Role of seed-borne inoculum of Rhizoctonia solani in sheath blight of ...

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ABSTRACT: Rhizoctonia solani causing sheath blight of rice is reported to survive ... investigation seed samples were collected from sheath blight infected field.
Indian Phytopath. 59 (4) : 445-452 (2006)

Role of seed-borne inoculum of Rhizoctonia solani in sheath blight of rice P.N. SIVALINGAM, S.N. VISHWAKARMA and U.S. SINGH* Centre of Advanced Studies in Plant Pathology, Department of Plant Pathology, College of Agriculture, G.B. Pant University of Agriculture and Technology, Pantnagar 263 145

ABSTRACT: Rhizoctonia solani causing sheath blight of rice is reported to survive through soil and collateral hosts. Although it infects seed in field, role of seed-borne inoculum in disease development is not properly understood. During the present investigation seed samples were collected from sheath blight infected field (>90 %) and were stored at room temperature. Symptoms induced by the pathogen on seeds were recorded. No correlation was observed in degree of seed discolouration and isolation frequency of the pathogen. Survival of pathogen and its transmission from seed to emerging seedlings was observed at monthly intervals using paper towel, agar plate and sterilized soil medium methods separately treated with biocontrol agents (Trichoderma harzianum, T. virens and Pseudomonas fluorescens) and fungicide (Carbendazim) treatment. The results showed that (i) paper towel method was most efficient method followed by sterilized soil medium method to assess the transmission of pathogen from seed to seedlings and (ii) the inoculum survived well in seeds from one season to another at room temperature. Biocontrol agents and carbendazim treatments proved that R. solani was internally seed-borne in nature. Inspite of good survival of the pathogen, its transmission to rice plants under field conditions was very poor. Key words: Rhizoctonia solani, Trichoderma harzianum, T. virens, Pseudomonas fluorescens, internally seedborne

Due to intensive and extensive cropping system, and also cultivation of high yielding varieties, a number of diseases and pests have attained serious proportions. Sheath blight of rice, caused by R. solani is one of the most widely spreading diseases of rice (Lee and Rush, 1983; Gangopadhyay and Chakrabarti, 1982). Miyaki (1910) reported this disease first time from Japan. But in India, Paracer and Chahal reported this disease from Gurdaspur (Punjab) only in 1963. It causes yield loss ranging from 5.2 to 69 percent (Hori, 1969; Kannaiyan and Prasad, 1978; Naidu, 1992). This disease is distributed in most of the rice growing countries of the world like Bangladesh, China, Colombia, Cuba, Germany, India, Indonesia, Iran, Japan, Korea, Malaysia, Russia, The Netherlands, Nigeria, The Philippines, Senegal, Sri Lanka, Taiwan, Thailand, Trinidad and Tabago, UK (Manchester), USA (Louisiana, Mississippi) and Vietnam (Dasgupta, 1992). The pathogen has a wide host range and in *Corresponding author: [email protected]

rice it infects leaf sheath but symptoms may be produced on any aerial part of the plant. The lesions first appear on the leaf sheath at or above water level as water soaked, circular to oblong, ellipsoid to ovoid shapes and somewhat irregular greenish grey in colour and they spread into discrete lesions with narrow blackish or dark brown margins. Sclerotia are formed on or near those spots depending on the weather conditions. The fungus spreads rapidly to other parts of the plant including leaf blade and panicles including seeds. Pathogen is reported to survive in the soil during winter as sclerotia or as mycelium. The fungus also survives on a number of collateral hosts during off-season (Kannaiyan and Prasad, 1981). However, only little information is available on the role of seed-borne inoculum in disease development in the field (Roy, 1989; Acharya and Gupta, 1996). Here, we are describing the role of seed-borne inoculum in transmission of R. solani from seed to seedling and its survival in seed.

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MATERIALS AND METHODS

Transmission test

Collection of seed sample

Transmission test was carried out under in vitro, glass house and field condition.

Seed samples (cultivar- Sarju 52) were collected from sheath blight infected (>90 percent disease incidence) rice field and stored at room temperature. Temperature measurement Daily max. and min. room temperature was measured by using max. and min. thermometer from January to June end. Monthly mean average max. and min. temperatures were calculated. Survival test Test for survival of R. solani in rice seed was carried out in monthly interval from January to June. Each month total 5 replications were used and each replication contained 25 seeds, which were selected randomly from the sample and were surface sterilized with 70% ethanol and washed twice with sterile double distilled water (SDW) and then seeds were dipped in a 1000 ppm of streptopenicillin solution to arrest the bacterial growth from seed. Surface sterilized seeds were placed on a Petriplate containing potato dextrose agar (PDA) medium. Plates were kept in an incubator and the temperature was maintained at 25 ± 1oC. After 48h the plates were observed and the total number of infected seeds was counted on the basis of presence of R. solani mycelium. Same procedure was followed in all the replications and percent inoculum survival was calculated by following formula. Total number of seeds having R. solani mycelium Percent inoculum survival = ————————————x 100 Total number of seeds used

Survival of pathogen in different grades of discoloured seeds Six different categories of dicoloured seeds were separated; 0-Normal seed (no spot); 1 – Regular brownish black dots; 2 – Irregular brownish black spots; 3 – Black patch (1/2 seed); 4 – Full black colour and 5 – Ashy grey colour. In each category 5 replications were used in each month from January to June and survival test was carried out as explained above.

Transmission test in vitro Collected rice seeds were surface sterilized with 70% ethanol and twice washed with SDW. The surface sterilized seeds were treated with following biocontrol agents and fungicide. They are, (i) Trichoderma virens + - each 3 g/kg of Carbon Methyl Cellulose seed (ii) T. harzianum

- 3 g/kg of seed

(iii) Pseudomonas fluorescens

- 3 g/kg of seed

(iv) T. harzianum + P. fluorescens

- 3 g/kg of seed

(v) Carbendazim @ 0.1% (Bavistin 50 WP)

- 1 g/kg of seed

Surface sterilized seeds were coated with spore powder of above biocontrol agents and carbendazim by using sterile SDW. Coated seeds were kept in a polythene bag separately overnight. Next day the seeds were used to test the transmission of pathogen from seed to seedling. The following methods were used for transmission test in vitro. 1. Paper towel method 2. Agar medium in test tube method 3. Sterilized soil medium in Petriplate method Paper towel method Paper towels were soaked in running tap water overnight; two moist towel papers of 23 cm x 30 cm size were kept over a butter paper of 25 cm x 37 cm size, the seeds were covered with another towel moist paper and rolled up. The rolls were kept in inclined position in an incubator at 25±1oC. Number of seedling infection of shoot, root and both were counted after 7 days, and symptoms on seedlings also were observed. Small piece of symptomatic leaf were grown on PDA to confirm the R. solani pathogen. Total of five replications were used for each treatment and for each replication

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25 seeds were used. Seeds treated with SDW served as control. Agar medium in test tube method Two percent agar medium was prepared and poured in to 1/3rd of a test tubes and sterilized. The fungicide and biocontrol agents treated seeds were put singly into a test tube containing agar medium under aseptic condition. The test tubes were kept in an incubator at 25 ± 1oC. Recording on symptom and presence of pathogen were done as described above. Sterilized soil medium in Petriplate method The soil was taken in a polypropylene bag and sterilized. Sterilized soil was put in a Petriplate and treated seeds were placed on it, over which the SDW was sprinkled slightly. The plates were kept in an incubator at 25±1oC for 7 days. All the work was done under aseptic condition. After 7 days the seedlings were uprooted, washed thoroughly and shoot, root and both infected seedlings were counted. Recording on symptom and presence of pathogen were done as described above. Transmission test in glass house The soil was taken in a polypropylene bag and sterilized. Biocontrol agents and fungicide treated seeds were sown in pots. For each treatment 5 pots were used and 25 seeds were sown in each pots. Seeds treated with SDW were used as control. Pots were kept in glass house where temperature and humidity was maintained in normal condition and watered normally. After 7 days, seedlings were uprooted, washed thoroughly with tap water and number of seedling showing symptoms on shoot, root and both were recorded. The presence of pathogen was confirmed as described above. Transmission test in field The seedlings were raised in nursery and 28 days old seedlings were transplanted to main field. Fertilizer application and watering was done normally. At flowering stage, 25 hills were selected randomly for each replication and totaly 3 replications were used. Number of hill tillers showing sheath blight symptoms were counted and the disease incidence was calculated by following formula.

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Number of hills showing symptoms Disease incidence = –––––––––––––––––––– × 100 Total number of observed hills

RESULTS Survival of R. solani in rice seed In order to assess survival of R. solani in rice seed, infected seeds were stored at room temperature and pathogen was isolated from these seeds at monthly interval. Survival was estimated as frequency of isolation of R. solani. (Table 1). Frequency of isolation of pathogen declined with time. However, it was still 69.6% in June as compared to 87.2% in January. Decline in survival was more from January to April (87.2 to 74.4%) and from February to June (80.8 to 69.6%). But between January to March and March to May there was no reduction in isolation frequency. Monthly average max. and min, and monthly lowest and highest temperatures were also recorded (Table 2). Survival of R. solani in different categories of discoloured seeds Presence of pathogen was observed in both discoloured and non-discoloured (i.e. normal) seeds (Table 3; Fig. 1). Discolouration of seeds could be Table 1. Survival of R. solani in rice seed Month

% Survival

January February March April May June CD (P = 0.05)

87.2 (69.7) 80.8 (64.4) 80.0 (63.7) 74.4 (59.8) 72.0 (58.2) 69.6 (56.8) (7.4)

Each value is a mean of five replications and each replication contains 25 seeds. Figures in parentheses are angular transformed values.

correlated to the survival of pathogen in seeds. In normal seeds and discoloured seeds belonging to category 1 and 2 there was significant decline in isolation frequency of R. solani in June as compared

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In vitro transmission test

Table 2. Monthly average of max and min room temperature and lowest and highest temperature (0C) from January to June, 2000 Month

Min (0C)

Max (0C)

Lowest (0C)

Highest (0C)

January

12.12

17.54

11.00

18.00

February

15.41

20.55

13.50

23.00

March

19.03

22.61

18.50

27.00

April

27.73

31.10

25.00

35.50

May

31.48

34.61

30.00

37.00

June

30.33

33.40

29.50

35.00

Paper towel method In this method, most (99%) of the seedlings raised from infected seeds exhibited lesions either on shoot (17.1%), root (55.4%) or on both shoot and root (26.5%). Significant reduction in shoot infection was observed in both bioagents and carbendazim treated seeds. Carbendazim also significantly reduced root infection; but bioagents had little effect (Table 4; Fig. 2). Combination of T. harzianum + P. fluorescens was most effective in reducing shoot infection and in enhancing seed germination.

Table 3. Survival of R. solani in discoloured seeds of different categories Month

January February March April May June CD (P=0.05)

Grades* 0

1

2

3

4

5

61.6 (51.8) 58.0 (49.7) 42.4 (40.6) 44.4 (41.8) 46.8 (43.1) 38.8 (38.4) (5.6)

82.8 (65.7) 87.2 (69.3) 80.4 (63.8) 75.2 (60.3) 71.6 (58.0) 67.2 (55.2) (5.5)

90.0 (72.0) 88.8 (70.8) 89.2 (71.5) 79.4 (63.1) 70.2 (59.9) 72.4 (58.4) (5.7)

77.8 (62.0) 77.2 (61.7) 90.6 (72.8) 74.6 (60.1) 81.0 (64.4) 72.6 (58.6) (6.3)

80.4 (63.8) 84.4 (67.0) 91.4 (73.2) 74.8 (60.0) 87.6 (69.6) 84.4 (67.4) (5.5)

79.8 (63.4) 79.6 (63.5) 86.6 (69.2) 78.8 (62.7) 80.0 (63.7) 81.2 (64.5) (6.6)

*0 = Normal seed, 1 = Regular brownish black dots, 2 = Irregular brownish black spots, 3 = Black patch, 4 = Full black colour, 5 = Ashy grey colour Each value is a mean of five replications and each replication contains 25 seeds. Figures in parentheses are angular transformed value.

to January. In other three categories (3 to 5) there was no decline in survival of the pathogen (Table 3). Transmission tests Seedling symptom observation Seven days old seedlings raised from R. solani infected seeds showed brownish black or blackish discoloured lesions on coleoptile, first leaf and/or radical (Fig. 2 and 3). These symptoms were occasionally present on second leaf and sheath also. From these lesions R. solani was invariably isolated which were former confirmed with microscopic observation. These symptoms were observed both in in vitro and glasshouse transmission tests.

Fig. 1. Different categories of discolouration on rice seed

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Agar medium in test tube method In agar medium method 45.7% seedlings exhibited lesions either on shoot (22.2%), root (11.6%) or on both (11.9%). Carbendazim was effective in reducing infection on root and/or shoot but only T. harzianum increased seed germination (Table 5). Sterilized soil medium method In this method the seed treated with either carbendazim or T. virens significantly enhanced seed germination. However, only carbendazim suppressed lesions on shoot and/or root as compared to control. Among bioagents, T. harzianum alone or in combination with P.

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fluorescens significantly reduced root infection (Table 6; Fig. 3). Transmission Test in Glass House When infected seeds were sown in pots under glass house conditions almost 50% emerging seedlings exhibited symptoms on root, shoot or on both (Table 7; Fig. 4). None of the treatments significantly increased germination. However, infection on root, shoot and root + shoot was reduced by seed treatment with carbendazim. Among different treatments highest germination percentage was observed in Pseudomonas fluorescens treated seeds but it was not significantly different from control (Table 7).

Table 4. Effect of different treatments on seed germination and seedling infection by R. solani as observed by paper towel method Treatment

Germination (%)

Shoot infection (%)

Root infection (%)

Root + shoot infection (%)

Control Trichoderma virens T. harzianum Pseudomonas fluorescens T. harzianum + P. fluorescens 0.1% carbendazim CD (P=0.05)

84.0 (66.9) 70.6 (57.3) 78.6 (62.5) 76.0 (63.1) 90.0 (63.2) 79.0 (63.2) NS

17.1 (23.9) 4.2 (7.5) 4.1 (9.1) 4.3 (9.2) 9.5 (15.6) 9.5 (15.6) (10.7)

55.4 (49.0) 46.9 (43.2) 49.2 (43.6) 40.6 (38.2) 7.6 (15.8) 7.6 (15.8) (19.4)

26.5 (30.7) 29.1 (32.5) 24.3 (26.7) 23.4 (28.8) 5.5 (10.4) 5.5 (10.4) (10.9)

Each value is a mean of 5 replications and each replication contains 25 seeds. Figure in parentheses are angular transformed value.

Transmission test in field When infected seeds were sown in field, transmission to the seedling was quite low (6.2%). Seed treatment with either T. virens or carbendazim reduced disease incidence on seedlings but difference was significant only in the case of carbendazim (Table 8). DISCUSSION

Fig. 2. Sheath blight symptoms on seedlings observed in paper towel method (infected portion of seedling indicated by arrow)

R. solani was isolated from 87.2% of surface sterilized seeds, collected from field severely infected by sheath blight (incidence > 90%) after harvest, indicating internal seed-borne nature of the pathogen. In past R. solani is reported to be isolated from 9 to 39% of rice seeds (COPR, 1976; Soave et al., 1983; Binesh and Torabi, 1985).

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Table 5. Effect of different treatments on seed germination and seedling infection by R. solani as observed in test tube by agar medium method Treatment

Germination (%)

Shoot infection (%)

Root infection (%)

Root + shoot infection (%)

Control Trichoderma virens T. harzianum Pseudomonas fluorescens T. harzianum + P. fluorescens 0.1% carbendazim CD (P=0.05)

77.0 (61.6) 78.0 (62.1) 83.0 (65.7) 77.0 (61.4) 78.0 (62.1) 76.0 (60.9) NS

22.2 (25.1) 11.2 (16.2) 14.5 (20.0) 14.2 (21.8) 16.8 (23.5) 10.1 (14.4) NS

11.6 (19.8) 8.9 (15.3) 7.2 (13.8) 7.8 (16.0) 9.0 (17.2) 1.5 (3.2) (8.2)

11.9 (18.1) 5.2 (10.1) 4.9 (9.8) 6.6 (11.5) 5.1 (11.7) 2.8 (6.1) NS

Each value is a mean of 5 replications and each replication contains 25 seeds. Figure in parentheses are angular transformed value.

Table 6. Effect of different treatments on seed germination and seedling infection by R. solani as observed in sterilized soil medium in Petriplate method. Treatment

Germination (%)

Shoot infection (%)

Root infection (%)

Root + shoot infection (%)

Control Trichoderma virens T. harzianum Pseudomonas fluorescens T. harzianum + P. fluorescens 0.1% carbendazim CD (P = 0.05)

76.0 (61.1) 87.0 (71.3) 65.0 (53.9) 77.0 (61.6) 74.0 (59.7) 87.0 (69.8) (10.3)

38.2 (38.0) 26.0 (30.6) 18.4 (22.7) 30.9 (33.3) 17.9 (24.0) 8.8 (15.2) (11.8)

28.3 (32.1) 28.5 (32.1) 16.0 (22.7) 30.7 (33.4) 11.3 (19.3) 5.2 (11.8) (7.5)

16.4 (23.6) 15.3 (22.8) 16.6 (23.8) 27.3 (31.4) 10.7 (16.8) 3.3 (8.2) (8.1)

Each value is a mean of 5 replications and each replication contains 25 seeds. Figure in parentheses are angular transformed value.

May and June, which were higher than optimum for pathogen i.e. 30 – 320C (Gangopathyay, 1983; Ou, 1985). Since R. solani was isolated from normal as well as different types of discoloured seeds, no specific seed discolouration symptom could be correlated with sheath blight infection. Isolation frequency of R. solani was lower from apparently normal seeds, indicating that pathogen may cause seed discoloration. Fig. 3. Sheath blight symptoms on seedlings observed in sterilized soil medium method. (infected portion of seedling indicated by arrow)

When stored at room temperature survival declined but it was still quite high (i.e. 69.6 %) in June, i.e. at sowing time during the next crop season. Decline in survival could be because of temperature encountered in the months of April,

There was decline in isolation frequency of R. solani during storage at room temperature but it remained quite high (~70%) at the time of sowing of crop during the next crop season. It indicates critical role of seed-borne inoculum in disease development in field. However, transmission efficiency was much poor under field condition as compared to in vitro or green house.

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Table 7. Effect of different treatments on seed germination and seedling infection by R. solani under glass house condition Treatment

Germination (%)

Shoot infection (%)

Root infection (%)

Root + shoot infection (%)

Control Trichoderma virens T. harzianum Pseudomonas fluorescens T. harzianum + P. fluorescens 0.1% carbendazim CD (P = 0.05)

78.0 (62.2) 85.0 (67.5) 71.0 (57.7) 86.0 (71.2) 72.0 (58.6) 85.0 (67.4) (9.9)

26.3 (30.3) 18.5 (25.2) 14.6 (19.8) 22.5 (27.9) 16.4 (20.9) 8.4 (15.0) (12.6)

15.7 (22.9) 10.8 (17.2) 11.4 (19.2) 10.0 (17.7) 11.4 (19.4) 3.5 (8.4) (8.9)

7.8 (14.3) 3.4 (8.3) 6.9 (13.5) 6.2 (12.6) 11.3 (19.4) 1.2 (2.8) (9.5)

Each value is a mean of 5 replications and each replication contains 25 seeds. Figure in parentheses are angular transformed value.

roots are submerged in medium (agar or soil) while they were exposed in paper towel method.

Fig. 4. Sheath blight symptoms during seedling stage Table 8. Effect of different treatments on disease incidence under field condition Treatments Control T. virens 0.1% carbendazim CD (P=0.05)

Disease incidence (%) 6.2 (14.5) 2.2 (8.5) 0.2 (1.5) (4.7)

Each value contains 3 replications and each replication contains 25 hills. Figure in parentheses are angular transformed value.

Among different in vitro techniques used to assess the transmission of pathogen from seed to seedling, paper towel method was most efficient followed by sterilized soil medium in Petriplate medium method. Root infection was higher in paper towel method, while in other methods shoot infection was more prevalent. It could be due to the fact that

Carbendazim provided better protection of seedlings, irrespective of method used, as compared to bioagents. Since treated seeds were surface sterilized before bioagent application it may be because of the fact that biocontrol agents control the externally seed borne and soil borne pathogen but not internally seed-borne and systemic pathogen (Cook and Baker, 1983). It again strengthens our conclusion that R. solani is internally seed-borne as speculated by Acharya and Gupta (1996). In field, the transmission from seed to plant was only 6.2%. Ou (1985) and Mathur (1983) also reported quite low (4-6.6%) transmission from infected seeds to plant under field conditions. This may be because of inability of R. solani to cause systemic infection (Naidu, 1983) and/or unfavourable environmental conditions for disease development in field. But, this little amount of inoculum present in seed may build up the inoculum load where the pathogen is already present and may also spread from one area to other, where the disease is absent. ACKNOWLEDGEMENTS Authors wish to thank Dr. H.S. Chaube, Dr. V.K. Agarwal and Dr. V.S. Pundhir for various help received during the course of investigation and thank Indian Council of Agricultural Research for Junior Research Fellowship awarded to the first author to conduct the research.

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Kannaiyan, S. and Prasad, N.N. (1981). Effect of organic amendments on seedling infection of rice caused by R. solani. Plant and Soil. 62: 131. Lee, F.N. and Rush, M.C. (1983). Rice sheath blight: a major disease. Pl. Dis. 67: 829-832 Mathur, S.C. (1983). Fungal diseases of rice in India. In: Recent Advances in Plant Pathology edited by Hussain A., Singh, K., Singh, B.P and Agnihortri, V.P. Lucknow, Print House. pp. 368. Miyaki, I. (1910). Studies uber die reisptlance in Japan. Jl. Cell. Agric. Imp.Univ. 2: 237-276

Cook, R.J. and Baker, K.F. (1983). The nature and practice of biological control of plant pathogens. St. Paul: Ann. Phytopathol. Soc. pp. 589.

Naidu, V.D. (1983). Sheath blight occurrence in rice nurseries. International Rice Research News letter. 8: 10.

Dasgupta, M.K. (1992). Rice Sheath Blight: The challenge continues. In: Plant Diseases of International Importance: Diseases of Cereals and Pulses. Vol.I. (eds. U.S.Singh, A.N.Mukhopadhyay, J.Kumar and H.S. Chaube). Prentice Hall. Englewood chiffs, New Jersery. pp 130-157.

Naidu, V.D. (1992). Influence of sheath blight of rice on grain and straw yield in some popular local varieties. J. Res. Publ. 10: 78-80

Gangopadhyay, S. (1983). Current concept of fungal diseases of rice. Today and Tommorrow’s publishing company. New Delhi: pp. 349. Gangopadhyay, S and Chakrabarti, N.K. (1982). Sheath blight of rice. Rev. Pl. Pathol. 61: 451-460 Hori, M. (1969). On forecasting the damage due to sheath blight of rice plants and the critical point for judging the necessity of chemical control of the disease. Rev. Pl. Protect. Res. 2: 70-73 Kannaiyan, S. and Prasad, N.N. (1978). Studies on the variability of sclerotia of R. solani Kuhn in soil and water. Madras Agric. J. 65: 741-742

Ou, S.H. (1985). Rice Diseases. 2nd ed., Common sheath blight of rice. International Rice Research News letter. 6: 14. Paracer, C.S. and Chahal, D.S. (1963). Sheath blight of rice caused by Rhizoctonia solani Kunn. A new record of India. Curr. Sci. 32: 328-329 Roy, A.K. (1989). Source of seed-borne infection of sheath blight of rice. Oryza. 26: 111-112 Soave, J., Azzini, l.E., Villela, O.V. and Gallo, P.B. (1983). Selection of irrigated rice cultivars for low incidence of spotted seeds. Summa Phytopathol. 9: 179. Received for publication July 15, 2006