Development of Species/Genus speci c Primers for Identi cation of Three Trichoderma Species and for Detection of Trichoderma Genus

You Zhou Chinese Academy of Tropical Agricultural Sciences https://orcid.org/0000-0002-3153-5222 Jun Wang Environment and plant protect institute, Chinese academy of tropical agricultural sciences Laying Yang Eniviroment and plant protection insitute, Chinese academy of tropical agricultural sciences Lijia Guo Environment and plant protection insitute, Chinese academy of tropical agricultural sciences Shuting He Qiongzhong lvyuan ecological agriculture development Co., Ltd. Wei Zhou Biotechnology research institute, guangxi academy of agricultural sciences Junsheng Huang (  h888111@126.com ) Chinese Academy of Tropical Agricultural Sciences


Introduction
Trichoderma is a widely used microorganism, which can be used not only for the production of industrial enzymes, but also for controlling plant diseases and promoting plant growth (Vinale et al. 2008;Nawrocka and Małolepsza 2013;Lehmann et al. 2016). At present, many pesticides and fertilizer products made of Trichoderma have been applied (Woo et al. 2014). Among Trichoderma species, T. harzianum, T. virens, T. koningiopsis have been widely exploited for prevention and control a variety of plant diseases (Moreno et al. 2009;Daguerre et al. 2014;Contreras-Cornejo et al. 2016).
Correct identi cation of Trichoderma species is a prerequisite for the use of Trichoderma. It is di cult to identify Trichoderma species by morphological characteristics (Błaszczyk et al. 2011;Sun et al. 2016;Kredics et al. 2018). The current method of identifying Trichoderma is mainly a combination of morphological and molecular characteristics (Jaklitsch 2009;Zhu and Zhuang 2015). The genes currently used for molecular identi cation of Trichoderma mainly include internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1) gene, RNA polymerase II subunit (rpb2) gene, and ATP citrate lyase (acl1) gene (Jaklitsch and Voglmayr 2015;Du Plessis et al. 2018). In a laboratory without a sequencing instrument, this is a time-consuming procedure for obtaining PCR products sequence information. In addition, identi cation of Trichoderma species based on BLAST searches may cause misidenti cation (Meincke et al. 2010). For the rapid identi cation of Trichoderma species, the ITS region, tef1 gene, and rpb2 gene are using to be the barcode locus for designing speci c primers. So far, several species-speci c primers have been developed for Trichoderma species identi cation (Friedl and Druzhinina 2012;Prabhakaran et al. 2015;Saroj et al. 2015).
Trichoderma are applied to the soil for controlling a variety of soil-borne plant diseases, such as tomato fusarium wilt and groundnut collar rot disease (Gajera et al. 2015;Taghdi et al. 2015). The amount (density) of Trichoderma in soil was related to the e ciency of inhibiting to soil-borne disease, high densities of Trichoderma spp. can effectively suppress pathogens in soil (Kataoka et al. 2010;Huang et al. 2011;Oskiera et al. 2017). However, the viability of Trichoderma in soil is affected by various factors such as soil water content, nutrients, and pH (Daryaei et al. 2016a;Daryaei et al. 2016b). So, monitoring the quantity of Trichoderma in soil can provide a scienti c basis for increasing or no application of Trichoderma to soil. And, it is also important to determine Trichoderma where it extends in the soil. So, Hagn et al. (2007) developed genus-speci c primers to identify and quantify Trichoderma spp. in soils.
At presently, a number of species/genus-speci c primers have published for using to identify T. harzianum, T. virens and Trichoderma genus (Kredics et al. 2018). However, after our tests, it was found that not all published speci c primers can accurately identify the target species of Trichoderma, and some genus speci c primers of Trichoderma produce non-speci c ampli cation products, so these primers can't be used to detect the amount of Trichoderma in environmental samples. Therefore, in order to obtain more alternative speci c primers for identi cation of T. harzianum, T. virens, T. koningiopsis, and for detection the quantity of Trichoderma genus. The aims of the study were to: 1) establish easy and reproducible PCR methods with species-speci c primers base on tef1 gene for rapid identi cation T. harzianum, T. virens, and T. koningiopsis; 2) utilizing the ITS region to design a genus-speci c primers used for future detecting, monitoring and quantifying Trichoderma.

Materials And Methods
Fungal strains DNA extraction 11 Trichoderma species and 13 Non-Trichoderma strains utilized in this study were obtained from Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, and Department of Plant Pathology, College of Agriculture, Sichuan Agricultural University (Table 1). The GenBank numbers of the strains see Table 1. Total DNA of these strains was extracted from pure cultures using the method as described by Thambugala et al. (2015). DNA concentrations were adjusted to 50 ng/µl and 10 ng/µl.  If the speci c primer does not produce non-speci c products in conventional PCR ampli cation, the primer will be used for quantitative PCR ampli cation to further test its speci city. The real-time PCR were performed in the QuantStudio™ 6 Flex real-time PCR system (Life Technologies), and the reaction mixtures (25 µl) contained: 9 µl of nuclease-free water, 12.5 µl of SYBR-green PCR Master mix (Tiangen Biotech, Beijing, China), 1 µl of genomic DNA (10 ng/µl), 1 µl of each primer (10 Mm), 0.5 µl of 50X ROX dye.
The real-time PCR parameters of ITricF/ITricR were 95 ℃ for 15 min, followed by 40 cycles of 95 ℃ for 10 s, 62 ℃ for 30 s, and a melting curve of 95 ℃ for 15 s followed by 1 min at 60 ℃ with a nal ramp to 95 ℃ with continuous data collection to test for primer dimers and non-speci c ampli cation.

Results
In this study, three sets species-speci c primers were designed based on tef1 genes for rapid identi cation of Trichoderma harzianum, T.virens, and T. koningiopsis. The genus-speci c primers were designed based on ITS sequences for detection and quanti cation of Trichoderma spp. under soil or other natural conditions.
PCR with the EHarF2/EHarR2 primers gave a single band of 253 bp for T. harzianum (Fig. 1a), and other Trichoderma species and fungi failed to amplify band on this set. But, the EHarF2/EHarR2 could amplify a more than 500 bp band from T. ghanense (Fig. 1a, Lane 7). The EKoisF/EKoisR primers could amplify a target amplicons of 255 bp from only the T. koningiopsis (Fig. 1b). However, the primers also could produce clear and obvious DNA bands of more than 500 bp from Cladosporium tenuissimum, Colletotrichum scovillei, and Diaporthe sp. (Fig. 1b, Lane 18, Lane 19 and Lane 20). The T. virens-speci c primers EVireF/EVireR gave an amplicon of 263 bp for T. virens, and no amplicon was observed with the other representative strains on this set (Fig. 1c), but, this primer pair also amplify a more than 500 bp band from T. sinense (Fig. 1c, Lane 9).
The experimental results show that although EHarF2/EHarR2, EKoisF/EKoisR and EVireF/EVireR can only amplify target bands on the target Trichoderma species, however these primers can also amplify non-target bands on other fungi. Therefore, these three primers pairs can only be used for species identi cation and not for quantitative analysis.
The ITricF/ITricR primers could amplify a single band of 103-111 bp for 11 Trichoderma species, and none of these other fungal taxa produced any visible band (Fig. 2a). The melting curve analysis showed that there was only a single peak at Real-Time PCR ampli cation with ITricF/ITricR primers pair (Fig. 2b). The Real-Time PCR products were checked by agarose gel electrophoresis, and no band was observed with the no-Trichoderma genus fungi. The result was shown that the ITricF/ITricR primers could amplify DNA only from Trichoderma genus, and could use for Real-Time PCR ampli cation.

Discussion
Most Trichoderma species were as biocontrol fungus widely using to control soil-borne diseases. In order to rapidly identify Trichoderma species, species-speci c primers pairs such as Th1F/Th1R for T. harzianum, Z04_2F/Z04_2R for T. atroviride, and Tc_RIF/ T_RIR for T. citrinoviride were developed in recently years (Prabhakaran et al. 2015;Skoneczny et al. 2015;Saroj et al. 2015).
In the present study, we designed 3 speci c primer pairs EVireF/EVireR for T. virens, EHarF2/EHarR2 for T. harzianum and EKoisF/EKoisR for T. koningiopsis based on the tef1 gene, and designed primers ITricF/ITricR according to the ITS region which can amplify all species of Trichoderma but no other genera. These primers were analyzed by a primer-blast online tool and found to be cross-reactivity with non-target species/genera. The operation is simple, and only one routine PCR is needed to obtain a stable experimental effect, which provides a possibility for rapid identi cation of T. harzianum, T. virens, T. koningiopsis and Trichoderma genus, respectively. High densities of Trichoderma spp. can effectively suppress pathogens in soil (Qiu et al. 2012;Oskiera et al. 2017). So, monitoring the quantity of Trichoderma in soil can provide a scienti c basis for increasing or no to apply Trichoderma on soil. At present, Real-Time PCR is a popular and economical tool to quantify microorganism in samples. In this study, ITricF/ITricR primers produced only one single peak at RT-PCR reaction, which indicating that a system for detecting the quantity of Trichoderma in soil or other environment can be developed in the future using the primers and its RT-PCR reaction conditions.