SJAR Spanish Journal of Agricultural Research SJAR 2171-9292 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) 13789 10.5424/sjar/2019172-13789 Research article Identification and pathogenicity of Rhizoctonia solani AG-4 causing root rot on chickpea in Turkey Basbagci Gurkan Directorate of Plant Protection Research Institute Bornova, Izmir, Turkey. Unal Filiz Directorate of Plant Protection Central Research Institute Ankara, Turkey. Uysal Ayse Directorate of Plant Protection Research Institute Bornova, Izmir, Turkey. Dolar Fatma S. Ankara University, Faculty of Agriculture, Dept. Plant Protection, 06110, Dışkapı, Ankara, Turkey. should be addressed to Gurkan Basbagci: gurkanbasbagci07@hotmail.com 01 06 2019 2019 17 2 10.5424/sjar/2019172-13789 03 08 2018 28 05 2019 © 2019 INIA 2019 This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC-by 4.0) License. Abstract

In the 2016-17 growing seasons, surveys were conducted in the Isparta, Uşak, KÜtahya and Denizli provinces of Turkey to identify the Rhizoctonia solani AG-4 associated with root and crown rot of chickpea. A total of 75 isolates of Rhizoctonia were obtained from surveyed areas. Visual diagnostic, isolation and microscopic observation identified the causal organism as R. solani . Sequence data of the ITS rDNA region confirmed the species identity and revealed that the anastomosis group of the 23 isolates were AG-4 HGII. The isolates were variable in their morphological characters. The sequences generated during this study were clustered in the same branch with the reference isolates of R. solani AG-4 HGII based on their ITS sequencing on chickpea and the isolate grouping was not related to their geographic origins or virulence pattern. Pathogenicity tests revealed that all AG-4 isolates were pathogenic on chickpea and the disease severity values of 23 isolates varied between 42.8% and 100%. Based on the virulence, the isolates were grouped into two categories: 5 of them exhibited moderately virulence and 18 of them exhibited highly virulence reaction on chickpea. The high virulent isolate level (>50% disease severity) was determined as 78.2% of all 23 isolates. This is the first report of R. solani AG-4 as a pathogen of chickpea in Turkey.

Additional key words: anastomosis group; ITS sequence; morphological characters; phylogenetic tree; radical assay. Additional key words: AG (anastomosis group); DS% (percentage of disease severity); HV (highly virulent); ITS (internal transcribed spacer); LSD (least significance difference); LV (less virulent); MV (moderately virulent); PCR (polymerase chain reaction); PDA (potato dextrose agar). Ministry of Food, Agriculture and Livestock, Republic of Turkey (TAGEM/BSAD/16/1/02/05).

Author's contributions: Conception and design, critical revision of the manuscript for important intellectual content: GB, FSD. Data acquisition and interpretation: GB, AU, FSD. Coordinating the research project: FSD. Technical and material support: FÜ, AU. All authors read and approved the final manuscript.

Citation Basbagci, G.; Unal, F.; Uysal, A.; Dolar, F. S. (2019). Identification and pathogenicity of Rhizoctonia solani AG-4 causing root rot on chickpea in Turkey. Spanish Journal of Agricultural Research, Volume 17, Issue 2, e1007. https://doi.org/10.5424/sjar/2019172-13789

Competing interests: The authors have declared that no competing interests exist.

Introduction

Chickpea ( Cicer arietinum L.) is one of the most extensively grown legume crops in Turkey. However, chickpea production and yield level are limited because of biotic and abiotic stress factors in Turkey. Root rot pathogens are very important within biotic factors including plant diseases. To date, more than 50 pathogens have been reported on chickpea from different parts of the world. However, only a few of them cause serious economic losses, such as Ascochyta blight caused by Ascochyta rabiei (Pass.) Labr., Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris (Pad.), and root rot caused by a number of fungi, including Rhizoctonia bataticola (Taub.) Butler [ Macrophomina phaseolina (Tassi) Goid] and Rhizoctonia solani Kuhn [teleomorph: Thanatephorus cucumeris (Frank)] ( Nene & Reddy, 1987 ; Dolar, 1996 ; Bayraktar & Dolar, 2009 ). For management of the root rot pathogens firstly it is necessary to determine the disease agents which dominate and destroy the plant. Rhizoctonia species, causal agent of root rot of chickpea are wide spread on chickpea crops in the world where it is reported to cause considerable damage ( Hwang et al. , 2003 ; Gonzalez et al. , 2006 ). The dry root rot of chickpea caused by necrotrophic fungus R. bataticola has emerged as a serious threat to the chickpea production worldwide. If the plant is exposed to moisture stress conditions, the disease severity is higher ( Sharma et al. , 2010 ). The disease generally appears during late flowering and podding stages and the infected plants appear completely dried ( Pande et al. , 2004 ). R. solani is a soil and seed borne pathogen which has a wide host range and causes various diseases in important agricultural and horticultural crops due to its polyphagic nature and high saprophytic ability ( Anderson, 1982 ; Nelson et al. , 1996 ). R. solani , which causes wet root rot, can be seen in early periods when soil moisture is high, and it can cause infection in every growth stage of a plant. It usually causes root rot which starts on the tip of young roots, and gradual yellowing and wiltings of the leaves ( Dubey & Dwivedi, 2000 ). Rhizoctonia is typically a sterile fungal genus and has been characterized by division into binucleate and multinucleate groups. Hyphal anastomosis concept was introduced by Parmeter & Whitney (1970) for identification and characterization of Rhizoctonia isolates. Ogoshi (1987) classified R. solani primarily based on anastomosis behaviour; at present, 14 anastomosis groups (AGs) are recognised: AG-1-13 and AG-BI, and some that include several subgroups and isolates of R. zeae and R. oryzae have been assigned to WAG-Z and WAG-O, respectively ( Sneh et al. , 1991 , 1996 ; Carling et al. , 1999 , 2002 ; Yang & Li, 2012 ). Various molecular markers have been used for characterization and grouping of Rhizoctonia species. The genetic diversity of Rhizoctonia isolates has been studied using RAPD-PCR, SSR-PCR, rDNA-RFLP, rDNA-ITS sequence analysis, universally primed-PCR and rep-PCR ( Sharon et al. , 2008 ). Currently, the rDNA-ITS sequence analysis is the most appropriate method for classification of Rhizoctonia spp. and sequence analysis of the ITS-5.8S rDNA region has been used as a suitable molecular tool for identification of R. solani subgroups ( Hyakumachi et al. , 1998 ; Salazar et al. , 2000 ; Priyatmojo et al. , 2001 ; Carling et al. , 2002 ). Genetic heterogenicity between, and within, anastomosis groups was evaluated by Fenille et al. (2003) , using sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA. Comparison of the ITS region is significant in the determination of anastomosis groups, and also these sequences are useful for verifying subsets. Ganeshamoorthi & Dubey (2013) firstly used ITS region to determine anastomosis grouping of R. solani isolates from chickpea by using ITS1 and ITS4 primers. Different anastomosis groups such as AG-1, AG-2-2, AG-2-2LP, AG-2-3, AG-3, AG-4 and AG-5 were reported on chickpea in the world ( Dubey et al. , 2011 ). Chickpea is sensitive to infection by several anastomosis groups (AG-4 and AG-5) ( Hwang et al. , 2003 ; Dubey et al. , 2011 ; Ganeshamoorthi & Dubey, 2015 ). The presence of pathogenic isolates of AG-4 of R. solani in chickpea has been reported by many researchers in different country ( Hwang et al. , 2003 ; Dubey et al. , 2011 , 2014 ; Ganeshamoorthi & Dubey, 2013 , 2015 ). In Turkey, isolates of R. solani AG-5 have already been determined to be pathogenic on chickpea ( Tuncer & Erdiller, 1990 ; Demirci et al. , 1998 ), but so far the presence and pathogenicity of R. solani AG-4 have been not reported.

The objectives of the present study were to (i) identify and characterize the R. solani AG-4 isolates associated with root and crown rot of chickpea in Turkey, and (ii) determine the pathogenicity of the isolates.

Material and methods Survey and fungal isolation

Survey studies were conducted in 2016 and 2017 during the chickpea production seasons in the Isparta, Uşak, KÜtahya and Denizli provinces in Turkey (Table 1, Fig. 1).

Location of survey area in Turkey and number of samples (2016-17 growing seasons). Location of survey area (orange) in Turkey.

During these surveys, the infected plants, which had dark-brown lesions on the roots and crown, were collected. Depending on the size of the fields, at least 5 plants were collected from each field. To isolate the pathogen, root parts of diseased plants were washed in tap water and dried on sterile blotter paper. Symptomatic root and crown tissues were cut into 0.2-0.5 cm pieces, then surface-sterilized with 1% sodium hypochloride (NaOCl) for 2-3 min. After rinsing in sterile distilled water five times and subsequently drying on blotter paper, 5 or 6 tissue pieces were placed in Petri dishes containing potato dextrose agar (PDA, Merck) and amended with streptomycin sulphate (50 mg/L). Petri dishes were incubated at 23±1°C with a 12-h photoperiod for 7-10 days. For the purification of the isolated fungus, single hyphal tips were transferred onto new PDA plates from the fungal cultures on 1.5% water agar. Purified isolates were stored on PDA, filter papers in eppendorf tubes at +4°C and colonized wheat seed at -20°C.

 Morphological characterization and AG typing of isolates

All isolates were characterized based on cultural mor­phology, cellular nuclei number in young vegetative hyphae, and anastomosis reactions with tester isolates. Cultural characteristics were determined by growing each isolate on PDA and incubating at 25°C for 14 days ( Sneh et al. , 1991 ). Cultures were examined for colony color, sclerotia formation and color, and aerial mycelia formation. Observations were recorded visually by the method described by Burpee et al. (1980) . Nuclear condition of isolates was determined using the Safranin O staining technique ( Bandoni, 1979 ). For anastomosis groups determination, the slide technique coupled with Safranin O staining was used for observing hyphal fusion reactions between testers and unknown isolates ( Kronland & Stanghellini, 1988 ). Briefly, Rhizoctonia isolates and testers initially were grown on PDA at 25 °C in the dark. A sterilized coverslip was coated with a thin layer of 0.5% PDA and placed on Petri dishes containing 1.5% water agar medium. Agar disks of Rhizoctonia isolates and the tester isolates were cut from the growing edge of the plate and transferred to opposite ends of the coverslip on the water agar plates. After incubation at 25°C for 24-72 h in the dark, when overlapping mycelia of two isolates were observed, the coverslip was removed from the plate and placed on a microscope glass slide, and stained with safranin O and 3% KOH. Sites of hyphal interaction were examined under a light microscope and occurrence of anastomosis was determined when the hyphae of paired isolates were fused and exchanged cytoplasms. Reaction types observed between interacting hyphae were assigned to one of the four categories (C0, C1, C2 or C3) described by Carling et al. (1988) . For the anastomosis testing, pairing for each unknown isolate was replicated three times.

ITS-rDNA gene sequencing

For molecular characterization, each isolate was grown on PDA (Merck) at 25±1°C. After 5-7 days, about 300 mg mycelium was harvested by removing excess of the solid media using a sterile scalpel, and stored at -20°C until used. Genomic DNA was extracted using the Plant/Fungi DNA Isolation Kit (Norgen, Biotek) following the manufacturer's recommendations for fun­­gal DNA isolation. The ITS region of the rDNA was amplified using ITS 1 (5' TCC GTA GGT GAA CCT GCGG 3') and ITS 4 (5' TCC TCC GCT TAT TGA TATGC 3'), primers described by White et al. (1990) . The PCR reactions were carried out in a 50 µL final volume containing 25 µL of PCR Master Mix (Norgen Biotek Corporation, Canada), 2 µL of each primer (concentration 10 pmol/µL), 5 µL of DNA template and 16 µL of PCR-grade water. The DNA amplifications were performed in a thermocycler (Eppendorf AG, Hamburg, Germany) using the following cycle para­meters: initial denaturation at 95°C for 2 min; 35 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s; and a final extension step at 72°C for 5 min. Following the PCR reaction, the amplified products were loaded in a 1.5% agarose gel stained with GelRed, together with 100 bp DNA marker (Norgen Biotek Corporation, Canada). Before loading, both samples and marker were stained with Blue/Orange 6X Loading Dye (Norgen Biotek Cor­poration, Canada) used for tracking migration du­ring electrophoresis. Electrophoresis was run at 100 V for 1 hour. The DNA bands were visualised using a Quantum Capt. ST4 Imaging System (Quantum Corporation, France). They were sequenced by ALTIGENBIO Life Science (International Biotechnology Company, Izmir, Turkey). ITS sequence analysis was performed using BLAST via http://www.ncbi.nlm.nih.gov . Sequences of each isolate were deposited in GenBank (see accession numbers in Table 3).

Pathogenicity tests

According to the results of AG typing and ITS-rDNA gene sequencing, isolates identified as AG-4 of R. solani were tested for pathogenicity by radicle assay in petri dishes under in vitro conditions, then virulence scales were determined based on the percentage of disease severity. The isolates were allowed to incubate at 25±2°C for 10-15 days, then mycelial discs were taken with a 4 mm diameter cork borer from an actively growing edge of the fungal culture. Each piece was placed in the center of a petri plate containing 2% water agar and incubated at 25±2°C for 48 h. Susceptible chickpea cultivar seeds (cv. ILC 482) were immersed in 1% NaOCl for 5 min for surface disinfection and washed with sterile distilled water 3 times. Then 7 seeds were placed at equal distances around the fungus piece. As a control treatment, seeds were placed around the sterile PDA discs. All petri plates were wrapped with parafilm and then incubated at 25±2°C with 12 h photoperiod for 10-12 days. Subsequently, by examining the hypocotyls of germinated seeds, the disease development was assessed according to the scale of 0 to 5 (Fig. 2) based on the size of the necrotic area in the hypocotyl ( Ichielevich-Auster et al. , 1985 ). Three plates were used for each isolate.

The 0-5 scale used in disease development assessment: 0=healthy, 1=1-10% infection of hypocotyls, 2=11- 30% infection of hypocotyls, 3=31-50% infection of hypocotyls, 4=51-80% infection of hypocotyls and 5=plant dead.

For evaluation of each isolate, scale values were given to all the seeds in each replicate. No scale value was given as no infections were seen in the control treatment. Percentage of disease severity (DS%) was calculated from the Townsend-Heuberger's (1943) formula based on scale values obtained by pathogenicity tests.

According to the results of the in vitro tests, pathogenicity was also tested on seed in pots with the most virulent isolate on susceptible chickpea cultivar (cv. ILC482). Inoculum was prepared on the sterile wheat grains in tests tubes. The mixture of perlite: silt loam+fine sand (2:1) was sterilized and filled in each plastic pot (13 cm in diameter). Susceptible chickpea cultivar seeds were immersed in 1% NaOCl for 5 min for surface disinfection and washed with sterile distilled water three times. Five chickpea seeds were placed in each pot and five pathogen-colonized wheat grains were placed near the seeds to serve as inoculum. The control consisted of pots without inoculum. There were five replicate pots per treatment. The pots were incubated at 25±2°C with 12 h photoperiod for 10-12 days in a growth chamber. Re-isolations were made from the roots of diseased plants to confirm the identity of the causal agent.

 Data analyses

Radicle assay test for pathogenicity was carried out in a completely randomized design of three replicates. For all pathogenic isolates, the mean DS% was used to categorize their relative virulence, where isolates were categorized as highly virulent (HV) if they showed a mean DS% between 50.1 and 100%, moderately virulent (MV) between 20.1 and 50%, and less virulent (LV) between 0 and 20%.

Percentage data was transformed into angular va­lues to produce an approximately constant variance before carrying out the analysis of variance (ANOVA). The statistical significance was assessed at p < 0.05 and Fisher's least significance difference (LSD) test was used to separate means. Statistical analyses were performed using the JMP 14.0 (SAS Institute, Cary, NC, USA) software package. A phylogenetic tree of the R. solani isolates was constructed based on ITS sequencing using MEGA 10.0.5 via the neighbor-joining bootstsap method. The tester isolate of R. solani AG-4 which was obtained from Japan and the reference isolates of R. solani AG-4 HGI (AY15270­4.1, AB000007.1), HGII (AY154308.1, AB000006.1) ( Kuninaga et al. , 1997 ; Kuramae et al. , 2003 ) and HGIII (DQ102449.1, AY154659.1) ( Kuramae et al. , 2003 ; Sharon et al. , 2007 ) were also used on the tree for comparison with the isolates generated in this study.

Results Morphological and molecular characterization of isolates

Two-hundred-sixty-eight plant samples were collec­ted from chickpea growing areas in the Isparta, Uşak, KÜtahya and Denizli provinces in Turkey (Table 1). A total of 75 isolates of Rhizoctonia were obtained from damaged tissues and twenty-three of them were identified as R. solani AG-4 according to cultural morphology and anastomosis reactions with tester isolates. Microscopic inspection of twenty-three R. solani AG-4 isolates revealed a buff-colored to dark-brown, regularly septate mycelium with a slight constriction at the septum. Each hypha cell contained more than two nuclei (Fig. 3a).

Multinucleate hyphal cell (a) and colony growth of <italic>R. solani</italic> AG-4 on PDA (b), C1 anastomosis reaction (contact) between hyphae (c), and C3 anastomosis (hyphal fusion) reaction between hyphae (d).

The color of the colony varied from whitish brown (Fig. 3b) to dark brown. Among the 23 isolates studied, 4 isolates were whitish brown, 16 isolates were light brown and 3 isolates were dark brown. On the basis of growth pattern, 5 isolates produced abundant mycelium, while 11 isolates had moderate mycelium and the remaining 7 isolates recorded slight mycelium. Although 13 isolates produced sclerotia, 10 isolates did not. Among the 13 isolates that produced sclerotia, 9 of them showed light brown sclerotia color while the rest of them were dark brown. Based on the formation of sclerotia, 8 of them had scattered form, 4 of them had central form and only 1 of them had peripheral form (Table 2). On the other hand, C1 and C3 anastomosis reactions were observed between AG-4 isolates and test isolates diagnosed in anastomosis reaction studies (Fig. 3c, d).

Cultural and sclerotial characteristics and pathogenic behaviour of AG-4 isolates of <italic>R. solani.</italic>

After the pathogenicity tests, re-isolated fungi were characterized based on cultural morphology and anas­tomosis reactions with tester isolate of AG-4 of R. solani.

The ITS sequences of 23 isolates of R. solani , which belonged to AG-4 were recovered from the GenBank (Acc. no. MH231493 to MH231515). The sequences were compared to reference sequences of R. solani anastomosis groups in GenBank, and were found to be most similar to group AG-4, the intraspecific group HG-II at 99 to 100% identity (Table 3). DNA bands of 23 R. solani AG-4 isolates varied from 615 to 667 bp long.

ITS sequencing data of AG-4 isolates of <italic>R. solani.</italic>

The phylogenetic tree was constructed from the nucleotide sequence similarity of 23 isolates along with the tester isolate of R. solani AG-4 which was obtained from Japan and the reference isolates of R. solani AG-4 HGI, HGII and HGIII (Fig. 4). The sequences generated during this study were clustered in the same branch with the tester isolate of R. solani AG-4 (MK280743.1) and the reference isolates of R. solani AG-4 HGII. The reference isolates of R. solani AG-4 HGI and the reference isolates of R. solani AG-4 HGIII clustered seperately. Grouping the isolates based on their ITS sequencing was not related to their geographic origins or virulence patterns.

Neighbor-joining tree showing the phylogenetic relationship among isolates of <italic>R. solani</italic> AG-4 based on their ITS sequences. The tester isolate of <italic>R. solani</italic> AG-4 obtained from Japan were labelled square “■”; the reference isolates of <italic>R. solani</italic> AG-4 HGI, HGII and HGIII were labelled triangle “▲”. Pathogenicity of the isolates

According to pathogenicity test under in vitro conditions, all AG-4 isolates were found to be pathogenic on chickpea. The control seeds did not develop symptoms (Fig. 5). The results showed that the disease severity values of 23 isolates varied between 42.8% and 100% (Table 2).

Pathogenicity test: healthy hypocotyls in control plate (a), hypocotyl infection at 11 days after inoculation (b).

According to the virulence of isolates, 23 isolates of R. solani AG-4 were grouped into two categories: MV and HV. Five isolates were found to be MV and 18 isolates were HV on chickpea (Table 2). No isolate was in the 0-20% (LV) range. According to the values, HV isolate level (>50% disease severity) was determined as 78.2% of all 23 isolates.

The pathogenicity of U12, K9 and IS29 differed significantly ( p < 0.05) from the other isolates, while there were no statistically significant differences ( p > 0.05) among other isolates according to Fisher's LSD test (Table 2). Isolate U12 was the most virulent whereas isolate IS29 was the least virulent (100% and 42.8%, respectively). According to the pot trial results, U12 caused 100% pre-emergence damping-off in chickpea (Fig. 6).

Inoculated pot with isolate U12 (left) and control pot (right) (a); pre-emerging damping-off in chickpea (b). Discussion

A total of 75 isolates of Rhizoctonia were obtained from surveyed areas in Turkey. Twenty-three of them were multinucleate and belonged to anastomosis group 4 (AG-4 HGII). The isolates were variable in their morphological characters. Based on the colony color, 23 isolates of R. solani were assigned into three categories and most of them had a light brown colony color. Ganeshamoorthi & Dubey (2015) characterized 50 isolates in terms of cultural variability obtained from chickpea and among four isolates of AG-4 of R. solani two isolates showed light brown, while two isolates had dark brown colony color. In the present study, moderate growth pattern was the dominant character among the isolates. Sclerotia were central, peripheral or scattered, and light brown to dark brown. According to the study reported by Ganeshamoorthi & Dubey (2015) , three isolates of AG-4 showed dark and scattered, one isolate showed dark and peripheral sclerotia formation. These results were parallel with our findings.

The frequency of AG-4 in all isolates collected was found to be quite high with a value of 30.6% whereas Mikhail et al. (2010) , Ganeshamoorthi & Dubey (2013) , and Dubey et al. (2014) revealed that the frequency of AG-4 obtained from chickpea was 13.7%, 8% and 7% respectively. On the other hand, Hwang et al. (2003) determined that all the isolates obtained from chickpea belonged to AG-4 with a value of 100% frequency, which indicates that the frequency of R. solani AG-4 isolates obtained from chickpea varies in different countries.

The present study shows that ITS sequencing is a powerful tool in understanding and determinating the relationship between anastomosis groups and subgroups of R. solani . All isolates belong to AG-4 HGII at 99 to 100% identity. DNA bands of 23 R. solani AG-4 isolates varied from 615 to 667 bp long in this study. Dubey et al. (2014) reported that R. solani isolates were variable respect to their nucleotide sequences of ITS region (650-750 bp). According to Ganeshamoorthi & Dubey (2013) , ITS region is very useful to study intra-specific diversity of the pathogen and helps in the development of species level diagnostic molecular markers. They found that, using ITS1 and ITS4 primers, 50 isolates of R. solani produced bet­ween 572 and 715 bp long bands.

In this study, the generated sequences were clustered into the same branch of the reference isolates of R. solani AG-4 HGII. However, grouping of the isolates was not related to their geographic origins or virulence pattern. Boysen et al. (1996) , also observed sequence variations in ITS region of nine R. solani isolates of AG-4. The method was also used to express the genetic similarity among 52 isolates of R. solani ( El-Samawaty et al. , 2008 ). Also, Ganeshamoorthi & Dubey (2013) analysed 50 R. solani isolates obtained from chickpea by the phylogenetic tree method and they grouped into two categories. In the present study, the findings clearly indicate that the chickpea populations of R. solani AG-4 are highly variable in their ITS region. The results are also in agreement with Kuninaga et al. (1997) , who found a highly variable ITS 5.8s rDNA sequence in 45 isolates of R. solani .

The results of the pathogenicity studies clearly show that all the AG-4 HGII isolates tested are pathogenic on chickpea. AG-4 of R. solani has also been reported many times to be pathogenic to chickpea worldwide ( Hwang et al. , 2003 ; Dubey et al. , 2011 , 2014 ; Ganeshamoorthi & Dubey, 2013 , 2015 ). According to the virulence of isolates, 23 isolates of R. solani AG-4 have been categorized into two groups. Five of them were MV and 18 of them were HV on chickpea. In this study, the isolates had high pathogenic behaviour on chickpea, with a value of 78.2% which was the HV category. This also supports the findings of Dubey et al. (2011) who found that the disease incidence caused by isolates, including AG-4, varied from 11 to 100% and the HV isolate level was 75.6% on chickpea. However, Ganeshamoorthi & Dubey (2015) found 4 out of 50 R. solani isolates which belonged to AG-4 and all of them were MV on chickpea. In the present study, as a result of the pathogenicity test in pots, isolate U12 showed pre-emergence damping-off with 100% disease severity value. Hwang et al. (2003) revealed that five isolates of R. solani collected from Saskatchewan (Canada) belonged to AG-4 and caused 100% pre-emergence damping-off in chickpea in the pathogenicity test. Results of these studies are in parallel with our findings.

So far, five AG groups (AG-1, AG-2, AG-3, AG-4 and AG-5) of R. solani have been detected in chickpea in the world ( Hwang et al. , 2003 ; Mikhail et al. , 2010 ; Youssef et al. , 2010 ; Dubey et al. , 2011 , 2014 ; Ganeshamoorthi & Dubey, 2013 , 2015 ). In Turkey, AG-4 of R. solani has been reported to be pathogenic to barley ( Demirci, 1998 ; Ünal & Kara, 2017 ), common bean ( Eken & Demirci, 2004 ; Kılıçoğlu & özkoç, 2013 ), pepper ( Tuncer & Eken, 2013 ), tomato ( Yıldız & Döken, 2002 ), soybean ( Erper et al. , 2011 ), cotton ( Kural et al. , 1994 ), Johnsongrass ( Demirci et al. , 2002 ) and wheat ( Demirci, 1998 ; Ünal et al. , 2015 ) but this group had not yet been reported in chickpea in Turkey. The present study shows then that both the morphological and molecular analyses of R. solani isolates indicate that this is the first identification of AG-4 on chickpea in Turkey.

Acknowledgements

The authors are thankful to Transitional Zone Agricultural Research Institute, Eskişehir, Turkey and Directorate of Plant Protection Research Institute, Bornova, Izmir, Turkey for technical support. The authors would like to thank Prof. Erkol Demirci (Karadeniz Technical University, Turkey) and Dr. A. Ogoshi (Hokkaido University, Japan) for the AG tester strains and for permission to include them in our analyses.

 References Anderson NA , 1982 . The genetics and pathology of Rhizoctonia solani. Ann Rev Phytopathol 20 : 329 - 347 . https://doi.org/10.1146/annurev.py.20.090182.001553 Bandoni RJ , 1979 . Safranin-O as a rapid nuclear stain for fungi. Mycologia 71 : 873 - 874 . https://doi.org/10.1080/00275514.1979.12021088 Bayraktar H , Dolar FS , 2009 . Genetic diversity of wilt and root rot pathogens of chickpea, as assessed by RAPD and ISSR. Turk J Agr For Sci 33 : 1 - 10 . Boysen M , Borja M , del Moral C , Salazar O , Rubio V , 1996 . Identification at strain level of Rhizoctonia solani AG-4 isolates by direct sequence of asymmetric PCR products of the ITS regions. Curr Genet 29 : 174 - 181 . https://doi.org/10.1007/BF02221582 Burpee LL , Sander PL , Sherwood RT , 1980 . Anastomosis group among isolates of Ceratobasidium cornigerum (Bourd) Rogers and related fungi. Mycologia 72 : 689 - 701 . https://doi.org/10.1080/00275514.1980.12021238 Carling DE , Kuninaga S , Leiner RH , 1988 . Relatedness within and among intraspecific groups of Rhizoctonia solani: A comparison of grouping by anastomosis and by DNA hybridization. Phytoparasitica 16 : 209 - 210 . Carling DE , Pope EJ , Brainard KA , Carter DA , 1999 . Characterization of mycorrhizal isolates of Rhizoctonia solani from an orchid, including AG-12, a new anastomosis group. Phytopathology 89 ( 10 ): 942 - 946 . https://doi.org/10.1094/PHYTO.1999.89.10.942 Carling DE , Baird RE , Gitaitis RD , Brainard KA , Kuninaga S , 2002 . Characterization of AG-13, a newly reported anastomosis group of Rhizoctonia solani. Phytopathology 92 ( 8 ): 893 - 899 . https://doi.org/10.1094/PHYTO.2002.92.8.893 Demirci E , 1998 . Rhizoctonia species and anastomosis groups isolated from barley and wheat in Erzurum, Turkey. Plant Pathol 47 ( 1 ): 10 - 15 . https://doi.org/10.1046/j.1365-3059.1998.00214.x Demirci E , Eken C , Kantar F , 1998 . Wilt and root rot pathogens of chickpea cv. "Aziziye-94". J Plant Pathol 80 2 175 Demirci E , Eken C , Zengin H , 2002 . First report of Rhizoctonia solani and binucleate Rhizoctonia from Johnsongrass in Turkey. Plant Pathol 51 391 https://doi.org/10.1046/j.1365-3059.2002.00704.x Dolar FS , 1996 . Survey of chickpea diseases in Ankara, Turkey. Int Chickpea Pigeonpea Newslet 3 : 33 - 35 . Dubey SC , Dwivedi RP , 2000 . Diseases of leguminous crops caused by Rhizoctonia solani and their management. Adv Publ Concept, New Delhi, India, 61 76 In: Advances in plant disease management; Udit N, Kumar K, Srivastava M (eds). Dubey SC , Tripathi A , Upadhyay BK , Thakur M , 2011 . Pathogenic behaviour of leguminous isolates of Rhizoctonia solani collected from different Indian agro-ecological regions. Ind J Agr Sci 81 : 64 - 69 . Dubey SC , Tripathi A , Upadhyay BK , Deka UK , 2014 . Diversity of Rhizoctonia solani associated with pulse crops in different agro-ecological regions of India. World J Microbiol Biotechnol 30 : 1699 - 1715 . https://doi.org/10.1007/s11274-013-1590-z Eken C , Demirci E , 2004 . Anastomosis groups and pathogenicity of Rhizoctonia solani and binucleate Rhizoctonia isolates from bean in Erzurum, Turkey. J Plant Pathol 86 ( 1 ): 49 - 52 . El-Samawaty AMA Amal A Asran MR Omar Abd-Elsalam KA 2008 Anastomosis groups, pathogenicity, and cellulase production of Rhizoctonia solani from cotton. Pest Technol 1 2 117 124 Erper İ , Karaca GH , özkoç İ , 2011 . Identification and pathogenicity of Rhizoctonia species isolated from bean and soybean plants in Samsun, Turkey. Archiv Phytopathol Plant Prot Abingdon, 44 : 78 - 84 . https://doi.org/10.1080/03235400903395427 Fenille RC , Ciampi MB , Kuramae EE , Souza NL , 2003 . Identification of Rhizoctonia solani associated with soybean in Brazil by rDNA-ITS sequences. Fitopatol Bras 28 ( 4 ): 413 - 418 . https://doi.org/10.1590/S0100-41582003000400011 Ganeshamoorthi P , Dubey SC , 2013 . Anastomosis grouping and genetic diversity analysis of Rhizoctonia solani isolates causing wet root rot in chickpea. Afr J Biotechnol 12 ( 43 ): 6159 - 6169 . https://doi.org/10.5897/AJB2013.12439 Ganeshamoorthi P , Dubey SC , 2015 . Morphological and pathogenic variability of R. solani isolates associated with wet root rot of chickpea in India. Legume Res 383 389 395 https://doi.org/10.5958/0976-0571.2015.00123.X González D , Cubeta MA , Vilgalys R , 2006 . Phylogenetic utility of indels within ribosomal DNA and beta-tubulin sequences from fungi in the Rhizoctonia solani species complex. Mol Phylogen Evol 40 : 459 - 470 . https://doi.org/10.1016/j.ympev.2006.03.022 Hwang SF , Gossen BD , Chang KF , Turnbull GD , Howard RJ , Blade SF , 2003 . Etiology and impact of Rhizoctonia seedling blight and root rot of chickpea on the Canadian prairies. Can J Plant Sci 83 : 959 - 967 . https://doi.org/10.4141/P02-165 Hyakumachi M , Mushika T , Ogiso Y , Toda T , Kageyama K , Tsuge T , 1998 . Characterization of a new cultural type (LP) of Rhizoctonia solani AG-2-2 isolated from warm-season turfgrasses, and its genetic differentiation from other cultural types. Plant Pathol 47 : 1 - 9 . https://doi.org/10.1046/j.1365-3059.1998.00212.x Ichielevich-Auster M , Sneh B , Koltin Y , Barash I , 1985 . Suppression of damping-off caused by Rhizoctonia species by a nonpathogenic isolate of R. solani. Phytopathology 75 : 1080 - 1084 . https://doi.org/10.1094/Phyto-75-1080 Kılıçoğlu MÇ , özkoç İ , 2013 . Phylogenetic analysis of Rhizoctonia solani AG-4 isolates from common beans in Black Sea coastal region, Turkey, based on ITS-5. 8S rDNA, Turk J Biol 37 : 18 - 24 . Kronland WC , Stanghellini ME , 1988 . Clean slide technique for the observation of anastomosis and nuclear condition of Rhizoctonia solani. Phytopathology 78 : 820 - 822 . https://doi.org/10.1094/Phyto-78-820 Kuninaga S , Natsuaki T , Takeuchi T , Yokosawa R , 1997 . Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani. Curr Genet 32 : 237 - 243 . https://doi.org/10.1007/s002940050272 Kural İ , Sagır A , Tatli F , 1994 . Characterization and pathogenicity of anastomosis groups of Rhizoctonia solani isolated from cotton in Southeastern Turkey. 9th Congr Mediterr Phytopath Union, Kuşadası Aydın, Turkey, 117 120 Kuramae EE , Buzeto AL , Ciampi MB , Souza NL , 2003 . Identification of Rhizoctonia solani AG 1-IB in lettuce, AG 4 HG-I in tomato and melon, and AG 4 HG-III in broccoli and spinach, in Brazil. Eur J Plant Pathol 109 : 391 - 395 . Mikhail MS , Sabet KK , Omar MR , Amal AA , Kasem KK , 2010 . Current Rhizoctonia solani anastomosis groups in Egypt and their pathogenic relation to cotton seedlings. Afr J Microbiol Res 4 : 386 - 395 . Nelson B , Helms T , Christianson T , Kural I , 1996 . Characterization and pathogenicity Rhizoctonia from soybean. Plant Dis 80 : 74 - 80 . https://doi.org/10.1094/PD-80-0074 Nene YL , Reddy MV , 1987 . Chickpea diseases and their control. CABWallingford, Oxon, UK, 233 270 In: The chickpea; Saxena, MC & Singh KB (eds.), Ogoshi A , 1987 . Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani KÜhn. Ann Rev Phytopathol 25 : 125 - 143 . https://doi.org/10.1146/annurev.py.25.090187.001013 Pande S , Kishore GK , Rao JN , 2004 . Evaluation of chickpea lines for resistance to dry root rot caused by Rhizoctonia bataticola. Int Chickpea Pigeonpea Newslet 11 : 37 - 38 . Parmeter JR , Whitney HS , 1970 . Taxonomy and nomenclature of the perfect state. Univ of California Press, Berkeley, USA 6 19 In: Rhizoctonia solani biology and pathology; Parmeter JR Jr (ed.), Priyatmojo A , Yamauchi R , Kageyama K , Hyakumachi M , 2001 . Grouping of binucleate Rhizoctonia anastomosis group d (ag-d) isolates into subgroups i and ii based on whole-cell fatty acid compositions. J Phytopathol 149 : 421 - 426 . https://doi.org/10.1046/j.1439-0434.2001.00652.x Salazar O , Julian MC , Rubio V , 2000 . Primer based on specific rDNA-ITS sequences for PCR detection of Rhizoctonia solani, R. solani AG 2 subgroups and ecological types, and binucleate Rhizoctonia. Mycol Res 104 : 281 - 285 . https://doi.org/10.1017/S0953756299001355 Sharma M , Mangla UN , Krishnamurthy M , Vedez V , Pande S , 2010 . Drought and dry root rot of chickpea 5th Int Food Legumes Res Conf (IFLRCV) & Eur Conf on Grain Legumes (AEP VII), Antalya, Turkey. Sharon M , Freeman S , Kuninaga S , Sneh B , 2007 . Genetic diversity, anastomosis groups and virulence of Rhizoctonia spp. from strawberry. Eur J Plant Pathol 117 : 247 - 265 . https://doi.org/10.1007/s10658-006-9091-7 Sharon M , Kuninaga S , Hyakumachi M , Naito S , Sneh B , 2008 . Classification of Rhizoctonia spp. using rDNA-ITS sequence analysis supports the genetic basis of the classical anastomosis grouping. Mycoscience 49 : 93 - 114 . https://doi.org/10.1007/S10267-007-0394-0 Sneh B , Burpee L , Ogoshi A , 1991 . Identification of Rhizoctonia species. Am Phytopath Soc Press, St. Paul, MN, USA. Sneh B , Jabaji-Hare S , Neate S , Dijst G , 1996 . Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and diseases control Springer Sci & Business Media. https://doi.org/10.1007/978-94-017-2901-7 Townsend GK , Heuberger JW , 1943 . Methods for estimating losses caused by diseases in fungicide experiments. Plant Dis Reptr 27 : 340 - 343 . Tuncer G , Erdiller G , 1990 . The identification of Rhizoctonia solani Kuhn anastomosis groups isolated from potato and some other crops in Central Anatolia. J Turk Phytopathol 19 ( 2 ): 89 - 93 . Tuncer S , Eken C , 2013 . Anastomosis grouping of Rhizoctonia solani and binucleate Rhizoctonia spp. isolated from pepper in Erzincan, Turkey. Plant Prot Sci, Prague, 49 : 130 - 134 . https://doi.org/10.17221/77/2012-PPS Ünal F , Bayraktar H , Yıldırım AF , Akan K , Dolar FS , 2015 . Kayseri, Kırşehir, Nevşehir ve Aksaray illeri buğday ekim alanlarındaki Rhizoctonia tÜr ve anastomosis gruplarının belirlenmesi. Bitki Koruma BÜlteni 55 ( 2 ): 107 - 122 . Ünal F , Kara ME , 2017 . Molecular characterization of Rhizoctonia species and anastomosis groups in barley production areas in Ankara province. J Turk Phytopath 46 ( 2 ): 61 - 67 . White TJ , Bruns T , Lee S , Taylor JW , 1990 . Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Acad Press, Inc., NY, 315 322 In: PCR protocols: A guide to methods and applications; Innis MA et al. (eds). https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Yang G , Li C , 2012 . General description of Rhizoctonia species complex. InTech, Croatia, 41 52 In: Plant Pathology; Cumagun CJR (ed). https://doi.org/10.5772/39026 Yıldız A , Döken MT , 2002 . Anastomosis group determination of Rhizoctonia solani Kuhn (Telomorph: Thanatephorus cucumeris) isolates from tomatoes grown in Aydın, Turkey and their disease reaction on various tomato cultivars. J Phytopathol 150 ( 10 ): 526 - 528 . https://doi.org/10.1046/j.1439-0434.2002.00785.x Youssef NOB , Rhouma A , Krid S , Kharrat M , 2010 . First report of Rhizoctonia solani AG 2-3 on chickpea in Tunisia. Phytopathol Mediterr 49 : 253 - 257 .