RHIZOCTONIA SPECIES FROM STRAWBERRY PLANTS IN ERZINCAN, TURKEY: ANASTOMOSIS GROUPS AND PATHOGENICITY

T. G. Kesimci¹, E. D. Durak² and E. Demirci³

¹Department of Plant Protection, Faculty of Agriculture, Igdir University, Igdir, TURKEY

²Department of Plant Protection, Faculty of Agriculture, Yuzuncu Yil University, Van, TURKEY

³Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, Trabzon, TURKEY

Corressponding author’s email: tubagenc25@hotmail.com

ABSTRACT

This study was carried out to determine anastomosis groups and pathogenicity of 166 Rhizoctonia isolates obtained from strawberry plant samples in Erzincan province, Turkey during 2009 and 2010. Rhizoctonia species and anastomosis groups (AGs) of all the isolates were determined using classical techniques. Molecular characterizations of the selected Rhizoctonia isolates were performed by sequence analysis of the internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA). Of these, 155 isolates were identified as binucleate (BN) Rhizoctonia spp., and 11 isolates were as multinucleate (MN) Rhizoctonia solani. Three BN Rhizoctonia isolates were identified as Ceratobasidium albasitensis, the remaining BN Rhizoctonia isolates were assigned to AG-A (54 isolates), AG-E (11 isolates), AG-G (37 isolates), AG-H (6 isolates), AG-K (44 isolates). Rhizoctonia solani isolates were classified as AG-2-1 (4 isolates), AG-4 HGII (3 isolates) and AG-5 (4 isolates). Pathogenicity test was conducted on strawberry plants (cv. Fern), and AG-G isolates were found to constitute the highest disease severity than other species or AGs of Rhizoctonia obtained in this study. BN Rhizoctonia AG-E, R. solani AG-5 and C. albasitensis were first time reported on strawberry plants in Turkey.

Keywords: Anastomosis group, black root rot, Ceratobasidium albasitensis, rDNA-ITS region, Rhizoctonia, strawberry.

http://doi.org/10.36899/JAPS.2022.3.0473
Published first online October 19. 2021

INTRODUCTION

The cultivated strawberry (Fragaria x ananassa Duchesne) is one of the most important berry crops and has increasingly become economically important in the world (Fang et al., 2013). Turkey was the fourth largest strawberry producer after China, United States of America and Mexico, respectively in 2018 (FAO, 2020). Strawberry yields and fruit quality are influenced by diseases, pests and unsuitable environmental factors. Fungal diseases have caused economic losses and decreased fruit quality in strawberry cultivation (Mirmajlessi et al., 2018). Black root rot is considered to be a complex disease, caused by the interaction of several pathogens (Pythium spp., Fusarium spp., Cylindrocarpon spp. and Rhizoctonia spp.), enviromental factors and nematodes on strawberry (Botha et al., 2003; Manici et al., 2005; Matsumoto and Yoshida, 2006). Typical symptoms of black root rot are shorter root system, presence of darker areas on the root, extensive death of feeder rootlets and death of some parts or all of the main roots (Yılmaz, 2006). The pathogens also infect other parts of strawberry plants, causing stunting, wilting, necrosis and death of plant (Vico, 1994; Sharon et al., 2007). The genus Rhizoctonia is one of the most important pathogens of strawberry black root rot disease (Martin, 1988; Sneh et al., 1991; Manici and Bonora, 2007; Sharon et al., 2007).

In several studies, Fusarium spp. (Demirer-Durak and Demirci, 2014; Sarıgül Ertek et al., 2018), Verticillium dahliae (Genc Kesimci and Demirci, 2020), Rhizoctonia spp., (Dinler, 2014; Demirer-Durak and Demirci, 2018; Dinler et al., 2018; Sarıgül Ertek et al., 2018), Alternaria spp., Macrophomina phaseolina and Phytophthora spp. (Sarıgül Ertek et al., 2018) were isolated from underground parts of strawberry plants in Turkey.

Rhizoctonia species were divided into three main groups including multinucleate (MN) Rhizoctonia, binucleate (BN) Rhizoctonia and uninucleate (UN) Rhizoctonia (Stalpers and Andersen, 1996). Multinucleate Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) isolates have been divided into 13 anastomosis groups (AG-1 to AG-13) (Sneh et al., 1991; Carling et al., 1994; Ogoshi, 1996; Carling et al., 1996; 1999; 2002; Sharon et al., 2006). According to the results of the latest publications, BN Rhizoctonia isolates (teleomorph: Ceratobasidium spp.) have been designated to be 19 anastomosis groups (AG-A to AG-I, AG-K, AG-L, AG-O to AG-S, AG-U to AG-W) (Sharon et al., 2008; Yang et al., 2015; Dong et al., 2017; Misawa and Kurose, 2019). Furthurmore, some AGs of R. solani and BN Rhizoctonia are categorized into different subgroups (Sharon et al., 2006; 2008). Ceratobasidium albasitensis (teleomorph; there is no anamorphic name) was isolated and described as a new species on Crocus sativus and Pinus halepensis (Gonzalez et al., 2002).

Although BN Rhizoctonia AG-A, AG-G, AG-I and/or AG-K are frequently isolated from strawberry plants, AG-B, AG-C and AG-F are infrequently isolated. At the same time, R. solani AG 4 HGI, AG-5 and AG-6 were obtained from strawberry plants in various studies (Martin 1988, 2000; Botha et al., 2003; Manici and Bonora, 2007; Sharon et al., 2007; Fang et al., 2013). In Turkey, BN Rhizoctonia AG-A, AG-G, AG-H, AG-K and R. solani AG-2-1, AG-3, AG-4 were isolated from strawberry plants or seedlings (Dinler, 2014; Demirer-Durak and Demirci, 2018; Dinler et al., 2018).

Anastomosis grouping is frequently used for identification and classification of R. solani and BN Rhizoctonia isolates. Conventionally, anastomosis grouping using test isolates is principally the most helpful method to identify Rhizoctonia AGs, although some subgroups of AGs are not distinguishable on the basis of hyphal anastomosis reactions from other members of their AGs (Carling, 1996). Therefore, the classical method is supported by molecular techniques (Botha et al., 2003; Sharon et al., 2007; 2008). Internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) are widely used for molecular identification of species, AGs and subgroups of Rhizoctonia isolates (Gonzalez et al., 2001; Sharon et al., 2006; 2008).

There are very few studies about AGs and pathogenicity of Rhizoctonia spp. on strawberry in Turkey. We aimed to investigate Rhizoctonia isolates obtained from strawberry in Erzincan province. This study was conducted to determine the species and/or AGs of Rhizoctonia isolates using the classical and/or molecular techniques, and to determine the pathogenicity of the selected isolates from each species and/or AGs on strawberry. In addition, another research question was whether the results had similarities or differences with those obtained from studies conducted in other countries.

MATERIALS AND METHODS

Isolation and Identification of Rhizoctonia species: Diseased strawberry plants were collected from 16 fields of three districts (Table 1) in Erzincan province during 2009-2010. Depending on the growth area, 10 to 40 diseased plant samples were taken from one field. Collected plants were taken in polyethylene bags, brought to the laboratory in an ice box cooler and kept at 4ºC until isolation.

Before isolation, plant samples were washed using tap water to remove soil particles. Plant segments (aproximatelly 1.5 cm long) were cut from basal stems. The segments were surface-disenfected for 1 min in 1% sodium hypochlorite (NaOCl), rinsed with steril water, and dried on steril filter paper. Superficially disinfected segments were placed on 1.5% Water Agar (WA) containing 50 mg/l streptomycin sulfate in Petri plates. The plates were incubated at 25 ºC in the dark for 2-3 days. After the period, cultures were purified from colony growth of Rhizoctonia hyphae and transferred to plates WA and Potato Dextrose Agar (PDA). Purified isolates were stored at 5 ºC on PDA slant in the laboratory (Demirci and Döken, 1993).

Rhizoctonia isolates obtained from strawberry plants were characterized on the basis of characteristics of vegetative hyphae under the light microscope (Ogoshi, 1975). Anastomosis groups of Rhizoctonia isolates were determined using classical and/or molecular method. In the classical method, AGs of the isolates were determined by using standardized technique (Demirci and Döken, 1995). Rhizoctonia isolates from strawberry plants were compared with R. solani and BN Rhizoctonia tester isolates in our culture collection (Eken and Demirci, 2004). Tester isolates and unknown isolate were placed 2-4 cm apart from each other in Petri plates including %1.5 WA. Then, the plates were incubated at 25 ºC for 2-3 days, and hypahal interaction between the isolates was investigated under the light microscope. If hyphal anastomosis was observed, the isolates were identified as the same AG.

In the molecular method, a total of fifteen isolates, at least one or more isolates representing each Rhizoctonia species and AG, were subjected to molecular diagnosis using PCR and sequence analysis. To obtain mycelia to be used in DNA isolation, Rhizoctonia isolates were grown on the PDA Petri plate at 25 ºC for 48-72 hours, and transferred to flask containg 100 ml of Potato Dextrose Broth (PDB) media, and incubated for 4-7 days at 25 ºC in the dark without shaking. Fungal mycelium was harvested onto sterile ependorf tubes. Total genomic DNA isolation, PCR amplification and sequence analysis of the rDNA-ITS regions (ITS1, 5.8, ITS2) of Rhizoctonia isolates were performed by REFGEN (Ankara University Technopolis, Ankara, Turkey). Universal primers ITS1 and ITS4 were used to amplify the rDNA-ITS regions. The sequences obtained from ITS regions of Rhizoctonia isolates were queried against the National Center for Biotechnology Information (NCBI) sequence database using the online BLAST (Basic Local Alignment Search Tool) analysis to determine sequence identity and find the closest match based on maximal percent identity. Only sequence homologies of 97 to 100% were considered in assigning a species, AG, and/or subgroup to each Rhizoctonia isolate. All sequences were edited using BioEdit software, version 7 (Hall, 1999), and aligned using the Clustal W algorithm (Thompson et al., 1994). A phylogenetic tree was inferred using the neighbor-joining method (Saitou and Nei, 1987) implemented in MEGA software, version 6 (Tamura et al., 2013) and 1000 bootstrap replicates. The sequences of 15 Rhizoctonia isolates were deposited in NCBI database as accession numbers of MT380166 to MT380180).

Pathogenicity test: Pathogenicity test of 59 Rhizoctonia isolates was carried out on strawberry seedlings cv. Fern. (Table 3). The isolates were grown on PDA in 9 cm diam Petri dish for 7 days at 25 ºC in the dark. Wheat grains were soaked and boiled with steril water (Bandy et al., 1984). The bottles containg wheat grains were autoclaved at 121 ºC for 1 h on two consecutive days. For each isolate, PDA plugs with Rhizoctonia mycelium were transferred to bottle including steril grains (Martin, 1988), and bottles were incubated for 1 month at 22-24 ºC. Sterile PDA plugs were used for control treatment.

Roots of strawberry seedlings were cut in length of 5 cm before planting (Sharon et al., 2007). Subsequently, the seedlings were transplanted into plastic pots where containing soil, manure and perlite at 1:1:1 (v:v:v). The mix were autoclaved at 121 ºC for 60 min for two consecutive days. Fifteen infected wheat grain were added to each potting soil (Ichielevich-Auster et al., 1985; Botha et al., 2003; Sharon et al., 2007). Control pots were inoculated with sterile wheat grains. Four strawberry seedlings were inoculated for each isolate, and each inoculation test was replicated twice. All inoculated plants were maintained for 75 days at 24 ºC with a 12 h:12 h L:D photoperiod. At the end of this period, plant roots were removed and washed. The diseases severity was evaluated according to modified 0-4 descriptive scale (Muyolo et al 1993), where: 0= healthy plant; 1 = localized tissue discoloration without necrosis; 2 = nearly complete root necrosis, partially restricted root length; 3 =root rot, root length severely restricted and 4=dead plant. Re-isolations were made from plant roots as described previously, and each recovered isolate was confirmed with anastomosis test by original isolate to complete Koch’s postulates.

Root lengths (LaMondia and Martin, 1989) and the fresh weights of plants (Porras et al., 2007; Sharon et al., 2007) were measured. The plants were dried at 60 ºC for 72 h, and their dry weights were weighed (Botha et al., 2003; Sharon et al., 2007). The data obtained from the experiment results were analyzed by Duncan's multiple comparison test using SPSS (P<0.05).

RESULTS AND DISCUSSION

Rhizoctonia species and anastomosis groups: Isolation was performed from 438 plant samples, and 166 Rhizoctonia isolates (Table 1) were successfully obtained from basal stems of strawberry plants in Erzincan province. Initially, the isolates were examined for their characteristic feature such as typical hyphal branching, dolipore septum, and no clamp connection (Ogoshi, 1975). According to classical technique (hyphal anastomosis reactions), R. solani and BN Rhizoctonia isolates were separated into species and AGs. With hyphal anastomosis tests, R. solani and BN Rhizoctonia isolates grouped to three AGs (AG-2, AG-4 and AG-5) and five AGs (AG-A, AG-E, AG-G, AG-H and AG-K), respectively. Three Rhizoctonia isolates that hyphal fusion with each other failed to anastomose with all R. solani and BN Rhizoctonia tester isolates.

Table 1. Number of isolates of Rhizoctonia species and anastomosis groups (AGs) determined by classical and molecular tecniques.

Species and/or AGs of selected 15 Rhizoctonia isolates (Table 2) in this study were determined according to analysis of the rDNA-ITS sequences using BLAST. Phylogenetic identification of these isolates against known sequences from GenBank corresponded consistently to the results of AG tester isolates. Finally, R. solani isolates were identified as AG-2-1, AG-4 HGII and AG-5, BN Rhizoctonia isolates were identified as AG-A, AG-E, AG-G, AG-H, AG-K and C. albasitensis (Table 2). A neighbor-joining phylogenetic tree that was produced from the rDNA-ITS sequence data and percent sequence similarity of 15 Rhizoctonia isolates are given in Fig.1. The subgroups of the two AGs (AG-2 and AG-4) of R. solani and three isolates of C. albasitensis were only determined using the molecular technique.

As a results, R. solani isolates were classified as AG-2-1 (2.41%), AG-4 HGII (1.81%) and AG-5 (2.41%), BN Rhizoctonia isolates were as AG-A (32.53%), AG-E (6.63%), AG-G (22.29%), AG-H (3.60%), AG-K (26.51%) and C. albasitensis (1.81%). Binucleate Rhizoctonia AG-A (54 isolates), AG-G (37 isolates) and AG-K (44 isolates) were obtained from all locations in this research, and the isolate numbers (135/166 isolates, 81.33%) of these three AGs are very high when compared to the total number of isolates (Table 1). However, the isolation frequency of R. solani AG-2-1, AG-4 HGII, AG-5, BN Rhizoctonia AG-E, AG-H and C. albasitensis were found relatively close to each other.

The results obtained from this study showed that BN Rhizoctonia isolates from strawberry plants were frequently isolated than R. solani isolates. Similarly, BN Rhizoctonia isolates were more commonly reported from strawberry plants in many studies as AG-A, AG-G and AG-I in Connecticut and California (Martin, 1988; Martin, 2000), AG-A, AG-G and AG-I in South Africa (Botha et al., 2003), AG-A, AG-F, AG-G and AG-K in Israel (Sharon et al., 2007), AG-A, AG-G, AG-I and AG-F in Italy (Manici and Bonora, 2007), AG-A, AG-B, AG-C, AG-G, AG-I and AG-K in Australia (Fang et al., 2013), AG-A, AG-G, AG-K and AG-H in Turkey (Dinler, 2014; Demirer-Durak and Demirci, 2018; Dinler et al., 2018). In previous studies, R. solani AG-2-1, AG-3, AG-4, AG-4 HGI, AG-5 and AG-6 have also been reported on strawberry plants (Martin, 1988; Botha et al., 2003; Sharon et al., 2007; Dinler, 2014; Demirer-Durak and Demirci, 2018). Therefore, the findings of us and other researchers supports that BN Rhizoctonia AGs are the major pathogen related to black root rot of strawberry plants in the world.

Table 2. Species, anastomosis groups (AGs), subgroups, isolate code, sampling location, sampling date and GenBank accession number of selected 15 Rhizoctonia isolates used to molecular diagnosis.

Binucleate Rhizoctonia AG-H and AG-E isolates were isolated only from strawberry plants in Refahiye, but AG-A, AG-G and AG-K obtained from three locations (Center, Üzümlü and Refahiye) in this study (Table 1). Binucleate Rhizoctonia AG-A, AG-G and/or AG-K have been identified on strawberry plants in many previous studies (Martin, 1988, 2000; Botha et al., 2003; Manici and Bonora, 2007; Sharon et al., 2007, Fang et al., 2013; Demirer-Durak and Demirci, 2018; Dinler et al., 2018 ). The diversity of species or AGs of Rhizoctonia obtained from a cultivated plant can be affected by the mainly host, also geographical location, enviromental conditions as temperature and moisture, or previously grown plant species in the same location (Manici and Bonora, 2007; Demirer-Durak and Demirci, 2018). For example, AG-I was found more virulent than AG-A or AG-G at 10 or 15 ºC, whereas AG-G was more virulent than AG-A or AG-I at 24 ºC; AG-G isolates were isolated more frequently than AG-A or AG-I in spring samples, whereas AG-I isolates were isolated more frequently than AG-A or AG-G in fall samples (Martin, 1988; LaMondia and Martin, 1989). On the other hand, R. solani AG-3 has been reported as the major group causing disease on potato plants (Bandy et al., 1988; Demirci and Döken, 1993; Woodhall et al., 2007). Demirer-Durak and Demirci (2018) reported that R. solani AG-3 widely isolated from strawberry plants and suggested that this could be related to potato grown before strawberry.

Pathogenicity test: All R. solani isolates and randomly selected 48 BN Rhizoctonia isolates (Table 3) were used to in the pathogenicity test on strawberry cv. Fern seedlings. The inoculated strawberry plants showed symptoms including brown discoloration or necrosis on the roots, the presence of short roots or dead plants. No symptoms were observed in the control plants.

As a result of pathogenicity test was found virulence differences among the species or AGs of Rhizoctonia (Table 3). Moreover, there were also differences in virulence of isolates belonging to the same AGs (data not shown), and similar results were observed by Martin (2000). In this study, AG-G isolates which caused moderate disease severity (2.17 according to 0-4 scale) had the highest disease severity among the tested Rhizoctonia species or AGs. Whereas isolates of BN Rhizoctonia AG-A, AG-H, AG-K, R. solani AG-2-1, AG-4, and C. albasitensis were weakly pathogenic. On the other hand, BN Rhizoctonia AG-E and R. solani AG-5 were not pathogenic on strawberry seedlings when compared to control plants. Binucleate Rhizoctonia isolates obtained frequently from strawberry plants are generally more pathogenic on strawberry seedlings in Turkey and other countries (Martin, 1988; 2000; Botha et al., 2003; Manici and Bonora, 2007; Sharon et al., 2007; Fang et al., 2013; Demirer-Durak and Demirci, 2018; Dinler et al., 2018). It has been reported that R. solani AG-3, AG-4 and AG-6 are also virulent on strawberry seedlings (Botha et al., 2003; Sharon et al., 2007; Demirer-Durak and Demirci, 2018). Fang et al (2013) were demonstrated that the BN Rhizoctonia isolates showed a wide variation in virulence on strawberry plants.

Table 3. Pathogenicity of Rhizoctonia isolates on strawberry plants.

*: Disease severity was on a scale of 0-4; 0= healthy plant; 1 = localized tissue discoloration without necrosis; 2 = nearly complete root necrosis, partially restricted root length; 3 = root rot, root length severely restricted and 4=dead plant.

**: Mean±Standard Deviation. There are differences between the averages indicated by different letters within columns according to Duncan’s multiple range test (P<0,05).

In the pathogenicity test, it was observed that all isolates of R. solani and BN Rhizoctonia led to significantly reduced fresh weight, dry weight and root length of strawberry plants when compared with the control plants. Similar results were previously reported by other researchers (Demirer-Durak and Demirci, 2018; Borrero et al., 2019). Isolates of Rhizoctonia were recovered from all inoculated plant roots to complete Koch’s postulate and each isolate was confirmed with original isolate. Rhizoctonia was never obtained from control plants.

Conclusıon: This study provided a basis for increasing our knowledge about species or AGs and pathogenicity of BN Rhizoctonia and R. solani on strawberry plants. The data obtained from this study showed the diversity and significance of Rhizoctonia species and/or AGs on strawberry. In this study, it has been determined that the main problem in strawberry root rot is caused by BN Rhizoctonia. Thus, it has been confirmed that BN Rhizoctonia is one of the important causes of black root rot disease of strawberry. Understanding genetic diversity of Rhizoctonia may help to understand and will also facilitate the control of black root rot disease.

Acknowledgments: A part of this article was published at the 1st International Conference on Food Agriculture and Animal Sciences (ICOFAAS 2018) in Erzincan, Turkey.

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