Nested PCR methodology for detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples

Nested PCR methodology for detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Nested PCR methodology for detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples Laura Valentina Laverde-Arias, Yeirme Yaneth Jaimes-Suárez, Adriana González-Almario This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6506698/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Persea americana and Theobroma cacao plants can be infected by fungal species of the genus Lasiodiplodia, which cause the disease known as dieback. However, T. cacao can also be infected by Ceratocystis sp., which causes cocoa wilt. These fungi cause necrosis of the vascular tissue, leading to wilting and death of the plant. However, these symptoms are not observed in the early stages of infection, making timely diagnosis of these pathogens difficult. Therefore, in this study, a nested PCR technique was developed for the early detection of Ceratocystis sp. and Lasiodiplodia sp. from infected and asymptomatic seedling stem samples. Results: A standardized pre-treatment with glycerol and 96% ethanol to soften cocoa and avocado seedling stem tissue was effective before DNA isolation. Subsequent amplification of the actin gene from pre-treated stem tissues confirmed that this step is necessary to obtain nucleic acids free of PCR inhibitors. Furthermore, the Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R primer sets designed in this study demonstrated to be specific for the detection of Ceratocystis sp. and Lasiodiplodia sp., respectively, with a sensitivity ranging from 2-4 ng/μL. By using the primers ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for Ceratocystis sp., and ITS1/ITS4, and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia sp. in a nested-PCR, both pathogens were successfully detected directly from infected stem samples without a fungal isolation step in culture media. The results were easily interpreted by agarose electrophoresis according to fragment size differences, a 279 bp amplicon to samples artificially infected with C. cacaofunesta and a 397 bp amplicon to samples with L. theobromae and L. subglobosa . Conclusion: The use of the nested PCR protocol standardized in this study with the primers sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for Ceratocystis sp.; and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia sp., allowed early diagnosis of both pathogens in asymptomatic cocoa and avocado stem seedlings. stem pre-treatment sensitivity asymptomatic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Cocoa ( Theobroma cacao L.) and avocado ( Persea americana Mill.) plantations are significant crops in tropical regions, especially in South Asia, Latin America, and West Africa, because the climatic conditions are optimal for their development [ 20 , 34 ]. In Colombia, the area planted with cocoa has increased by 13% in the last five years, and its production has increased by 127% in less than 15 years [ 19 ]. In 2024, Colombia reached its highest production with 67,678 tons, and exports (cocoa beans and derivatives) represented US $ 265 million for the country [ 12 ]. Concerning avocado cultivation, Colombia has approximately 32,132 hectares dedicated to the cultivation of the Hass variety [ 32 ]. A 15% increase in Hass avocado exports has been observed, generating revenues of US $ 218.6 million, with a 42% increase in exports reported [ 1 ]. However, the productivity of T. cacao and P. americana plantations in Colombia has been adversely affected by various pests and plant diseases, the latter mostly caused by fungi [ 8 , 27 ]. Fungal species of the genus Lasiodiplodia sp. have been reported as pathogens of both crops and are known to cause a variety of symptoms, such as leaf spots, fruit rot, or necrosis in different plant tissues. However, one of the most relevant diseases it causes is dieback, characterized by the wilting of the upper branches of the plants and internal necrosis of the vascular system [ 30 ]. In Colombia, L. theobromae (Pat.) Griffon & Maubl. has been described as a pathogen of P. americana since the late 1990s [ 29 ], and it was reported for the first time in Colombia as a causal agent of dieback in T. cacao [ 25 ]. On the other hand, the species of Ceratocystis sp. may also cause vascular necrosis, particularly in T. cacao , which leads to general wilting and even death of the whole plant as the disease progresses [ 5 , 36 ]. This pathogen can be transmitted by pruning tools or Xileborus beetles [ 23 ]. In Colombia, C. cacaofunesta Engelbr. & T. C. Harr. has been reported as the causal agent of cocoa wilt [ 6 ]. Nonetheless, the symptoms caused by both Lasiodiplodia sp. and Ceratocystis sp. are not evident in the early stages of infection, making an opportune diagnosis difficult. Traditional diagnosis based on pathogen isolation in culture media is not successful in asymptomatic tissues due to low inoculum density [ 16 ] and may fail to detect dormant spores [ 22 ]. Therefore, the use of PCR-based molecular tools applied to diagnosis increases the specificity and sensitivity for pathogen detection in different plant tissues [ 3 ], especially during early intracellular colonization, which may not produce detectable symptoms, as is the case with Lasiodiplodia sp. and Ceratocystis sp. infections, since the damage starts in the internal stem tissues. For this reason, this study standardized a genus-specific nested PCR technique for the early detection of Lasiodiplodia sp. and Ceratocystis sp., which can be used to verify the phytosanitary health of T. cacao and P. americana propagation material. Results Stem pre-treatment for DNA extraction - standardized protocol Due to the difficulty of grinding lignified tissues and promoting cell lysis of stems, a pre-treatment based on the methodology of Lu et al. [18] was standardized. The sample preparation protocol is as follows: 1. Using disinfected and sterilized pruning shears, cut the stems into approximately 5 mm thick pieces 2. Transfer the cut pieces to a sterile glass flask containing a mixture of 96% ethanol and pure glycerol (1:1) to a volume sufficient to cover the entire sample 3. Cover the flask with aluminum coil to avoid the volatilization of ethanol and incubate for 6 hours at 40°C in a water bath 4. Filter the sample through a sieve and wash with abundant sterile distilled water to remove excess ethanol and glycerol 5. Dry the stem pieces on a sterile absorbent paper sheet and transfer them to a sterile mortar 6. Add an equal amount of autoclaved white sand to the sample volume and grind until it turns into a fine powder 7. Fill ⅕ of a sterile 15 mL falcon tube with the macerated stem and start the DNA extraction according to the CTAB protocol [10] with the following modifications: extending the original sample incubation time with CTAB buffer from 30 minutes to overnight; adding an extra incubation step of the supernatant with chloroform and finally, including an additional washing step of the DNA with 70% ethanol to optimize the nucleic acid purity. DNA extraction with standardized protocol Plant DNA was extracted from pre-treated stems of T. cacao and P. americana seedlings infected under nursery conditions. The actin gene was amplified to confirm the quality of the DNA and to exclude the possible presence of PCR inhibitors in the diagnostic test (fig. 1). The results showed that the amplification of the actin gene from cacao plants infected with Ceratocystis cacaofunesta was successful in the positive control (DNA from healthy cacao plant) and the samples infected in nursery. A band of approximately 200 bp was evident, which was consistent with the expected amplicon size (171 bp). Regarding actin gene amplification in avocado plants, a PCR product of approximately 100 bp was obtained in the positive control (DNA from healthy avocado plants) and in plant samples infected with Lasiodiplodia subglobosa , which was consistent with the expected amplicon size of 121 bp. These results indicate that the pretreatment successfully induced cell lysis, and the DNA extracted was free of PCR inhibitors. Designed primers for detection of Lasiodiplodia sp. and Ceratocystis sp. – performance evaluation in vitro The designed primers Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R only amplified DNA from Ceratocystis sp. and Lasiodiplodia sp. respectively, when tested with DNA from other fungal genera and from plant species, demonstrating specificity (Fig. 2). Furthermore, the observed amplicons corresponded to the expected fragment sizes predicted in silico , 279 bp for Ceratocystis sp. and 397 bp for Lasiodiplodia sp. to differentiate between the two fungal pathogens. Based on serial dilutions of the initial concentration of DNA extracted from L. theobromae and C. cacaofunesta , performed to determine the minimum detectable amount of fungal DNA, it was found that PCR could detect DNA concentrations as low as 2-4 ng/μL for both pathogens (fig. 3). For L. theobromae , amplification was observed up to a 1:1000 dilution, although the band was weak and indistinct. Therefore, the detection limit was estimated to be at a 1:100 dilution, corresponding to a DNA concentration of 3.869 ng/μL (Figure 3a). For C. cacaofunesta , a faint band was observed at a dilution of 1:1000 and therefore the reliable detection limit for this pathogen was estimated at a dilution of 1:100, corresponding to 2.215 ng/μL to avoid any doubtful interpretation of the results (Figure 3b). Direct detection of Lasiodiplodia sp. and Ceratocystis sp. in cocoa and avocado seedling stems To improve the sensitivity of the molecular diagnosis, a nested PCR was performed to increase microbial DNA copies through a first round of amplification with the universal ITS1/ITS4 primers [13], and the resulting PCR products were then used as template DNA for a second amplification with the primers Las.ITS77-F/Las.ITS452-R and Cer.ITS119-F/Cer.ITS379-R, designed in this study. The results showed that amplifications to detect Lasiodiplodia sp. were successful only in the positive controls and in the stem samples from the P. americana and T. cacao , which were infected but showed no wilting symptoms at 7 dpi. For the detection of Ceratocystis sp. in plant tissues, a nested PCR was performed using DNA from C. cacaofunesta as a positive control and DNA extracted from five apparently healthy seedlings sampled at 9 dpi, and from control plant inoculated with a culture-free agar square. A band of ~300 bp was obtained in the positive control and the nursery stem samples, indicating that the primers successfully detected the pathogen. As expected, the negative controls and stem samples from healthy, uninfected plants showed no amplification band, confirming the robustness and consistency of the diagnostic test. To confirm the results, the PCR products were sent for sequencing and their identity to the fungal genera Lasiodiplodia and Ceratocystis was confirmed. Discussion The standardized pre-treatment to soften lignified tissues and facilitate DNA extraction from stems was confirmed to be successful and ensured that nucleic acids are free from PCR inhibitors. The chemical principle underpinning this method is that glycerol and heat promote the cleavage of β-O-4 bonds in lignin [ 7 , 38 ] accelerating the loss of structural stiffness, and ethanol disrupts intercellular cohesion and breaks the bonds present between the cell layers that comprise the lignified tissue [ 4 ]. Additionally, the Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R designed primers demonstrated to be specific and sensitive detecting Ceratocystis sp. and Lasiodiplodia sp., respectively. This is particularly relevant in the context of early diagnosis, as these primer sets can be used without the need for sequencing the PCR products to determine the identity of the pathogen; instead, the result can be interpreted only based on differences in fragment size. Moreover, the diagnosis can be performed directly from stem samples and does not require prior purification of the fungus on a Petri dish since no cross-annealing was found with the plant DNA. Although other molecular methods have been reported for the diagnosis of plant diseases and PCR-based tests have been widely used as sensitive tools [ 9 ], nested PCR does not require a real-time thermal cycler or reagents such as probes or intercalants that emit a fluorescent signal as required in qPCR, nor specific enzymes such as Bst -polymerase or recombinase as required in LAMP or RPA techniques [ 17 , 21 ]. As a result, the use of nested PCR is less expensive and sensitive enough to detect pathogens at low inoculum densities and turns into a useful methodology to screen grafting and propagation material of perennial crops. The results of this study also demonstrate that performing two rounds of amplification improves sensitivity; for example, nested PCR for the detection of Phytophthora sp. from soil, root, and stem samples increase the sensitivity by 10 to 1000 times compared to PCR with a single round of amplification [ 14 , 31 ]. Consequently, the technique presented in this study can be fine-tuned in the future to detect species within the same fungal genus and even multiple detections within the same reaction tube. This will require the use of more informative molecular markers and further analysis of the genetic data to identify regions that distinguish species, particularly for genera such as Lasiodiplodia and Ceratocystis , in which the occurrence of cryptic species and species complexes is described [ 28 ]. However, this tool can also be used to screen asymptomatic tissues or seedlings, and even to confirm microbiological diagnoses to get a more rapid and robust result. Conclusion In this work, a molecular protocol was developed to detect Lasiodiplodia sp. and Ceratocystis sp. in asymptomatic but infected seedlings stems of P. americana and T. cacao . To ensure the high quality and purity of the plant DNA, the tissue must be pre-treated with glycerol and 96% ethanol to soften it. For fungal molecular identification, this DNA must be subjected to a nested PCR with the primer sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for Ceratocystis sp. and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia sp. to obtain a product of 279 bp for Ceratocystis and 397 pb for Lasiodiplodia sp. Materials and methods Fungal isolation and DNA extraction A total of 18 isolates were selected from the strain bank of the La Suiza Research Center – AGROSAVIA (Corporación Colombiana de Investigación Agropecuaria), located in Santander, Colombia. Six isolates of the genus Ceratocystis sp., eight of Lasiodiplodia sp., and two isolates of Rosellinia sp., one isolate of Moniliophthora roreri , and two isolates of Fusarium sp. were included as specificity controls. The isolates of Ceratocystis sp., Lasiodiplodia sp., and Fusarium sp. were grown on PDA (Papa Dextrose Agar) supplemented with chloramphenicol (50 mg/L); the isolates of Rosellinia sp. were reactivated on acidified PDA medium (pH 4.0) and M. roreri on V8 (V8 Juice Agar). The Petri dishes were incubated at 26°C for 8 days. In the case of Rosellinia sp. the dishes were covered with aluminum foil to block light exposure for 30 days. For biomass production, five agar plugs were cut over the colony edges and transferred to a sterile tube containing 25 mL of modified V8 broth (100 mL Campbell´s V8 tomato juice, 40 g of glucose, and 30 g of peptone per litre). They were incubated at 26°C on a shaker at 120 rpm for 5 days for Ceratocystis sp., Lasiodiplodia sp., and Fusarium sp., and 15 days for Rosellinia sp. and M. roreri . For the DNA extraction, an optimized variant of the protocol proposed by Doyle & Doyle [ 10 ] was used. This variant consisted of the addition of β-mercaptoethanol absolute (1.5% relative to the final buffer volume) to induce cell lysis more rapidly, together with the CTAB extraction buffer (TRIS 2.0 M, EDTA 0.5 M, NaCl 4.0 M and 0.3% CTAB per litre) supplemented with proteinase K at 20 mg/mL [ 26 ]. Plant material inoculation and sample collection Six-month-old seedlings of Theobroma cacao L. (CCN 51 genotype) and Persea americana Mill. (Santa Cruz variety) were infected under nursery conditions at a temperature of 28 ± 2°C and relative humidity of 75%. Inoculation with Lasiodiplodia sp. was performed on cacao seedlings with the isolate Las003 and on avocado seedlings with the isolate Las001. The infection with Ceratocystis cacaofunesta (isolate NCF15) was performed on cacao plants only, following the method described by Argôlo-Magalhães et al. [ 2 ]. For each pathogen, a group of 25 seedlings was infected, and negative controls were inoculated with sterile agar plugs. Five destructive samplings were taken at 7, 14, 25, 36, and 45 days post inoculation (dpi) to monitor the progression of the dieback disease caused by Lasiodiplodia sp. in both cacao and avocado plants, and at 9, 16, 27, 38 and 60 dpi to monitor the cacao wilt caused by Ceratocystis sp. At each sampling, the stems were cut longitudinally, and the length of the necrotic area (cm) was measured with a ruler. The plant material was dried in a Biocool FD-1C-80 Freeze Dryer (Beijing, China), then vacuum-packed and kept frozen until the time of DNA extraction for validation of the diagnostic nested PCR. Primer design Primers were designed to target genus-specific regions between the Internal Transcribed Spacer 1 and 2 (ITS 1 and 2). Bioinformatic analyses were performed using the ITS sequences available in GenBank (accession numbers MK811125.1, KY031620.1, NR_174713.1, MK368389.1, MH107831.1, and NR_111174.1) and the ITS sequences of the eight isolates of Lasiodiplodia sp. and six of Ceratocystis sp. selected from the strain bank of the La Suiza Research Center. Once the specific regions were identified for each pathogen, the primers were generated using Primer3Plus [ 35 ] and the tools available on the IDT (Integrated DNA Technologies) website. Primer specificity was checked in silico using Primer-BLAST and Benchling, and no nontarget annealing was detected. The primer sequences are shown below: Primer set for the detection of Lasiodiplodia sp. Las.ITS77-F: 5′ -ACCTCTGTTGCTTTGGCGGCTC- 3′ Las.ITS452-R: 5′ -GTTCAGAAGGTTCGTCCGGCGG- 3′ Primer set for the detection of Ceratocystis sp. Cer.ITS119-F: 5′ -CCAGCAGTATTAGTCTCACCAC- 3′ Cer.ITS379-R: 5′ -CCTCCAACGCCAAGAAAGA- 3′ Evaluation of the sensitivity and specificity of the primers To test the specificity of the designed primers, a PCR was performed using DNA from Ceratocystis sp. and Lasiodiplodia sp. as positive controls and DNA from three different fungal genera ( Rosellinia sp., Fusarium sp., and Moniliophthora roreri ) and DNA from the plant species T. cacao and P. americana as negative controls. Amplification was performed using the PCR conditions proposed by Tanović et al. [ 33 ] for the ITS region, replacing the annealing temperature by 65°C for the Cer.ITS119-F/ Cer.ITS379-R primer set and 68°C for Las.ITS77-F/ Las.ITS452-R set. Primer sensitivity was evaluated by serial dilutions of DNA from the isolates Las003 ( L. theobromae ) and NCF15 (C. cacaofunesta ) in Mili Q ultrapure water by factors of 1:10 up to a dilution of 1x10 − 5 . The DNA concentration of the primary aliquots was quantified using a DeNovix spectrophotometer (Wilmington, USA) configured with the double-stranded DNA (dsDNA) readout parameter. Finally, PCR amplifications were performed in triplicate using the DNA at its initial concentration and each of the dilutions to determine the minimum DNA concentration that could be detected. PCR amplification of DNA from cacao and avocado stems To verify that vegetal genomic DNA was free of PCR inhibitors, the actin gene was amplified with the primers Tc.ACT-F/Tc.ACT-R [ 24 ] and Pa.ACT-F/Pa.ACT-R [ 11 ] for T. cacao and P. americana , respectively. The amplification program consisted of an initial denaturation at 95°C for 3 minutes, followed by 35 cycles consisting of a denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 30 seconds, and finally an extension cycle at 72°C for 5 minutes. Aliquots of DNA from T. cacao provided by the Tibaitatá Research Center and from P. americana provided by the Turipaná Research Center, both belonging to AGROSAVIA, were used as positive controls for the PCR reactions. Direct detection of Lasiodiplodia sp. and Ceratocystis sp. in stems Lyophilized plant material from nursery infections was used to evaluate the efficacy of molecular detection directly from stems using the Las.ITS77-F/Las.ITS452-R and Cer.ITS119-F/Cer.ITS379-R primers. DNA extraction was performed on T. cacao and P. americana stems from plants collected at 7 dpi for Lasiodiplodia sp. and 9 dpi for Ceratocystis sp., following the standardized protocol. PCR reactions were then performed with Biohelix PCR SuperMix at 1X, each primer at 0.2 µM, DNA at concentrations ranging from 30 to 50 ng/µL) and Mili Q type I water to adjust the final reaction volume, and the results were electrophoresed on a 1% agarose gel at 100 V for 45 minutes. To increase the sensitivity of the diagnostic test, a nested PCR was standardized, which consisted of a first round of amplification using the primers ITS1/ITS4 [ 13 , 37 ] and the program proposed by Tanović et al. [ 33 ]. The PCR products derived from the first amplification were used as templates for the second PCR round. The primer set Las.ITS77-F/Las.ITS452-R was used to detect Lasiodiplodia sp. and Cer.ITS119-F/ Cer.ITS379-R to detect Ceratocystis sp., and the amplification was performed according to the conditions proposed by Tanović et al. [ 33 ] changing the annealing temperature to 68 o C and 68 o C, respectively. PCR products were separated on a 1% agarose gel electrophoresis at 100 V for 45 minutes and visualized in a transilluminator. All experiments were conducted in triplicate. Diagnostic nested PCR - protocol description Molecular detection of Lasiodiplodia sp. and Ceratocystis sp. was made directly from infected seedling asymptomatic stems. For this, a nested PCR reaction was developed for each pathogen. Each PCR round consisted of each primer at a concentration of 0.2 µM, BioHelix PCR Supermix at 1X, template DNA in the range of 30–50 ng/µl, and double-distilled water (ddH2O) to adjust the final volume. The amplification conditions were the following: First amplification round - ITS1/ITS4 primer set: ITS1: 5′ - TCC GTA GGT GAA CCT GCG G − 3′ ITS4: 5′ - TCC TCC GCT TAT TGA TAT GC − 3′ The PCR program consisted of initial denaturation at 94 o C for 1:30 minutes, followed by 29 cycles at 94 o C for 30 seconds, alignment at 55 o C for 30 seconds, extension at 72 o C for 30 seconds and finally one cycle at 72 o C for 9 minutes and 30 seconds [ 33 ]. Second amplification round - Cer.ITS119-F/Cer.ITS379-R or Las.ITS77-F/Las.ITS452-R primer set: From the first PCR, a volume between (1–2 µL), which ensured a DNA concentration of 300–400 ng/µL was used as the template for the next PCR round. For detection of Ceratocystis sp. the amplification program was the same used in the first PCR round, changing the annealing temperature to 65 o C, and using the following primers: Cer.ITS119-F: 5′ -CCAGCAGTATTAGTCTCACCAC- 3′ Cer.ITS379-R: 5′ -CCTCCAACGCCAAGAAAGA- 3′ For the detection of Lasiodiploida sp., the amplification program was the same used in the first PCR round, changing the annealing temperature to 68 o C and using the following primers: Las.ITS77-F: 5′ -ACCTCTGTTGCTTTGGCGGCTC- 3′ Las.ITS452-R: 5′ -GTTCAGAAGGTTCGTCCGGCGG- 3′ PCR products were separated on a 1% agarose gel electrophoresis at 100 V for 45 minutes and visualized in a transilluminator. DNA from Ceratocystis sp. and Lasiodiplodia sp. were used as positive controls. All steps required to perform the molecular identification of Ceratocystis sp. and Lasiodiplodia sp. from infected and asymptomatic seedlings of tobacco and avocado are shown in Fig. 5 . Declarations Supplementary information Acknowledgments We thank Dr. Claudia Holguín Aránzazu for submitting the research project to a call for proposal funding. We also thank Andrea Berroterán, Donald Galvis, Jesús Cano and Dinael Balaguera for their contributions in preparing materials to carry out the experiments. Finally, we thank the staff of Tibaitatá and Turipaná laboratories at Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA) for providing plant DNA aliquots. Author contributions L.V.L.A carried out the experimental work, the data analysis, picture taking, and the writing of the manuscript. A.G.A and Y.Y.J.S contributed to the experimental planning, writing, and revising of the manuscript. Funding This work was funded by the Ministerio de Agricultura y Desarrollo Rural (MADR) [Colombian Ministry of Agriculture and Rural Development] via Agreements implemented through Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA) under the project “ Fortalecimiento de las capacidades en ciencia, tecnología e innovación del Centro de Investigación La Suiza para el desarrollo de proyecto de fitosanidad y agroindustria en sistemas de producción priorizados en el departamento de Santander ” Availability of data and materials All data generated in this study are included in the publication. All materials are available through the corresponding authors. Ethics approval and consent to participate Not applicable. Consent for publication The authors agreed to publish this manuscript. Competing interests The authors declare that they have no competing interests. Author details 1. Agricultural Sciences Faculty. Universidad Nacional de Colombia. Bogotá, Colombia. 2. 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Establishing references for gene expression analyses by RT-qPCR in Theobroma cacao tissues. Genetics and Molecular Research. 2011;10(4):3291–305. Pisco‐Ortiz C, Rodríguez E, Dávila‐Mora L, Villabona‐Gelvez A, Zuluaga P. First report of Lasiodiplodia theobromae causing dieback on Theobroma cacao in Colombia. New Disease Reports. 2024;49(2). Porebski S, Bailey LG, Baum BR. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter. 1997;15(1):8–15. Ramírez-Gil JG, Peterson AT. Current and potential distributions of the most important diseases affecting Hass avocado in Antioquia Colombia. Journal of Plant Protection Research. 2023;59(2), 214-228 Rathnayaka A, Chethana K, Manawasinghe I, Wijesinghe S, De Silva N, Tennakoon D, Phillips A, Liu J, Jones E, Wang Y, Hyde K. Lasiodiplodia : Generic revision by providing molecular markers, geographical distribution and haplotype diversity. Mycosphere. 2023;14(1):1254-1339. Saltarén LF, Varón de Agudelo F, Marmolejo F. Patógenos radicales en material de propagación de aguacate ( Persea americana Mill.). Fitopatología Colombiana. 1998;22(2), 52-58. Salvatore MM, Andolfi A, Nicoletti R. The Thin Line between Pathogenicity and Endophytism: The Case of Lasiodiplodia theobromae . Agriculture. 2020;10(10):488. Silvar C, Duncan JM, Cooke DEL, Williams NA, Díaz J, Merino F. Development of specific PCR primers for identification and detection of Phytophthora capsici Leon. European Journal of Plant Pathology. 2005;112(1):43–52. Suarez A. Critical overview of the expansion of Hass avocado plantations in Salamina, northern Caldas, Colombia. Journal of Land Use Science. 2024;19(1):230–8. Tanović B, Koščica M, Hrustić J, Mihajlović M, Trkulja V, Delibašić G. Botrytis squamosa - the causal agent of onion leaf blight in Bosnia and Herzegovina. Pesticidi I Fitomedicina. 2019;34(1), 9–17. Traoré D. Cocoa and Coffee Value Chains in West and Central Africa: Constraints and Options for Revenue-Raising Diversification Food and Agriculture Organization of the United Nations 2. FAO - AAACP Paper Series. 2009;3:1-116. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012;40(15):e115. Van Wyk M, Wingfield BD, Marin M, Wingfield MJ. New Ceratocystis species infecting coffee, cacao, citrus and native trees in Colombia. Fungal Diversity. 2010;40(1):103–17. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols. 1990;315–22. Zhang W, Noppadon Sathitsuksanoh, Simmons BA, Frazier CE, Barone JR, Renneckar S. Revealing the thermal sensitivity of lignin during glycerol thermal processing through structural analysis. RSC Advances. 2016;6(36):30234–46. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6506698","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":446518500,"identity":"7aa65c7a-7c50-4e93-9b89-c7c381a59a3d","order_by":0,"name":"Laura Valentina Laverde-Arias","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACPnYwxczAwN4ApAuA+AABLWzMMC08IKUGJGmRSCBaC/MziY97rOX4Z74xe/DDgEGO70YC44cPeLWwmUnOeJZuLHE7x9ywx4DBWPJGArPkDPwOMzbmOXA4seF2jpkEjwFD4oYbCWzMPHi1sH82/nPgcP38m2fMJP8YMNQToYXH8DHDgcMJBjd4zKSBtgAZhLUUPuw5kG648UxambSMgYThzDMPm/H6hZ+9fcOBHwes5eWOH94m+abCRp7vePJBvCGGDiSAmLGBBA2jYBSMglEwCrABAN7ORQCfo71bAAAAAElFTkSuQmCC","orcid":"","institution":"National University of Colombia","correspondingAuthor":true,"prefix":"","firstName":"Laura","middleName":"Valentina","lastName":"Laverde-Arias","suffix":""},{"id":446518501,"identity":"d98d27df-f919-4c65-94c0-c6aa9a67adbe","order_by":1,"name":"Yeirme Yaneth Jaimes-Suárez","email":"","orcid":"","institution":"Colombian Corporation for Agricultural Research","correspondingAuthor":false,"prefix":"","firstName":"Yeirme","middleName":"Yaneth","lastName":"Jaimes-Suárez","suffix":""},{"id":446518502,"identity":"098652da-dd6d-42d8-b0c1-fb6e54ef8923","order_by":2,"name":"Adriana González-Almario","email":"","orcid":"","institution":"National University of Colombia","correspondingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"","lastName":"González-Almario","suffix":""}],"badges":[],"createdAt":"2025-04-22 18:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6506698/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6506698/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81291405,"identity":"74e87830-7800-4cd6-a42a-72e28ba311c5","added_by":"auto","created_at":"2025-04-24 12:05:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":189226,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel to visualize PCR products of the actin gene.\u003cstrong\u003e a) \u003c/strong\u003ePCR products for \u003cem\u003eT. cacao\u003c/em\u003e; (-) control: negative control; T1-T2: positive controls; T4-T7: DNA extracted from seedlings inoculated in the nursery with \u003cem\u003eL. theobromae\u003c/em\u003e. \u003cstrong\u003eb)\u003c/strong\u003ePCR products for \u003cem\u003eP. americana\u003c/em\u003e; (-) control: negative control; T1: positive control; T2- T4: DNA extracted from seedlings inoculated in nursery with \u003cem\u003eL. subglobosa\u003c/em\u003e. Negative controls show no amplification.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6506698/v1/a3c87fd0bc805492009f607b.png"},{"id":81291406,"identity":"f2442393-e002-4162-b9b0-d8ca494622ad","added_by":"auto","created_at":"2025-04-24 12:05:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":309080,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel of PCR products to check the specificity of the primers designed for the diagnostic tests.\u003cstrong\u003e a) \u003c/strong\u003eAmplification with Cer.ITS119-F/Cer.ITS379-R. (-) control: negative control;\u003cstrong\u003e \u003c/strong\u003eNCF15: DNA of \u003cem\u003eC. cacaofunesta\u003c/em\u003e; P1-P8: DNA of \u003cem\u003eLasiodiplodia\u003c/em\u003espp.;\u003cstrong\u003e R1-R2: \u003c/strong\u003eDNA of \u003cem\u003eRosellinia \u003c/em\u003esp.; Fus: DNA of \u003cem\u003eFusarium\u003c/em\u003esp.; Mr: DNA of \u003cem\u003eMoniliophthora roreri\u003c/em\u003e; Tc: DNA of \u003cem\u003eTheobroma cacao\u003c/em\u003e. \u003cstrong\u003eb) \u003c/strong\u003eAmplification with Las.ITS77-F/Las.ITS452-R.\u003cstrong\u003e \u003c/strong\u003e(-) control: negative control; Las003: DNA of \u003cem\u003eL. theobromae\u003c/em\u003e; R1-R2: DNA of \u003cem\u003eRosellinia \u003c/em\u003esp.; Mr: DNA of \u003cem\u003eMoniliophthora roreri\u003c/em\u003e;\u003cstrong\u003e \u003c/strong\u003eFus: DNA of\u003cem\u003e Fusarium \u003c/em\u003esp.;\u003cstrong\u003e \u003c/strong\u003eTc: DNA of \u003cem\u003eTheobroma cacao\u003c/em\u003e; Pa: DNA of \u003cem\u003ePersea americana\u003c/em\u003e; P1-P6: DNA of \u003cem\u003eCeratocystis\u003c/em\u003e spp.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6506698/v1/180f6937077423b3e16c881c.png"},{"id":81292169,"identity":"8cac4b5d-9956-44c8-8c60-51af5e0869da","added_by":"auto","created_at":"2025-04-24 12:13:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":200033,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel of PCR products obtained in the sensitivity test for the detection of \u003cem\u003eCeratocystis\u003c/em\u003esp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp.\u003cstrong\u003e a)\u003c/strong\u003e Amplifications using Cer.ITS119-F/Cer.ITS379-R; (-) control: negative control; NCF15: DNA of \u003cem\u003eC. cacaofunesta\u003c/em\u003e; 1:10-1:100000: serial dilutions of original DNA aliquot. \u003cstrong\u003eb)\u003c/strong\u003eAmplifications using Las.ITS77-F/Las.ITS452-R; (-) control: negative control; Las003: DNA of \u003cem\u003eL. theobromae\u003c/em\u003e; 1:10-1:100000: serial dilutions of original DNA aliquot.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6506698/v1/21d7da9f3a1cdc8491efbbc7.png"},{"id":81291408,"identity":"8e19efed-1d68-4400-b9ab-516d9676c599","added_by":"auto","created_at":"2025-04-24 12:05:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":277362,"visible":true,"origin":"","legend":"\u003cp\u003eNested PCR products obtained in the detection of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and \u003cem\u003eCeratocystis\u003c/em\u003esp. in stem samples. \u003cstrong\u003ea)\u003c/strong\u003e Diagnosis of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. on \u003cem\u003eP. americana\u003c/em\u003e stems sampled at 7 dpi. (-) control: PCR negative control; Las001: DNA from \u003cem\u003eL. subglobosa\u003c/em\u003e isolate used as positive control; M1-M5: DNA from avocado seedlings inoculated with \u003cem\u003eL. subglobosa\u003c/em\u003e collected at 7 dpi. M(-): DNA from seedlings inoculated with sterile agar disc as negative control, T1-T2: DNA extracted from stems of healthy seedlings. \u003cstrong\u003eb)\u003c/strong\u003e diagnosis of \u003cem\u003eLasiodiplodia\u003c/em\u003esp. in \u003cem\u003eT. cacao\u003c/em\u003e stems sampled at 7 dpi. (-) control: PCR negative control; Las003, Las005, Las011, Las013 and Las014: DNA from isolates of \u003cem\u003eLasiodiplodia\u003c/em\u003espp. used as positive controls; T1-T6: DNA from healthy cocoa seedling stems, M(-): DNA from seedling inoculated with sterile agar disc as negative control; M1, M2, M4 and M5: DNA from cocoa seedlings inoculated with \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e collected at 7 dpi. \u003cstrong\u003ec)\u003c/strong\u003e diagnosis of \u003cem\u003eCeratocystis\u003c/em\u003esp. in \u003cem\u003eT. cacao\u003c/em\u003e stems sampled at 9 dpi. (-) control: PCR negative control, NCF15: positive control, DNA from \u003cem\u003eC. cacaofunesta\u003c/em\u003e isolate. M1-M5: DNA from cocoa seedlings inoculated with \u003cem\u003eC. cacaofunesta\u003c/em\u003ecollected at 9 dpi. M(-): DNA from seedling inoculated with sterile agar disc as negative control, T1: DNA from healthy cacao seedling stem.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6506698/v1/9f708cae4a2fa27e9e3b960b.png"},{"id":81291412,"identity":"321edc87-b915-4a5b-a8fd-98a47218634d","added_by":"auto","created_at":"2025-04-24 12:05:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":244896,"visible":true,"origin":"","legend":"\u003cp\u003eStages of molecular detection of \u003cem\u003eCeratocystis\u003c/em\u003e sp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. from lignified stem seedlings of \u003cem\u003eT. cacao\u003c/em\u003e and \u003cem\u003eP. americana\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6506698/v1/7a43712007a6608181fdf201.png"},{"id":81415201,"identity":"5cfd7ab2-99ca-4f50-bfe6-8fdc47993d3d","added_by":"auto","created_at":"2025-04-25 23:46:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1870586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6506698/v1/9d224915-6203-40ea-901f-2a593e52a153.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nested PCR methodology for detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples","fulltext":[{"header":"Background","content":"\u003cp\u003eCocoa (\u003cem\u003eTheobroma cacao\u003c/em\u003e L.) and avocado (\u003cem\u003ePersea americana\u003c/em\u003e Mill.) plantations are significant crops in tropical regions, especially in South Asia, Latin America, and West Africa, because the climatic conditions are optimal for their development [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In Colombia, the area planted with cocoa has increased by 13% in the last five years, and its production has increased by 127% in less than 15 years [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In 2024, Colombia reached its highest production with 67,678 tons, and exports (cocoa beans and derivatives) represented US \u003cspan\u003e$\u003c/span\u003e 265\u0026nbsp;million for the country [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Concerning avocado cultivation, Colombia has approximately 32,132 hectares dedicated to the cultivation of the Hass variety [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A 15% increase in Hass avocado exports has been observed, generating revenues of US \u003cspan\u003e$\u003c/span\u003e 218.6\u0026nbsp;million, with a 42% increase in exports reported [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the productivity of \u003cem\u003eT. cacao\u003c/em\u003e and \u003cem\u003eP. americana\u003c/em\u003e plantations in Colombia has been adversely affected by various pests and plant diseases, the latter mostly caused by fungi [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFungal species of the genus \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. have been reported as pathogens of both crops and are known to cause a variety of symptoms, such as leaf spots, fruit rot, or necrosis in different plant tissues. However, one of the most relevant diseases it causes is dieback, characterized by the wilting of the upper branches of the plants and internal necrosis of the vascular system [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In Colombia, \u003cem\u003eL. theobromae\u003c/em\u003e (Pat.) Griffon \u0026amp; Maubl. has been described as a pathogen of \u003cem\u003eP. americana\u003c/em\u003e since the late 1990s [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and it was reported for the first time in Colombia as a causal agent of dieback in \u003cem\u003eT.\u003c/em\u003e cacao [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. On the other hand, the species of \u003cem\u003eCeratocystis\u003c/em\u003e sp. may also cause vascular necrosis, particularly in \u003cem\u003eT. cacao\u003c/em\u003e, which leads to general wilting and even death of the whole plant as the disease progresses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This pathogen can be transmitted by pruning tools or \u003cem\u003eXileborus\u003c/em\u003e beetles [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In Colombia, \u003cem\u003eC. cacaofunesta\u003c/em\u003e Engelbr. \u0026amp; T. C. Harr. has been reported as the causal agent of cocoa wilt [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nonetheless, the symptoms caused by both \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and \u003cem\u003eCeratocystis\u003c/em\u003e sp. are not evident in the early stages of infection, making an opportune diagnosis difficult.\u003c/p\u003e \u003cp\u003eTraditional diagnosis based on pathogen isolation in culture media is not successful in asymptomatic tissues due to low inoculum density [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and may fail to detect dormant spores [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, the use of PCR-based molecular tools applied to diagnosis increases the specificity and sensitivity for pathogen detection in different plant tissues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], especially during early intracellular colonization, which may not produce detectable symptoms, as is the case with \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and \u003cem\u003eCeratocystis\u003c/em\u003e sp. infections, since the damage starts in the internal stem tissues. For this reason, this study standardized a genus-specific nested PCR technique for the early detection of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and \u003cem\u003eCeratocystis\u003c/em\u003e sp., which can be used to verify the phytosanitary health of \u003cem\u003eT. cacao\u003c/em\u003e and \u003cem\u003eP. americana\u003c/em\u003e propagation material.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStem pre-treatment for DNA extraction - standardized protocol\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the difficulty of grinding lignified tissues and promoting cell lysis of stems, a pre-treatment based on the methodology of Lu et al. [18] was standardized. The sample preparation protocol is as follows:\u003c/p\u003e\n\u003cp\u003e1.\u0026nbsp; \u0026nbsp;\u0026nbsp;Using disinfected and sterilized pruning shears, cut the stems into approximately 5 mm thick pieces\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp; \u0026nbsp;\u0026nbsp;Transfer the cut pieces to a sterile glass flask containing a mixture of 96% ethanol and pure glycerol (1:1) to a volume sufficient to cover the entire sample\u003c/p\u003e\n\u003cp\u003e3.\u0026nbsp; \u0026nbsp;\u0026nbsp;Cover the flask with aluminum coil to avoid the volatilization of ethanol and incubate for 6 hours at 40\u0026deg;C in a water bath\u003c/p\u003e\n\u003cp\u003e4.\u0026nbsp; \u0026nbsp;\u0026nbsp;Filter the sample through a sieve and wash with abundant sterile distilled water to remove excess ethanol and glycerol\u003c/p\u003e\n\u003cp\u003e5. Dry the stem pieces on a sterile\u0026nbsp;absorbent paper sheet and transfer them to a sterile mortar\u003c/p\u003e\n\u003cp\u003e6.\u0026nbsp; \u0026nbsp;\u0026nbsp;Add an equal amount of autoclaved white sand to the sample volume and grind until it turns into a fine powder\u003c/p\u003e\n\u003cp\u003e7.\u0026nbsp; \u0026nbsp;\u0026nbsp;Fill ⅕ of a sterile 15 mL falcon tube with the macerated stem and start the DNA extraction according to the CTAB protocol [10] with the following modifications: extending the original sample incubation time with CTAB buffer from 30 minutes to overnight; adding an extra incubation step of the supernatant with chloroform and finally, including an additional washing step of the DNA with 70% ethanol to optimize the nucleic acid purity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction with standardized protocol\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant DNA was extracted from pre-treated stems of \u003cem\u003eT. cacao\u003c/em\u003e and \u003cem\u003eP. americana\u003c/em\u003e seedlings infected under nursery conditions. The actin gene was amplified to confirm the quality of the DNA and to exclude the possible presence of PCR inhibitors in the diagnostic test (fig. 1). The results showed that the amplification of the actin gene from cacao plants infected with \u003cem\u003eCeratocystis cacaofunesta\u0026nbsp;\u003c/em\u003ewas successful in the positive control (DNA from healthy cacao plant) and the samples infected in nursery. A band of approximately 200 bp was evident, which was consistent with the expected amplicon size (171 bp). Regarding actin gene amplification in avocado plants, a PCR product of approximately 100 bp was obtained in the positive control (DNA from healthy avocado plants) and in plant samples infected with \u003cem\u003eLasiodiplodia\u003c/em\u003e \u003cem\u003esubglobosa\u003c/em\u003e, which was consistent with the expected amplicon size of 121 bp. These results indicate that the pretreatment successfully induced cell lysis, and the DNA extracted was free of PCR inhibitors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDesigned primers for detection of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and\u003cem\u003e\u0026nbsp;Ceratocystis\u003c/em\u003e sp. \u0026ndash; performance evaluation \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe designed primers Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R only amplified DNA from \u003cem\u003eCeratocystis\u0026nbsp;\u003c/em\u003esp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. respectively, when tested with DNA from other fungal genera and from plant species, demonstrating specificity (Fig. 2). Furthermore, the observed amplicons corresponded to the expected fragment sizes predicted \u003cem\u003ein silico\u003c/em\u003e, 279 bp for \u003cem\u003eCeratocystis\u003c/em\u003e sp. and 397 bp for \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. to differentiate between the two fungal pathogens.\u003c/p\u003e\n\u003cp\u003eBased on serial dilutions of the initial concentration of DNA extracted from \u003cem\u003eL. theobromae\u003c/em\u003e and \u003cem\u003eC. cacaofunesta\u003c/em\u003e, performed to determine the minimum detectable amount of fungal DNA, it was found that PCR could detect DNA concentrations as low as 2-4 ng/\u0026mu;L for both pathogens (fig. 3). For \u003cem\u003eL. theobromae\u003c/em\u003e, amplification was observed up to a 1:1000 dilution, although the band was weak and indistinct. Therefore, the detection limit was estimated to be at a 1:100 dilution, corresponding to a DNA concentration of 3.869 ng/\u0026mu;L (Figure 3a). For \u003cem\u003eC. cacaofunesta\u003c/em\u003e, a faint band was observed at a dilution of 1:1000 and therefore the reliable detection limit for this pathogen was estimated at a dilution of 1:100, corresponding to 2.215 ng/\u0026mu;L to avoid any doubtful interpretation of the results (Figure 3b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDirect detection of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and \u003cem\u003eCeratocystis\u003c/em\u003e sp. in cocoa and avocado seedling stems\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo improve the sensitivity of the molecular diagnosis, a nested PCR was performed to increase microbial DNA copies through a first round of amplification with the universal ITS1/ITS4 primers [13], and the resulting PCR products were then used as template DNA for a second amplification with the primers Las.ITS77-F/Las.ITS452-R and Cer.ITS119-F/Cer.ITS379-R, designed in this study. The results showed that amplifications to detect \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. were successful only in the positive controls and in the stem samples from the \u003cem\u003eP. americana\u003c/em\u003e and \u003cem\u003eT. cacao\u003c/em\u003e, which were infected but showed no wilting symptoms at 7 dpi. For the detection of \u003cem\u003eCeratocystis\u003c/em\u003e sp. in plant tissues, a nested PCR was performed using DNA from \u003cem\u003eC. cacaofunesta\u003c/em\u003e as a positive control and DNA extracted from five apparently healthy seedlings sampled at 9 dpi, and from control plant inoculated with a culture-free agar square. A band of ~300 bp was obtained in the positive control and the nursery stem samples, indicating that the primers successfully detected the pathogen. As expected, the negative controls and stem samples from healthy, uninfected plants showed no amplification band, confirming the robustness and consistency of the diagnostic test. To confirm the results, the PCR products were sent for sequencing and their identity to the fungal genera \u003cem\u003eLasiodiplodia\u003c/em\u003e and \u003cem\u003eCeratocystis\u003c/em\u003e was confirmed.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe standardized pre-treatment to soften lignified tissues and facilitate DNA extraction from stems was confirmed to be successful and ensured that nucleic acids are free from PCR inhibitors. The chemical principle underpinning this method is that glycerol and heat promote the cleavage of β-O-4 bonds in lignin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] accelerating the loss of structural stiffness, and ethanol disrupts intercellular cohesion and breaks the bonds present between the cell layers that comprise the lignified tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, the Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R designed primers demonstrated to be specific and sensitive detecting \u003cem\u003eCeratocystis\u003c/em\u003e sp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp., respectively. This is particularly relevant in the context of early diagnosis, as these primer sets can be used without the need for sequencing the PCR products to determine the identity of the pathogen; instead, the result can be interpreted only based on differences in fragment size.\u003c/p\u003e \u003cp\u003eMoreover, the diagnosis can be performed directly from stem samples and does not require prior purification of the fungus on a Petri dish since no cross-annealing was found with the plant DNA. Although other molecular methods have been reported for the diagnosis of plant diseases and PCR-based tests have been widely used as sensitive tools [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], nested PCR does not require a real-time thermal cycler or reagents such as probes or intercalants that emit a fluorescent signal as required in qPCR, nor specific enzymes such as \u003cem\u003eBst\u003c/em\u003e-polymerase or recombinase as required in LAMP or RPA techniques [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As a result, the use of nested PCR is less expensive and sensitive enough to detect pathogens at low inoculum densities and turns into a useful methodology to screen grafting and propagation material of perennial crops.\u003c/p\u003e \u003cp\u003eThe results of this study also demonstrate that performing two rounds of amplification improves sensitivity; for example, nested PCR for the detection of \u003cem\u003ePhytophthora\u003c/em\u003e sp. from soil, root, and stem samples increase the sensitivity by 10 to 1000 times compared to PCR with a single round of amplification [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Consequently, the technique presented in this study can be fine-tuned in the future to detect species within the same fungal genus and even multiple detections within the same reaction tube. This will require the use of more informative molecular markers and further analysis of the genetic data to identify regions that distinguish species, particularly for genera such as \u003cem\u003eLasiodiplodia\u003c/em\u003e and \u003cem\u003eCeratocystis\u003c/em\u003e, in which the occurrence of cryptic species and species complexes is described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, this tool can also be used to screen asymptomatic tissues or seedlings, and even to confirm microbiological diagnoses to get a more rapid and robust result.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work, a molecular protocol was developed to detect \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and \u003cem\u003eCeratocystis\u003c/em\u003e sp. in asymptomatic but infected seedlings stems of \u003cem\u003eP. americana\u003c/em\u003e and \u003cem\u003eT. cacao\u003c/em\u003e. To ensure the high quality and purity of the plant DNA, the tissue must be pre-treated with glycerol and 96% ethanol to soften it. For fungal molecular identification, this DNA must be subjected to a nested PCR with the primer sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for \u003cem\u003eCeratocystis\u003c/em\u003e sp. and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. to obtain a product of 279 bp for \u003cem\u003eCeratocystis\u003c/em\u003e and 397 pb for \u003cem\u003eLasiodiplodia\u003c/em\u003e sp.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003cp\u003eFungal isolation and DNA extraction\u003c/p\u003e\n \u003cp\u003eA total of 18 isolates were selected from the strain bank of the La Suiza Research Center \u0026ndash; AGROSAVIA (Corporaci\u0026oacute;n Colombiana de Investigaci\u0026oacute;n Agropecuaria), located in Santander, Colombia. Six isolates of the genus \u003cem\u003eCeratocystis\u003c/em\u003e sp., eight of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp., and two isolates of \u003cem\u003eRosellinia\u003c/em\u003e sp., one isolate of \u003cem\u003eMoniliophthora roreri\u003c/em\u003e, and two isolates of \u003cem\u003eFusarium\u003c/em\u003e sp. were included as specificity controls. The isolates of \u003cem\u003eCeratocystis\u003c/em\u003e sp., \u003cem\u003eLasiodiplodia\u003c/em\u003e sp., and \u003cem\u003eFusarium\u003c/em\u003e sp. were grown on PDA (Papa Dextrose Agar) supplemented with chloramphenicol (50 mg/L); the isolates of \u003cem\u003eRosellinia\u003c/em\u003e sp. were reactivated on acidified PDA medium (pH 4.0) and \u003cem\u003eM. roreri\u003c/em\u003e on V8 (V8 Juice Agar). The Petri dishes were incubated at 26\u0026deg;C for 8 days. In the case of \u003cem\u003eRosellinia\u003c/em\u003e sp. the dishes were covered with aluminum foil to block light exposure for 30 days.\u003c/p\u003e\n \u003cp\u003eFor biomass production, five agar plugs were cut over the colony edges and transferred to a sterile tube containing 25 mL of modified V8 broth (100 mL Campbell\u0026acute;s V8 tomato juice, 40 g of glucose, and 30 g of peptone per litre). They were incubated at 26\u0026deg;C on a shaker at 120 rpm for 5 days for \u003cem\u003eCeratocystis\u003c/em\u003e sp., \u003cem\u003eLasiodiplodia\u003c/em\u003e sp., and \u003cem\u003eFusarium\u003c/em\u003e sp., and 15 days for \u003cem\u003eRosellinia\u003c/em\u003e sp. and \u003cem\u003eM. roreri\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eFor the DNA extraction, an optimized variant of the protocol proposed by Doyle \u0026amp; Doyle [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e] was used. This variant consisted of the addition of \u0026beta;-mercaptoethanol absolute (1.5% relative to the final buffer volume) to induce cell lysis more rapidly, together with the CTAB extraction buffer (TRIS 2.0 M, EDTA 0.5 M, NaCl 4.0 M and 0.3% CTAB per litre) supplemented with proteinase K at 20 mg/mL [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003ePlant material inoculation and sample collection\u003c/p\u003e\n\u003cp\u003eSix-month-old seedlings of \u003cem\u003eTheobroma cacao\u003c/em\u003e L. (CCN 51 genotype) and \u003cem\u003ePersea americana\u003c/em\u003e Mill. (Santa Cruz variety) were infected under nursery conditions at a temperature of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity of 75%. Inoculation with \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. was performed on cacao seedlings with the isolate Las003 and on avocado seedlings with the isolate Las001. The infection with \u003cem\u003eCeratocystis cacaofunesta\u003c/em\u003e (isolate NCF15) was performed on cacao plants only, following the method described by Arg\u0026ocirc;lo-Magalh\u0026atilde;es et al. [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. For each pathogen, a group of 25 seedlings was infected, and negative controls were inoculated with sterile agar plugs. Five destructive samplings were taken at 7, 14, 25, 36, and 45 days post inoculation (dpi) to monitor the progression of the dieback disease caused by \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. in both cacao and avocado plants, and at 9, 16, 27, 38 and 60 dpi to monitor the cacao wilt caused by \u003cem\u003eCeratocystis\u003c/em\u003e sp. At each sampling, the stems were cut longitudinally, and the length of the necrotic area (cm) was measured with a ruler. The plant material was dried in a Biocool FD-1C-80 Freeze Dryer (Beijing, China), then vacuum-packed and kept frozen until the time of DNA extraction for validation of the diagnostic nested PCR.\u003c/p\u003e\n\u003cp\u003ePrimer design\u003c/p\u003e\n\u003cp\u003ePrimers were designed to target genus-specific regions between the Internal Transcribed Spacer 1 and 2 (ITS 1 and 2). Bioinformatic analyses were performed using the ITS sequences available in GenBank (accession numbers MK811125.1, KY031620.1, NR_174713.1, MK368389.1, MH107831.1, and NR_111174.1) and the ITS sequences of the eight isolates of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and six of \u003cem\u003eCeratocystis\u003c/em\u003e sp. selected from the strain bank of the La Suiza Research Center. Once the specific regions were identified for each pathogen, the primers were generated using Primer3Plus [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e] and the tools available on the IDT (Integrated DNA Technologies) website. Primer specificity was checked \u003cem\u003ein silico\u003c/em\u003e using Primer-BLAST and Benchling, and no nontarget annealing was detected. The primer sequences are shown below:\u003c/p\u003e\n\u003cp\u003ePrimer set for the detection of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp.\u003c/p\u003e\n\u003cp\u003eLas.ITS77-F: 5\u0026prime; -ACCTCTGTTGCTTTGGCGGCTC- 3\u0026prime;\u003c/p\u003e\n\u003cp\u003eLas.ITS452-R: 5\u0026prime; -GTTCAGAAGGTTCGTCCGGCGG- 3\u0026prime;\u003c/p\u003e\n\u003cp\u003ePrimer set for the detection of \u003cem\u003eCeratocystis\u003c/em\u003e sp.\u003c/p\u003e\n\u003cp\u003eCer.ITS119-F: 5\u0026prime; -CCAGCAGTATTAGTCTCACCAC- 3\u0026prime;\u003c/p\u003e\n\u003cp\u003eCer.ITS379-R: 5\u0026prime; -CCTCCAACGCCAAGAAAGA- 3\u0026prime;\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003cp\u003eEvaluation of the sensitivity and specificity of the primers\u003c/p\u003e\n \u003cp\u003eTo test the specificity of the designed primers, a PCR was performed using DNA from \u003cem\u003eCeratocystis\u003c/em\u003e sp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. as positive controls and DNA from three different fungal genera (\u003cem\u003eRosellinia\u003c/em\u003e sp., \u003cem\u003eFusarium\u003c/em\u003e sp., and \u003cem\u003eMoniliophthora roreri\u003c/em\u003e) and DNA from the plant species \u003cem\u003eT. cacao\u003c/em\u003e and \u003cem\u003eP. americana\u003c/em\u003e as negative controls. Amplification was performed using the PCR conditions proposed by Tanović et al. [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] for the ITS region, replacing the annealing temperature by 65\u0026deg;C for the Cer.ITS119-F/ Cer.ITS379-R primer set and 68\u0026deg;C for Las.ITS77-F/ Las.ITS452-R set.\u003c/p\u003e\n \u003cp\u003ePrimer sensitivity was evaluated by serial dilutions of DNA from the isolates Las003 (\u003cem\u003eL. theobromae\u003c/em\u003e) and NCF15 (C. \u003cem\u003ecacaofunesta\u003c/em\u003e) in Mili Q ultrapure water by factors of 1:10 up to a dilution of 1x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. The DNA concentration of the primary aliquots was quantified using a DeNovix spectrophotometer (Wilmington, USA) configured with the double-stranded DNA (dsDNA) readout parameter. Finally, PCR amplifications were performed in triplicate using the DNA at its initial concentration and each of the dilutions to determine the minimum DNA concentration that could be detected.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003cp\u003ePCR amplification of DNA from cacao and avocado stems\u003c/p\u003e\n \u003cp\u003eTo verify that vegetal genomic DNA was free of PCR inhibitors, the \u003cem\u003eactin\u003c/em\u003e gene was amplified with the primers Tc.ACT-F/Tc.ACT-R [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] and Pa.ACT-F/Pa.ACT-R [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e] for \u003cem\u003eT. cacao\u003c/em\u003e and \u003cem\u003eP. americana\u003c/em\u003e, respectively. The amplification program consisted of an initial denaturation at 95\u0026deg;C for 3 minutes, followed by 35 cycles consisting of a denaturation at 95\u0026deg;C for 30 seconds, annealing at 50\u0026deg;C for 30 seconds, extension at 72\u0026deg;C for 30 seconds, and finally an extension cycle at 72\u0026deg;C for 5 minutes. Aliquots of DNA from \u003cem\u003eT. cacao\u003c/em\u003e provided by the Tibaitat\u0026aacute; Research Center and from \u003cem\u003eP. americana\u003c/em\u003e provided by the Turipan\u0026aacute; Research Center, both belonging to AGROSAVIA, were used as positive controls for the PCR reactions.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDirect detection of\u003c/strong\u003e \u003cstrong\u003eLasiodiplodia\u003c/strong\u003e \u003cstrong\u003esp. and\u003c/strong\u003e \u003cstrong\u003eCeratocystis\u003c/strong\u003e \u003cstrong\u003esp. in stems\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eLyophilized plant material from nursery infections was used to evaluate the efficacy of molecular detection directly from stems using the Las.ITS77-F/Las.ITS452-R and Cer.ITS119-F/Cer.ITS379-R primers. DNA extraction was performed on \u003cem\u003eT. cacao\u003c/em\u003e and \u003cem\u003eP. americana\u003c/em\u003e stems from plants collected at 7 dpi for \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and 9 dpi for \u003cem\u003eCeratocystis\u003c/em\u003e sp., following the standardized protocol. PCR reactions were then performed with Biohelix PCR SuperMix at 1X, each primer at 0.2 \u0026micro;M, DNA at concentrations ranging from 30 to 50 ng/\u0026micro;L) and Mili Q type I water to adjust the final reaction volume, and the results were electrophoresed on a 1% agarose gel at 100 V for 45 minutes.\u003c/p\u003e\n \u003cp\u003eTo increase the sensitivity of the diagnostic test, a nested PCR was standardized, which consisted of a first round of amplification using the primers ITS1/ITS4 [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e] and the program proposed by Tanović et al. [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. The PCR products derived from the first amplification were used as templates for the second PCR round. The primer set Las.ITS77-F/Las.ITS452-R was used to detect \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and Cer.ITS119-F/ Cer.ITS379-R to detect \u003cem\u003eCeratocystis\u003c/em\u003e sp., and the amplification was performed according to the conditions proposed by Tanović et al. [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] changing the annealing temperature to 68 \u003csup\u003eo\u003c/sup\u003eC and 68 \u003csup\u003eo\u003c/sup\u003eC, respectively. PCR products were separated on a 1% agarose gel electrophoresis at 100 V for 45 minutes and visualized in a transilluminator. All experiments were conducted in triplicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003cp\u003eDiagnostic nested PCR - protocol description\u003c/p\u003e\n \u003cp\u003eMolecular detection of \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. and \u003cem\u003eCeratocystis\u003c/em\u003e sp. was made directly from infected seedling asymptomatic stems. For this, a nested PCR reaction was developed for each pathogen. Each PCR round consisted of each primer at a concentration of 0.2 \u0026micro;M, BioHelix PCR Supermix at 1X, template DNA in the range of 30\u0026ndash;50 ng/\u0026micro;l, and double-distilled water (ddH2O) to adjust the final volume. The amplification conditions were the following:\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003cp\u003eFirst amplification round - ITS1/ITS4 primer set:\u003c/p\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003cp\u003eITS1: 5\u0026prime; - TCC GTA GGT GAA CCT GCG G \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\n \u003cdiv id=\"Sec16\" class=\"Section4\"\u003e\n \u003cp\u003eITS4: 5\u0026prime; - TCC TCC GCT TAT TGA TAT GC \u0026minus;\u0026thinsp;3\u0026prime;\u003c/p\u003e\n \u003cp\u003eThe PCR program consisted of initial denaturation at 94 \u003csup\u003eo\u003c/sup\u003eC for 1:30 minutes, followed by 29 cycles at 94 \u003csup\u003eo\u003c/sup\u003eC for 30 seconds, alignment at 55 \u003csup\u003eo\u003c/sup\u003eC for 30 seconds, extension at 72 \u003csup\u003eo\u003c/sup\u003eC for 30 seconds and finally one cycle at 72 \u003csup\u003eo\u003c/sup\u003eC for 9 minutes and 30 seconds [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003cp\u003eSecond amplification round - Cer.ITS119-F/Cer.ITS379-R or Las.ITS77-F/Las.ITS452-R primer set:\u003c/p\u003e\n \u003cp\u003eFrom the first PCR, a volume between (1\u0026ndash;2 \u0026micro;L), which ensured a DNA concentration of 300\u0026ndash;400 ng/\u0026micro;L was used as the template for the next PCR round. For detection of \u003cem\u003eCeratocystis\u003c/em\u003e sp. the amplification program was the same used in the first PCR round, changing the annealing temperature to 65 \u003csup\u003eo\u003c/sup\u003eC, and using the following primers:\u003c/p\u003e\n \u003cp\u003eCer.ITS119-F: 5\u0026prime; -CCAGCAGTATTAGTCTCACCAC- 3\u0026prime;\u003c/p\u003e\n \u003cp\u003eCer.ITS379-R: 5\u0026prime; -CCTCCAACGCCAAGAAAGA- 3\u0026prime;\u003c/p\u003e\n \u003cp\u003eFor the detection of \u003cem\u003eLasiodiploida\u003c/em\u003e sp., the amplification program was the same used in the first PCR round, changing the annealing temperature to 68 \u003csup\u003eo\u003c/sup\u003eC and using the following primers:\u003c/p\u003e\n \u003cp\u003eLas.ITS77-F: 5\u0026prime; -ACCTCTGTTGCTTTGGCGGCTC- 3\u0026prime;\u003c/p\u003e\n \u003cp\u003eLas.ITS452-R: 5\u0026prime; -GTTCAGAAGGTTCGTCCGGCGG- 3\u0026prime;\u003c/p\u003e\n \u003cp\u003ePCR products were separated on a 1% agarose gel electrophoresis at 100 V for 45 minutes and visualized in a transilluminator. DNA from \u003cem\u003eCeratocystis\u003c/em\u003e sp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. were used as positive controls.\u003c/p\u003e\n \u003cp\u003eAll steps required to perform the molecular identification of \u003cem\u003eCeratocystis\u003c/em\u003e sp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. from infected and asymptomatic seedlings of tobacco and avocado are shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Claudia Holguín Aránzazu for submitting the research project to a call for proposal funding. We also thank Andrea Berroterán, Donald Galvis, Jesús Cano and Dinael Balaguera for their contributions in preparing materials to carry out the experiments. Finally, we thank the staff of Tibaitatá and Turipaná laboratories at Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA) for providing plant DNA aliquots.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.V.L.A carried out the experimental work, the data analysis, picture taking, and the writing of the manuscript. A.G.A and Y.Y.J.S contributed to the experimental planning, writing, and revising of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003cbr\u003e\u003c/strong\u003eThis work was funded by the Ministerio de Agricultura y Desarrollo Rural (MADR) [Colombian Ministry of Agriculture and Rural Development] via Agreements implemented through Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA) under the project “\u003cem\u003eFortalecimiento de las capacidades en ciencia, tecnología e innovación del Centro de Investigación La Suiza para el desarrollo de proyecto de fitosanidad y agroindustria en sistemas de producción priorizados en el departamento de Santander\u003c/em\u003e”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated in this study are included in the publication. All materials are available through the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003cbr\u003e\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003cbr\u003e\u003c/strong\u003eThe authors agreed to publish this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003cbr\u003e\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1.\u0026nbsp; \u0026nbsp; \u0026nbsp;Agricultural Sciences Faculty. Universidad Nacional de Colombia. Bogotá, Colombia.\u003c/p\u003e\n\u003cp\u003e2. \u0026nbsp; \u0026nbsp; Corporación Colombiana de Investigación Agropecuaria - AGROSAVIA, Centro de Investigación La Suiza, Rionegro, Santander, Colombia.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgronegocios. Se han exportado 102.376 toneladas de aguacate Hass desde enero hasta septiembre | Agronegocios.co. AGRONEGOCIOS. 2024; https://www.agronegocios.co\u003c/li\u003e\n\u003cli\u003eArg\u0026ocirc;lo M, Dora E, Lopes UV, Rosa A, Virg\u0026iacute;nia Oliveira Damaceno. Leaf disc method for screening\u003cem\u003e Ceratocystis\u003c/em\u003e wilt resistance in cacao. Tropical Plant Pathology. 2016;41(3):155\u0026ndash;61\u003c/li\u003e\n\u003cli\u003eAzevedo-Nogueira F, Rego C, Gon\u0026ccedil;alves HMR, Fortes AM, Gramaje D, Martins-Lopes P. The road to molecular identification and detection of fungal grapevine trunk diseases. Frontiers in Plant Science. 2022;13.\u003c/li\u003e\n\u003cli\u003eBossu J, Le Moigne N, Corn S, Trens P, Di Renzo F. Sorption of water\u0026ndash;ethanol mixtures by poplar wood: swelling and viscoelastic behaviour. Wood Science and Technology. 2018;52(4):987\u0026ndash;1008.\u003c/li\u003e\n\u003cli\u003eCabrera OG, Patricia E, Jos\u0026eacute; J, \u0026Aacute;lvarez JC, Amarante G. Ceratocystis Wilt Pathogens: History and Biology\u0026mdash;Highlighting \u003cem\u003eC. cacaofunesta\u003c/em\u003e, the Causal Agent of Wilt Disease of Cacao. Springer eBooks. 2016;383\u0026ndash;428.\u003c/li\u003e\n\u003cli\u003eCarrero-Guti\u0026eacute;rrez ML, Gonz\u0026aacute;lez-Sayer S, Yeirme Jaimes-Su\u0026aacute;rez, Gonz\u0026aacute;lez-Almario C, Gonz\u0026aacute;lez-Almario A. \u003cem\u003eCeratocystis cacaofunesta\u003c/em\u003e is responsible for cocoa crops wilt in Colombia: morphological and molecular characterization of isolates. Journal of Plant Pathology. 2024.\u003c/li\u003e\n\u003cli\u003eChen S, Liu R, Lei Y, Morrell JJ, Yan L. Accelerating thermal decomposition of wood cell wall with glycerol. Journal of Materials Research and Technology. 2021;11:1637\u0026ndash;44. \u003c/li\u003e\n\u003cli\u003eCubillos G. Main diseases and pests of cocoa in Colombia. Horticult Int J. 2024;8(4):123‒128\u003c/li\u003e\n\u003cli\u003eDemeuse KL, Grode AS, Szendrei Z. Comparing qPCR and Nested PCR Diagnostic Methods for Aster Yellows Phytoplasma in Aster Leafhoppers. Plant Disease. 2016;100(12):2513\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eDoyle JJ, Doyle JL. A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochemical Bulletin. 1987;19: 11-15.\u003c/li\u003e\n\u003cli\u003eEngelbrecht J, Duong TA, van den Berg N. Development of a Nested Quantitative Real-Time PCR for Detecting \u003cem\u003ePhytophthora cinnamomi\u003c/em\u003e in \u003cem\u003ePersea americana \u003c/em\u003eRootstocks. 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A Panoramic View on Grapevine Trunk Diseases Threats: Case of Eutypa Dieback, \u003cem\u003eBotryosphaeria\u003c/em\u003e Dieback, and Esca Disease. Journal of Fungi. 2022;8(6):595.\u003c/li\u003e\n\u003cli\u003eLobato IM, O\u0026rsquo;Sullivan CK. Recombinase polymerase amplification: Basics, applications and recent advances. TrAC Trends in Analytical Chemistry. 2018;98:19\u0026ndash;35. \u003c/li\u003e\n\u003cli\u003eLu Y, Jiao L, He T, Zhang Y, Jiang X, Yin Y. An optimized DNA extraction protocol for wood DNA barcoding of \u003cem\u003ePterocarpus erinaceus\u003c/em\u003e. IAWA Journal. 2020;41(4):644\u0026ndash;59.\u003c/li\u003e\n\u003cli\u003eMinisterio de Agricultura y Desarrollo Rural. Cadena de cacao. Direcci\u0026oacute;n de Cadenas Agr\u0026iacute;colas y Forestales. 2021. https://sioc.minagricultura.gov.co\u003c/li\u003e\n\u003cli\u003eMinoli S, Monta\u0026ntilde;o M, Castro JP, Zabala A, Hodgetts G, Ortega Pacheco DV. Carbon Market Opportunities in livestock production, and cocoa and coffee agroforestry systems. An analysis of opportunities in Latin America and the Caribbean. 2023.https://hdl.handle.net/11324/21980\u003c/li\u003e\n\u003cli\u003eMori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. Journal of Infection and Chemotherapy. 2009;15(2):62\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eMuniz CR, Freire FCO, Viana FMP, Cardoso JE, Correia D, Jalink H, et al. Polyclonal antibody-based ELISA in combination with specific PCR amplification of internal transcribed spacer regions for the detection and quantitation of \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e, causal agent of gummosis in cashew nut plants. Annals of Applied Biology. 2012;160(3):217\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003ePaladines-Rezabala A, Moreira-Morrillo AA, Mieles AE, Garc\u0026eacute;s-Fiallos FR, Paladines-Rezabala A, Moreira-Morrillo AA, et al. Avances en la comprensi\u0026oacute;n de la interacci\u0026oacute;n entre \u003cem\u003eCeratocystis cacaofunesta\u003c/em\u003e y \u003cem\u003eXyleborus ferrugineus\u003c/em\u003e (Coleoptera: Curculionidae: Scolytinae) en \u0026aacute;rboles de cacao. Scientia Agropecuaria. 2022;13(1):43\u0026ndash;52. \u003c/li\u003e\n\u003cli\u003ePinheiro TT, Litholdo Jr. CG, Sereno ML, Leal Jr. GA, Albuquerque PSB, Figueira A. Establishing references for gene expression analyses by RT-qPCR in \u003cem\u003eTheobroma cacao\u003c/em\u003e tissues. Genetics and Molecular Research. 2011;10(4):3291\u0026ndash;305.\u003c/li\u003e\n\u003cli\u003ePisco‐Ortiz C, Rodr\u0026iacute;guez E, D\u0026aacute;vila‐Mora L, Villabona‐Gelvez A, Zuluaga P. First report of \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e causing dieback on \u003cem\u003eTheobroma cacao\u003c/em\u003e in Colombia. New Disease Reports. 2024;49(2).\u003c/li\u003e\n\u003cli\u003ePorebski S, Bailey LG, Baum BR. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter. 1997;15(1):8\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez-Gil JG, Peterson AT. Current and potential distributions of the most important diseases affecting Hass avocado in Antioquia Colombia. Journal of Plant Protection Research. 2023;59(2), 214-228\u003c/li\u003e\n\u003cli\u003eRathnayaka A, Chethana K, Manawasinghe I, Wijesinghe S, De Silva N, Tennakoon D, Phillips A, Liu J, Jones E, Wang Y, Hyde K. \u003cem\u003eLasiodiplodia\u003c/em\u003e: Generic revision by providing molecular markers, geographical distribution and haplotype diversity. Mycosphere. 2023;14(1):1254-1339. \u003c/li\u003e\n\u003cli\u003eSaltar\u0026eacute;n LF, Var\u0026oacute;n de Agudelo F, Marmolejo F. Pat\u0026oacute;genos radicales en material de propagaci\u0026oacute;n de aguacate (\u003cem\u003ePersea americana\u003c/em\u003e Mill.). Fitopatolog\u0026iacute;a Colombiana. 1998;22(2), 52-58.\u003c/li\u003e\n\u003cli\u003eSalvatore MM, Andolfi A, Nicoletti R. The Thin Line between Pathogenicity and Endophytism: The Case of \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e. Agriculture. 2020;10(10):488.\u003c/li\u003e\n\u003cli\u003eSilvar C, Duncan JM, Cooke DEL, Williams NA, D\u0026iacute;az J, Merino F. Development of specific PCR primers for identification and detection of \u003cem\u003ePhytophthora capsici\u003c/em\u003e Leon. European Journal of Plant Pathology. 2005;112(1):43\u0026ndash;52.\u003c/li\u003e\n\u003cli\u003eSuarez A. Critical overview of the expansion of Hass avocado plantations in Salamina, northern Caldas, Colombia. Journal of Land Use Science. 2024;19(1):230\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eTanović B, Ko\u0026scaron;čica M, Hrustić J, Mihajlović M, Trkulja V, Deliba\u0026scaron;ić G. \u003cem\u003eBotrytis squamosa \u003c/em\u003e- the causal agent of onion leaf blight in Bosnia and Herzegovina. Pesticidi I Fitomedicina. 2019;34(1), 9\u0026ndash;17. \u003c/li\u003e\n\u003cli\u003eTraor\u0026eacute; D. Cocoa and Coffee Value Chains in West and Central Africa: Constraints and Options for Revenue-Raising Diversification Food and Agriculture Organization of the United Nations 2. FAO - AAACP Paper Series. 2009;3:1-116. \u003c/li\u003e\n\u003cli\u003eUntergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012;40(15):e115.\u003c/li\u003e\n\u003cli\u003eVan Wyk M, Wingfield BD, Marin M, Wingfield MJ. New \u003cem\u003eCeratocystis\u003c/em\u003e species infecting coffee, cacao, citrus and native trees in Colombia. Fungal Diversity. 2010;40(1):103\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eWhite TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols. 1990;315\u0026ndash;22.\u003c/li\u003e\n\u003cli\u003eZhang W, Noppadon Sathitsuksanoh, Simmons BA, Frazier CE, Barone JR, Renneckar S. Revealing the thermal sensitivity of lignin during glycerol thermal processing through structural analysis. RSC Advances. 2016;6(36):30234\u0026ndash;46.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"stem, pre-treatment, sensitivity, asymptomatic","lastPublishedDoi":"10.21203/rs.3.rs-6506698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6506698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003ePersea americana\u003c/em\u003e and \u003cem\u003eTheobroma cacao\u003c/em\u003e plants can be infected by fungal species of the genus \u003cem\u003eLasiodiplodia,\u003c/em\u003e which cause the disease known as dieback. However, \u003cem\u003eT. cacao\u003c/em\u003e can also be infected by \u003cem\u003eCeratocystis \u003c/em\u003esp., which causes cocoa wilt. These fungi cause necrosis of the vascular tissue, leading to wilting and death of the plant. However, these symptoms are not observed in the early stages of infection, making timely diagnosis of these pathogens difficult. Therefore, in this study, a nested PCR technique was developed for the early detection of \u003cem\u003eCeratocystis \u003c/em\u003esp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. from infected and asymptomatic seedling stem samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eA standardized pre-treatment with glycerol and 96% ethanol to soften cocoa and avocado seedling stem tissue was effective before DNA isolation. Subsequent amplification of the actin gene from pre-treated stem tissues confirmed that this step is necessary to obtain nucleic acids free of PCR inhibitors. Furthermore, the Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R primer sets designed in this study demonstrated to be specific for the detection of \u003cem\u003eCeratocystis\u003c/em\u003esp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp., respectively, with a sensitivity ranging from 2-4 ng/μL. By using the primers ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for \u003cem\u003eCeratocystis\u003c/em\u003e sp., and ITS1/ITS4, and Las.ITS77-F/Las.ITS452-R for \u003cem\u003eLasiodiplodia\u003c/em\u003esp. in a nested-PCR, both pathogens were successfully detected directly from infected stem samples without a fungal isolation step in culture media. The results were easily interpreted by agarose electrophoresis according to fragment size differences, a 279 bp amplicon to samples artificially infected with \u003cem\u003eC. cacaofunesta\u003c/em\u003e and a 397 bp amplicon to samples with \u003cem\u003eL. theobromae\u003c/em\u003e and \u003cem\u003eL. subglobosa\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe use of the nested PCR protocol standardized in this study with the primers sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for \u003cem\u003eCeratocystis\u003c/em\u003esp.; and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for \u003cem\u003eLasiodiplodia\u003c/em\u003e sp., allowed early diagnosis of both pathogens in asymptomatic cocoa and avocado stem seedlings.\u003c/p\u003e","manuscriptTitle":"Nested PCR methodology for detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-24 12:05:17","doi":"10.21203/rs.3.rs-6506698/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"012f7892-628f-479f-99b3-2b440898b463","owner":[],"postedDate":"April 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-25T23:38:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-24 12:05:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6506698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6506698","identity":"rs-6506698","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}