Duplex Real-Time PCR Assays for the Simultaneous Detection and Quantification of Botryosphaeriaceae Species Causing Canker Diseases in Woody Crops

Woody canker diseases caused by fungi of the Botryosphaeriaceae family are producing increasing losses in many economically important woody crops, including almond. To develop a molecular tool for the detection and quantification of the most aggressive and threatening species is of main importance. This will help to prevent the introduction of these pathogens in new orchards and to conveniently apply the appropriate control measures. Three reliable, sensitive and specific duplex qPCR assays using TaqMan probes have been designed for the detection and quantification of (a) Neofusicoccum parvum and the Neofusicoccum genus, (b) N. parvum and the Botryosphaeriaceae family and (c) Botryosphaeria dothidea and the Botryosphaeriaceae family. The multiplex qPCR protocols have been validated on artificially and naturally infected plants. Direct systems to process plant materials, without DNA purification, allowed high-throughput detection of Botryosphaeriaceae targets even in asymptomatic tissues. These results validate the qPCR using the direct sample preparation method as a valuable tool for Botryosphaeria dieback diagnosis allowing a large-scale analysis and the preventive detection of latent infection.


Introduction
Almond (Prunus dulcis (Mill.) D.A. Webb) is one the most important nut crops in the world. California (USA) is the leader in almond production followed by Spain and Australia (FAOSTAT, 2021; http://www.fao.org/faostat/en/#data (accessed on 10 March 2023). The implementation of new management techniques, such as high-density cultivation, prune intensification, drip irrigation and fertilization, mechanical harvest, use of more productive varieties and the cultivation in agronomically and environmentally more favorable cropping areas, has increased the fruit production in the last decades [1]. However, this new scenario, together with the current climate change situation, have favored the increase in almond diseases [2][3][4]. Among them, woody canker diseases, associated with fungi of the Botryosphaeriaceae family, affect a great number of agronomically important woody crops such as olive, grapevine, avocado, blueberry, stone fruit, citrus and nut crops, including almond [5][6][7][8][9][10][11][12]. These diseases affect the trunk and branches of young and mature almond trees causing cankers, extensive gumming, dieback, discoloration and necrosis of internal tissues and, especially in severe cases and young trees, plant death. Almond canker diseases have been described in California [13][14][15][16][17], Iran [18], Turkey [19], Morocco [20] and Spain [5,[21][22][23], causing important economic losses [24].
Fungi of the Botryosphaeriaceae family are one of the most prevalent pathogens in almond orchards [13,16,21]. Many Botryosphaeriaceae species have been associated with with FAM fluorophore and TaqMan probes for the detection of Neofusicocum spp. and Botryosphaeriaceae family were marked with SUN fluorophore.

Analytical Specificity-Inclusivity and Exclusivity-And Limit of Detection
Simplex qPCR protocols for the specific detection of N. parvum and B. dothidea did not detect other Neofusicoccum or Botryosphaeria species used in the specificity tests. Furthermore, BLAST analyses showed that N. parvum-specific qPCR protocol would not detect Neofusicoccum species associated to trunk diseases of walnut (N. luteum, N. mediterraneum, N. nonquaesitum, N. vitifusiforme) and pistachio (N. australe, N. hellenicum, N. mediterraneum, N. pistaciae N. vitifusiforme) [24,46] nor Neofusicoccum species affecting other Mediterranean crops, such as Citrus spp. and olive [47]. However, primers and probe for the detection of N. parvum could detect some of the phylogenetically closest species in the N. parvum species complex, such as N. ribis and N. kwambonambiense, which affect Mediterranean woody crops (Table S2). The B. dothidea simplex qPCR protocol could also detect B. corticis and B. fabicerciana described in Prunus and other woody crops in Mediterranean areas but not reported in almond [48,49] (Table S2). On the contrary, primers and probes designed for the specific detection of the genus Neofusicoccum and the Botryosphaeriaceae family could detect, respectively, all the Neofusicoccum and Botryosphaeriaceae species described in almond and Mediterranean crops. No amplification was detected using DNA from non-target species or negative controls.
Simplex and duplex qPCR reactions showed efficiencies between 90 and 110% and R 2 about 0.99 ( Figure 1). Duplex qPCR reactions did not decrease the sensitivity of detection nor decrease R 2 values compared with simplex qPCR reactions.
The limit of detection of N. parvum, B. dothidea, Neofusicoccum spp. and Botryosphaeriaceae family was 10 fg of genomic DNA from pure fungal colonies (see Material and Methods). Although it was possible to detect as low as 1 fg of genomic DNA in some of the replicates, the limit of detection was established from 10 fg of genomic DNA due to the reliability of the repetitions (Figure 1). dothidea and Botryosphaeriaceae family. N. parvum and B. dothidea were detected with FAM fluorophore. Neofusicoccum genus and Botryosphaeriaceae family were detected with SUN fluorophore. Tenfold dilutions of genomic DNA from pure colonies of N. parvum and B. dothidea (1 ng to 10 fg) were amplified in three or six (10 fg point, limit of detection) replicates. Efficiency (E), coefficient of determination (R 2 ) and regression equations of standard curves are shown for each qPCR reaction.
The limit of detection of N. parvum, B. dothidea, Neofusicoccum spp. and Botryosphaeriaceae family was 10 fg of genomic DNA from pure fungal colonies (see Material and Methods). Although it was possible to detect as low as 1 fg of genomic DNA in some of the replicates, the limit of detection was established from 10 fg of genomic DNA due to the reliability of the repetitions (Figure 1).

Detection of Botryosphaeria Species in Naturally and Artificially Infected Plants
Thirteen out of eighteen samples from naturally infected almonds cvs. Soleta , Lauranne , Marcona and Belona rendered positive results by using the developed qPCR

Detection of Botryosphaeria Species in Naturally and Artificially Infected Plants
Thirteen out of eighteen samples from naturally infected almonds cvs. 'Soleta', 'Lauranne', 'Marcona' and 'Belona' rendered positive results by using the developed qPCR methods ( Table 2). B. dothidea and N. parvum were detected in 62.5% and 12.5% of the affected trees, respectively, by simplex and duplex qPCRs. Mixed infections of Botryosphaeriaceae species were not detected in any of the analyzed trees. Three 'Soleta' and two 'Lauranne' trees which presented canker symptoms (Sol 5, Sol 6, Lau 3 and Lau 4) rendered negative results for Botryosphaeriaceae species detection by qPCR, but Cytospora sp. was isolated from these trees by isolation and sequence identification. The simplex and the duplex qPCR for detection of N. parvum and Neofusicoccum genera yielded negative results when applied to almond trees naturally infected with B. dothidea. Similarly, simplex B. dothidea-specific qPCR yielded negative results when applied to trees naturally infected with N. parvum. Asymptomatic trees from 'Soleta' (Sol 10) and 'Lauranne' (Lau 6) cultivars were negative for simplex and duplex qPCR detection. The detection of Botryosphaeriaceae species by simplex or duplex qPCR was 100% consistent with the results obtained by isolation and sequencing ( Table 2). Table 2. Concordance between molecular and traditional methods (simplex and duplex qPCR using extracted DNA as sample and isolation methods) for the detection of Botryosphaeriaceae species in subcortical tissue of naturally infected almond trees.
Artificially inoculated almond twigs and naturally infected samples were used to compare qPCR detection of Botryosphaeriaceae species by using DNA extracted with a commercial kit or directly prepared plant crude extracts. Detection of N. parvum, B. dothidea, Neofusicoccum genus and Botryosphaeriaceae family by direct qPCR using 1:10 dilution of plant crude extracts of artificially and naturally infected samples had 100% of coincident results with qPCR using DNA extractions, demonstrating high agreement between the two sample preparation techniques. The sensitivity of detection when using plant crude extracts was one order of magnitude lower than when using DNA extracted with a commercial kit. Additionally, plant crude extracts used without dilution (1:1; v:v) did not always give amplification signal. Therefore, a 1:10 (v:v) dilution of the plant crude extract is advisable for direct qPCR detection (Table 3). Table 3. Comparison of real-time PCR (qPCR) sensitivities for the detection of Botryosphaeriaceae fungi in serial dilutions of DNA from an artificially inoculated almond twig extracted with a commercial kit (DNA extraction) or by direct sample preparation method (plant crude extracts).

DNA Extraction Plant Crude Extracts
Neofusicoccum

Detection of Botryosphaeriaceae Species on Asymptomatic Plant Tissues
The qPCR protocols developed using dilutions of plant crude extracts as sample could detect N. parvum and B. dothidea in artificially inoculated asymptomatic plant tissues. The first necrotic lesions appeared on some of the inoculated twigs 10 days after inoculation (dai) for N. parvum and B. dothidea but only the asymptomatic twigs were selected for the qPCR analysis. N. parvum was detected from 3 dai to 16 dai in all asymptomatic twigs assayed. B. dothidea was detected in 33% of the asymptomatic twigs analyzed at 3 dai and in 100% of the asymptomatic twigs from 10 to 16 dai ( Figure 2). The amount of fungal DNA quantified in the asymptomatic twigs ranged from 11.6 fg at 3 dai to 1.09 ng at 16 dai for N. parvum and from 18.2 fg at 3 dai to 1.1 pg at 16 dai for B. dothidea. Both fungi were respectively detected from 10 dai in symptomatic twigs.

Discussion
Wood canker diseases are a major threat to global nut crop productivity. Among them, canker diseases caused by Botryosphaeriaceae fungi have not been given the importance they deserve and the role of these fungi in wood diseases has been overlooked. The fact that Botryosphaeriaceae species have a wide host range and produce latent infec-

Discussion
Wood canker diseases are a major threat to global nut crop productivity. Among them, canker diseases caused by Botryosphaeriaceae fungi have not been given the importance they deserve and the role of these fungi in wood diseases has been overlooked. The fact that Botryosphaeriaceae species have a wide host range and produce latent infections has led to their "silent" introduction into crop fields through adjacent infected crops and infected but asymptomatic nursery plant material, to later disperse through rain, wind, insects or pruning and harvest tools. In fact, the number of reports associated with trunk diseases caused by Botryosphaeriaceae in numerous woody crops worldwide has significantly increased in the past years [50,51]. The preventive detection of the causal agents is one of the most important and effective strategies for the control of fungal diseases. For that reason, the development of an accurate, sensitive and rapid diagnostic method is of great importance. In this work, molecular tools based on TaqMan-qPCR protocols have been developed for the specific detection and quantification of the two most important Botryosphaeriaceae species of the almond crop: N. parvum and B. dothidea. However, as these two species are not the only ones that infect almond, and more than one species is usually detected in a producing orchard and in a single infected plant, duplex qPCR have been designed to detect the genus Neofusicoccum and the family Botryosphaeriaceae, thus giving information about the presence of other unknown Botryosphaeriacae species.
One of the main advantages of the designed qPCR protocols is their high sensitivity of detection compared with other PCR-based methods previously reported. Conventional and nested PCR performed in multiplex reached a detection limit of 1 pg and 0.1 pg of N. parvum gDNA, respectively [38][39][40] and 100 pg of B. dothidea gDNA [38]. These methods are time consuming and require visualization of the results in agarose gels. This entails the risk of cross contamination and, therefore, the appearance of false positives. Previously reported qPCR methods for detection of canker-causing pathogens generally used SYBR Green chemistry [42,44]. Those using hydrolysis probes were more sensitive and were able to detect as low as 200 fg of N. parvum gDNA [43]. Our methods have improved the sensitivity of detection, even allowing the detection of Botryosphaeriaceae species in asymptomatic tissues. In addition, the implementation of a duplex qPCR for the simultaneous detection of several targets at the same time and the use of a direct sample preparation method allow the analysis of a large number of samples, saving costs. One of the main constraints of the designed qPCR protocols is the impossibility to distinguish between N. parvum and some of the phylogenetically closest species within the N. parvum species complex such as N. ribis and N. kwambonambiense or between B. dothidea and B. corticis and B. fabicerciana. These species are not described in almond but affect some Mediterranean woody crops such as blueberry, grapevine, citrus and avocado [25,49,52,53]. If a precise identification is required, isolation of the pathogen and subsequent morphological identification or sequencing of another genome locus would be necessary.
Several studies have evidenced the endophytic phase of Botryosphaeriaceae fungi in nursery plant material [54][55][56][57]. In this sense, the high sensitivity of the developed qPCR tools allows for the early detection of these pathogens even before the appearance of disease symptoms, providing evidence of the latent infection of these canker-causing pathogens in woody plant tissues. Even more, using the direct sample preparation method described in this study allows the friendly and more economic analysis of a large number of samples. Detection of the target fungi failed when using non diluted plant crude extracts probably due to the presence in the reaction of high amounts of PCR inhibitors of plant origin. Using bovine serum albumin (BSA, 0.1 mg/mL) in the qPCR reaction helped to detect more true positives, but not all. Therefore, the possibility of detecting false negatives makes it advisable to use 1:10 dilutions and BSA which showed high agreement with the DNA extraction method. Therefore, these tools could be applied for the preventive detection of Botryosphaeriaceae fungi in recently planted young trees and in nursery plant material, even being certified as free of the aforementioned pathogens, ensuring the sale of healthy plants to the farmer and, at the same time, avoiding the introduction and dispersion of these pathogens in production fields.
The qPCR protocols described in this work are useful not only for the accurate pathogen identification and disease diagnostics in almond, but also for their application in epidemiological studies to determine the source of inoculum (air, irrigation water, neighboring crops) and quantify the seasonal variation of the inoculum level and the patterns of dispersion of Botryosphaeriaceae spores in almond orchards as previously described [43]. Additionally, this knowledge will permit to establish the correct schedules for the application of control measures, thus avoiding economic losses and damage to the environment. Validation of these qPCR protocols for detection of Botryosphaeriaceae groups in environmental samples should be addressed. In addition, these molecular tools could be applied to evaluate the efficacy of fungicide or biological control agents treatments, by quantifying the time-course concentration of fungal inoculum before and after treatments, as previously reported in other pathosystems [58].
In conclusion, this study provides an accurate, sensitive and easy-to-use molecular tool for the detection of the most important botryosphaeriaceous canker-causing pathogens, N. parvum and B. dothidea, along with any other species from the Neofusicoccum genus or Botryosphaeriaceae family in almond. These qPCR protocols could be potentially also applied to other woody crops affected by these diseases, after appropriate validation, i.e., the verification that the variation in the specific matrix does not affect the test performance.

Surveys of Almond Orchards and Fungal Isolation
Three commercial almond orchards affected by almond trunk diseases located in Alcalá del Río, La Rinconada and Jerez de la Frontera locations (southwestern Spain) were surveyed in 2021 and 2022. Symptomatic plant material exhibiting trunk cankers, gummosis and internal necrosis were taken to be analyzed in the laboratory. Samples were used for both the isolation of Botryosphaeriaceae fungi in culture medium and for the detection of Botryosphaeriaceae species using qPCR protocols. For the isolation of fungal isolates, plant tissues were surface-disinfected in 1.5% sodium hypochlorite solution for 2 min, rinsed twice in sterile distilled water, and left to air dry in a laminar flow cabinet. Small pieces were plated on potato dextrose agar supplemented with 0.5 g/L of streptomycin sulphate (Sigma-Aldrich, St. Louis, MO, USA) (PDAS). Petri dishes were incubated at 25 • C in darkness for 7-10 d and any Botryosphaeriaceae-like colony was individually transferred to PDA plates. All isolates were hyphal-tipped and maintained at −80 • C in cryovials containing 20% glycerol and at 4 • C in vials containing sterile distilled water, in the fungal collection of IFAPA Centro Las Torres (Seville, Spain).
The rest of fungal isolates used in this study were Botryosphaeriaceae species isolated from other woody crops, such as avocado and blueberry affected by trunk diseases [8,10], fungal genera phylogenetically close to the target species, and non-Botryosphaeriaceae pathogenic or endophytic species from almond (Table 4). For identification, sequencing of the internal transcribed spacer (ITS) nuclear rDNA using ITS1 and ITS4 primers [59] and a portion of the tef1 gene using EF446f and EF1035r primers [60] was performed. The sequences generated were deposited in the GenBank (Table S1) and compared with available sequences by BLAST analysis.

Design of qPCR Protocols
Firstly, simplex qPCR protocols were designed for the single detection of N. parvum, B. dothidea, Neofusicoccum spp. and the Botryosphaeriaceae family, respectively. Then, conditions for duplex qPCR for the simultaneous detection of N. parvum/Neofusicoccum spp., N. parvum/Botryosphaeriaceae family and B. dothidea/Botryosphaeriaceae family were optimized:

Design of Specific Primers and Probes
Sequences of the ITS region, tef1 gene, and tub2 gene from Botryosphaeriaceae species were retrieved form the GenBank and aligned using Mega 7.0 software [61]. Nucleotide sequences among the three loci that showed the highest specific consensus for each fungal target and not for the remaining species were selected and used to design forward/reverse primers and TaqMan probes using the software from Integrated DNA Technologies, Inc. (IDT) under its default settings. A BLASTn query against the NCBI GenBank database was used to ensure the specificity of the primers and probes in silico (Table S2). TaqMan probes were labeled with 6-carboxy-fluorescein, FAM (for N. parvum and B. dothidea) and SUN (for Neofusicoccum spp. and Botryosphaeriaceae family) reporter dyes at the 5 -end. TaqMan probes also harbored an internal ZEN quencher and an Iowa Black FQ quencher (IBFQ) at the 3 -end.

Optimization of qPCR Conditions
Combination of several primers and probes concentrations were tested to obtain the best efficiency reaction and higher sensitivity. Final concentrations of 300, 600 and 900 nM of primers and 100, 150, 250 nM of probes were assayed. Each DNA sample was run in simplex and duplex reactions to compare reaction efficiency and sensitivity. qPCR assays were performed in 96-well plates using a CFX Connect thermocycler (Bio-Rad, Hercules, CA, USA) in a 20 µL reaction volume. Reaction cocktails contained sample template (

Analytical Specificity and Analytical Sensitivity of the qPCR Reactions
The analytical specificity, inclusivity and exclusivity of each individual and duplex qPCR were tested on DNA from 81 isolates including species of the Botryosphaeriaceae family and other fungal species pathogenic and non-pathogenic to almond (Table 4). In addition, BLAST analysis was also performed using forward and reverse primers and probe sequences of each Botryosphaeriaceae target separately on the corresponding sequences of all described Botryosphaeriaceae species affecting almond and other Mediterranean crops (Table S2).
The analytical sensitivity of the qPCR assays was evaluated using eight ten-fold decreasing concentrations of DNA obtained from pure colonies of N. parvum NpALM2 isolate and B. dothidea BdALM2 isolate (0.2 ng/µL to 0.2 fg/µL) diluted in a background of DNA (10 ng/µL) extracted from the subcortical tissue of a healthy almond plant. An amount of 5 µL of each dilution was used as template in the qPCR reactions (1 ng to 1 fg of total genomic DNA/qPCR reaction). For each dilution point, we amplified three replicates (from 1 ng to 100 fg/qPCR reaction) and six replicates (for 10 and 1 fg/qPCR reaction) and the values obtained for each dilution series were used to generate standard curves for quantification of the respective fungal targets in simplex and duplex qPCR reactions. Each qPCR assay was repeated at least three times. Linear regression between the quantification cycle (Cq) and the log value of DNA concentration was performed to obtain the corresponding quantification values.

Plant DNA Extraction and Direct Sample Preparation Method
Samples from almond trees (small pieces of inoculated twigs and trunk subcortical tissue) were divided into two halves: one half was grounded in a mortar in the presence of liquid nitrogen and 0.1 mg of the homogenized tissue were used for DNA isolation following the instructions of the DNeasy Plant Pro kit (Qiagen, Hilden, Germany). DNA concentration was measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and stored at −20 • C for further analysis; the other half was introduced into plastic bags containing a soft net (Bioreba, Reinach, Switzerland) and grounded with the help of a manual homogenizer (or a hammer in the case of trunk subcortical tissues), in the presence of 1:20 (w:v) PBS buffer, pH 7.2 supplemented with 2% (w:v) polyvinyl pyrrolidone and 0.2% (w:v) sodium diethyl dithiocarbamate to obtain a plant crude extract. An amount of 5 µL of kit-extracted DNA and 5 µL of plant crude extracts were used in the qPCR reactions. Serial dilutions (1:1 to 1:10 7 v:v) of extracted DNA and plant crude extracts from the same tissue were used for qPCR approaches to compare the sensitivities of the two methods.

Validation of the Assays in Naturally and Artificially Infected Tissues
For validation of the qPCR protocols in naturally infected plants, subcortical tissue from the trunk of symptomatic and asymptomatic (negative control) almond trees were collected, surface sterilized in 1.5% sodium hypochlorite for 2 min, rinsed twice in sterile distilled water and air-dried. Small pieces (0.5 cm) were taken for both, the reisolation of inoculated fungi in PDA plates and qPCR detection. The pieces selected for fungal reisolation were placed on PDA plates and incubated at 25 • C for 7-10 days in darkness. The pieces selected for qPCR detection were divided into two halves, one for DNA extraction and the other for the preparation of plant crude extracts (see Section 4.6). For all samples, qPCR reactions were performed for the simplex detection of N. parvum and B. dothidea and the duplex detection of N. parvum + Neofusicoccum spp., N. parvum + Botryosphaeriaceae family and B. dothidea + Botryosphaeriaceae family.
For validation of the qPCR assays in artificially infected almonds, one-year-old twigs from the 'Guara' cultivar were inoculated with representative isolates of N. parvum (isolate NpALM2) and B. dothidea (isolate BdALM2). For this, the almond twigs were cut to 12 cm in length, surface sterilized by spraying with 75% ethanol to run off and air dried in a laminar flow cabinet under sterile conditions. Two wounds per twig were made with a 5-millimeter-diameter cork borer. Five-millimeter mycelium plugs from colonies actively growing on PDA were placed in the wounds and sealed with parafilm. Inoculated twigs were individually inserted in glass tubes containing 2 mL of sterile water. Tubes were capped and incubated in a growth chamber at 25 • C. Eight twigs per isolate were inoculated and twigs with non-colonized PDA plugs were used as negative controls. Two weeks after inoculation, the twigs were observed and small pieces from the margin of necrotic lesions were taken for both the reisolation of inoculated fungi in PDA plates and DNA extraction for qPCR detection, as explained above.
To confirm the specificity of the qPCR protocols, plant crude extracts and gDNA extracted form plant material artificially and naturally infected with N. parvum or B. dothidea were used as template in all simplex and duplex qPCR reactions.

Detection of Botryosphaeriaceae Fungi in Asymptomatic Tissues
To ascertain whether the qPCR protocols designed were able to detect Botryosphaeriaceae fungi in asymptomatic plant tissues, N. parvum NpALM2 and B. dothidea BdALM2 isolates were artificially inoculated on respective detached twigs of almond cv. Guara as explained above. Thirty twigs were inoculated per pathogen isolate. Successive sampling was performed at 3, 6, 10, 13 and 16 dai. At each sampling time, 5 twigs without symptoms were selected. Samples consisted of pieces (1 × 0.5 mm) of subcortical tissue without epidermis taken from the left and right of the inoculation site, 2 mm apart from the inoculation point. The sampled tissue fragments were processed to extract DNA and prepare plant crude extracts, as described above. Duplex qPCR protocols were applied to detect N. parvum and B. dothidea, respectively, and the percentage of detection in asymptomatic twigs was calculated.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12112205/s1, Table S1. GenBank Accession number of rRNA internal transcribed spacer (ITS) and translation elongation factor 1 alpha (tef1) gene of Botryosphaeriaceae isolates from almond, avocado and blueberry. Table S2. In silico analysis of the specificity of qPCR primers and probes for the detection of Neofusicoccum parvum and Botryosphaeria dothidea affecting Mediterranean woody crops. Reference [62] is cited in the supplementary materials.