Ninety-eight isolates of Rhizoctonia spp. were obtained from barley and wheat grown in Erzurum, Turkey. Of these, 78% were Rhizoctoniasolani (AG-2 type 1, AG-3, AG-4, AG-5 and AG-11), 10% were binucleate Rhizoctonia (AG-I and AG-K) and the remainder were Waitea circinata var circinata (Rhizoctonia sp.). Among the binucleate Rhizoctonia, AG-I was not recovered from barley. In pathogenicity tests on barley and wheat, the highest disease severity was caused by isolates of AG-4 and AG-11, whereas isolates of AG-2 type 1, AG-3, AG-5 and W. c. var circinata were moderately virulent. Isolates of binucleate Rhizoctonia were all nonpathogenic. This is the first report of R. solani AG-11 and W. c. var circinata from Turkey.
Barley (Hordeum vulgare) and wheat (Triticum aestivum) have been widely grown for many years in Erzurum province. Diseases caused by soilborne pathogens are a serious problem in this area (Eken & Demirci, in press). However, no studies have been carried out to determine the species and anastomo-sis groups of Rhizoctonia on barley and wheat in the region.
Rhizoctonia solani (teleomorph Thanatephorus cucumeris) and binucleate Rhizoctonia (teleomorph Ceratobasidium) are divided into anastomosis groups (AGs) based on hyphal anastomosis reactions between isolates. Twelve AGs of R. solani have been reported, including AG-1–11 and AG-BI (Sneh et al., 1991; Carling et al., 1994). Binucleate Rhizoctonia isolates are grouped into AG-A to S (Sneh et al., 1991). The teleomorph of Rhizoctonia oryzae and Rhizoctonia zeae has been described as Waitea circinata (Warcup & Talbot, 1962). Gunnell (1986) described three varieties of W. circinata based on differences in colony morphology of the vegetative state: W. c. var circinata, W. c. var oryzae (anamorph R. oryzae) and W. c. var zeae (anamorph R. zeae). The anamorphic name of W. c. var circinata has not yet been assigned, but the teleomorphic name may be useful, although the teleomorph is rarely observed (Leiner & Carling, 1994).
This study was initiated to determine the species and anastomosis groups of Rhizoctonia present on barley and wheat in Erzurum, and to assess virulence of these isolates on barley and wheat seedlings.
Materials and methods
Collection, isolation and identification of Rhizoctonia
Barley and wheat plant samples were collected in 11 districts of Erzurum during 1992–95. Isolations were made from crown and subcrown internode tissues. The tissue sections were surface disinfected in 0.5% sodium hypochlorite for 1 min and placed on 1.5% water agar containing 50 mg L−1 streptomycin sulfate (Demirci & Döken, 1993). After 48–72 h incubation at 20–25°C, hyphae from the margin of each developing colony were placed on water agar or potato dextrose agar (PDA). Rhizoctonia isolates were transferred to PDA slants and stored at 10°C.
Isolates of Rhizoctonia obtained in this manner were identified on characteristics of their vegetative hyphae (Ogoshi, 1975), nuclear condition (Bandoni, 1979) and hyphal anastomosis with known tester isolates (Table 1) of R. solani, binucleate Rhizoctonia and W. circinata, using standardized techniques for anastomosis group determination (Parmeter et al., 1969).
Table 1. Designation and origin of isolates of Rhizoctonia species used in these studies aAnastomosis groups for Rhizoctonia solani. Tester isolates included: AG-1, AG-2 type 1, AG-2 type 2, AG-3, AG-4, AG-5, AG-6, AG-7, AG-8, AG-9, AG-10, AG-11 and AG-BI, provided by Dr A. Ogoshi (Hokkaido University, Japan), Dr D. E. Carling (University of Alaska, Fairbanks, USA) and Dr S. M. Neate (CSIRO, Division of Soils, Australia).bAnastomosis groups for binucleate Rhizoctonia. Tester isolates included: AG-A, AG-Ba, AG-Bb, AG-C, AG-D, AG-E, AG-F, AG-G, AG-H, AG-I, AG-K, AG-L, AG-N, AG-O, AG-P and AG-Q, all provided by Dr A. Ogoshi (Hokkaido University, Japan).cThree subspecies (isolate no W616, 231 and 590) of Waiteacircinata provided by R. H. Leiner (University of Alaska, Fairbanks, USA).
Colony morphology of Waitea
W. circinata isolates obtained in this study (Table 1) and W. c. var circinata (W616), W. c. var oryzae (231) and W. c. var zeae (590) from R. H. Leiner (University of Alaska, Fairbanks, USA) were grown on PDA in the dark at 25°C and evaluated for 3 weeks for colour of mycelium and size, shape and colour of sclerotia. The variety of W. circinata isolates was determined from differences in colony morphology of the vegetative state (Gunnell, 1986; Leiner & Carling, 1994).
Hyphal diameter and number of nuclei per cell were determined for cultures grown at 25°C on cellophane overlaying 1.5% water agar in petri plates. Mycelium was stained with 3% KOH and safranin O (Bandoni, 1979) and examined at ×400 using phase contrast microscopy. Hyphal diameter was determined for each isolate by measuring 10 cells at right angles to the longitudinal cell wall. Nuclei were counted in 10 cells per isolate selected at random from a location in the stain gradient where nuclei and septa could be distinctly observed.
Three replications of 22 isolates (Table 1) were arranged at four temperatures: 20, 25, 30 and 35°C. These isolates include 12 of W. circinata obtained in this study, one each of W. c. var circinata (W616), W. c. var oryzae (231) and W. c. var zeae (590), one isolate from each of five anastomosis groups of R. solani (WS1 from AG-2 type 1, WA7 from AG-3, WH1 from AG-4, WT3 from AG-5 and WN3 from AG-11), and one isolate from each of two anastomosis groups of binucleate Rhizoctonia (WN13 from AG-I and BO2 from AG-K). To determine rates of radial growth of the isolates, 4 mm diameter agar disks from the edge of colonies growing on PDA were placed in the centre of 9 cm diameter petri plates containing PDA (Leiner & Carling, 1994). The radius of each colony was determined daily until the colony reached the edge of the petri plate. The last radial measurement before the hyphal growth reached the edge was used to calculate radial growth rate in mm per 24 h.
Since the optimum temperature for growth of W. circinata was not reached in the initial range of tem-peratures, the growth rate of seven isolates (BN2, BK2, WA6, WN5, W616, 231 and 590) was determined over a narrower range of temperatures: 26, 28, 30, 32 and 34°C. Radial measurements used in calculation of growth rates were made according to the procedure above.
Pathogenicity of 36 isolates (Table 1) was determined on barley (cv. Tokak) and wheat (cv. Kırik) seedlings. Each inoculation treatment was replicated four times. Inoculum was prepared on moistened wheat kernels (Bandy et al., 1984) in petri dishes (10 g of wheat kernels plus 20 mL of distilled water per dish), which were autoclaved twice at 24-h intervals, inoculated with plugs of mycelium from cultures grown on PDA and incubated at 25°C for 10 days.
For seedling tests, plants were grown in 14 cm diameter pots containing sterile soil mix of topsoil (coarse sandy loam) and sand (builder's sand) (1:1, v/v) in growth chambers maintained at 20 ± 2°C with a 12-h photoperiod. Ten seeds of barley or wheat were sown to a depth of 2 cm in each pot, and inoculated immediately by placing 10 colonized wheat kernels in contact with the seeds. In control treatments, sterile wheat kernels were used. Thirty days after sowing, the plants were harvested, washed and rated for disease on crown and subcrown internode tissues, using a scale of 0–4, where 0 = no symptoms, 1 = traces of superficial discolouration, 2 = one or more small lesions (< 0.5 cm), 3 = one or more large lesions (> 0.5 cm) and 4 = girdling lesions.
Rhizoctonia species and anastomosis groups
The number of isolates of Rhizoctonia species and anastomosis groups obtained from barley and wheat plants is given in Table 2. Among the isolates of Rhizoctonia recovered from barley, 71% were R. solani, 9% were binucleate Rhizoctonia and 20% were W. circinata (Rhizoctonia sp.), whereas from wheat, 81% of the isolates were R. solani, 11% were binucleate Rhizoctonia and 8% were W. circinata. The isolates of W. circinata from barley and wheat anastomosed with W. c. var circinata (W616), W. c. var oryzae (231) or W. c. var zeae (590).
Table 2. Number of isolates of Rhizoctonia species and anastomosis groups obtained from barley and wheat near Erzurum, Turkey
Colony morphology of Waitea
Like the isolate of W. c. var circinata (W616), colonies of W. circinata from barley and wheat were white at first, and changed colour from orange to light brown with increasing age. Colonies of W. c. var oryzae (231) and W. c. var zeae (590) were orange when young, but later turned to salmon and brown, respectively. Sclerotia of W. circinata isolates from barley and wheat were irregular in shape, similar to those of W. c. var circinata and W. c. var oryzae, but differing from the more or less globose and regular sclerotia formed in W. c. var zeae. In colour they were orange during formation, later darkening to brown as in W. c. var circinata and W. c. var zeae, whereas old sclerotia of W. c. var oryzae were orange. Nuclear number, hyphal diameter and size of sclerotia of these isolates are given in Table 3. The diameter of sclerotia of W. c. var zeae isolates was half that of other isolates.
Table 3. Diameter and number of nuclei in mature hyphal cells and diameter of sclerotia of Waiteacircinata isolates obtained in this study and three subspecies of W. circinataaIsolate no. in parantheses.bTen observations per isolate.cMean values followed by standard error of the mean.dRange.
With the exception of W. c. var zeae, the growth rates of Rhizoctonia isolates increased as temperature increased up to 25°C and decreased above 30°C (Fig. 1). Growth of W. c. var zeae increased up to 30°C, then decreased at 35°C. The optimum temperature for growth of W. c. var circinata and W. c. var oryzae as well as for W. circinata from barley and wheat,was near 28°C, but that for W. c. var zeae was near 32°C (Fig. 2).
Isolates of W. circinata and isolates representing different anastomosis groups of R. solani varied in virulence (Table 4). The highest levels of disease on barley and wheat seedlings resulted from inoculation with isolates of AG-4 and AG-11 of R. solani. Isolates from other anastomosis groups of R. solani and the isolates of W. circinata were moderately virulent. The most common symptoms on barley and wheat seedlings were discoloured or necrotic local lesions on crown and subcrown internode tissues. All isolates of binucleate Rhizoctonia were nonpathogenic.
Table 4. Pathogenicity of Rhizoctonia species on barley and wheat seedlings aDisease index 0–4; 0, no symptoms; 1, traces of superficial discolouration; 2, one or more small lesions (< 0.5 cm); 3, one or more large lesions (> 0.5 cm); 4, girdling lesions.bMeans of four replications followed by standard error of the mean.
Of all the Rhizoctonia isolates from barley and wheat in Erzurum, 78% were R. solani (AG-2 type 1, AG-3, AG-4, AG-5 and AG-11), 10% were binucleate Rhizoctonia (AG-I and AG-K) and 12% were W. circinata (Rhizoctonia sp.). Among the binucleate Rhizoctonia, AG-I was not isolated from barley. In a study carried out in Central Anatolia of Turkey, only R. solani AG-4 isolates were recovered from barley and wheat (Tuncer & Erdiller, 1990). In the present study, species and anastomosis groups of Rhizoctonia other than AG-4 were isolated from barley and wheat for the first time in Turkey.
The colony colour of the W. circinata isolates from barley and wheat tended to be similar to both W. c. var circinata and W. c. var zeae in old cultures, and to W. c. var circinata in young cultures. The isolates of W. circinata were similar in sclerotial shape to W. c. var circinata and W. c. var oryzae; however, they resembled W. c. var zeae and W. c. var circinata in colour. Leiner & Carling (1994) reported that in the shape and colour of sclerotia, W. c. var circinata was similar to W. c. var oryzae and W. c. var zeae, respectively. However, Gunnell (1986) reported that W. c. var circinata isolates produced globose sclerotia. On the other hand, the diameter of sclerotia of the W. c. var zeae isolate was nearly half that of sclerotia of W. c. var circinata and W. c. var oryzae and of those of W. circinata isolates obtained in this study. The optimum growth temperature recorded for W. circinata, W. c. var circinata and W. c. var oryzae was 28°C, and for W. c. var zeae was 32°C. The optimum for W. circinata varieties was quite close to the temperatures given by Leiner & Carling (1994). In the anastomosis tests, hyphal fusions were observed when isolates of W. circinata were paired with W. c. var circinata, W. c. var oryzae and W. c. var zeae. Gunnell (1986) and Burton et al. (1988) also reported occasional anastomosis between R. oryzae and R. zeae. The taxonomic identity of the isolates of W. circinata obtained in this study was defined as W. c. var circinata according to colony morphology and colour. The occurrence of R. solani AG-11 and W. c. var circinata in Turkey was determined for the first time in this study.
In pathogenicity tests among the Rhizoctonia species, isolates of R. solani AG-4 and AG-11 were the most virulent on barley and wheat, whereas AG-2 type 1, AG-3 and AG-5 isolates were moderately virulent. Isolates of AG-4 and AG-5 have been reported to cause root rot on wheat (Rush et al., 1994). Virulence of AG-5 on wheat was also determined by Xia & Li (1990) in addition to its virulence on barley (Xia & Li, 1990; Zhao & Li, 1993). In contrast to these reports, Ogoshi et al. (1990) found that isolates of AG-4 and AG-5 were nonpathogenic on barley and wheat. R. solani AG-8, which is responsible for bare patch and root rot on barley and wheat (Neate & Warcup, 1985; Ogoshi et al., 1990), was not recovered in the areas of Turkey where surveys were conducted. In addition, Carling et al. (1994) stated that R. solani AG-11 was shown to damage wheat in greenhouse and growth chamber studies.
I thank Professor M. T. Döken of the Department of Plant Protection, Faculty of Agriculture, Adnan Menderes University, Aydın, Turkey for critical reading and suggestions, and C. Eken of the Department of Plant Protection, Faculty of Agriculture, Atatürk University, 25240 Erzurum, Turkey, for collecting the 1995 isolates.