Dual labelled probe assays for differentiation of Botryosphaeria dothidea , Neofusicoccum mediterraneum and Neofusicoccum parvum , based on polymorphisms in the MAT1-2-1 gene

Summary. Botryosphaeriaceous fungi are widespread, and cause serious diseases in many economically important crops. Botryosphaeria dothidea , Neofusicoccum mediter-raneum and N. parvum are the most important members of this family in the Mediterranean region. These fungi are frequently isolated from the same host, which together with their extensive and increasing host range necessitates development of rapid and reliable diagnostic tools. Species boundaries within the Botryosphaeriaceae have been defined based on phylogenetic analyses of multiple gene sequences, including those of mating type genes. The MAT1-2-1 gene displayed high sequence variability between Botryosphaeriaceous species, so was selected as the target for development of a definitive diagnostic tool. This paper outlines a new and robust molecular tool, composed of three TaqMan assays based on polymorphisms located in the MAT1-2-1 gene of B. dothidea , N. mediterraneum and N. parvum . Each assay differentiated the target species from other Botryosphaeriaceae , and from non-target fungi.

Species differentiation within the Botryosphaeriaceae has been mainly based on phylogenetic analyses of multiple gene sequences, such as the internal transcribed spacer region (ITS), the elongation factor 1-alpha, (TEF1-a), the beta tubulin, and the RNA polymerase II subunit (RPB2) (Pavlic et al., 2009;Chen et al., 2015).Although an RFLP analysis tool based on the ITS region could distinguish the main Botryosphaeriaceous species, this could not differentiate five species, including N. parvum (Slippers et al., 2007).More rapid molecular tools, such as PCR primers, have been developed for detection of Botryosphaeriaceous species only at the genus level, or for differentiation of Botryosphaeria dothidea from Neofusicoccum spp.(Ridgway et al., 2011;Palavouzis et al., 2022).Pathogen phylogenies based on mating type genes were comparable to multigene analyses for species differentiation.Notably, the MAT1-2-1 gene displayed high sequence variability between Botryosphaeriaceous species (Lopes et al., 2017).
With many species causing disease in an increasing range of hosts (Vakalounakis et al., 2019;Batista et al., 2020), rapid and accurate detection at the species level is important for the effective management of these pathogens, including quarantine measures and epidemiological studies.The present study reports development of robust and dual labelled probe molecular tools ("TaqMan" technology), targeting the MAT1-2-1 gene, for differentiation among B. dothidea, N. mediterraneum (MAT1-2-1 strains) and N. parvum, from fungal cultures and in planta.
Initially, gene sequences of MAT1-2-1 from N. mediterraneum, N. parvum and B. dothidea retrieved from the NCBI database, and sequences for each species derived from isolates of our own collection (deposited in GenBank under the Accession Numbers OQ632937 for B. dothidea, OQ632936 for N. parvum and OQ596433 for N. mediterraneum), were aligned using Clustal Omega (Madeira et al., 2019).Differences between sequences of the three species were detected using the MEGA software (Kumar et al., 2018).Based on sequence polymorphisms, dual labelled probes and primers were designed for MAT1-2-1 genes of B. dothidea (homothallic), N. mediterraneum MAT1-2-1 strain and N. parvum (homothallic).Primers were designed so that nucleotide polymorphisms specific for the target sequence were positioned at the 3' end of each primer, and PCR amplicons were smaller than 200 bp.Dual labelled probes were designed to have a Tm 8-10°C higher than the primers with a sequence length of 15-30 bp, avoiding a G nucleotide at the 5' end of the probe so that quenching of the 5' fluorophore was prevented.Nucleotide polymorphisms conferring probe specificity were placed close to the middle of the probe sequence.Primers and probes were screened for self-dimers, heterodimers and hairpins, using primer 3 plus software.To check target specificity, blast search against non-target Diplodia seriata MAT 1-2-1 sequences showed low homology with designed primers and probes.Primers and probes were then synthesized by eurofins genomics, labelling the 5' end of all probes (for B. dothidea, N. mediterraneum and N. parvum) with fluorescein (FAM) and the 3' end with the Black Hole Quencher-1 (BHQ1) (Table 1).
Table 1.List of primers and probe sequences for TaqMan qPCR assays, developed for detection and quantification of Botryosphaeria dothidea, Neofusicoccum mediterraneum and Neofusicoccum parvum, based on polymorphisms in the corresponding MAT1-2-1 genes.Polymorphisms of primers and probes between the different species are indicated with nucleotides letters in bold font.
Primer/probe name Sequence (5'->3') Target Gene Product size (bp) For the three dual labelled probe assays, the PCR mixture contained 1× KAPA Probe Fast Universal qPCR kit (KK4701) with fungal DNA of different concentrations as template, 300 nM of each of the forward and reverse and probe, and 1× Rox dye.The qPCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles each of 3 sec at 95°C and 30 sec at 60°C, without a final extension period.All reactions were performed in a Step Plus (Applied Biosystems) real time PCR machine cycler.As a first step, the quality of all genomic DNA samples was assessed using universal primers ITS4 and ITS5.Amplification was performed with KAPA Taq polymerase using the manufacturer's instructions.PCR conditions consisted of initial denaturation at 95°C for 3 min, followed by 35 cycles each of 30 s at 95°C, 30 s at 55°C, and 60 s at 72°C, and a final extension period of 10 min at 72°C.DNA from the following species was used: B. dothidea, N. parvum, N. mediterraneum MAT1-2-1 strain, while Diplodia seriata, P. chlamydospora and N. mediterraneum MAT1-1-1 strain were included as non-target species.
All three TaqMan assays differentiated the target species from the other two species under study, with no cross-reactions.In particular, the B. dothidea dual labelled probe assay reliably detected target DNA up to 2 pg μL -1 , at mean Ct 31.3.The N. mediterraneum MAT1-2-1 specific assay detected target DNA up to 20 pg μL -1 , at mean Ct 31.9.Non-target DNA at 8 ng μL -1 from Diplodia seriata, P. chlamydospora and N. mediterraneum MAT1-1-1 strain was not detected, while N. parvum and B. dothidea DNA occasionally produced amplification signal at Ct above 35.The N. parvum specific assay detected target DNA up to 2 pg μL -1 at mean Ct 34.5.Non-target DNA at 8 ng μL -1 from Diplodia seriata, P. chlamydospora or N. mediterraneum was not detected, while B. dothidea produced a non-specific signal at Ct 36 (Table 2).For all assays, linear relationships were observed between log of DNA concentrations (serial dilution) and Ct values (Figure 1).Target DNA at 200 pg μL -1 for all three assays was detected at similar Ct, even if spiked with 8 ng μL -1 DNA from pistachio (average Ct = 24 for B. dothidea and 27 for N. mediterraneum and N. parvum assays).Pistachio DNA at 8 ng μL -1 was detected over Ct 35 for all assays.
Due to the high number of species in the Botryosphaeriaceae causing similar symptoms, molecular tools that enable rapid and accurate detection at the pathogen species level are considered to be important for disease management.The present study developed a diagnostic tool to differentiate among B. dothidea, N. mediterraneum and N. parvum.For detection of N. mediterraneum, the assay described here allowed detection only of MAT1-2-1 strains.Another diagnostic tool targeting the MAT1-1-1 gene has yet to be designed.The present study focused on detection of these pathogens because they prevail among species isolated from pistachio, grapevine, citrus (Rumbos and Rumbou, 2001;Vakalounakis et al., 2019;Gusella et al., 2021), and other hosts (including olive, pomegranate, and white willow) (personal communications, Dr Tsopelas; Dr E.J. Paplomatas).
The present study developed three TaqMan assays based on sequence variation of the MAT1-2-1 gene (Lopes et al., 2017) that enabled differentiation between B. dothidea, N. mediterraneum (MAT1-2-1 strains), and N. parvum.The developed diagnostic tool is superior to other differentiation methods for Botryosphaeriaceae, as it requires no time-consuming steps such as RLFP-PCR (Slippers et al., 2007) or polyacrylamide for SSCP analyses (Ridgway et al., 2011).Furthermore, its practical application will be important, as it is possible to quantify species within infected plant tissues, potentially contributing to studies of pathogen prevalence and species interactions, and epidemiology of the diseases they cause.FUNDING This Research Project is co-financed by Greece and the European Union (European Social Fund) through the Operational Program "Human Resources Development, Education and Lifelong Learning 2014-2020", and Program EDBM103, "Support for researchers with an emphasis on young researchers-cycle B'", in the context of the project "Risk evaluation of development of a resistant form of disease via transfer of mating type genes from the Botryosphaeria dothidea species to species of the genus Neofusicoccum" (MIS 5048471).

AVAILABILITY OF DATA AND MATERIAL
Sequence data that support part of the findings of this study are available at the GenBank database under the accession numbers listed in Table 3.
Table 3. List of MAT1.2.1 sequences (Accession number, fungal strain information, host, origin and reference) from species of Botryosphaeriaceae, that were aligned using Clustal Omega, in order to detect polymorphisms unique for each of Botryosphaeria dothidea, Neofusicoccum mediterraneum (MAT1-2-1 strains) and Neofusicoccum parvum to design species-specific dual labelled probe qPCR assays.

Figure 1 .
Figure 1.Standard curves for Botryosphaeria dothidea (A), Neofusicoccum mediterraneum (B) and N. parvum (C) specific dual labelled probe (TaqMan) assays.Mean cycle threshold (Ct) values of DNA dilution series for each pathogen were plotted against the log of the DNA concentration (three replicates per concentration of fungal DNA tested).Reactions of undetermined values were set at 40.
NA: not applicable -the target was detected in one replicate.