- Email: [email protected]
Light microscopic observation of the process of perfect hyphal anastomosis of Rhizoctonia oryzae (Ryker & Gooch)
•
Volume 8, Part 3, August 1994
LIGHT MICROSCOPIC OBSERVATION OF THE PROCESS OF PERFECT HYPHAL ANASTOMOSIS OF RHIZOCTONIA ORYZAE (RYKER & GOOCH) K.A. BHUIY AN & K. ARAI Laboratory of Plant Pathology, Kagoshima University, Kagoshima 890, Japan Rhizoctonia spp. are readily isolated from diseased plants and soils, with isolates differing in pathogenicity and morphology, as well as cultural and physiological characteristics. Hyphal fusion or anastomosis is considered the most useful method for grouping different Rhizoctonia isolates (Ogoshi, 1987). When isolates of Rhizoctonia are paired 2-3 cm apart on a medium in a Petridish or glass slides, their mycelia grow and overlap, which can be observed under microscope. If hyphaI fusion occurs, the isolates belong to the same anastomosis group (AG), and often, attraction of hyphae and death of fused cells are observed (Yokoyama & Ogoshi, 1984). Usually two types of fusion have occurred: (1) perfect fusion which brought about complete dissolution of the cell walls and connection of the protoplasm; (2) imperfect fusion which resulted in the imperfect dissolution of the cell walls and an imperfect connection of protoplasm and caused killing reaction between the fused hyphae. Several anastomosis group have been reported in the case of Rhizoctonia solani and binucleate Rhizoctonia (Burpee, 1980; Ogoshi, 1976; Parmeter et al., 1969). Only a limited amount of work has been done on the mechanism or process of hyphaI anastomosis. Yokoyama et al. (1983 and 1985) observed the perfect fusion of hyphae of R. solani with both light and electron microscopy. They documented mainly the process of self anastomosis, but gave no information regarding the frequency of different positions of fusion. Moreover, there has been no investigation regarding the process of hyphaI fusion with species of Rhizoctonia other than R. solani. Based on the formation of sclerotial crust, colony types and appearance different isolates of R. oryzae are grouped into 11 cultural types (Bhuiyan, 1994) We attempted to investigate the detailed process of perfect anastomosis of R. oryzae and also attempted to classify a
total of 106 isolates of R. oryzae isolated from bent grass, paddy soils and rice plants from different locations of Kagoshima and Saga Prefectures of Japan on the basis of hyphaI anastomosis. Isolates were identified on the basis of mature colonies on PSA (Potato-Sucrose-Agar) medium in Petri dishes. Isolates that conformed to the criteria for Rhizoctonia spp. (Ogoshi, 1975) were retained in culture and were further sub-divided into multinucleate and binucleate isolates according to the procedure as described by Bandoni (1979). Among the 860 isolates of Rhizoctonia tested, 106 were identified as R. oryzae. Identification of R. oryzae was confirmed after anastomosis with the tester isolate R01 of R. oryzae (WAG-O), obtained from the stock culture of the laboratory of Plant Pathology, Saga University, Japan. The method of observing hyphal fusion and the formula for calculating the mean distance of hyphaI attraction and frequency of hyphaI fusion was the same as described by Yokoyama et al. (1983) with slight modifications. Mean distance of attraction = 2: Distance of hyphal attraction Number of hyphaI contacts Distance of hyphal attraction means the distance from the turning point of one hypha to the point of contact with another hypha. Number of hyphal contacts includes points of crossing of hyphae from opposite directions and points of fusion. Fusion frequency was determined by the following formula: % FF = A(100) B
A = the sum of fusion points; B = the sum of contact points; fusing, crossing and side by side hyphaI adhering points are included in the term contact points. Frequency of hyphal fusion was
Volume 8, Part 3, August 1994
Fig 1 Time lapse series showing hyphal attraction and the process of perfect fusion between two growing hyphal tips in Rhizoctonia oryzae (scale: 20 [.tm). (A-D) One growing hyphaI tip became attracted to the side of another growing hyphal tip and made contact eight rriinutes after first observation. (E) The contact point became swollen (arrow). (F) Initiates dissolution of the cell wall of two hyphae at the contacted point. (G) Dissolution of the hyphal walls at the contacted point progressed and a new septum was initiated (arrow). (H) Hyphal fusion was completed within 17 minutes after contact and developed a new septum in both the hyphae (arrows).
counted as an average of 10 microscopic eye fields (1.8 mm2;ef). All the 106 isolates tested were anastomosed with the tester isolate R01 by opposing the individual isolates 2.5 cm apart on glass slides covered with 2% water agar medium. The frequency of hyphal fusion differed among different isolates with a range of 10-60%. All the tested isolates that anastomosed perfectly with the tester isolate R01, were considered as the same anastomosis group as the tester isolate WAG-O. The highest fusion frequency (60%) was observed with isolate KA 57 and this was used to observe the process of perfect anastomosis which is documented in Fig 1 (A-H). With two hyphal growing tips, one tip became attracted to the side of another tip; the tips was attracted from a distance of 12.5 [.tm (Fig 1A). A growing hyphal tip came in contact with the side of the other hyphal tip 8
minutes after the initial attraction (Fig 1D). The contact points became swollen (Fig IE, arrow) and 8 minutes after contact, dissolution of the hyphaI wall was observed (Fig IF). Twelve minutes after contact, a new septum was initiated and incomplete dissolution of hyphal walls at the contact point was observed (Fig 1G). Complete fusion and connection of protoplasm was observed at 17 minutes after contact and two new septa were developed on both the fused hyphae (Fig 1H, arrows). Two growing hypha I tips, when attracted to each other from a distance of 10 [.tm, came in contact with each other within 3 minutes. Dissolution of hyphal cell walls at the point of contact started 4 minutes after contact and fusion was completed by the protoplasm being connected within 7 minutes after contact (photographs not given as the process of attraction, contact, dissolution and connection of protoplasm or complete
•
Volume 8, Part 3, Augu t 1994
Fig 2 Perfect fusion of Rhizoctonia oryzae at different contact points ( cale: 20 ~lm; arrows indicate the point of fusion). (A) Fusion at hypha I tip to tip position. (B) Fusion at hyphal tip to side of a tip position. (C) Fusion at side of hyphal branch to hyphal tip position and a branch-like projection at the point of fusion (arrow). (D) Side to side fusion between two parallel growing hyphae.
fusion was similar to Fig 1, except the time required for completion of anastomosis). The perfect fusion of hyphae of R. oryzae occurred at four different points: hyphal tip to hyphaI tip position, hyphaI tip to side of tip position, hyphaI tip to side of hyphaI branch position and side to side fusion between two parallel growing hyphae (Fig 2 A-D). Among the total perfect fusion, the fusion at tip to tip, tip to side of a tip, tip to side of a hyphal branch and fusion between two parallel growing hyphae were 21, 35, 32 and 12%, respectively. Before fusion the opposing hyphae become attracted to each other and the distance of attraction varied widely. In the case of tip to tip fusion, one tip attracted another tip when both the hyphae approached within 2-125 ~m. In the case of tip to side of a tip fusion, attraction occurred from a distance of1-10 ~m whereas tip to side of a lateral wall of hyphaI branch fusion, the distance of attraction varied from 2-150 ~lm. The process of anastomosis could be summarized as hyphaI growth, secretion of one or more attracting substances, attraction to the substances, contact of hyphae, cessation of hyphal growth, formation of branch like projections, dissolution of hyphaI walls and finally connection of protoplasm. The attraction may be caused by substances secreted by hyphae of the same anastomosis group, certain metabolites might be secreted from which the first step of hyphal anastomosis may be started (Yokoyama et al., 1983;
Bhuiyan & Arai, 1993). The difference of cultural types and appearance and pathogenicity among the isolates of R. oryzae (Bhuiyan, 1994) suggested that R. oryzae could be divided into several anastomosis groups when studied with a large number of isolates from different geographic locations and with various sources of host plants. However, as this study confirmed that the process of perfect anastomosis of R. oryzae is similar to that of R. solani (Yokoyama et al., 1983) it might be possible that different anastomosis groups of intraspecific groups are contained in R. oryzae. Further taxonomic and epidemiological examinations of R. oryzae are needed.
References
Bandoni, RJ. (1979) Safranin 0 as a rapid stain for fungi. Mycologia 71: 873-874. Bhuiyan, K.A. & Arai, K. (1993) Physiological factors affecting hypha I growth and fusion of Rhizoc-
tonia oryzae. Transactions of the Mycological Society of Japan 34: 389-397.
Bhuiyan, K.A. (1994) Pathological and physiological studies of Rhizoctonia oryzae (Ryker & Gooch) causing rice bordered sheath spot disease. A Ph.D. dissertation submitted to the Vnited Graduate School of Agricultural Science, Kogoshima, Japan 148 pp. Burpee, L. (1980) Rhizoctonia cerealis causes yellow patch of turf grass. Plant Disease 64: 1114-1116. Ogoshi, A. (1976) Studies on the grouping of Rhizoctonia solani Kuhn with hyphal anastomosis and on the perfect stages of the groups. Bulletin of the National Institute of Agricultural Science, Series C 30,1-63. Ogoshi, A. (1987) Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kuhn. Annual Review of Phytopathology 25: 125-143. Parmeter, J.R Jr., Sherwood, RT. & Platt, W.D. (1969) Anastomosis grouping among isolates of
Thanatephorus
cucumeris.
Phytopathology
59:
1270-1278. Yokoyama, K. & Ogoshi, A. (1984) Studies on hyphal anastomosis of Rhizoctonia solani IV. Observation of imperfect fusion by light and electron microscopy.
Transactions of the Mycological Society of Japan 27:
399-413. Yokoyama, K. Ogoshi, A. & Vi, T. (1983) Studies on hyphal anastomosis of Rhizoctonia solani I. Observation of perfect fusion with light microscopy.
Transactions of the Mycological Society of Japan 24:
329-340. Yokoyama, K., Ogoshi, A. & Vi, T. (1985) Studies on hyphal ana tomo is of Rhizoctonia solani II. The ultra-structural changes of hyphal cells during perfect fusion. Transactions of the Mycological Society of Japan 26: 199-207.
Volume 8, Part 3, August 1994
LIGHT MICROSCOPIC OBSERVATION OF THE PROCESS OF PERFECT HYPHAL ANASTOMOSIS OF RHIZOCTONIA ORYZAE (RYKER & GOOCH) K.A. BHUIY AN & K. ARAI Laboratory of Plant Pathology, Kagoshima University, Kagoshima 890, Japan Rhizoctonia spp. are readily isolated from diseased plants and soils, with isolates differing in pathogenicity and morphology, as well as cultural and physiological characteristics. Hyphal fusion or anastomosis is considered the most useful method for grouping different Rhizoctonia isolates (Ogoshi, 1987). When isolates of Rhizoctonia are paired 2-3 cm apart on a medium in a Petridish or glass slides, their mycelia grow and overlap, which can be observed under microscope. If hyphaI fusion occurs, the isolates belong to the same anastomosis group (AG), and often, attraction of hyphae and death of fused cells are observed (Yokoyama & Ogoshi, 1984). Usually two types of fusion have occurred: (1) perfect fusion which brought about complete dissolution of the cell walls and connection of the protoplasm; (2) imperfect fusion which resulted in the imperfect dissolution of the cell walls and an imperfect connection of protoplasm and caused killing reaction between the fused hyphae. Several anastomosis group have been reported in the case of Rhizoctonia solani and binucleate Rhizoctonia (Burpee, 1980; Ogoshi, 1976; Parmeter et al., 1969). Only a limited amount of work has been done on the mechanism or process of hyphaI anastomosis. Yokoyama et al. (1983 and 1985) observed the perfect fusion of hyphae of R. solani with both light and electron microscopy. They documented mainly the process of self anastomosis, but gave no information regarding the frequency of different positions of fusion. Moreover, there has been no investigation regarding the process of hyphaI fusion with species of Rhizoctonia other than R. solani. Based on the formation of sclerotial crust, colony types and appearance different isolates of R. oryzae are grouped into 11 cultural types (Bhuiyan, 1994) We attempted to investigate the detailed process of perfect anastomosis of R. oryzae and also attempted to classify a
total of 106 isolates of R. oryzae isolated from bent grass, paddy soils and rice plants from different locations of Kagoshima and Saga Prefectures of Japan on the basis of hyphaI anastomosis. Isolates were identified on the basis of mature colonies on PSA (Potato-Sucrose-Agar) medium in Petri dishes. Isolates that conformed to the criteria for Rhizoctonia spp. (Ogoshi, 1975) were retained in culture and were further sub-divided into multinucleate and binucleate isolates according to the procedure as described by Bandoni (1979). Among the 860 isolates of Rhizoctonia tested, 106 were identified as R. oryzae. Identification of R. oryzae was confirmed after anastomosis with the tester isolate R01 of R. oryzae (WAG-O), obtained from the stock culture of the laboratory of Plant Pathology, Saga University, Japan. The method of observing hyphal fusion and the formula for calculating the mean distance of hyphaI attraction and frequency of hyphaI fusion was the same as described by Yokoyama et al. (1983) with slight modifications. Mean distance of attraction = 2: Distance of hyphal attraction Number of hyphaI contacts Distance of hyphal attraction means the distance from the turning point of one hypha to the point of contact with another hypha. Number of hyphal contacts includes points of crossing of hyphae from opposite directions and points of fusion. Fusion frequency was determined by the following formula: % FF = A(100) B
A = the sum of fusion points; B = the sum of contact points; fusing, crossing and side by side hyphaI adhering points are included in the term contact points. Frequency of hyphal fusion was
Volume 8, Part 3, August 1994
Fig 1 Time lapse series showing hyphal attraction and the process of perfect fusion between two growing hyphal tips in Rhizoctonia oryzae (scale: 20 [.tm). (A-D) One growing hyphaI tip became attracted to the side of another growing hyphal tip and made contact eight rriinutes after first observation. (E) The contact point became swollen (arrow). (F) Initiates dissolution of the cell wall of two hyphae at the contacted point. (G) Dissolution of the hyphal walls at the contacted point progressed and a new septum was initiated (arrow). (H) Hyphal fusion was completed within 17 minutes after contact and developed a new septum in both the hyphae (arrows).
counted as an average of 10 microscopic eye fields (1.8 mm2;ef). All the 106 isolates tested were anastomosed with the tester isolate R01 by opposing the individual isolates 2.5 cm apart on glass slides covered with 2% water agar medium. The frequency of hyphal fusion differed among different isolates with a range of 10-60%. All the tested isolates that anastomosed perfectly with the tester isolate R01, were considered as the same anastomosis group as the tester isolate WAG-O. The highest fusion frequency (60%) was observed with isolate KA 57 and this was used to observe the process of perfect anastomosis which is documented in Fig 1 (A-H). With two hyphal growing tips, one tip became attracted to the side of another tip; the tips was attracted from a distance of 12.5 [.tm (Fig 1A). A growing hyphal tip came in contact with the side of the other hyphal tip 8
minutes after the initial attraction (Fig 1D). The contact points became swollen (Fig IE, arrow) and 8 minutes after contact, dissolution of the hyphaI wall was observed (Fig IF). Twelve minutes after contact, a new septum was initiated and incomplete dissolution of hyphal walls at the contact point was observed (Fig 1G). Complete fusion and connection of protoplasm was observed at 17 minutes after contact and two new septa were developed on both the fused hyphae (Fig 1H, arrows). Two growing hypha I tips, when attracted to each other from a distance of 10 [.tm, came in contact with each other within 3 minutes. Dissolution of hyphal cell walls at the point of contact started 4 minutes after contact and fusion was completed by the protoplasm being connected within 7 minutes after contact (photographs not given as the process of attraction, contact, dissolution and connection of protoplasm or complete
•
Volume 8, Part 3, Augu t 1994
Fig 2 Perfect fusion of Rhizoctonia oryzae at different contact points ( cale: 20 ~lm; arrows indicate the point of fusion). (A) Fusion at hypha I tip to tip position. (B) Fusion at hyphal tip to side of a tip position. (C) Fusion at side of hyphal branch to hyphal tip position and a branch-like projection at the point of fusion (arrow). (D) Side to side fusion between two parallel growing hyphae.
fusion was similar to Fig 1, except the time required for completion of anastomosis). The perfect fusion of hyphae of R. oryzae occurred at four different points: hyphal tip to hyphaI tip position, hyphaI tip to side of tip position, hyphaI tip to side of hyphaI branch position and side to side fusion between two parallel growing hyphae (Fig 2 A-D). Among the total perfect fusion, the fusion at tip to tip, tip to side of a tip, tip to side of a hyphal branch and fusion between two parallel growing hyphae were 21, 35, 32 and 12%, respectively. Before fusion the opposing hyphae become attracted to each other and the distance of attraction varied widely. In the case of tip to tip fusion, one tip attracted another tip when both the hyphae approached within 2-125 ~m. In the case of tip to side of a tip fusion, attraction occurred from a distance of1-10 ~m whereas tip to side of a lateral wall of hyphaI branch fusion, the distance of attraction varied from 2-150 ~lm. The process of anastomosis could be summarized as hyphaI growth, secretion of one or more attracting substances, attraction to the substances, contact of hyphae, cessation of hyphal growth, formation of branch like projections, dissolution of hyphaI walls and finally connection of protoplasm. The attraction may be caused by substances secreted by hyphae of the same anastomosis group, certain metabolites might be secreted from which the first step of hyphal anastomosis may be started (Yokoyama et al., 1983;
Bhuiyan & Arai, 1993). The difference of cultural types and appearance and pathogenicity among the isolates of R. oryzae (Bhuiyan, 1994) suggested that R. oryzae could be divided into several anastomosis groups when studied with a large number of isolates from different geographic locations and with various sources of host plants. However, as this study confirmed that the process of perfect anastomosis of R. oryzae is similar to that of R. solani (Yokoyama et al., 1983) it might be possible that different anastomosis groups of intraspecific groups are contained in R. oryzae. Further taxonomic and epidemiological examinations of R. oryzae are needed.
References
Bandoni, RJ. (1979) Safranin 0 as a rapid stain for fungi. Mycologia 71: 873-874. Bhuiyan, K.A. & Arai, K. (1993) Physiological factors affecting hypha I growth and fusion of Rhizoc-
tonia oryzae. Transactions of the Mycological Society of Japan 34: 389-397.
Bhuiyan, K.A. (1994) Pathological and physiological studies of Rhizoctonia oryzae (Ryker & Gooch) causing rice bordered sheath spot disease. A Ph.D. dissertation submitted to the Vnited Graduate School of Agricultural Science, Kogoshima, Japan 148 pp. Burpee, L. (1980) Rhizoctonia cerealis causes yellow patch of turf grass. Plant Disease 64: 1114-1116. Ogoshi, A. (1976) Studies on the grouping of Rhizoctonia solani Kuhn with hyphal anastomosis and on the perfect stages of the groups. Bulletin of the National Institute of Agricultural Science, Series C 30,1-63. Ogoshi, A. (1987) Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kuhn. Annual Review of Phytopathology 25: 125-143. Parmeter, J.R Jr., Sherwood, RT. & Platt, W.D. (1969) Anastomosis grouping among isolates of
Thanatephorus
cucumeris.
Phytopathology
59:
1270-1278. Yokoyama, K. & Ogoshi, A. (1984) Studies on hyphal anastomosis of Rhizoctonia solani IV. Observation of imperfect fusion by light and electron microscopy.
Transactions of the Mycological Society of Japan 27:
399-413. Yokoyama, K. Ogoshi, A. & Vi, T. (1983) Studies on hyphal anastomosis of Rhizoctonia solani I. Observation of perfect fusion with light microscopy.
Transactions of the Mycological Society of Japan 24:
329-340. Yokoyama, K., Ogoshi, A. & Vi, T. (1985) Studies on hyphal ana tomo is of Rhizoctonia solani II. The ultra-structural changes of hyphal cells during perfect fusion. Transactions of the Mycological Society of Japan 26: 199-207.