A Ready-to-Use Single- and Duplex-TaqMan-qPCR Assay to Detect and Quantify the Biocontrol Agents Trichoderma asperellum and Trichoderma gamsii

Trichoderma asperellum strain icc012 and Trichoderma gamsii strain icc080, the microbial active ingredients of RemedierTM (ISAGRO, Novara, Italy), are biocontrol agents (BCAs) employable for crop protection against a wide range of fungal pathogens, including soil-borne pathogens and fungi involved in grapevine trunk disease. In this study, single and duplex real-time quantitative PCR (qPCR) methods to detect and quantify T. asperellum and T. gamsii were developed. Primers/probe sets were designed on the T. asperellum and T. gamsii rpb2 genes and tested for specificity on a panel of microorganisms commonly associated with grape wood and soil. No differences were observed comparing single- and duplex-qPCR assays on different BCAs, 1 pg of target DNA was detected approximately at Cq = 34. R2-values and the efficiency were always equal to 0.99 and >80%, respectively. The detection limit of the duplex-qPCR assay on artificially inoculated samples was 2 × 103 and 4 × 104 conidia g-1 of grape wood tissue and soil, respectively. The methods will be useful to better schedule BCA application in the field and in grapevine nurseries, as well as for investigating the dynamic of BCA populations.


INTRODUCTION
The application of biocontrol agents (BCAs) in sustainable agriculture model represents an ecofriendly strategy compared with the use of synthetic plant protection products (PPPs) for managing weeds, insects and fungal pathogens including fungicide-resistant mutants (Jensen et al., 2016;Bruce et al., 2017;Rotolo et al., 2018).
Since combining two or more beneficial microbes in a biopesticide would be advantageous to BCA management (Raupach and Kloepper, 1998), the mixture of T. asperellum strain icc012 and T. gamsii strain icc080 is used in Remedier TM to increase the activity and widening the environmental adaptability (Liguori, 2016). This microbial pesticide is registered against soil-borne pathogens affecting horticultural crops and turfs, and it is the only BCA-based PPP allowed in Italy to control pathogens associated with grapevine trunk diseases (GTDs).
To date, 133 fungal species belonging to 34 genera have been associated with GTDs affecting, singularly or simultaneously, table and wine grapes as well as rootstocks. PPPs effective in controlling GTDs-associated fungi are still lacking, and the BCAs Trichoderma atroviride and Trichoderma harzianum were the ones most studied for their effectiveness (Gramaje et al., 2018). On the other hand, information on T. asperellum and T. gamsii refer mostly to their use for cutting wounds protection while the population dynamics has been scarcely studied and appropriate monitoring systems are lacking. Yet, the monitoring of these BCAs in natural environments is essential to evaluate their effectiveness and scheduling their applications (Torsvik and Øvreås, 2002).
This study aimed at developing a molecular qPCR tool for an easy detection and quantification of T. asperellum strain icc012 and T. gamsii strain icc080. Comparisons between singleand duplex-qPCR assays were performed, then the assays were validated on fungal DNA extracts from grapevine wood tissue and soil samples contaminated with different concentrations of BCAs conidia.
Non-target species of fungi, yeasts and bacteria used were from the microbial culture collection of our Department.
Fungi and yeasts were routinely grown on potato dextrose agar (PDA: infusion from 200 g peeled and sliced potatoes kept at 60 ± 1 • C for 1 h, 20 g dextrose per liter of distilled water, pH adjusted to 6.5, and 20 g agar Oxoid No. 3) at 21 ± 1 • C in the darkness. Alternatively, bacteria were routinely grown on Luria-Bertani medium (LB: 10 g tryptone-peptone, 5 g yeast extract, pH adjusted to 7.0, and 14 g agar per liter of distilled water) at 25 ± 1 • C in the darkness.

Primers/Probe Sets Design
Sequences of the genes translation elongation factor 1-alpha (tef1), endochitinase 42 (ech42) and RNA polymerase B subunit II (rpb2) of target and non-target Trichoderma species were retrieved from GenBank 1 , and aligned using the SeqMan Pro software (DNASTAR, Lasergene, Madison, WI, United States). Based on the highest presence of speciesspecific single-nucleotide polymorphisms (SNPs), the rpb2 gene was selected and the sequences of different Trichoderma species (Supplementary Figure S1), including 64 sequences of T. asperellum and 10 sequences of T. gamsii, were aligned and examined in silico using the SeqMan Pro software (DNASTAR). The SNPs identified in T. asperellum and T. gamsii were used. The primers/probe sets ( Table 1) were manually designed primarily in order to include the specific SNPs in the 3 position of the primer forward (base 504 for T. asperellum reference sequence GenBank accession No. GU198278.1) and probe (base 806 for T. gamsii reference sequence GenBank accession No. KJ665270.1). Other SNPs in different positions of T. asperellum and T. gamsii primers/probe sets were also recorded. The absence of secondary structures and dimers and the feasibility of the use of Taq Man R -qPCR were verified using the Primer Express 3.0 software (Applied Biosystems, Foster City, CA, United States). Primers/probe sets were custom-synthesized (Macrogen, Seoul, South Korea) including FAM (6-carboxyfluorescein) and HEX (6-hexachlorofluorescein) 1 http://www.ncbi.nlm.nih.gov/

DNA Extraction From Trichoderma and qPCR Conditions
Genomic DNA of both BCAs and non-target fungi and yeasts was extracted from 5-day-old colonies grown at 21 ± 1 • C on cellophane disks overlaid on PDA, according to the protocol of De Miccolis Angelini et al. (2010). DNA from bacteria was extracted according to Rotolo et al. (2016). Quantity and quality of DNA was assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Amplifications were performed in a CFX96 TM Real-Time PCR Detection System Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, United States) whereas the CFX Manager TM version 1.0 software (Bio-Rad Laboratories, Hercules, CA, United States) was used for experimental setup and data analysis.
PCR mixes consisted of 6.25 µL of Sso Advanced TM Universal Probes Supermix (Bio-Rad Laboratories, Hercules, CA, United States), 250 nM of each primer, 150 nM of each probe, 1 (single-qPCR) or 2 (duplex-qPCR) µL of DNA template, and ultrapure water to 12.5 µL. Thermal cycling conditions were 95 • C for 2 min, followed by 40 cycles of 95 • C for 5 s and 64.5 • C for 30 s. All qPCR assays were run with appropriate controls, including the non-template control (NTC). Two replicates of each sample were analyzed, and reactions were repeated at least twice.

Specificity and Sensitivity Assays
The specificity of the TaqMan-based duplex-qPCR was assessed on a panel of microorganisms commonly associated with grapevines ( Table 2). Genomic grapevine and soil DNAs were also used as external negative control to exclude cross-reaction of the primers/probe sets, and all qPCR assays were run with appropriate controls, including the NTCs.
Ten-fold serial dilutions (100 ng to 0.01 pg) of genomic DNA of T. asperellum icc012 and T. gamsii icc080 were used in sensitivity assays. The standard curves for each BCA were generated in both single-and duplex-qPCR by plotting the quantification cycle (C q ) values vs. the Log 10 of 10-fold serial dilutions of DNA. Comparison between single-and duplex-qPCR was done for each species. Two replicates of each dilution were analyzed, and reactions were repeated at least twice. qPCR reactions were positive if C q value was ≤35.

Preparation of Grape Wood and Soil Samples
Conidia of T. asperellum and T. gamsii were scraped from the surface of 7-days-old colonies grown on PDA at 25 ± 1 • C in the dark and suspended in sterile distilled water containing 0.05% Tween 20. Mycelial fragments were removed through Miracloth (Calbiochem, Darmstadt, Germany). Aliquots (1 mL) of diluted conidial suspension (from 10 8 to 10 0 conidia mL −1 ) were used to infest 50 mg of grape wood chips (protocol 1) and 50 or 250 mg of clay soil (protocol 1 and protocol 2, respectively) previously sterilized, then ground and sieved at 2 mm to separate the gravel fraction. Samples were centrifuged (Eppendorf, Hamburg, Germany) at 14,000 rpm for 30 min and the pellet was subjected to DNA extraction. Five wood and soil samples artificially infested with T. asperellum or/and T. gamsii conidia were analyzed by qPCR and in the meantime, samples of T. asperellum-or T. gamsii-infested soil were placed on PDA and CFU were counted.

DNA Extraction and Purification, qPCR From Wood and Soil Samples
DNA extraction from wood chips and clay soil (protocol 1) was done using the CTAB method (Cullen et al., 2001), slightly modified as described below. Samples were homogenized in 600 µL extraction buffer (0.12 M Na 2 HPO 4 , 1.5 M NaCl, 2% CTAB) with 0.5 g acid-washed glass beads 425-600 µm (Sigma-Aldrich, St. Louis, MO, United States) and 2 steel spheres (5 mm diam.). The suspension was strongly shaken for 5 min at 1,500 oscillations min −1 using a Mixer Mill (MM301, Retsch GmbH, Haan, Germany). The supernatant, collected after centrifugation at 14,000 rpm for 15 min, was transferred in a new 2 mL microtube. Extraction was carried out in 750 µL of chloroform. Nucleic acids were collected by centrifugation for 15 min at 14,000 rpm, precipitated with 750 µL of isopropanol at −80 ± 3 • C for 30 min, and recovered by centrifugation at 14,000 rpm for 15 min. The pellet, washed with 200 µL of ethanol (70%), was suspended in 50 µL of ultrapure water.
DNA extraction from soil was carried out according to Martin-Laurent et al. (2001, protocol 2).
Wood-DNA extract was purified using Sepharose 6B (Sigma-Aldrich, St. Louis, MO, United States)−columns, while the soil-DNA extract was purified on both Sepharose 6B-and polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, St. Louis, MO, United States) columns, prepared as reported by Rotolo et al. (2016). DNA concentration and purity were estimated as described above and DNA was stored at −80 • C until use. To verify the success of DNA extraction, the extract was amplified by using the universal primers ITS5/ITS4, and the PCR mixture and conditions previously described. DNA extracted was directly amplified in single-and duplex-qPCR. Curves were generated by plotting the C q values vs. the Log 10 of the number of conidia added to the samples and BCAs were finally quantified as conidia per g −1 of grapevine wood tissue or soil.

Primers/Probe Sets Specificity
SNPs identified in intra-and external species alignments of the rpb2 gene sequences were used for species-specific primers/probe sets design. The 142 and 113 bp amplicons were confirmed for T. asperellum and T. gamsii, respectively (Figure 1). 2 | Quantification cycle (C q ) values of T. asperellum (TA) and T. gamsii (TG) primers/probe sets tested in the specificity assay through duplex-qPCR.

Species
Host Geographic origin C q (TA/TG) *  For duplex-qPCR assays, the best conditions to avoid unspecific amplification products were 64.5 • C (annealing temperature), 250 nM/150 nM (primers/probes concentrations) and 35 cycles. The specificity of the assay was also tested against genomic DNA from 66 non-target organisms as well as from grapevines and soil. No amplicons were generated using non-target DNA from some Trichoderma spp. (T. atroviride, T. paraviridescens, and T. polysporium) not carrying the 3 SNP in the primer forward (T. asperellum) or in the probe (T. gamsii) ( Table 2 and Supplementary Figure S1).

Primers/Probe Sets Sensitivity
A linear response was observed from 100 ng to 1 pg of T. asperellum and T. gamsii DNA in single-qPCR (Figures 2A,B). R 2 and efficiency of the standard curve were always equal to 0.99 and >80%, respectively, and the linear regression slopes were -3.10 and -3.11, respectively, for T. asperellum and T. gamsii. Both T. asperellum and T. gamsii primers/probe sets showed the same sensitivity when duplexed (Figure 2C). In the duplex-qPCR assay, 1 pg of target DNA for both species was also detected approximately at C q 34. Unspecific amplification occurred beyond the 35th cycle. In duplex-qPCR, R 2 and FIGURE 1 | Amplicons obtained with Trichoderma asperellum and T. gamsii primers/probe sets. M: 100 bps marker; Amplification mixtures were: 1-3, T. asperellum primers/probe set; 4-6, T. gamsii primers/probe set. Samples analyzed was: 1 and 4, T. asperellum DNA; 2 and 5, T. gamsii DNA; 3 and 6, no template controls. efficiency of the standard curves were also equal to 0.99 and >80% for both primer/probe sets, while the linear regression slopes were −3.21 and −3.13 for T. asperellum and T. gamsii, respectively.

Validation on Grapevine Wood and Soil
To validate the single-and duplex-qPCR assays, wood chips, and soil samples were artificially infested with a 10-fold dilution of conidial suspensions of T. asperellum and T. gamsii used singularly and in mixture. BCAs were only detected in all samples expected to be positive.
The detection limit in single-and duplex-qPCR was 2 × 10 3 conidia g −1 of grape wood chips for both BCAs. In single-qPCR, R 2 and efficiency of the standard curves were 0.99 and 131.4% for T. asperellum and 0.99 and 137.3% for T. gamsii. In the same assays, slopes values were −2.74 and −2.66 for T. asperellum and T. gamsii, respectively (Table 3). In duplex-qPCR, R 2 and efficiency of the standard curves were 0.97 and 127.3% for T. asperellum and 0.99 and 137.6% for T. gamsii, while slope values were −2.09 for T. asperellum and −2.66 for T. gamsii (Table 3).
Two protocols for DNA extraction from soil were preliminarily compared using different concentrations of T. asperellum and T gamsii conidia and protocol 2 worked better than protocol 1 (Supplementary Table S1).
In both single-and duplex-qPCR, the detection limit for T. asperellum and T gamsii was 4 × 10 4 conidia g −1 of soil (Table 3). In single-qPCR, R 2 and efficiency of the standard curves were 0.99 and 84.7% for T. asperellum and 0.96 and 81.2% for T. gamsii. In the same assays, slopes values were −3.75 and −3.87 for T. asperellum and T. gamsii, respectively (Table 3).
On the other hand, in duplex-qPCR, R 2 and efficiency of the standard curves were 0.95 and 100.1% for T. asperellum and 0.97 and 80.0% for T. gamsii. Slopes values were, respectively, −3.32 and −3.92 for T. asperellum and T. gamsii, respectively ( Table 3).
BCAs quantification obtained by qPCR assays always agreed with the CFU formed on PDA medium.

DISCUSSION
A fast reliable and sensitive species-specific method for detecting and quantifying the BCAs T. asperellum and T. gamsii, that are also the bioactive ingredients of the biofungicide Remedier TM (strains icc012 and icc080, respectively), used to control pathogens associated with GTDs as well as soil-borne and turf pathogens, was developed.
qPCR represents an alternative tool for an efficient quantification of individual fungal species through the estimation of DNA (Lievens and Thomma, 2005). In the current study, single-and duplex-qPCRs based on the uniqueness of SNPs identified on the single-copy gene encoding the rpb2 were set up and standardized. rpb2 is a widely studied gene, whose many sequences are available in GenBank for homology comparisons. According to the in silico analysis, SNP in the base position 504 of T. asperellum and 806 of T. gamsii were recognized to specifically discriminate the target BCAs from the nontarget fungi, including T. harzianum and T. viride, that are commonly detected in soil and currently used for crop protection (Druzhinina et al., 2011). Less specific SNPs were detected on the other two examined gene sequences corresponding to ech42 and tef1. However, the latter gene proved unsuitable for our purpose, although species-specific primers based on the tef1 gene had been proposed for the identification of T. asperellum, T. longibrachiatum and T. virens (Devi et al., 2012).
The specificity against fungal and bacterial species associated with different crops, with special reference to GTD pathogens, was tested in silico and by qPCR. The 142 and 113 bp amplicons identified for T. asperellum and T. gamsii, respectively, discriminated both the BCAs present in the biopesticide Remedier. The single-base mismatch in 3 position of the primer forward or of the probe were sufficient for avoiding false negatives and for discrimination from the majority of other Trichoderma species and other fungi. Some Trichoderma species, which are occasionally found (Samuels et al., 2010;Braithwaite et al., 2017), did not carry the single-base mismatch in 3 position (Supplementary Figure S1). In these species other SNPs present in different positions of the T. asperellum and T. gamsii primers/probe sets could allow the discrimination from the target species as observed for T. atroviride, T. paraviridescens, and T. polysporium analyzed in duplex-qPCR ( Table 2).
Starting from DNA extracted from pure culture, the detection limit was 1 pg, in agreement with other studies using singlecopy nuclear genes for qPCR (Ridgway and Stewart, 2000;Bluhm et al., 2002;Dodd et al., 2004;Fredlund et al., 2008;Scauflaire et al., 2012). According to provision for quantitative real-time PCR experiments, the linear dynamic range was extended to six different Log10 DNA amounts (Bustin et al., 2009). Slopes of the linear regression of qPCR assay ranged between -3.1 and -3.6, corresponding to a PCR efficiency of between 80 and 100%, and the R 2 -value was always ≥0.99.
When BCAs added to grapevines or soil samples were quantified in single-and duplex-qPCR, the R 2 -values were always ≥0.95, but the slope values (-2.0 to -2.7) indicated a slight decrease in the efficiency of the qPCR, caused by the presence of potential inhibitors (i.e., polyphenols, polysaccharides, humic acids, and metals) co-extracted with the DNA and inhibiting PCR reactions by decreasing the Taq polymerase activity or by limiting template's availability (Kermekchiev et al., 2009;Opel et al., 2010). Assuming 100% DNA recovery, the protocol quantified up to 2 × 10 3 and 4 × 10 4 conidia g −1 in plants and soil, respectively. These results agree with those reported for fungi other than Trichoderma (Selma et al., 2008;Garrido et al., 2009) and for other Trichoderma species, such as T. virens (Dodd et al., 2004;Oskiera et al., 2017).

CONCLUSION
The duplex-qPCR assay represents a useful tool for the simultaneous detection and quantification of T. asperellum and T. gamsii and can assist in learning more about fungal activity, survival and spread in large-scale monitoring. Accordingly, BCA populations can be monitored on grapevines and different crops, both in the field and nursery.

AUTHOR CONTRIBUTIONS
DG, SP, RDMA, and FF conceived and planned the experiments. DG and CR performed the experiments. DG and SP took the lead in writing the manuscript. SP, RDMA, and FF supervised the research. All authors contributed to the interpretation of the results, provided critical feedback and helped to shape the research, analysis, and manuscript.

FUNDING
This research was partially carried out in the framework of the Projects: (i) "Laboratory network for the selection, characterization, and conservation of germplasm and for preventing the spread of economically-relevant and quarantine pests (SELGE) No. 14, " funded by the Apulia Region, PO FESR 2007-2013 -Axis I, Line of intervention 1.2., Action 1.2.1 and (ii) Epidemiology, genetics of plant pathogens and development of molecular diagnostic methods granted by the University of Bari.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02073/full#supplementary-material FIGURE S1 | SNPs identification in T. asperellum (A) and T. gamsii (B) rpb2 gene sequences for primers/probe sets design. * For each Trichoderma spp., GenBank accession number of representative rpb2 gene sequences is included between parenthesis. SNPs as compared to T. asperellum (A) and T. gamsii (B) are displayed with a different color.
TABLE S1 | Detection of T. asperellum DNA extracted with two extraction protocols by qPCR. * 50 mg (protocol 1) and 250 mg (protocol 2). n.a., no amplification. Values are mean ± standard error of three biological replicates.