Introduction:

Rhizoctonia solani Kühn is considered a species complex comprising weak plant pathogenic and nonpathogenic fungi, as well as some of the most economically important soil-borne fungal pathogens causing diseases of various crops and wild plant species [1, 2]. The pathogen consists of distinct genetically isolated populations called anastomosis groups (AG 1 to AG 13 and AG-BI) including some intraspecific subgroups [3, 4]. Differentiation of anastomosis groups and subgroups within R. solani is originally based on frequency of hyphal fusions between isolates of the same anastomosis group or subgroup [5, 6, 7, 8 and many others].

Rhizoctonia solani, as a causative agent of root and stem base necrosis of summer savory (Satureja hortensis L.), was for the first time identified in Poland in 2010 [9]. In the same year, web blight of winter savory (Satureja montana L.) caused by AG 1-IA of R. solani was first diagnosed in Italy [10]. To our knowledge there are no other studies in the literature to identify a particular AG of R. solani as a causative agent of disease in summer or garden savory (S. hortensis L.).

The objective of this study was to document the occurrence of web blight; a new disease in summer savory (S. hortensis L.) caused by R. solani AG 1-IA.

Materials and methods:

Observation sites and hostplants:

Disease surveys were carried out in August 2019, July and August 2020 at three neighboring, non-profit kitchen gardens in the village of Aleksandrovo, Lovech District, Central North Bulgaria. The geographical coordinates of the three observation sites were as follows: 43^o15’10.30” N 24^o56’27.67” E; 43^o15’11.94” N 24^o56’30.35 E; 43^o15’12.86” N 24^o56’33.57” E, respectively, all situated in the second river terrace, the middle part of the Osam River Basin, at an altitude of about 98 m above sea level. The soil type of the three investigated sites was Alluvial-Meadow (Eutric Fluvi­sol) with deep profile depth and pH ranging between 5.9 and 6.5. According to the local management practice, the crop was planted at a spacing of 40 to 50 cm in a single row at the middle of about 30 to 40 cm wide raised pathways between sunken beds in which vegetable crops, such as sweet pepper or onion, were grown.

Sampling and pathogen isolation:

Wilted savory plants showing root and basal stem rot as well as visually healthy plants were collected, placed in labeled plastic bags and taken to laboratory. Isolations were made either immediately or within 36 hours after sampling. Savory roots and stem bases were washed in running tap water for 60 min. Small pieces (2-3 mm) of necrotic tissue were excised from the edge of lesions or from respective parts of the visually healthy plants. After surface sterilization in 70% ethanol for 60 s, the host tissue segments were rinsed three times with sterile distilled water and blotted dry between two sterile filter papers. Sterilized root and stem-base segments were then placed on 90 mm Petri plates containing nonselective media such as Oatmeal agar (OA), Potato Dextrose agar (PDA) or Water agar (WA). Plates were incubated at 26˚C in the dark for 7 to 10 days. Hyphal tips of fungal colonies growing out from the plant tissues were aseptically transferred to new Petri plates with fresh OA medium to obtain pure cultures of individual fungal isolates.

Pathogen identification and morphological characterization:

Identification of the isolated fungi was conducted according to the cultural properties, morphological and microscopical characteristics of R. solani described by Sherwood [5], Sneh et al. [2], Ajayi-Oyetunde and Bradley [11] and others. Pure cultures of isolates were grown in 90 mm Petri plates containing PDA medium. After 10 days of cultivation at 26˚C in the dark, colonies were examined for colour, mycelium growth pattern – scarce, moderate or abundant, presence, colour, and distribution pattern of sclerotia – central, scattered or peripheral, and microsclerotia. Three to four-day cultures were used for microscopic examinations. Septa formation near the branching junction, constriction at the point of origin and the angle of branching were observed [12].

Anastomosis grouping:

The anastomosis grouping of R. solani isolates from summer savory was determined by subjecting each isolate to hyphal anastomosis pairing by using AG 1-IA, AG 1-IB, AG 1-IC, AG 2-1, AG 2-2, AG 3, AG 4, and AG 6, tester strains of multinucleate R. solani. A method described by Parmeter et al. [13] and Anderson [1] was followed. Six-cm mycelial discs were cut aseptically with stainless steel agar disc cutter from margins of colonies of the unknown isolates and known AG tester strains actively growing on PDA. One mycelial disc of unknown R. solani isolate and one disc of a tester strain were placed simultaneously 3-4 cm apart directly onto the surface of 2% water agar medium in 90 mm Petri plates. For each combination three replicate pairings were tested. The pairings were cultivated at 20-24 ˚C room temperature until advancing hyphae of both isolates made contact and slightly overlapped, and then directly examined microscopically for hyphal fusion under compound microscope at 400x magnification. The observed hyphal anastomosis reactions were classified in categories as determined by Parmeter et al. [13] and Carling [7]: C0: no anastomosis, paired isolates belong to different AGs; C1: hyphal contact is apparent but no fusion occurs, adjacent cell death is occasional, paired isolates belong to the same AG or different AGs; and C2: hyphal fusion is obvious, adjacent cells always die, paired isolates belong to the same AG. Anastomosis reaction between paired isolates was considered positive if C2 category occurred at five or more hyphal fusion points in each of all three replicates [7, 14].

Pathogenicity test:

For pathogenicity test, all R. solani isolates were cultured for 14 days in Petri plates on OA at 25-26 °C in the dark. When the cultures filled the plates, the content of each plate was thoroughly mixed with 1L of sterilized Alluvial-Meadow soil (Fluvisol) (30% sand, 17.5% silt, 52% clay, and 1.25% organic matter; pH 7.95) which was then placed in individual pot. Each pot was planted with three 21-day-old savory plants, cultivar Mestna. Six replicate pots were used for each isolate. Six pots prepared identically, but without pathogen served as a control. The experiment was carried out twice. Development of symptoms on inoculated savory plants was tracked for two-month period.

Results:

Sampling and pathogen isolation:

At the three observation sites disease was observed on individual summer savory (Satureja hortensis L.) plants, not exceeding 5% of the stands. Various disease symptoms were observed, including root and basal stem rot, leaf and stem blight; the latter two appeared as large leaf necrotic lesions. Roots, stems and leaves of the symptomatic plants almost entirely decayed and turned brown. All diseased plants exhibited varying degrees of wilt symptoms, then wilted irreversibly, withered and eventually died. Fungal hyphae often appear on infected plant parts as a spider’s web; hence we suggest the name web blight for the disease. A total of 11 Rhizoctonia spp. isolates were readily obtained from all inoculated plants showing disease symptoms as well as from tissues of symptomless savory plants.

Pathogen identification and morphological characterization:

All Rhizoctonia spp. isolates formed abundant mycelium on PDA medium, which initially was colorless or white, then, after 7-10 days of cultivation, became light brown. The isolates were identified by microscopic observation of their morphological characteristics. Hyphae initially appeared hyaline but turned light brown with the age. Branches originated from distal dolipore septum grew at approximately right angles to the main hyphae, are slightly curved at the junction, and had a crossed wall near the junction. Within 10 to 14 days after transfer of mycelia to PDA medium relatively few 1 to 3 mm in diameter sclerotia were formed, initially white, and then turning dark brown with maturation. In culture only “sasakii-type” sclerotia were observed, scattered in the colony, whereas no microsclerotia were detected. Based on cultural and morphological characteristics, all isolates were identified as Rhizoctonia solani Kühn. as described by Sherwood [5], Anderson [1], Sneh et al. [2].

Anastomosis grouping:

All savory isolates obtained from the three observation sites were assigned to R. solani AG 1 according to the hyphal anastomosis reactions with tester isolates of the same AG. Importantly, all isolates of R. solani from summer savory exhibited C2 reaction (obvious hyphal fusion at contact points and killing reaction of the adjacent cells) with AG 1-IA, AG 1-IB and AG 1-IC tester isolates. Taking into account that AG 1-IB and AG 1-IC generally exhibit C1 type reaction [14, 15] and that in the present study all R. solani isolates fused very well (C2 reaction) with the three subgroups of AG 1, it could therefore be concluded that the isolates of R. solani causing web blight of summer savory belong to AG 1-IA of the pathogen. No C3 reaction [8] was observed in this study between tested R. solani isolates and tester isolates from subgroups -IA, -IB or -IC of AG 1. No anastomosis reaction (C0) occurred between our field isolates and the tester isolates from the other AGs used in the test (AG 2-1, AG 2-2, AG 3, AG 4 and AG 6). Based on similarity of symptoms and signs, we propose the name “web blight” for the disease on summer savory after the name given to a disease caused by the same pathogen (R. solani AG 1-IA) but in winter savory (S. montana L.)

Pathogenicity test:

Diseased plants, exhibiting symptoms identical to those observed on naturally infected plants appeared in each inoculated pot. The inoculated pathogens were consistently reisolated from symptomatic tissues, thereby fulfilling Koch’s postulates. None of the uninoculated control plants developed symptoms of the disease.

Discussion:

In the present study, R. solani Kühn was identified as the disease-causing agent of web blight disease of summer savory (S. hortensis L.) in three small-scale kitchen gardens in Bulgaria. All isolates of R. solani obtained were assigned to AG 1-IA of the pathogen after paring of each isolate with a tester isolate of AG 1 (subgroups -IA, -IB, and -IC), AG 2-1, AG 2-2, AG 3, AG 4 and AG 6 on 2% WA in Petri plates. Cultural and morphological traits of the pathogen matched the descriptions of subgroup -IA of R. solani AG 1, but not that of AG 1-IB or AG 1-IC [14, 16, 17, 18]. Our observations are also comparable to the findings of more recent studies conducted by Yang and Li. [8], Ramos-Molina et al. [19], Ajayi-Oyetunde and Bradley [11], Yamamoto et al. [4] and  Sandoval et al. [12] giving identical description of AG 1-IA of R. solani.

In our literature search, we found no other published reports identifying particular AG of R. solani as a causative agent of disease in summer or as also known garden savory (S. hortensis). To the best of our knowledge, it was the first observation of the disease caused by AG 1-IA of R. solani in summer savory (S. hortensis) in Europe and elsewhere.

To date, R. solani Kühn AG 1-IA has been generally considered the predominant pathogen associated with sheath blight of rice (Oryza sativa L.) [19]. The disease is the second most destructive of the crop after rice blast, yet the most important under high fertility conditions in intensive rice production systems [20]. The pathogen, also known as sasakii form of R. solani AG 1 [21] or “cultural type” 2 of Sherwood [5] has been reported by different authors as a causal agent of various diseases of a wide range of plant species, associated with variety of symptoms and signs, most summarized by Stroube [22], Ogoshi [6], Snech et al. [2], Chavarro Mesa et al. [23], Nagaraj et al. [24].

Based on similarity of symptoms and signs, we propose the name “web blight” for the disease on summer savory (S. hortensis) after the name of a disease caused by the same pathogen (R. solani AG 1-IA) in winter savory (S. montana L.) [10].