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

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.


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.
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.  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].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.
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 speciesspecific 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.

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.  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).
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, 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.

Table 1 .
Isolates used to test primer-probe sets.

Table 2 .
Primer and probe target species and gene region, name, sequence and fluorophore, final concentration, anneal temperature, and amplicon size.