorVT0688VT0688grapeFrederick Co. VA
Monilinia fructicolaVT1110Mfa1Jonamac appleLancaster Co. PA [51,52]
Neonectria ditissimaVT1133EUC1-T-1appleFloyd Co. VA
Penicillium expansumVT1135TDL12.1—[49]
Pestalotiopsis maculansVT0746VT0746grapeFrederick Co. VA
Phomopsis viticolaVT0005VT0005VitisFrederick Co. VA
Plasmopara viticolaVT0693VT0693VitisShenandoah Co. VA
Plants
Malus domestica McIntoshVT0695——Frederick Co. VA
Vitis viniferaGRAPE DNA——Frederick Co. VA
Table 2. Primer and probe target species and gene region, name, sequence and fluorophore, final concentration, anneal temperature, and amplicon size.
Table 2. Primer and probe target species and gene region, name, sequence and fluorophore, final concentration, anneal temperature, and amplicon size.
SpeciesGene RegionPrimer and ProbeSequence (5′-3′)Final Concentration (nM)Anneal T (°C)Amplicon Size (bp)
C. chrysophilumladACHLADF2CAT CGT GGC TGT AAT TTT GGA TGT TTC30072164
CHLADRCTT GCC GAA TCC TTC GCT GGT GGT CAC GGC CGA T300
CHLADP6FAM-GAC ACC AGT CGC CTT GAC GTG G-MGBNFQ100
C. fioriniaecalmodulinFICALFTTT ACG CAG CAA CCA CTG GCA ACC ATC60069182
FICALRGTC TCT GAT TAG CAC TAT CTA CAT GC600
FICALPVIC-TTC AAG GTG AGA AGA TCT GGC GCA A-MGBNFQ200
C. fructicolaladAFRLADF2TCT CAT GAC AGG AGC TTC CGA GAT TTC60070164
FRLADRGCT GCC GAA CCC CTC ATT GGT GGT CAC GGC CGA C600
FRLADPVIC-AAC ACC AGT CGC CTT AAC GTG A-MGBNFQ200
C. gloeosporioides s.s.GAPDHGLGFCTC CAA GCT CGW CAT GAC TTC AC60068114
GLGRGAT TTC AAT TGG CAT TAA TTC ATR ATG GCC600
GLGP6FAM-GCC GCC CGC GTT TAG TAC AC-MGBNFQ200
C. henanenseApMatHEAPFTGA CTT GGT CAT CGA TTC GCT TCC CG30065141
HEAPRGCG AGG ATG GTT CTC GAT TCG300
HEAPPVIC-CCT TGC GCC AGA AAC CAA CCC ACC T-MGBNFQ100
C. noveboracenseladANOLADFGGG AAG TAT AGT CAG CGC ATT G30068357
NOLADRTAA TCG CCG TCT CTC GTT CGT TCG AC300
NOLADPVIC-CGT CAT GAC TGG AAT TTG TGA TGT TCC-MGBNFQ100
C. nymphaeaeGAPDHNYMGFGAT AAC ACC AGC TTC GTC GAT ATC30069132
NYMGRTCT GTC AGC AAG TTT TGT CTC GGC300
NYMGP6FAM-GAT TGG GCT TGT TGT AAC GAC ACG-MGBNFQ100
C. siamenseApMatSIAPFACT GAT ATC GGC GCT GCC AG30070168
SIAPRGAA GGG AAT CGA TGG CCA GAT GTG300
SIAPP6FAM-CGA CCT AAG GTT GTC TTT GTG TCC TAG-MGBNFQ100
C. theobromicolabeta-tubulinTHTUBFCTT TCA CCC GAG TTC CAT GTT CAC C60065181
THTUBRGCG AGA GAT TAG CCC TTA GCC CTG C600
THTUBP6FAM-CGT CAA TCC GAC CCC CTA CTG CG-MGBNFQ200
Other primer or primer–probe sets tested but produced too much non-specific amplification:
C. chrysophilumAPN2CHAPNF2GGC AAT CTA CAC CCG CAA CGC G30072131
CHAPNRGGT ACC CGC CGA TAT GCT G300
CHAPNPVIC-CGT GGC GCG ACC TGC CCC CG-MGBNFQ100
C. fioriniaeGAPDHFIGFTAC AAT AAC ACC AGC TTC ATC GGT AAC10065154
FIGRTCT GTC AGC AAA TTT TGT TTG GGC100
C. fructicolaAPN2FRAPNFGGC AAT CTA CAC CCG CAA CGC A10065131
FRAPNRGGT ACC CGC CGA TGT GCT G100
C. noveboracenseApMatNOAPFGTG AGG ACC ATT GAT TTG CCC ACA TGT T10065116
NOAPRGGA TCA GAC CTA GCT ATT CCC GTG ATG100
C. nymphaeaeACTNYACTFCGC AGA CCG CAA TCT TCT CCG TCA GG10065150
NYACTRGCA GGA GAT GGC ATT GCC GCA GC100
Table 3. Efficiency (E), R2, slope, y-intercept, Cq at 1000 pg, and limit of detection (LoD) of primer–probe sets.
Table 3. Efficiency (E), R2, slope, y-intercept, Cq at 1000 pg, and limit of detection (LoD) of primer–probe sets.
Primer–Probe Set (Colletotrichum Species, Gene)ER2slopey-InterceptCq at 1000 pgLoD in pg (Cq)
CHLAD (C. chrysophilum, ladA)94.6%0.992−3.54622.525230.5 (36)
FICAL (C. fioriniae, calmodulin)92.1%0.987−3.63022.867230.5 (36)
FRLAD (C. fructicola, ladA)99.5%0.963−3.34628.457295 (36)
GLG (C. gloeosporioides s.s., GAPDH)108.2%0.978−3.15822.104220.5 (33)
HEAP (C. henanense, ApMat)92.1%0.991−3.52922.851230.5 (35)
NOLAD (C. noveboracense, ladA)90.5%0.991−3.52922.9851241 (35)
NYMG (C. nymphaeae, GAPDH)92.6%0.991−3.52922.851230.5 (35)
SIAP (C. siamense, ApMat)93.2%0.983−3.71322.752220.5 (35)
THTUB (C. theobromicola, beta-tubulin)101.3%0.921−3.22627.215275 (35)
Table 4. Number of isolates per species that were amplified for each primer–probe set (n = total number of isolates, number amplified/number tested). Amplifications are in bold. Primer–probe sets: CHLAD is for C. chrysophilum in gene ladA, FICAL C. fioriniae in calmodulin, FRLAD C. fructicola in ladA, GLG C. gloeosporioides s.s. in GAPDH, HEAP C. henanense in ApMat, NOLAD C. noveboracense in ladA, NYMG C. nymphaeae in GAPDH, SIAP C. siamense in ApMat, and THTUB C. theobromicola in beta-tubulin.
Table 4. Number of isolates per species that were amplified for each primer–probe set (n = total number of isolates, number amplified/number tested). Amplifications are in bold. Primer–probe sets: CHLAD is for C. chrysophilum in gene ladA, FICAL C. fioriniae in calmodulin, FRLAD C. fructicola in ladA, GLG C. gloeosporioides s.s. in GAPDH, HEAP C. henanense in ApMat, NOLAD C. noveboracense in ladA, NYMG C. nymphaeae in GAPDH, SIAP C. siamense in ApMat, and THTUB C. theobromicola in beta-tubulin.
TaxonCHLADFICALFRLADGLGHEAPNOLADNYMGSIAPTHTUB
Colletotrichum acutatum species complex
C. acutatum s.s. (n = 1)0/10/10/10/10/10/10/10/10/1
C. fioriniae (n = 15)3/1413/150/140/150/150/142/150/150/15
C. godetiae (n = 2)0/20/20/20/20/20/20/20/20/2
C. johnstonii (n = 2)0/20/20/20/20/20/20/20/20/2
C. lupini (n = 2)0/20/20/20/20/20/22/20/20/2
C. nymphaeae (n = 5)0/50/50/50/50/52/55/50/50/5
C. pyricola (n = 1)0/10/10/10/10/10/10/10/10/1
C. salicis (n = 2)0/20/20/20/20/20/20/20/20/2
Colletotrichum gloeosporioides species complex
C. chrysophilum (n = 13)13/130/134/130/130/130/130/130/130/13
C. fructicola (n = 30)8/300/3030/300/300/300/300/300/300/30
C. gloeosporioides s.s. (n = 1)0/10/10/11/10/10/10/10/10/1
C. henanense (n = 2)0/20/20/20/22/20/20/20/20/2
C. kahawae clade (n = 2)0/20/20/20/20/20/20/20/20/2
C. noveboracense (n = 5)0/50/50/50/50/55/50/50/50/5
C. siamense (n = 4)0/40/40/40/40/40/41/44/40/4
C. theobromicola (n = 1)1/10/10/10/10/10/10/10/11/1
Other fungi
Botryosphaeria dothidea (n = 1)0/10/10/10/10/10/10/10/10/1
Diaporthe sp. (n = 1)0/10/10/10/10/10/10/10/10/1
Diplocarpon coronariae (n = 4)0/40/40/40/30/40/40/40/40/4
Erysiphe necator (n = 1)0/10/10/10/10/10/10/10/10/1
Monilinia fructicola (n = 1)0/10/10/10/10/10/10/10/10/1
Neonectria ditissima (n = 1)0/10/10/10/10/10/10/10/10/1
Penicillium expansum (n = 1)0/10/10/10/10/10/10/10/10/1
Pestalotiopsis maculans (n = 1)0/10/10/10/10/10/10/10/10/1
Phomopsis viticola (n = 1)0/10/10/10/10/10/10/10/10/1
Plasmopara viticola (n = 1)0/10/10/10/10/10/10/10/10/1
Plants
Malus domestica (n = 1)0/10/10/10/10/10/10/10/10/1
Vitis vinifera (n = 1)0/10/10/10/10/10/10/10/10/1
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McHenry, D.J.; Aćimović, S.G. New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple. Microorganisms 2024, 12, 878. https://doi.org/10.3390/microorganisms12050878

AMA Style

McHenry DJ, Aćimović SG. New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple. Microorganisms. 2024; 12(5):878. https://doi.org/10.3390/microorganisms12050878

Chicago/Turabian Style

McHenry, Diana J., and Srđan G. Aćimović. 2024. "New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple" Microorganisms 12, no. 5: 878. https://doi.org/10.3390/microorganisms12050878

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