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A Diagnostic TaqMan Real-Time PCR Assay for In Planta Detection and Quantification of Colletotrichum theobromicola, Causal Agent of Boxwood Dieback

    Affiliations
    Authors and Affiliations
    • Harleen Kaur
    • Raghuwinder Singh †
    • Vinson Doyle
    • Rodrigo Valverde
    1. Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803
    Published Online:21 Oct 2021https://doi.org/10.1094/PDIS-11-20-2439-RE

    Abstract

    Boxwood dieback, caused by Colletotrichum theobromicola, is spreading at an alarming rate in the boxwood industry in the United States. Although C. theobromicola has been accepted as a distinct species within the C. gloeosporioides species complex, it is difficult to distinguish it from other closely related species based on morphology. Moreover, molecular identification of C. theobromicola requires amplification and sequencing of multiple loci, which can be expensive and time consuming. Therefore, a diagnostic TaqMan real-time PCR assay was developed for early and accurate detection and quantification of C. theobromicola in boxwood. The study involved the design of species-specific primers and a TaqMan probe to differentiate C. theobromicola from other closely related Colletotrichum species. The primers and probe discriminate between C. theobromicola and other species in the C. gloeosporioides species complex and can detect C. theobromicola at very low concentrations, illustrating the high specificity and sensitivity of the assay. This TaqMan real-time PCR assay accurately and rapidly distinguishes boxwood dieback from other diseases with similar symptomatology, including Macrophoma blight, Phytophthora root rot, and Volutella blight, as well as some disorders produced by abiotic agents.

    Boxwood (Buxus spp. L.) is a common and popular landscape plant in the United States and other parts of the world. Although considered to be hardy by nature, boxwood is susceptible to several diseases and disorders such as foliar blights and root rots, including an emerging disease in the United States, boxwood dieback, caused by the fungus Colletotrichum theobromicola Delacr. It has now been reported in the United States from Louisiana, New York, North Carolina, South Carolina, Virginia (Singh and Ratcliff 2016; Singh et al. 2015), and Texas (Hawk et al. 2018). Boxwood dieback produces random dieback of twigs, and initial symptoms appear as light-green-colored foliage on affected twigs (Fig. 1A). As the disease progresses, leaves become tan-colored and tend to remain attached to the affected twigs. The infection causes dark-black discoloration of affected twigs beneath the bark (Fig. 1B). Moreover, diseased plants exhibit random shoot dieback that is restricted to the upper canopy, leaving a healthy crown and no noticeable impact on the root system (Fig. 1C). Because boxwood dieback produces aboveground symptoms similar to those reported for Phytophthora root rot caused by Phytophthora spp. (Singh and Doyle 2017), visual disease diagnosis may be inaccurate and result in costly and ineffective management. Although the pathogen can be easily isolated from affected twigs, previous studies have shown that it may take 2 months or more for symptoms to appear after inoculation under greenhouse conditions (Hawk et al. 2018; Singh et al. 2015). This delay in the onset of disease symptoms may lead to unwitting dissemination of the causal agent. It also precludes the development of a monitoring system that would be useful for quarantine purposes. Therefore, an accurate and rapid diagnostic tool to detect C. theobromicola in suspected boxwood plant material at early growth stages is required so that boxwood producers can implement effective disease management strategies and prevent the spread of the disease.

    Fig. 1.

    Fig. 1. Buxus microphylla (Baby Gem boxwood) infected with boxwood dieback caused by Colletotrichum theobromicola exhibiting A, early symptoms of light-green foliage in the middle of the canopy, B, bright black discoloration of internal tissue, and C, random dieback with tan-colored foliage.

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    The pathogen causing boxwood dieback, C. theobromicola, belongs to the C. gloeosporioides (Penz.) Penz. & Sacc. species complex, which consists of about 40 or more closely related species (Jayawardena et al. 2016; Khodadadi et al. 2020; Sharma et al. 2017; Weir et al. 2012). Differentiating among species within the species complex is challenging due to a lack of distinctive morphological features. A study was conducted previously to identify specific markers to differentiate Colletotrichum species and suggested that three loci (APN2/MAT-IGS, GAP2-IGS, and APN2) are most suitable for reliably assigning isolates to a species within the C. gloeosporioides species complex (Vieira et al. 2020). However, no particular marker has been found to target C. theobromicola specifically. The main objective of this study was to develop a diagnostic TaqMan real-time polymerase chain reaction (PCR) assay for early in planta detection and quantification of C. theobromicola in boxwood, because sequencing multiple loci for accurate diagnosis of the disease is more expensive and more effort than a single qPCR reaction. Moreover, this will eliminate an additional step of isolation of the pathogen, which is time consuming and delays effective management measures.

    Materials and Methods

    Fungal isolates.

    The C. theobromicola isolates used in this study were obtained from infected boxwood plants collected from eight states in the United States, including Alabama, Louisiana, Missouri, North Carolina, Oklahoma, South Carolina, Texas, and Virginia (Table 1). The pathogen was isolated from symptomatic tissue as described previously (Singh and Doyle 2017; Singh et al. 2015). Ten-day-old, single-spore cultures grown on quarter-strength potato dextrose agar media (1/4 PDA) at 28°C were used for DNA extractions followed by PCR testing. C. theobromicola isolated from hosts other than boxwood (i.e., Anacardium occidentale L. and Theobroma cacao L. from Brazil and Panama, respectively), were also included to confirm that the designed primers and probes are robust to genetic variation within C. theobromicola and not just to the known haplotypes from boxwood (Table 1). Other Colletotrichum species within the C. gloeosporioides species complex—including C. chrysophilum (PN3) W.A.S. Vieira, W.G. Lima, M.P.S. Cãmara & V.P. Doyle; C. fructivorum (21ss) V. Doyle, P.V. Oudem & S.A. Rehner; C. gloeosporioides (Coll20); C. nupharicola (NJ2B) D.A. Johnson, Carris & J.D. Rogers; C. rhexiae (CH6-2) Ellis & Everh.; and C. siamense (Coll126) Prihastuti, L. Cai & K.D. Hyde—were also tested in this study to determine primer and probe specificity. In addition to culture isolates, DNAs extracted from symptomatic plant (bark) tissues were also tested to detect C. theobromicola from boxwoods. Bark tissue DNA was extracted from healthy or asymptomatic boxwood plants, greenhouse plants inoculated with C. theobromicola, and nursery and landscape plants affected by boxwood dieback.

    Table 1. The threshold cycle (Ct) values of Colletotrichum theobromicola isolates tested from different locations and hosts with the TaqMan real-time PCR assay using species-specific primers CalCtF and CalCtR and probe CalCtP

    DNA extraction from single-spore isolates and infected plant material.

    DNA extractions from single-spore cultures of eight United States isolates were performed using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s guidelines with the following modifications. Single-spore isolates were grown on 1/4 PDA at 28°C for 10 days. Mycelium from each isolate was scraped and added individually to 2 ml of fast-prep lysing matrix A tubes (MP Biomedicals, Irvine, CA) for DNA extraction. DNA samples were stored at –17°C. The extracted DNA quality and quantity were determined using a NanoDrop 2000c spectrophotometer using 2 μl of DNA sample (Thermo Fisher Scientific, Wilmington, DE) at the ratio of absorbance at 260 and 280 nm. DNA from known C. theobromicola-infected boxwood collected from the landscape, nursery, and greenhouse and asymptomatic plants was extracted from 100 mg of bark tissue (not the woody tissue) using the DNeasy Plant Mini Kit. DNA was stored and quantified as described above. DNAs of C. theobromicola from cashew and cocoa along with the DNAs of other Colletotrichum species from the C. gloeosporioides complex were obtained from a collection in our laboratory.

    Primers and probe design.

    Known sequences of Colletotrichum species within the gloeosporioides complex were retrieved from the GenBank database (NCBI, Bethesda, MD) using accession numbers obtained from Jayawardena et al. (2016), Vieira et al. (2017), and Weir et al. (2012). Previous studies reported 11 different markers suitable for differentiating Colletotrichum species (Doyle et al. 2013; Rojas et al. 2010; Vieira et al. 2017; Weir et al. 2012). Previously published sequences representing 11 different markers, including nuclear ribosomal internal transcribed spacers (nrITS), β-tubulin (TUB2), actin (ACT), intergenic spacer between GAPDH and a hypothetical protein (GAP2-IGS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), manganese-superoxide dismutase (SOD2), DNA lyase (APN2), calmodulin (CAL), glutamine synthetase (GS), chitin synthase (CHS-1), and the intergenic spacer between DNA lyase and the mating type locus MAT1-2-1 (APN2/MAT-IGS), were used to design species-specific primers for detection of C. theobromicola. The sequences were aligned and compared using MAFFT Sequence Alignment online version 7 (https://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013; Katoh et al. 2019; Kuraku et al. 2013) with advanced iterative refinement settings of G-INS-i. The parameters chosen for aligning the target sequences were as follows: scoring matrix for nucleotide sequences = 200PAM/K = 2; gap opening penalty = 1.53; and offset value = 0.0. Aligned sequences were visualized using AliView (Larsson 2014). The number of sequences across the multiple sequence alignments of the 11 different markers ranged from 43 to 260 sequences per alignment (mean = 155), representing a range of 7 to 40 species (mean = 30).

    Comparative analysis of all the loci was carried out when data across multiple species were available. The alignments were analyzed visually as well as using a software toolset for deciphering and managing biological sequences using the R programming language (DECIPHER R package) (Wright 2015). The DECIPHER R package designed various primer sets from 11 selected markers based on several variables using the DesignPrimers function. Markers that produced an amplicon size less than 100 bp or greater than 300 bp and markers that amplified species other than C. theobromicola were excluded. Three loci (CAL, APN2/MAT, and APN2) that resulted in species-specific amplification were shortlisted based on the appropriate amplicon size and target specificity. Out of these three loci, CAL was selected based on empirical amplification of DNA from a range of isolates of C. theobromicola that were isolated from boxwoods as well as other hosts. The APN2/MAT and APN2 markers produced false negatives in some cases for confirmed C. theobromicola DNA and, therefore, were excluded from further testing.

    The target CAL fragments between the newly designed primers were analyzed with the PrimerQuest tool (Integrated DNA Technologies) to develop a TaqMan fluorescent probe. Based on the fragment length (up to 30 bp), GC content (30 to 80%), and melting temperature ∼10°C higher than the primer Tm (i.e., 63 to 66°C), a probe was designed. This TaqMan probe was labeled with a fluorescent reporter dye (6 FAM) at the 5′ end and a nonfluorescent quencher dye (3 BHQ) at the 3′ end.

    Primer specificity.

    Conventional PCR

    The PCRs were carried out using a Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). The reaction mix for PCR consisted of the following reagents for a single reaction forming a total reaction volume of 25 μl: 12.5 μl of Go Taq Green Master Mix (Promega, Madison, WI), 8.5 μl of nuclease-free water (Qiagen), 1 μl of each forward (10 μM) and reverse (10 μM) primers (i.e., CALCtF and CALCtR; Integrated DNA Technologies), and 1 μl of DNA template in 0.2-ml Eppendorf PCR Tubes (Eppendorf, Hamburg, Germany). Nuclease-free water was used as a negative control. The following PCR parameters were used to amplify target DNA: 4 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 50°C, and 45 s at 72°C, and a final extension at 72°C for 7 min. The PCR products were visualized on a 2% agarose gel run at 110 V for 30 to 40 min.

    Real-time TaqMan PCR assay.

    A Bio-Rad CFX real-time PCR detection system (Bio-Rad Laboratories) was used to perform quantitative PCR in 96-well PCR plates (Bio-Rad Laboratories). Each reaction was performed with a total volume of 25 μl, containing Omnimix HS lyophilized PCR master mix (Takara Bio, Shiga, Japan) (one bead for two reactions), 1.25 μl (10 μM) of each forward and reverse primers (CALCtF and CALCtR, respectively), 1 μl (10 μM) of TaqMan probe (CALCtP) (Integrated DNA Technologies), 19 μl of nuclease-free H2O, and 2.5 μl of template DNA using the following qPCR protocol: initial denaturation at 95°C for 2 min, followed by 40 cycles at 95°C for 15 s and 63.5°C for 30 s. Standard curves were generated by amplifying 10-fold serial dilutions of C. theobromicola isolate PDCColl I (DNA from culture) and PDC20027-P5 (DNA from C. theobromicola-infected boxwood plant tissue) ranging from 14,000 to 1.4 pg/μl and from 2,790 to 2.79 pg/μl, respectively.

    Results

    Primers and probe.

    The species-specific forward CALCtF (5′-AAGGTCAGCTACGAGAATGTT-3′) (Fig. 2B) and reverse CALCtR (5′-GACGCATACCAATTGTAATCGATACT-3′) (Fig. 2C) primers were designed, and the target region was analyzed with the IDT PrimerQuest tool to design a species-specific TaqMan probe CALCtP (5′-CTGCTGCGGTCGATGTTGACTCT-3′) (Fig. 2A). The TaqMan probe was labeled with a fluorescent reporter dye (6 FAM) at the 5′ end and a nonfluorescent quencher dye (3 BHQ) at the 3′ end.

    Fig. 2.

    Fig. 2. Screenshot of calmodulin alignments of Colletotrichum theobromicola and other closely related Colletotrichum species in gloeosporioides species complex highlighting A, the newly designed species-specific probe CALCtP sequence that binds selectively to C. theobromicola due to differences among species in this region; B, the newly designed species-specific forward primer CALCtF; and C, the newly designed species-specific reverse primer CALCtR.

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    Primer specificity.

    The newly designed primers CALCtF and CALCtR were tested for their specificity against the NCBI database using the Primer-BLAST global alignment algorithm. Top searches that aligned with the primers were C. theobromicola with 100% identity and 100% query coverage. This gave a confirmation that the primers do not bind to any distantly related species and validates primer specificity. The primers CALCtF and CALCtR amplified a 226-bp PCR product, confirmed through sequencing, with DNA extracted from C. theobromicola cultures as well as DNA extracted from symptomatic boxwood plant tissue. No amplification was observed for the nontemplate control. To further check the specificity of primers CALCtF and CALCtR, closely related Colletotrichum species from the C. gloeosporioides complex, including C. chrysophilum, C. fructivorum, C. gloeosporioides, C. nupharicola, C. rhexiae, and C. siamense, were tested using conventional PCR and did not result in any amplification (Fig. 3). The results from conventional PCR showed that the primers CALCtF and CALCtR are species specific and only amplified DNA of C. theobromicola, including those isolated from cashew, cocoa, boxwoods, and symptomatic boxwood plant tissue. No amplification was observed with DNAs extracted from healthy or asymptomatic boxwood bark tissues (Fig. 4).

    Fig. 3.

    Fig. 3. A 2% agarose gel showing amplification (amplicon size 226 bp) of Colletotrichum theobromicola DNA (lane [L] 2 to L12) isolated from 10-day-old cultures and nonamplification of other closely related Colletotrichum species in the C. gloeosporioides species complex (L13 to L18) using species-specific primers CALCtF/CALCtR. M = marker (50 to 1,000 bp); L1 = nontemplate control; L2 = PDCColl I (Buxus sp.: Alabama); L3 = PDC18454 (Buxus sp.: Louisiana); L4 = PDC17484 (Buxus sp.: Texas); L5 = PDC18029 (Buxus sp.: Missouri); L6 = PDC14485 (Buxus sp.: North Carolina); L7 = PDC19024-A (Buxus sp.: Oklahoma); L8 = PDC19042 (Buxus sp.: South Carolina); L9 = PDCColl D (Buxus sp.: Virginia); L10 = GJS08-48 (Theobroma cacao: Panama); L11 = Isolate 136 (Anacardium occidentale: Brazil); L12 = GJS08-43 (Theobroma cacao: Panama); L13 = PN3 (C. chrysophilum); L14 = CH6-2 (C. rhexiae); L15 = NJ2B (C. nupharicola); L16 = Coll126 (C. siamense); L17 = 21ss (C. fructivorum); and L18 = Coll20 (C. gloeosporioides).

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    Fig. 4.

    Fig. 4. A 2% agarose gel showing amplification (amplicon size 226 bp) of Colletotrichum theobromicola DNA (lane [L] 4 to L9) from symptomatic plant tissue and nonamplification of healthy or asymptomatic plant tissue (L2 and L3) using species-specific primers CALCtF/CALCtR. M = marker (50 to 1,000 bp); L1 = nontemplate control; L2 = PDC20026-P1 (healthy plant from nursery); L3 = PDC20026-P2 (healthy plant from greenhouse); L4 = PDC20027-P1 (symptomatic plant from nursery); L5 = PDC20027-P2 (symptomatic plant from nursery); L6 = PDC20027-P3 (symptomatic plant from landscape); L7 = PDC20027-P4 (symptomatic plant from landscape); L8 = PDC20027-P5 (symptomatic plant from greenhouse); and L9 = PDC20027-P6 (symptomatic plant from greenhouse).

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    Real-time TaqMan PCR assay.

    The real-time PCR conducted with primers CALCtF, CALCtR, and probe CALCtP amplified culture DNA from C. theobromicola isolates from 10 different locations listed in Table 1 (Fig. 5A). The no template control and other Colletotrichum species from the C. gloeosporioides complex did not fluoresce. The threshold cycle (Ct) value for C. theobromicola culture DNA ranged from 16.13 to 25.57 with an average of 21.35 cycles.

    Fig. 5.

    Fig. 5. A, Amplification curves of Colletotrichum theobromicola DNAs from 11 isolates using species-specific primers CALCtF/CALCtR and TaqMan CALCtP probe. No amplification was observed from DNA samples isolated from C. chrysophilum, C. fructivorum, C. gloeosporioides, C. nupharicola, C. rhexiae, and C. siamense. B, Amplification of a 10-fold serial dilution of C. theobromicola PDCColl I with DNA concentrations ranging from 14,000 to 1.4 pg. C, A standard curve generated by amplification of 10-fold serial dilution of C. theobromicola isolate PDCColl I with E = 93.5%, R2 = 0.999, and slope = –3.488.

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    A standard curve was generated by serially diluting PDCColl I isolate of C. theobromicola DNA with concentrations ranging from 14,000 to 1.4 pg/μl (Fig. 5B). A linear relationship between DNA quantity and the Ct value was observed with y = 14.820, R2 = 0.999, and a slope of –3.488 with a 93.5% PCR assay amplification efficiency (Fig. 5C). The lowest DNA concentration that the designed primers and probe were able to detect was 1.4 pg/μl.

    Additionally, the real-time PCR amplified C. theobromicola from DNA extracted from bark collected from the transition zone between healthy and symptomatic tissue from known boxwood dieback-affected plants collected from nursery (PDC20027-P1, PDC20027-P2), landscape (PDC20027-P3, PDC20027-P4), and artificially inoculated boxwoods in the greenhouse (PDC20027-P5, PDC20027-P6) (Fig. 6A). No amplification was observed from DNA extracted from asymptomatic boxwoods (PDC20026-P1, PDC20026-P2). The Ct value for plant tissue ranged from 22.32 to 27.16 with an average of 24.27 cycles.

    Fig. 6.

    Fig. 6. A, Amplification curves of Colletotrichum theobromicola DNAs tested from plant tissue (PDC20027-P1, PDC20027-P2, PDC20027-P3, PDC20027-P4, PDC20027-P5, and PDC20027-P6) using species-specific primers CALCtF/CALCtR and TaqMan CALCtP probe. No amplification was observed from DNA samples isolated from healthy or asymptomatic boxwood plant tissue (PDC20026-P1 and PDC20026-P2). B, Amplification of a 10-fold serial dilution of C. theobromicola DNA PDC20027-P5 extracted from plant tissue with DNA concentrations ranging from 2,790 to 2.79 pg. C, A standard curve generated by amplification of 10-fold serial dilution of C. theobromicola isolate PDC20027-P5 with E = 93%, R2 = 0.999, and slope = –3.501.

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    A standard curve for symptomatic boxwood plant tissue was generated by serial dilution of plant PDC20027-P5 DNA concentrations ranging from 2,790 to 2.7 pg/μl and resulted in a slope of –3.501 and R2 value of 0.999 with a 93% PCR efficiency (Figs. 6B and C). The lowest detection level with symptomatic plant DNA tissue was determined to be 2.7 pg/μl.

    Discussion

    Boxwood dieback caused by C. theobromicola is an emerging disease in the nursery and landscape industry in the United States. The pathogen is spreading at alarming rates and affecting the boxwood industry negatively. The exact impact of the disease is not known due to its recent discovery, but a number of boxwood growers and breeders have presented their concerns at various regional and national horticulture industry meetings. Detection of boxwood dieback in the early stages of disease development is an important step toward implementing the disease management strategies and preventing further spread in the surrounding plantations or boxwood liners in the nurseries. Delayed onset of symptoms produced by boxwood dieback is a challenge for early and accurate detection. Under greenhouse conditions, artificial inoculation of healthy boxwoods resulted in initial symptoms of light-green or chlorotic foliage in as long as 6 weeks after inoculation followed by dieback symptoms after 12 weeks of inoculation (Singh et al. 2015). Previously, De Silva et al. (2017) summarized the quiescence or latent behavior of various species in the C. gloeosporioides species complex. The same phenomenon may explain the delayed symptom development by C. theobromicola infections in boxwoods.

    Current diagnosis of boxwood dieback relies on isolation of the pathogen followed by amplification and sequencing of multiple loci, including ACT, CHS-1, and ITS (Singh et al. 2015). Morphological characteristics to identify C. theobromicola are not considered reliable, because these features can change with successive subculturing, variable growing conditions, and time (Weir et al. 2012). These methods are expensive and time consuming and may take up to 3 to 4 weeks for accurate identification. Additionally, delayed diagnosis may lead to the accidental spread of the pathogen through pruning with contaminated tools, overhead irrigation practices, and other poor cultural practices. In order to avoid delayed diagnosis and further transmission of the disease in nurseries and landscapes, a rapid and reliable disease detection method is critically warranted.

    Therefore, the main goal of our study was to develop a diagnostic assay for rapid, accurate, and reliable detection and quantification of C. theobromicola in the early stages of disease development. The diagnostic TaqMan real-time PCR assay developed in this study successfully detected C. theobromicola from DNA isolated from both culture isolates as well as symptomatic boxwood bark tissue. This assay is species specific, because it only amplified C. theobromicola from boxwood, cashew, and cocoa. The primers CALCtF/CALCtR and the probe CALCtP did not amplify C. chrysophilum, C. fructivorum, C. gloeosporioides, C. nupharicola, C. rhexiae, and C. siamense (Fig. 5C). The assay is highly sensitive and detected C. theobromicola at early infection stages from boxwood liners and fully grown boxwood plants with a quantification limit of 1.4 and 2.79 pg of fungal DNA from culture and plant tissue, respectively. The newly designed calmodulin primers CALCtF and CALCtR amplified a 226-bp fragment of C. theobromicola both from culture isolates and from plant bark tissue. Calmodulin, along with other markers, has been tested in previous studies for identification of Colletotrichum species within the C. gloeosporioides complex (Vieira et al. 2020) and was not considered as the best marker for species differentiation. However, the calmodulin gene does contain unique sequence motifs that allowed us to successfully distinguish C. theobromicola from other closely related species in this study.

    The efficiency of the newly developed TaqMan real-time PCR is within acceptable limits between 90 and 100% (−3.6 ≥ slope ≥ −3.3) (qPCR Efficiency Calculator, Thermo Fisher Scientific: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/qpcr-efficiency-calculator.html; https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/poor-pcr-efficiency.html). These parameters indicate that the primers and probe we have designed enable the accurate detection as well as quantification of C. theobromicola in boxwoods, thereby leading to a newly designed approach for boxwood dieback disease diagnosis. The specificity, sensitivity, and PCR efficiency discussed above validate the new TaqMan real-time PCR to detect C. theobromicola, the causal agent of boxwood dieback. This molecular diagnostic assay can serve as an essential tool in plant diagnostic centers for easy and rapid detection of boxwood dieback as well as in research laboratories to conduct advanced studies on this pathogen using the species-specific primers and probe. The results of this study will not only help in the diagnosis of affected boxwoods with boxwood dieback but also will help in identifying C. theobromicola in/from other host plants.

    Early and accurate detection is critical in managing this pathogen known to cause delayed visible symptoms. Management of boxwood liners infected with C. theobromicola at the early stages of disease development may stop the disease in nurseries and prevent it from getting introduced into landscapes. Accurate detection will help landscapers and nursery growers implement effective disease management strategies and reduce the costs associated with boxwood cultivation. Currently, boxwood dieback has been officially and unofficially reported from several states in the United States and has led to significant losses. This newly developed assay provides the boxwood industry with a rapid and reliable diagnostic tool to detect boxwood dieback at early stages of disease development.

    Acknowledgments

    The authors acknowledge Tim Burks and Monique De Souza for their valuable help in the lab procedures. The authors extend their gratitude to Kassie Cooner, Loraine Graney, Mike Munster, Kevin Ong, Jen Olson, and Meg Williams for sharing C. theobromicola isolates.

    The author(s) declare no conflict of interest.

    Literature Cited

    The author(s) declare no conflict of interest.