Rice blast-mycoflora, symptomatology and pathogenicity Jamal-u-ddin Hajano, Mumtaz A. Pathan 1, Qayoom A. Rajput 2 and Mubeen A. Lodhi * Sindh Agriculture University Tandojam, 1 Lasbela University of Agriculture, Water and Marine Sciences (LUAWMS), Uthal, 2 EFUPVS and NPP, MINFAL, Islamabad * Corresponding author, E- mail: mubeenlodhi@gmail.com Abstract Rice (Oryza sativa L.) is the second most important cereal crop of the world as well as of Pakistan. Rice blast caused by a Magnaporthe oryzae (syn: Pyricularia oryzae Cav.) is an infectious fungal disease which is distributed worldwide and prevailing in more than 85 countries of the world. Therefore, present studies were carried out to isolated different fungi associated with seeds and leaves of commercially grown rice varieties and to test the pathogenicity of predominantly isolated species. Seven fungi namely Magnaporthe oryzae, Curvularia lunata, Helminthosporium oryzae, Fusarium moniliforme, Alternaria alternata, Nigrospora oryzae and Aspergillus niger were isolated from seeds and affected leaves of five rice varieties viz., IRRI-6, IRRI-8, DR-82, KS-282 and Shua. N. oryzae was isolated only from IRRI-6 and IRRI-8 rice varieties. Among these fungi, M. oryzae was predominantly isolated both from seeds and leaves of the rice varieties studied. Rice variety IRRI-6 followed by KS-282 appeared as most susceptible rice cultivars to rice blast fungus. M. oryzae was detected with highest frequency from leaves and seeds of IRRI-6. All other fungi were isolated either occasionally or with low frequency. Pathogenicity test of M. oryzae conducted on apparently most susceptible variety IRRI-6 has confirmed the pathogenic nature of the fungus. Plant growth was significantly decreased in inoculated plants as compared to un-inoculated plants. Moreover, typical rice blast disease symptoms were also produced in inoculated plants. Key words: Rice blast, fungi, Magnaporthe oryzae, symptomatology, pathogenicity Introduction Rice (Oryza sativa L.) is the second most important cereal crop of the world. It is staple food for more than half of the world s population. Approximately 90% of global rice production is contributed by Asia continent alone 15. Rice is also important staple food and cash crop of Pakistan. Rice crop is subjected to attack of 50 diseases that including 6 bacterial, 21 fungal, 4 nematodes, 12 viral and 7 miscellaneous diseases and disorders 12,31. However, major diseases are rice blast, brown spot, bacterial leaf blight and leaf streak, sheath blight, sheath rot, Fusarium wilt or Bakanae, stem rot, Tungro virus, false smut and post-harvest diseases 23. These diseases either attack at any growth stage of rice plant or infect rice grains after harvest, causing considerable losses in both quality and quantity of the produce. It is estimated that about 14-18% yield reduction was caused by these diseases worldwide 17.
Rice blast caused by a filamentous, ascomycete fungus Magnaporthe oryzae (syn: Pyricularia oryzae Cav.) is an infectious fungal disease which is distributed worldwide and prevailing in more than 85 countries of the world 9,22. Blast disease occurs in a wide range of climatic conditions from temperate to tropics and the pathogen is spread by wind and disseminated by infected plant debris or seeds left in the fields 31. Rice blast can be appeared at any stage of growth and produced various symptoms 13. Several rice blast epidemics have occurred in different parts of the world, resulting in yield losses in these areas ranging from 50 to 90 % of the expected crop 1. Under usual conditions yield losses due to blast ranged from 1-50% in different rice growing regions of the world depending upon the type of cultivars grown and environmental conditions prevailed 10. However, under favourable environmental conditions the disease caused heavy losses and yield may reach upto 90% 16. In south and south East Asia the losses due to blast was estimated about US$55 million annually 21. Rice blast is also recognized as one of the important disease in Pakistan causing considerable losses in yield 11,24. Studies were, therefore, carried out to study the symptomatology and isolation of fungi associated seeds and leaves of commonly growing rice varieties. Pathogenicity of the most frequently isolated fungal pathogen was also tested which is presented herein. Materials and methods Collection of samples: Diseased samples consisting of seeds and leaves of IRRI-6, IRRI-8, KS-282, DR-82 and Shua rice varieties, were collected from different locations of district Badin. These rice varieties are commonly cultivated in district Badin. These samples were placed in paper bags, which were properly labeled and brought to the laboratory for isolation of disease causing fungi. Isolation and purification of fungi: For isolation, affected seeds and leaves were surface sterilized with 5% commercial bleach (Sodium hypochlorite) for 1-1.5 minutes and then placed in sterilized Petri dishes containing freshly prepared potato dextrose agar (PDA) medium. Five seeds/pieces of diseased plant parts were placed in each Petri dish. These Petri dishes were incubated at 25 0 C for five days to induce sporulation of the fungi. Different fungal colonies were appeared, which were purified and multiplied on PDA. The isolated fungal species were identified on the basis of their morphological characteristics with the help of keys by Barnett and Hunter 3, Booth 5, Domsch et al., 7, Ellis 8, Singh 26 and Sutton 28. The data on frequency of isolated fungi from seeds and leaves of different rice varieties were recorded using the following formula: Colonization % = Number of seeds/pieces colonized by a fungus x 100 Total number of seeds/pieces studied Pathogenicity test: Pathogenicity test of most frequently isolated fungus, Magnaporthe oryzae Couch was carried out to confirm the etiology of the disease. For this purpose nursery of IRRI-6 was raised in earthen pots thoroughly washed with spirit containing 2 kg sterilized
soil. After 20 days nursery was transplanted in earthen pots containing sterilized soil at one plant per pot. Preparation of conidial suspension: For inoculation, conidial suspension was prepared from 14 days old culture of Magnaporthe oryzae. 10 ml of sterilized water was added in PDA plates containing culture of the test fungus. To remove the conidia, the culture was rubbed with sterilized hair brush gently and the spore suspension was collected in a sterilized glass beaker. The suspension was adjusted to 10 5 conidia per milliliter of water 6. This was done with the help of hemocytometer 30. Inoculation of pathogen inoculums: The prepared conidial suspension of the fungus was inoculated at fourth leaf stage of rice plant by spraying approximately at 0.2 ml/plant 6. The inoculated plants were incubated at 25 to 28 0 C under 100% humidity for 72 hours under darkness for penetration of conidia and disease development. The humidity was maintained by using humidifier and then was exposed to open air. The un-inoculated plants were served as control. There were 15 replications (each earthen pot containing one plant) of both inoculated and un-inoculated. After 15 days of inoculation, data on disease incidence, severity, plant height and weight was recorded. Re-isolation of the fungus: Re-isolation was also done from inoculated and un-inoculated plants as described above to confirm the pathogenic nature of the test fungus. For this purpose, leaves were washed thoroughly with tap water and cut into small pieces. After surface sterilization with 5% bleach solution, pieces were placed on PDA plates. The recovery of inoculated fungus was recorded and infection percent was calculated with the help of following formula. Infection % = Number of pieces colonized by the fungus x 100 Total number of pieces studied While disease incidence and severity were calculated with following formulas; Disease incidence % = Number of infected plants with the disease x 100 Total number of plants studied Disease severity % = Area of plant covered by the disease x 100 Total area of the plant studied Finally the data was analyzed by ANOVA using Statistix 8.1 software. Least significant differences (LSD) were calculated using significant level at P = 0.05. Results Disease symptoms: The rice blast fungus caused infection on all growth stages of rice plant. On leaves of susceptible rice variety the disease initially appeared as whitish or grayish specks along
the leaf margins (Fig. 1a). Later on they turned into elliptical spots which are elongated and diamond shaped with pointed ends (Fig. 1b). These spots became necrotic in the center with brown or reddish-brown margins (Fig. 1c). These spots collapse each other and forms large lesions (Fig. 1c). On stem, fungus produced elongated, grayish to black colour lesions (Fig. 1d). Disease also appeared on rice seeds as brown diamond shaped spot (Fig. 1e). Table- 1. Frequency of isolated fungi associated with leaves of different rice varieties. Isolated fungi Frequency (%) IRRI-6 IRRI-8 DR-82 KS-282 SHUA Magnaporthe oryzae 52 46 40 50 36 Curvularia lunata 16 12 18 14 26 Helminthosporium oryzae 10 10 18 8 14 Fusarium moniliforme 8 6 4 10 10 Alternaria alternata 6 4 10 6 4 Nigrospora oryzae - 16 - - - Aspergillus niger 8 6 10 12 10 Isolation of fungi from leaves of different rice varieties: Seven fungi namely Magnaporthe oryzae, Curvularia lunata, Helminthosporium oryzae, Fusarium moniliforme, Alternaria alternata, Aspergillus niger and Nigrospora oryzae were isolated from the leaves of five different rice varieties (Table 1). N. oryzae was isolated only from IRRI-8 rice variety (Table 1). The association of different fungi is greatly varied with rice cultivars. Among the seven fungi, the frequency of rice blast fungus, M. oryzae was significantly high and as compared to the other fungi. M. oryzae appeared as the pre-dominant fungus associated (Table 1). The maximum infection of M. oryzae is recorded in variety IRRI-6 (52%) followed by KS-282 (50%) and IRRI- 8 (46%). Whereas, minimum infection of M. oryzae was found on variety Shua (36%) followed by DR-82 (40%). As compared to M. oryzae, all other fungi were isolated in low frequencies from the infected leaves of rice varieties (Fig. 2).
In aggregate, the M. oryzae also appeared to be the pre-dominant fungus, which isolated in very high frequency (44.8%) from leaves of the all the five varieties (Fig. 2). The C. lunata stood second (17.2%) followed by H. oryzae (12%). All other fungi were rarely isolated from infected leaves of rice varieties. Table- 2. Frequency of fungi isolated from seeds of different rice varieties. Isolated fungi Frequency (%) IRRI-6 IRRI-8 DR-82 KS-282 SHUA Magnaporthe oryzae 23 20 21 20 19 Curvularia lunata 17.5 19 18 15 18 Helminthosporium oryzae 15 17 19 20 16 Fusarium moniliforme 12 14 11 13 8 Alternaria alternata 14 10 8 12 10 Nigrospora oryzae - 8 4 - - Aspergillus niger 13.5 11 6 16 12 Isolation of fungi from seeds of different rice varieties: Six fungi viz., C. lunata, H. oryzae, F. moniliforme, A. alternata, N. oryzae and A. niger were isolated from seeds of five rice varieties namely IRRI-6, IRRI-8, DR-82, KS-282 and Shua (Fig. 3). The rice blast pathogenic fungus, M. oryzae followed by C. lunata were the predominant fungi associated with seeds of all five varieties with high frequencies. N. oryzae was isolated from seeds in very low frequencies of only two rice varieties i.e. IRRI-8 and DR-82 (Table 2). The frequency of M. oryzae in all varieties was ranging from 19-23% with highest infection on IRRI-6 (23%) followed by DR-82 (21%). Whereas the frequency of C. lunata from seeds of rice varieties was ranging from15.0-19.0% (Table 2). However, N. oryzae was isolated from very few seeds of DR-82 (4.0%) and IRRI-8 (8.0%) respectively, (Table 2). Pathogenicity test of Magnaporthe oryzae: M. oryzae influenced significantly on the growth of rice plants and produced typical rice blast disease symptoms on inoculated plants of IRRI-6
variety. Plant height and weight were significantly reduced in plants inoculated with the fungus as compared to un-inoculated plants (Fig. 4). The inoculated plants produced typical symptoms of rice blast disease on the leaves of IRRI-6. The fungus produced typical mycelial growth on PDA medium when re-isolated from small pieces of inoculated leaves of IRRI-6 variety. After 15 days of inoculation, 80% disease incidence with 25.8% average disease severity was recorded in M. oryzae inoculated plants, whereas, disease was not developed in uninoculated plants. Inoculated plants showed 78% colonization by M. oryzae as compared to uninoculated plants (Fig. 4). Fig. 1. Disease symptoms, (a) whitish or grayish specks along the leaf margins, (b) elongated and diamond shape spots and (c) large lesion on the leaf. (d) large elongated lesions on stem and (e) brown diamond shaped spots on rice seeds.
Fig. 2. Fungi isolated from leaves of different rice varieties Fig. 3. Fungi isolated from seeds of different rice varieties.
Fig. 4. Effect of Magnaporthe oryzae on disease development and growth of inoculated plants of IRRI-6 variety. Discussion In the present studies, total seven fungi viz., Magnaporthe oryzae, Curvularia lunata, Helminthosporium oryzae, Fusarium moniliforme, Alternaria alternata, Nigrospora oryzae and
Aspergillus niger were isolated from the leaves and seeds of five rice varieties namely, IRRI-6, IRRI-8, DR-82, KS-282 and Shua. N. oryzae was isolated only from IRRI-8 variety. M. oryzae was the most predominant fungus that was isolated form leaves and seeds of all rice varieties. The infection of M. oryzae varied with the rice varieties IRRI-6 appeared to be the highly susceptible variety followed by KS-282 to M. oryzae, whereas minimum infection was observed on Shua. All other fungi such as, C. lunata, H. oryzae, F. moniliforme, A. alternata, N. oryzae and A. niger were isolated in comparatively with low frequencies. Our studies showed strong association of M. oryzae with affected plant parts of all rice varieties. The results are in accordance to those reported by other workers such as Naeem et al., 19 also recorded Pyricularia oryzae, Alternaria padwickii, Curvularia sp., Fusarium moniliforme and Bipolaris oryzae from seeds, shoot and root of different rice varieties. Similarly, Khan et al., 14 isolated F. semitectum, F. moniliforme, F. oxysporum, A. alternata, A. padwickii, C. oryzae, C. lunata, Drechslera oryzae and P. oryzae from rice seeds. Similar findings have been mentioned by Bhutta and Hussain 4, Whaid et al., 29, Misra and Dharam 18 and Mew and Gonzales 17. Inoculated healthy rice plants of IRRI-6 variety by M. oryzae produced typical symptoms of the disease. The leaves of plants inoculated with M. oryzae showed whitish to grayish specks initially, which enlarged and became spindle shaped necrotic spots with brown to reddish brown margins within 15 days of inoculation. M. oryzae appeared as the virulent and aggressive pathogen on the IRRI-6 variety, the disease appeared on 80 % of the inoculated plants after two weeks inoculation with 25.8 % average disease severity. It clearly indicated that blast disease caused by M. oryzae is one of the destructive diseases of rice and can cause severe damage and yield reduction under favourable environment conditions on susceptible variety. In the present studies the infection of M. oryzae also caused 25.5% and 55.4% reduction in plant height and plant weight, in inoculated plants as compared to un-inoculated plants. There are several reports described that M. oryzae can infect rice plant at any growth stage and caused considerable losses (Mew and Gonzales 17 ; Webster and Gunnell 31 ; Sirithunya et al., 27 and Phinyarat et al., 20 ). Similarly, Anthony 2 reported that 10-30 % of world rice crop was disturbed by blast disease caused by M. oryzae. In Nepal, rice blast caused by the same fungus is considered as the most serious disease of rice and caused 50% reduction in the yield on susceptible varieties. Similarly, Singh 25 reported 60-80 % yield losses from India on widely grown rice variety due to M. oryzae infection. References 1. Agrios GN, Plant Pathology (5th edition). Elsevier-Academic Press, San Diego, CA. 2005; p. 922. 2. Anthony T, Alain G, Michael T, Biomechanical model for appressorial design in Magnaporthe grisea. J. of Theoretical Biology, 2006; 240(1): 1-8. 3. Barnett HL, Hunter BB, Illustrated Genera of Imperfect Fungi. APS Press: St. Paul, MN, 1998.
4. Bhutta AR, Hussain SA, Seedborne fungi associated with rice seed lots in Pakistan. Int. Rice Res. Notes, 1998; 23: 26-27. 5. Booth C, The genus Fusarium. Commonwealth Mycological Institute, Kew Survey, England. 1971; 237 pp. 6. Chen HL, Chen BT, Zhang DP, Xie YF, Zhang Q, Pathotypes of Pyricularia grisea in Rice fields of central and Southern China. Plant Disease, 2001; 85(8): 843-850. 7. Domsch KH, Games W, Anderson TH, Compendium of Soil Fungi. Academic Press, NY, 1980. 8. Ellis MB, Dematiaceous Hyphomycetes. CAB. Commonwealth Mycological Institute, Kew Survey, England. 1971; 595 pp. 9. Gilbert MJ, Soanes DM, Talbot NJ, Functional Genomic Analysis of the Rice Blast Fungus Magnaporthe grisea. Applied Mycology and Biotechnology, 2004; 4: 331-352. 10. Greer CA, Scardaci SC, Webster RK, First report of Rice Blast caused by Magnaporthe grisea in California. Plant Disease, 1997; 81: 1094. 11. Hafiz A, Plant Diseases. PARC, Islamabad, 1986; p. 552. 12. Hollier CA, Groth DE, Rush MC, Webster RK, Common Names of Plant Diseases. APS, St. Paul, MN, 1993. 13. Izadyar M, 1985. Comparison of effectiveness of several fungicides to control rice blast. Iranian J.of Plt. Path. 1985; 20(1-4): 35-45. 14. Khan TZ, Gill MA, Nasir MA, Bukhari SA, Fungi associated with the seed of different rice varieties/lines. Pak. J. Phytopathol. 1999; 11: 22-24. 15. Mahy BWJ, Van Regenmortel MHV, Cereal viruses: Rice. Encyclopedia of Virology, 2008; 482-489. 16. Mehrotra RS, Plant Pathology. Tata MC Graw-Hill Co. Ltd., New Delhi, 1998. 17. Mew TW, Gonzales P, A Handbook of Rice Seedborne Fungi. International Rice Research Institute, Los Banós, Philippines, 20002; p. 83. 18. Misra AK, Dharam V, Efficacy of fungicides-xlvi: effect of fungicidal seed treatment against heavy inoculum pressure of certain fungi causing discolouration of paddy seeds. Ind. Phytopathol. 1990; 43: 175-178.
19. Naeem K, Anwar SA, Haque MI, Riaz A, Khan MSA, Seedborne fungi and bacteria of rice and their impact on seed germination. Pak. J. Phytopathol. 2001; 13: 75-81. 20. Phinyarat K, Alfonso CM, Patrick H, Vipa MH, Pattama S, Theerayuth T, Nitsri S, 2010. Four QTL in Rice Associated with Broad Spectrum Resistance to Blast Isolates from Rice and Barley. J. of Phytopathol. 2010; 158(2): 125-131. 21. Robert RS, Research Priorities for Rice Biotechnology. Pp.35-37, In: Rice Biotechnology: Toenniessen (eds.), Alden press Ltd., London, 1991. 22. Scardaci SC, Webster RK, Greer CA, Hill JE, William JF, Mutters RG, Brandon DM, McKenzie KS, Oster JJ, Rice blast: A new disease in California. Agronomy Fact Sheet Series. 1997-2. Departement of Agronomy and Range Science, University of California, Davis, 1997. 23. Sharma OP, Bambawale OM, Integrated management of key diseases of cotton and rice. Integrated Management of Plant Pest and Diseases, 2008; 4: 271-302. 24. Shazia I, Ahmad I, Ashraf M, A study on fungi and soil born diseases associated with rice-wheat cropping system of Punjab province of Pakistan. Pak. J. Biological Sci.2003; 6(1): 1-6. 25. Singh IN, Incidence of rice panicle stalk blast (Bl) in Manipur (India). Intl. R. R.Newsletter (Phili.). 1987; 12(4): 34-35. 26. Singh RS, Plant Pathogens (The Fungi). 2nd Ed. Oxford and IBH Publ. Co. PVT. Ltd. New Delhi, 1987; 443 pp. 27. Sirithunya P, Toojinda T, Pimpisitthavorn S, Sriprakon S, Genetic interaction between resistance genes of rice and diversity of blast pathogen in Thailand. In: Annual Research Conference on Rice and Temperate Cereals. 2001. 28. Sutton BC, The Coelomycetes. Commonwealth Mycological Institute, Kew Survey, England, 1980. 29. Wahid A, Saleem M, Khan MU, Tariq AH, Saleem A, Seedborne mycoflora of rice. J. Agri. Res. 1993; 31: 95-100. 30. Waller JM, Ritchie BJ, Holderness M, Plant Clinic Handbook. International Mycological Institute Bakeham Lane, Egham, Surrey TW20 9TY, 1998; 94 pp. 31. Webster RK, Gunnell PS, Compendium of Rice Diseases. APS, St. Paul, MN, 1992.