Conventional and real-time PCR assays for detection and identification of Rhizoctonia solani AG-2-2, the causal agent of root rot of sugar beet

SUMMARY Soil-borne fungi belonging to the genus Rhizoctonia are considered to be among the most destructive sugar beet pathogens. Although multinucleate R. solani AG-2-2 is frequently detected as the main causal agent of root rot of sugar beet worldwide, several binucleate (AG-A, AG-E and AG-K) and multinucleate Rhizoctonia ( R. solani AG-4, AG-5 and AG-8) have also been included in the disease complex. Due to their soil-borne nature and wide host range, the management of Rhizoctonia root rot of sugar beet is highly demanding. Identification of Rhizoctonia AG associated with root rot of sugar beet is the essential first step in determining a successful disease management strategy. In this paper we report a highly specific and sensitive real-time PCR protocol for detection of R. solani AG-2-2 which showed a high level of specificity after testing against 10 different anastomosis groups and subgroups, including AG-2-1 as the most closely related. Moreover, a similar conventional PCR assay showed the same specificity but proved to be at least a 100 times less sensitive. Future research will include further testing and adaptation of this protocol for direct detection and quantification of R. solani AG-2-2 in different substrates, including plant tissue and soil samples.


INTRODUCTION
Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop in European regions with moderate climate where it is grown for its high sugar content (Wibberg et al., 2016). As a crop with high monetary return (Wolf & Verreet, 2002), sugar beet was in 2016 cultivated on over 1.7 million ha in the EU (http:/fao.org/faostat). In Serbia, sugar beet is also considered to be a valuable and profitable crop. In 2017 it was grown on almost 50,000 ha (www.stat.gov.rs). Root diseases of sugar beet, most frequently caused by Rhizoctonia solani, Macrophomina phaeseolina, Fusarium oxysporum, Pythium spp., Phytophthora spp., and other agents, are an important constraint to profitable production worldwide, causing yield reductions of up to 50% (Jacobsen, 2006).
In Europe and the USA, the most important sugar beet root diseases are caused by worldwide distributed, soil-borne Rhizoctonia spp., subdivided into numerous anastomosis groups and subgroups (Ogoshi, 1976). Several multinucleate and binucleate Rhizoctonia have been described as sugar beet pathogens. In some sugar beet growing regions, binucleate AG-A, AG-E and AG-K (Strausbaugh et al., 2011;Miles et al., 2013), as well as multinucleate R. solani AG-4, AG-5 and AG-8 have been involved in the Rhizoctonia root rot disease complex (Olaya & Abawi, 1994;Strausbaugh et al., 2011), while multinucleate R. solani AG-2-2 is frequently detected as the main causal agent (Kiewnick et al., 2001;Jacobsen, 2006;Bolton et al., 2010, Abbas et al., 2014. Symptoms related to Rhizoctonia infections of sugar beet mainly include seedling damping-off and root rot with a characteristic presence of circular, necrotic lesions covering large portions of root (Harveson et al., 2009), as well as common above-ground symptoms of wilting and leaf chlorosis. Long-term persistence of R. solani is secured over many years via mycelium and sclerotia in crop debris. Under favorable conditions, sclerotia germinate to infect sugar beet roots (Harveson et al., 2009;Kiewnick et al., 2001;Jacobsen, 2006).
Managing Rhizoctonia root rot of sugar beet is difficult due to the soil-borne nature and wide host range of the pathogen (Allen et al., 1985;Kiewnick et al., 2001). Considering the known differences among AGs regarding host range and fungicide sensitivity, reliable and fast detection and identification of Rhizoctonia AG associated with root rot of sugar beet is the essential first step in developing a successful disease management strategy (Buhre et al., 2009;Lakshman et al., 2016). Determination of AG composition of Rhizoctonia spp. by morphology-based identification or by isolation, followed by sequencing and phylogenetic analyses, is labor-intensive and timeconsuming. Protocols for specific detection based on conventional (end point) PCR are available for several AGs, such as R. solani AG-1, AG-2 (Matsumoto, 2002;Salazar et al., 2000) and AG-3 (Lees et al., 2002). Realtime PCR based on specific detection is also available for several Rhizoctonia AGs, such as R. solani AG-1-IA (Sayler & Yang, 2007), AG-3 (Lees et al., 2002), AG-2-1 (Sturrock et al., 2015), AG-2-2 (Abbas et al., 2014), and AG-8, AG-10, AG-I, and genotypes of R. oryzae (Okubara et al., 2008).
In Serbia, root rot is one of the most important diseases of sugar beet. Until 2000, Fusarium spp. and Macrophomina phaesolina had been considered the most prevalent pathogens of sugar beet in Serbia (Marić et al., 1970). Since then, R. solani has been reported to be widely distributed (Stojšin et al., 2006), causing quantitative and qualitative reductions in yield of sugar beet (Vico et al., 2006). First data on molecular characterization confirmed the presence of R. solani AG 4-HGII (Stojšin et al., 2007) and R. solani AG 2-2 (Stojšin et al., 2011). As population diversity of Rhizoctonia spp. in any particular region may change due to different factors (Fenille et al., 2002;Hua et al. 2014), the main objectives of this study were to: (i) characterize Rhizoctonia isolates infecting sugar beet in Serbia based on morphological features and AG pairing; (ii) identify isolates by sequencing their ITS region of rDNA (ITS1, 5.8S rDNA and ITS2 regions), and (iii) develop a specific and sensitive conventional and real time PCR protocols for detection and identification of R. solani which is pathogenic to sugar beet in Serbia.

Fungal isolation
Isolates of Rhizoctonia were obtained from symptomatic sugar beet plants collected in the localities of Vašica and Adaševci (Srem District, Serbia) during 2015. Up to 10 randomly distributed samples with symptoms were collected per each locality, then stored at 5 o C and processed within 24-48 h. Tissue fragments from the border between necrotic and healthy root tissue were thoroughly washed with tap water for 2 h, surface sterilized for 2-3 min with 50% commercial bleach (2 % sodium hypochlorite), placed on potato dextrose agar (PDA; 200 g potato, 20 g dextrose, 17 g agar and 1 liter of distilled H 2 O) and incubated at 24 o C for 3-5 days. Rhizoctonia-like colonies were transferred onto fresh PDA and water agar (WA, 17 g agar and 1 liter of distilled H 2 O) in order to obtain hyphal tip isolates. The isolates were maintained on PDA slants at 4 o C in the Fungal Collection of the Department of Phytopathology, Institute of Phytomedicine, University of Belgrade -Faculty of Agriculture.

Morphological identification and AG determination
Morphological characterization of Rhizoctonia isolates was based on colony appearance and growth rate, as well as hyphal branching pattern and number of nuclei present in young hyphae. Colony appearance was assessed in 7-day old cultures grown on PDA at 24 o C in darkness, while growth rate was determined by measuring two perpendicular colony diameters in five cultures per isolate, and calculating an arithmetic average for each isolate. For the growth rate comparison, Student's t-test at 5% and 1% levels were performed (Sokal & Rohlf, 1995). Hyphal branching pattern was observed directly using a compound microscope Olympus CX41. The number of nuclei within hyphal cells was determined using a modified clean slide technique (Kronland & Stranghellini, 1988) after staining with aniline blue in lactophenol or safranin O (Herr, 1979).

Pathogenicity testing
Pathogenicity of two selected Serbian sugar beet Rhizoctonia isolates (270-15 and 275-15) was tested using inoculations of non-wounded sugar beet seedlings. Superficially sterilized commercial seeds of sugar beet were placed on PDA slants in 20 cm glass tubes and incubated at 23-25 o C in a cycle of 12 h light/12 h darkness. After seed germination and cotyledon development, mycelial plugs (5 mm in diameter) from 7-day old cultures were placed on the roots (mycelial surface face down), while seedlings inoculated with sterile PDA served as negative control. The pathogenicity of the isolates was estimated 7 days post inoculation (dpi). Each isolate was inoculated onto five seedlings, and the experiment was repeated twice. Re-isolations from symptomatic seedlings were performed using the same methods as for fungal isolation.

DNA amplification and sequencing
For molecular identification of two selected Rhizoctonia isolates (270-15 and 275-15), total genomic DNA was extracted from 100 mg of dry mycelium from 5-7-day old cultures grown in potato dextrose broth (PDB) by the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. The selected genome region ITS rDNA was amplified using the primers ITS1-F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990) (Table  1). Amplification reactions were performed in a total reaction volume of 25 μl, consisting of 12.5 μl 2 × PCR Master mix (Fermentas, Lithuania), 9 μl RNase-free water, 2.5 μl of both forward and reverse primers (100 pmol/μl, Metabion International, Deutschland) and 1 μl template DNA. Amplification conditions were as follows: initial denaturation step of 2 min at 95°C, followed by 35 cycles of 35s at 94°C, 1 min at 52°C and 2 min at 72°C, with final extension period of 10 min at 72°C. PCR products were stained in ethidium bromide, analyzed by 1% agarose gel electrophoresis and visualized using a UV transilluminator. The PCR products were ATA AAT TGG GTT TAT ATT AGA GTT GAG TAG ACA Budge et al., 2009 sequenced in both directions in an automated sequencer (ABI 3730XL Automatic Sequencer Macrogen Inc., Korea), using the same primers as for the amplification. Consensus sequences were computed using the ClustalW (Thompson et al., 1994), integrated in MEGA6 software (Tamura et al., 2013), and deposited in the GenBank (http://www.ncbi.nlm.nih.gov). Generated sequences were compared with each other by calculating nucleotide (nt) identities, as well as with previously deposited Rhizoctonia spp. isolates available in the GenBank, using the similarity search tool BLAST.

Conventional and real-time PCR detection
Protocols for specific detection of Rhizoctonia AG-2-2 based on conventional and real-time PCR were developed and compared. Both protocols are based on a previously published specific primer pair (Table 1) designed for detection of AG-2-2 using TaqMan chemistry (Budge et al., 2009).
Conventional PCR was performed in a total reaction volume of 5 μl, consisting of 2.5 μl 2 × PCR Master mix (Fermentas, Lithuania), 1.3 μl Nuclease-free water, 0.5 μl of both forward and reverse primers (100 pmol/μl, Metabion International, Deutschland) and 0.2 μl template DNA. Amplification conditions were as follows: initial denaturation step of 4 min at 94°C, followed by 35 cycles of 1 min at 95°C, 1 min at 49°C and 2 min at 72°C, with final extension period of 10 min at 72°C. PCR products were stained in ethidium bromide, analyzed by 1% agarose gel electrophoresis and visualized using a UV transilluminator.
Real-time PCR was conducted using a magnetic induction MIC qPCR cycler (Bio Molecular Systems, Australia) in the total reaction volume of 5 μl, containing 0.2 μl of target DNA, 2.5 μl FastGene IC Green 2x qPCR Universal mix (Nippon Genetics Europe Gmbh), 0.5 μl of both forward and reverse primers (100 pmol/ μl, Metabion International, Deutschland), and 1.3 μl Nuclease-free water. The amplification program consisted of 95°C for 2 min, followed by up to 40 cycles consisting of 95°C 15 s, 60°C 30 s, and 72°C 2 s. Fluorescence was monitored after each annealing step. Amplicon melting profiles were generated by increasing the temperature of the reaction from 72 to 95°C (0.3 0 C/s).
Specific detection using both protocols was tested against 10 different anastomosis groups and subgroups of binucleate (AG-A, AG-G, AG-F and AG-U), as well as multinucleate (AG-2-1, AG-3, AG-4-HGI, AG-4-2, AG-6 and Waitea) Rhizoctonia isolates from the Fungal Collection of the Department of Phytopathology, Institute of Phytomedicine, University of Belgrade -Faculty of Agriculture. The sensitivity of both conventional and real-time PCR were compared using 10 -1 to 10 -5 dilutions of target DNA. All samples in all experiments were amplified in duplicates or triplicates and all PCRs included NTCs (no template control).

Disease symptoms, pathogenicity and conventional identification
During 2015, samples were collected from two sugar beet crops with root rot disease incidence estimated at 30%. Only Rhizoctonia-like isolates were recovered from plants exhibiting typical symptoms of Rhizoctonia root rot (Fig 1A, B). A total of 17 hyphal-tip isolates from both localities (8 and 9 isolates from localities of Vašica and Adaševci, respectively) had uniform morphological features, and one isolate from each locality was selected for further studies, i.e. isolates designated as 270-15 (locality of Vašica) and 271-15 (locality of Adaševci) ( Table 2).
Pathogenicity testing revealed that both Serbian Rhizoctonia isolates were highly pathogenic, causing prominent root necrosis 7 dpi on all inoculated sugar beet seedlings. Control seedlings showed no symptoms. Isolates were successfully recovered from all symptomatic seedlings thus fulfilling Koch's postulates.

Molecular identification
Successful amplification of the ITS region of rDNA of both Serbian Rhizoctonia isolates resulted in obtaining amplicons of the predicted size of 700 bp, and after sequencing, manually edited consensus sequences of the isolates 270-15 and 271-15 were deposited in the GenBank database (Acc. Nos. MK123309 and MK123311, respectively). Sequence analyses revealed that the isolates share 99.6% similarity (with 3 bp differences), while BLAST analysis revealed the highest nt sequence homology of 99% (100% query coverage) of Serbian sugar beet isolates with over 50 sequences of R. solani mainly AG-2-2 but also with some of related AGs from different parts of the world and different host plants. Preliminary phylogenetic analyses (data not shown) confirmed that both Serbian sugar beet isolates belonged to R. solani AG-2-2.

Conventional and real time PCR specific detection
Using extracted DNA from pure cultures as the template, both conventional and real-time PCR protocols specifically amplified only the isolates of R. solani AG-2-2, exhibiting high specificity of both protocols. Using real-time PCR, the isolates 270-15 and 271-15 Rhizoctonia AG-2-2 were detected as early as in the 8-10 th cycles, which is at least 15 cycles earlier than any other detectable amplification (Figure 3). Melting temperatures of Tm 81.3 o C and 81.27 o C, respectively, provided clear differentiation from any other test Rhizoctonia AG at amplification, thus additionally confirming specific detection. Sensitivity of the conventional vs. real-time PCR differed significantly ( Figure 4). Real-time PCR detection was successful in all tested dilutions including the highest of 10 -5 , while no products were visible in conventional PCR after amplification of 10 -3 dilution and higher.
Rhizoctonia spp. are considered to be some of the most destructive sugar beet pathogens in Serbia and worldwide and so far the presence of R. solani AG-4 and AG-2-2 has been confirmed in Serbia (Stojšin et al., 2011). During 2015, we collected samples from two sugar beet crops with disease incidence estimated at 30% and plants exhibiting damping-off of sugar beet seedlings and roots, crown rot, and foliar blight. Two selected isolates representing a group of hyphal tip isolates from each of the localities were identified and characterized and only the presence of R. solani AG-2-2 was detected. In the majority of sugar beet production areas in Europe, R. solani AG-2-2 is also the most frequently isolated AG (Buddemeyer et al., 2004;Skonieczek et al., 2016) and it is considered to be favored by narrow crop rotation (Ithurrart et al., 2004).
Serbian isolates obtained in this study exhibited uniform morphological features consistent with published R. solani AG-2-2 isolates (Misawa et al., 2015). In pathogenicity testing, our isolates exhibited uniform pathogenicity with no differences in symptom appearance or intensity, and caused seedling necrosis similar to USA and Japan isolates (Carling et al., 2002;Figure 4. Primer sensitivity test for Rhizoctonia solani AG-2-2: template DNA dilutions of 0, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 of R. solani AG-2-2 Serbian isolate 270-15 and non-target control, amplified using conventional PCR (A) and real-time PCR (B). Bolton et al., 2010). Conventional identification by successful AG pairing with a known tester of AG-2-2 isolate, designated our isolates to be R. solani AG-2-2, which was further confirmed by ITS sequencing and sequence analyses. Two isolates originating from different localities and sugar beet crops exhibited a low level of 0.4% nt difference (3 bp), which correlates with a previously detected level of variability within the AG-2-2 population (Carling et al., 2002). Fast, specific and sensitive Rhizoctonia detection and AG identification is important and needed in practical, every day disease diagnosis, as well as in decision-making support for implementing control measures. In this paper we report a highly specific and sensitive real-time PCR protocol for the detection of R. solani AG-2-2 in which results can be obtained in a couple of hours. The use of already published primers for specific detection of R. solani AG-2-2 (Budge et al., 2009), and modification of the protocol by using SYBR Green chemistry instead of TaqMan significantly reduced the cost of analyses per sample. All real-time PCR reactions were conducted in a MIC thermal cycler in small reaction volumes of 5 μl recommended by the producer which additionally contributed to cost-effectiveness of the analyses. Abbas et al., (2014) also designed primers for detection of R. solani AG-2-2 in conventional, as well as in realtime PCR, with some limitations in amplicon size or cross-reactivity with closely and distantly related fungi including some Fusarium species. Budge et al. (2009) reported that primers designed in their research, which we also used in our protocols, exhibited a low level of cross-reactivity with R. solani AG-2-1. Nevertheless, in all our experiments, primers showed a high level of specificity after testing against 10 different anastomosis groups and subgroups, including the AG-2-1 isolate from our collection. Moreover, conventional PCR assay with the same primers showed in our study the same specificity but proved to be at least 100x less sensitive, probably due to the previously described limitation of post PCR visualization of amplicons (Lees et al., 2002).
Real-time PCR is a tool which can be successfully used for the fast and specific detection of Rhizoctonia at the species and AG levels, as well as for quantification of a wide range of pathogen DNA concentrations (Lees et al., 2002;Lievens et al., 2006). In this paper we are proposing a sensitive and specific real-time PCR protocol for direct identification and detection of R. solani AG-2-2. Future research will include further testing and adaptation of this protocol for direct detection and quantification of R. solani AG-2-2 in different substrates, including plant tissue and soil samples.