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Yongli. screening for resistances to rice disease

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Yongli. Screening for resistances to rice disease

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Yongli. screening for resistances to rice disease

  1. 1. Screening for Resistances to RiceScreening for Resistances to Rice DiseasesDiseases Yongli Zhou/Jauhar Ali*/Zhikang Li Institute of Crop Sciences/ National Key Facility for Crop Gene Resources and Genetic Improvement, CAAS * International Rice Research Institute
  2. 2. IntroductionIntroductionIntroductionIntroduction Methods of screening rice resistanceMethods of screening rice resistanceMethods of screening rice resistanceMethods of screening rice resistance to diseasesto diseasesto diseasesto diseasesto diseasesto diseasesto diseasesto diseases ◆◆◆◆ Rice blastRice blastRice blastRice blast ◆◆◆◆ Rice sheath blightRice sheath blightRice sheath blightRice sheath blight ◆◆◆◆ Rice bacterial blightRice bacterial blightRice bacterial blightRice bacterial blight ◆◆◆◆ Virus diseasesVirus diseasesVirus diseasesVirus diseases
  3. 3. Introduction of rice diseasesIntroduction of rice diseasesIntroduction of rice diseasesIntroduction of rice diseases
  4. 4. Fungus Diseases Blast, Sheath blight, Brown spot, Downy mildew, False smut… Bacterial Diseases Bacterial leaf blight, Bacterial leaf streak, Bacterial stripe… Rice Diseases Virus and Mycoplasma-like organism (MLO) Diseases Dwarf, Stripe, Yellow dwarf (MLO), Black-Streaked Dwarf, Orange leaf, Tungro and similar diseases, Yellow mottle virus… Disease Caused by Nematodes White tip, Stem nematode, Root nematode, Rice cyst nematode…
  5. 5. Screening Methods for Resistance to RiceScreening Methods for Resistance to RiceScreening Methods for Resistance to RiceScreening Methods for Resistance to Rice DiseasesDiseasesDiseasesDiseases Technique of Screening RiceTechnique of Screening RiceTechnique of Screening RiceTechnique of Screening Rice Resistance to BlastResistance to Blast
  6. 6. SymptomsSymptoms of Rice Blast Seedlings killed by leaf blast Lesions on leavesSeedlings killed by leaf blast Lesions on leaves Neck blast Infected panicle and spikelets
  7. 7. Infection Spread by Air-borne conidia Leaf blast Panicle blast Re-Infection Seedling blastNeck blast Causal agent Magnaporthe grisea (Pyricularia oryzae) Disease cycle of rice blast Mycelium and conidia on diseased straw and seeds are the principal over-wintering organs Leaf blast
  8. 8. Effects of environmental conditions on disease development Low night temperature (~20 ℃℃℃℃) and high humidity (>90%) are essential for infection and development of lesion ℃℃℃℃ The slight shading from sunlight at the early stage of lesion development fosters extension of the lesion A high nitrogen supply increases susceptibility of rice
  9. 9. Screening Methods for rice resistance to blastScreening Methods for rice resistance to blast 1. Screening in greenhouse by artificial inoculation - seedling blast 2. Screening at Blast Nursery – Leaf and panicle blast 3. Inoculation Method for Evaluating Panicle Blast (or Neck Blast) Resistance
  10. 10. To rapidly screen the resistant materials and identify Objective: Screening in greenhouse by artificial inoculation To rapidly screen the resistant materials and identify the race-specific resistance of rice cultivars, the resistance can be evaluated in greenhouse by artificial inoculation.
  11. 11. Isolate StockIsolate Stock Isolates for stock are revived in prune agar slants, and incubate at 28℃ for 10-14 days The slant is added with water and mycelia that grow well will be macerated with sterile needle. The suspension will be poured ℃ Paper disks cotton The suspension will be poured onto prune agar plates. Incubated at 28 ℃ for 7 days Scrap with glass slice to move the mycelia, add the sterilized paper disks, and induce sporulation in light for 4-10 days. Stock in small bottles. Kept in - 20℃ for years. Silica gel
  12. 12. Preparation of Isolates Stock cultures in paper disks are revived in prune agar slants, and incubate at 28℃℃℃℃ for 10 days Stocks Transplant to slant
  13. 13. The slant is added with water and mycelia that grows well will be macerated with sterile needle. The suspension will be poured onto prune agar plates. Incubated at 28 ℃ for 7 days
  14. 14. Scraping, scrap with glass slice to move the mycelia, and induce Incubate in sporulation cabinet in dark, 28℃℃℃℃ Scraping Plate full of spores Scraping, scrap with glass slice to move the mycelia, and induce sporulation in light for 4 days. About 10-20 ml sterilized water will be poured into Petri dishes containing conidia and will be gently scraped the surface lf the mycelia with glass slide. Conidial suspension is filtered through nylon mesh. The suspension’s concentration is determined using a hemacytometer and will be adjusted until around 5×10 conidia per ml.
  15. 15. Medium PreparationMedium Preparation A) Prune Agar (PA) Prunes: 3 pieces α- Lactose: 5g Yeast Extract: 1g Agar: 20g H2O: 1L B) Water Agar (WA) Agar: 40g H2O: 1L Add some Streptomycinsulfate sterile powder beforepouring for aqueous injection antibacterial
  16. 16. Preparation of Plant MaterialsPreparation of Plant Materials (( ----4~6 leaf )4~6 leaf )
  17. 17. Artificial inoculationArtificial inoculation ----SprayingSpraying
  18. 18. Kept in cages at 25℃, overnight
  19. 19. Kept in a mist room, 25℃ Diseased leafDiseased leaf
  20. 20. Process Time Isolates revival (From stock to prune slant, incubated at 28℃) 7 -10 days Germination, incubated in 25 ℃ 2 days sowing, 2reps, kept in greenhouse 14 days Plating the isolates, incubated at 28℃ 7 days Scraping, incubated in light 3 days Inoculation, kept in cages, 25℃ 1 day , overnight Kept in mist room, 25℃ 7 days scoring 1 day Totally 28 days
  21. 21. For greenhouse and field :For greenhouse and field : CODE (Predominant lesion type) 0 No lesions observed 1 Small brown specks of pinpoint size or larger brown specks without sporulating center 3 Small, roundish to slightly elongated necrotic sporulating spots, about 1-2 mm AssessmentAssessment 3 Small, roundish to slightly elongated necrotic sporulating spots, about 1-2 mm in diameter with a distinct brown margin or yellow halo 5 Narrow or slightly elliptical lesions, 1-2 mm in breadth, more than 3 mm long with a brown margin 7 Broad spindle-shaped lesion with yellow, brown, or purple margin 9 Rapidly coalescing small, whitish, grayish, or bluish lesions without distinct margins Note: Lesion type 5, 7, and 9 are considered typical susceptible lesions
  22. 22. AssessmentAssessment Lesion type 0 1 2 3 4 5 Disease Leaf Area (DLA) MAFF Microorganism Genetic Resources Manual No.18 ISSN 1344-1159
  23. 23. Objectives : • Encourage the use of genetically diverse sources of resistance in the development of varieties with durable blast resistance. Screening at Blast Nursery (BN) durable blast resistance. • Evaluate the varietal reaction of selected varieties and breeding lines against diverse populations of the fungus P. grisea around the world. • Monitor the type and distribution of virulence patterns of the blast pathogen in rice growing countries.
  24. 24. Spreader rows Breeding lines BN screening The nursery is composed of check varieties, blast monogenic lines, and improved breeding lines (test entries) Local resistant and susceptible cultivars should be selected and added to the test set by the cooperator.
  25. 25. Nursery Establishment and ManagementNursery Establishment and Management A. Testing seasonA. Testing season Blast symptoms may develop whenever proper environmental conditions exist. However, tests conducted during the rainy season are easier to manage as there is an adequate waterseason are easier to manage as there is an adequate water supply and the conditions are more favorable for disease development. The airborne spore population is generally highest about a month after the regular rice growing season begins. Considering these factors, an appropriate testing season should be selected to meet local conditions. The leaf blast and neck blast sets may be sown in a synchronized manner so that scoring for each can be done at about the same time.
  26. 26. B. Test PlotB. Test Plot Land for test plots should be of uniform fertility with shady surroundings and wind barriers. The plot should be built on upland soil; but if on lowland, it may be constructed one foot above the prevailingmay be constructed one foot above the prevailing water level during the rainy season. The convenient plot size is about 1.2 meters wide and 15-20 meters long. Alternate narrow alleys are useful footpaths.
  27. 27. C. FertilizersC. Fertilizers Abundant nitrogen is necessary to ensure adequate infection of Pyricularia grisea. A minimum of 100 to 120 kg N/ha in the form of ammonium sulfate may be applied with half at seeding 15 days after seeding. Also, superphosphate at the rate of 50 kg P205/haAlso, superphosphate at the rate of 50 kg P205/ha may be applied before seeding. Since under upland conditions rice frequently suffers from iron deficiency or other nutritional problems, we recommend that high levels of animal manure also be applied.
  28. 28. D.D. Planting methodPlanting method a) Testing rows and border rows. Testing rows of 30 or 50 cm long (one row per test entry) should be 10 cm apart. Two to three border rows lengthwise on both sides are to be planted at each end of the plot. Border rows serve as spreader rows by continuously supplying inoculum of blast pathogens to test entries. A mixture of several broadlyof blast pathogens to test entries. A mixture of several broadly susceptible local cultivars are planted in border rows to ensure presence of inoculum consisting of diverse races of the blast pathogen including those in farmer’s fields. Extremely susceptible cultivars are avoided as spreaders since plants are rapidly killed. b) Rate of seeding. Five grams of seed are required for each 50 cm row (1 gram/10cm).
  29. 29. E.E. Care of the blast nurseryCare of the blast nursery Since there is no replication, uniform distribution of inoculum and disease development by proper management are crucial for a precise evaluation. Plots are to be watered twice or more times each day if no rain falls. One watering period should extend to at least an hour or more, depending on prevailing weather conditions. Watering in the afternoon (about 5 or 6 p.m.) can be particularly effective in raising theafternoon (about 5 or 6 p.m.) can be particularly effective in raising the humidity during the night. During the dry season when it is windy, it is difficult to obtain a good blast development in the nursery. Blast can be induced by covering the plots with a plastic film just before sunset and removing it the following morning at about 7:00am to 9:00am. With plastic covering, the dew period is prolonged, thereby enabling the conidia that lodged on the leaves to germinate and penetrate the cells of the plant.
  30. 30. F. InoculumF. Inoculum Past experiments have shown that ordinary, natural airborne spores are present in sufficient quantity to start infection. In the dry season, when the population of airborne spores is low, inoculum may be provided by collecting diseased leaves, chopping them into pieces, 3 to 6 cm long, and uniformly scattering them over the plot about 10-15 days after sowing.scattering them over the plot about 10-15 days after sowing. Infected plants can also be transplanted between border rows. Spraying with the susceptible border rows with spore suspension two weeks after sowing may help initiate infection. Another method to insure sufficient inoculum is to plant susceptible varieties on a whole plot near the test plots 2-3 weeks before the test begins. This will serve as a bombardment plot.
  31. 31. ProtocolProtocol Germination of the seeds Sowing (3 reps, 5-10 grams/line/rep) 14 days later, introduce the sensitive lines 30 days later, scoring Select the resistant lines Transplant to the greenhouse or field Harvest the seeds
  32. 32. Data CollectionData Collection 0 No lesions 1 Small brown specks of pinpoint size or larger brown specks without sporulating center 2 Small roundish to slightly elongated, necrotic gray spots, about 1-2 mm in diameter, with a distinct brown margin. Lesions are mostly found on the lower leaves 3 Lesion type is the same as in scale 2, but a significant number of lesions are on the upper SCALE (for blast nursery, Leaf Blast ) The scales to be used in scoring are those from the 3rd edition of the "Standard Evaluation System for Rice" (1988), where a scale of 0-9 is adopted for classification of blast reactions as follows: 3 Lesion type is the same as in scale 2, but a significant number of lesions are on the upper leaves 4 Typical susceptible blast lesions, 3 mm or longer, infecting less than 2% of the leaf area 5 Typical blast lesions infecting 2-10% of the leaf area 6 Typical blast lesions infecting 11-25% of the leaf area 7 Typical blast lesions infecting 26-50% of the leaf area 8 Typical blast lesions infecting 51-75% of the leaf area and many leaves dead 9 More than 75% leaf area affected NOTE: Use this scale only for the nursery. Actual estimation of blast affected leaf area (%) is recommended for field assessment of blast disease together with predominant lesion type (see coding system for lesion type).
  33. 33. Inoculation Method for Evaluation of Panicle Blast (orInoculation Method for Evaluation of Panicle Blast (or Neck BlastNeck Blast) Resistance) Resistance The evaluation of large number of test entries for panicle (neck) blast resistance under field conditions is difficult due to different maturity of test entries.due to different maturity of test entries. Accordingly, the resistance of any entries showing low panicle infection under field conditions should be confirmed using artificial inoculation.
  34. 34. The method involves injection of aqueous spore suspension (2 x 105 spores/ml) with a syringe (Hamilton syringe, 1 ml capacity with removable and replaceable metal needles, point style 22°). ProtocolProtocol Inoculation is done soon after panicle emergence when the distance between the collar of the flag leaf and panicle base (neck node) is approximately 3 cm. Spore suspension (0.05 ml/panicle) is injected into the uppermost internode 2 cm below the panicle base. At least five panicles per test entry should be inoculated including both the primary and secondary tillers.
  35. 35. Injection Cotton surrounding
  36. 36. Typical blast lesions appear 7 days after inoculation. While lesions extend up to the neck in susceptible cultivars, lesions are necrotic and are restricted to the point of infection in resistant cultivars. This test showed clear-cut reaction. There were, however, differences in the sizes of the lesions. Due to variation in spikelet sterility in different susceptible cultivars, characterization of the lesion in the neck region iscultivars, characterization of the lesion in the neck region is preferred as a sole criterion. This method does not require plants to be inoculated in a humid chamber. All test entries with different heading dates can be inoculated at different times or planting can be adjusted to synchronize heading.
  37. 37. The results from this method showed that panicle blast reactions of some cultivars inoculated with 2 isolates of P. grisea were different from their leaf blast reactions. IB-1 IB-9 Cultivar Leaf blast Panicle blast Leaf blast Panicle blastblast blast blast blast Tres Marias R R R R Dawn S S R R IAC-47 S S S R Araguaia R S R R Carreon R S R R
  38. 38. The neck blast (panicle blast) nursery should be screened under conditions that favor the disease development. The entries should be planted in hills in a two-meter row plot. Two replications should be included. The entries should be in rectangular blocks surrounded by a row of the local susceptible check sown perpendicular to the test rows and two rows for both sides. Resistant local variety can also be included and should be planted side by side to the susceptibleincluded and should be planted side by side to the susceptible local varieties with different maturity. For each plot, record the number of panicles examined, and the number of panicles with severe neck node (panicle) infection or lesion covering completely around node, neck or the lower part of panicle axis (symptom type 7-9, see scale below). At least 100 panicles per entry should be scored.
  39. 39. Scoring Scale (based on symptoms)Scoring Scale (based on symptoms) 0 No visible lesion or lesions on only a few pedicels 1 Lesions on several pedicels or secondary branches 3 Lesions on a few primary branches or the middle part of panicle axis 5 Lesion partially around the panicle base (node) or the uppermost internode5 Lesion partially around the panicle base (node) or the uppermost internode or the lower part of panicle axis near the base 7 Lesion completely around panicle base or uppermost internode or panicle axis near the base with more than 30% of filled grain 9 Lesion completely around panicle base or uppermost internode or the panicle axis near the base with less than 30% of filled grains.
  40. 40. For the mass evaluation of neck blast (panicle blast) incidence may be computed as follows: Scoring Scale (based on symptoms)Scoring Scale (based on symptoms) Severe neck blast = No.of panicles with severe infection (Symptom type 7-9 only)× 100 Incidence (%) Total number of panicles observed Scoring for severe neck blast (panicle blast) incidence may be done as follows: 0 No incidence 1 Less than 5% 3 5 - 10% 5 11 - 25% 7 26 - 50% 9 More than 50%
  41. 41. Scoring Scale (based on symptoms)Scoring Scale (based on symptoms) 00 11 33
  42. 42. Scoring Scale (based on symptoms)Scoring Scale (based on symptoms) 77 9955
  43. 43. Technique of Screening Rice ResistanceTechnique of Screening Rice Resistance to Sheath Blightto Sheath Blight Methods of Screening Rice Resistance to Diseases to Sheath Blightto Sheath Blight
  44. 44. SymptomsSymptoms of rice sheath blight Sheath blight has become increasingly important because more fertilizers are being used and also because of the use new high- yielding cultivars which have large numbers of tillers, resulting in an increase in the humidity of plant layer.
  45. 45. Infection Re-infection Spread Symptom emergence Sclerotia floating on water may be carried away or drift off and finally come in contact with a rice plant, and then germinate Infection: the mycelium often grows from the outer surface of the sheath, taking a round-about way through the edge of the sheath, to the inner surface Disease cycle of rice sheath blight Survive in soil over winter as scerotia or as mycelium in diseased plants Spread
  46. 46. Inoculation method 1Inoculation method 1 The test populations are drill-seeded in three-row plots with 18 cm between rows and between plots. The plots are inoculated approximately 60 days after planting by broadcasting 100ml per plot of a 2:1 (V:V) mixture of rice hulls and un-hulled grains infested with the pathogens.with the pathogens. Preparation of Inoculum Isolate is Incubated in sterile mixture of rice hulls and un- hulled grains at 28 ℃℃℃℃ for 3-5 days.
  47. 47. Inoculation method 2Inoculation method 2 Inoculation was carried out with short woody toothpicks with a length of 0.8~1.0cm, autoclaved toothpicks were incubated with RH- 9 strain on PDA medium for 2-3 days, then placed behind the leaf collar of the third sheath, counting from the top, at the first stem elongation stage of growth.
  48. 48. AssessmentAssessment 6 5.5 5 4.5 7 6.5 8 7.5 8.5 6 5.5 5 4.5 7 6.5 8 7.5 8.5 Investigating the disease index the about 25 days after heading. 0 No infection observed 1 Lesion limited to lower 20% of the plant height 3 20-30% 1 2 3 4 4.5 3.5 1 2 3 4 4.5 3.5 Scale: Based on the reaching position of the lesion 0: No infection observed 5 31-45% 7 46-65% 9 More than 65% Scale (IRRI): Based on relative lesion height
  49. 49. Technique of Screening RiceTechnique of Screening Rice Resistance to Bacterial Leaf BlightResistance to Bacterial Leaf Blight Methods of Screening Rice Resistance to Diseases Resistance to Bacterial Leaf BlightResistance to Bacterial Leaf Blight
  50. 50. Symptoms: Lesions usually start neat the leaf tips or leaf margins or both, and extend down the outer edge(s). Yong lesions are pale green to grayish green, later turning yellow to gray (dead) with time. In very susceptible varieties, lesions may extend to the entire leaf length into the leaf sheath. Kresek or seedling blight caused wilting and death of the plants.
  51. 51. SusceptibleSusceptible cultivarcultivar ResistantResistant cultivarcultivar
  52. 52. Re-infectionSpread by wind and rain Infection Symptom emergence Invade through water pores on the leaf blade, growth cracks and wounds Xanthomonas oryzae pv. oryzae Spread The organism survives on diseased seed, straw and weed Disease cycle of rice blastDisease cycle of rice bacterial leaf blight
  53. 53. Races ofRaces of XooXoo and differentiation NILs used in IRRIand differentiation NILs used in IRRI
  54. 54. 15 20 25 30 Hua 564 Hua 565 Wanxian 763 Wanxian 77 Huanghuazhan Hexi 41 Yunjing 23 SAGC-4 SAGC-7 Zhonghua 1 Weed Tolerant Rice 1 Wuyujing 3 Wuyujing 20 Bacterialblightlesionlength(cm) Resistant Susceptible 0 5 10 0 5 10 15 Blast (%DLA) 053A-3 BD007 Cau 1 Cau 2 Yundao 1 Luyin 46 RC8 6527 JH15-1-1-1 PD29 D4098 Resistant Susceptible Bacterialblightlesionlength(cm) Vera Cruz IRRI 2011
  55. 55. PhenotypingPhenotyping of rice BB in screening housesof rice BB in screening houses Activities Time germination test of seeds 3 days Germination, incubated in 25 ℃℃℃℃ 2 days sowing 50 seeds, 3reps, kept in greenhouse 7-8 days transplant to screen house 40 days prepare Xoo, in 28℃℃℃℃ 6 days inoculation, in screen house 14 days scoring 1 day Totally 68 days
  56. 56. Preparation of Isolates stocks Methods Condition Tempera ture (℃℃℃℃) Time A 3-day-old slant 5 2 months (temporary) Storage methods forStorage methods for XooXoo slant (temporary) B Lyophiliz ed stock 5 Long term C Skmilk (nonfat) -20 5 years D Glycerol (30%) -70 - -80 10-20 years On PSA at 28 ℃℃℃℃for 2-3 day
  57. 57. Media forMedia for XooXoo A , WAKIMOTO’S MEDIM- Modified (WF-P) Sucrose 20g Peptone 5g Calcium Nitrate 0.5g Sodium Phosphate 0.75g Ferrous Sulfate 0.05g Agar 17g C, SUWAS’S MEDIUM Sodium Glutamate 2g MgCl2.6H2O 1g KH2PO4 0.1g Peptone 10g Sucrose 5g Agar 17g Distilled Water 1000ml Fe-EDTA *SS 1ml Stock Solution=0.657g/100ml Distilled Water 1000ml B, PEPTONE SUCROSE AGAR (PSA) Peptone 10g Sucrose 10g Sodium Glutamate 1g Agar 17g Distilled Water 1000ml D, XOS MEDIUM Monosodium Glutamate 5g Ca(NO3)2 1g K2HPO4 2g Peptone 2g Sucrose 20g Agar 17g Distilled Water 1000ml Fe-EDTA 1mg PH- 6.8-7.0
  58. 58. Preparing the plants for inoculationPreparing the plants for inoculation In a screening house Clipping methodClipping method In a green-house Cut upper portion of the leaf using sterile scissor dipped on bacterial suspension (107-8/ml) Two weeks after inoculation
  59. 59. AssessmentAssessment Scale (for greenhouse test, lesion area) 1 0-3% 2 4-6% 3 7-12% 4 13-25% 5 26-50% Scale (for field test, lesion area) 1 1-5% 3 6-12%5 26-50% 6 51-75% 7 76-87% 8 88-94% 9 95-100% 3 6-12% 5 13-25% 7 26-50% 9 51-100% Note: In both seedling and fields tests, folded young leaves should not be inoculated. Old or leaves with symptom of nutrient deficiency or other diseases should also be avoided for inoculation.
  60. 60. Population Cross Donor Gen. Number of lines (10 DS) HHZ5 Huang-Hua-Zhan*2/OM1723 OM1723 BC1F5 75 HHZ8 Huang-Hua-Zhan*2/Phalguna Phalguna BC1F5 56 HHZ9 Huang-Hua-Zhan*2/IR50 IR50 BC1F5 62 HHZ11 Huang-Hua-Zhan*2/IR64 IR64 BC1F5 56 Materials evaluated for blast and bacterial blight resistance HHZ12 Huang-Hua-Zhan*2/Teqing Teqing BC1F5 66 HHZ15 Huang-Hua-Zhan*2/PSBRC66 PSBRc66 BC1F5 45 HHZ17 Huang-Hua-Zhan*2/CDR22 CDR22 BC1F5 70 HHZ19 Huang-Hua-Zhan*2/PSBRC28 PSBRc28 BC1F5 82 by Dr. Xu in 2010-2011
  61. 61. 10 15 20 25 30 PXO 61 PXO 86 PXO 79 PXO 340 PXO 71 PXO 112 PXO 99 PXO 145 PXO 280 PXO 339 PXO 341 PXO 347 0 5 PXO 349 PXO 363 Interaction among 14 Xoo strains and selected lines in HHZ15 population.
  62. 62. Blast evaluation of virulent strainsBlast evaluation of virulent strains Evaluation of BB resistance of >500 lines (HHZ background) against 14 strains of 10Evaluation of BB resistance of >500 lines (HHZ background) against 14 strains of 10 XooXoo races, 2010 WSraces, 2010 WS HHZ PSBRc66 BC1F5 # 329 BC1F5 #350 Meirong Xu et al
  63. 63. ◇◇◇◇ Rice diseases caused by viruses and mycoplasma- like organism (MLO) Screening Rice Resistance to Virus Diseases ◇◇◇◇◇◇◇◇ Rice Tungro Disease (RTD)Rice Tungro Disease (RTD) mycoplasma- like organism (MLO)
  64. 64. Rice disease caused by viruses andRice disease caused by viruses and mycoplasmamycoplasma--like organism (MLO)like organism (MLO) The reaction of a certain genotype to rice virus infection can be assessed by a skilled worker based on visible symptoms after inoculation under natural conditions (in a field), or under controlled conditionsconditions (in a field), or under controlled conditions (in a greenhouse). The factors needed for a successful test are the presence of virus sources and insect vectors, inoculation at the susceptible growth stage of the test plants and favorable environmental conditions.
  65. 65. Field test:Field test: Screening of test materials, notably breeding lines, can be done in the fields and their reaction to virus infection can be assessed on a scale 0-9 based on the percentage of infection observed. Scale (% infection) 0 No symptom observed0 No symptom observed 1 1-10% 3 11-30% 5 31-50% 7 51-70% 9 71-100% However, field tests generally select vector resistance and are not appropriate for selecting virus resistance.
  66. 66. Greenhouse test:Greenhouse test: Resistance to the virus can be assessed in the greenhouse where factors needed for infection can be manipulated. Inoculation using a high number of vectors is desired and the susceptible check should have at least 90% ofsusceptible check should have at least 90% of infection. A healthy check would be also useful as a reference to measure plant height. Since some fertilizers might affect symptoms, it is recommended not to use any during the experiment.
  67. 67. Disease index (DI)Disease index (DI) A disease index (DI) for the genotype, which would represent both disease incidence and symptom severity, can be used as an indicator foe virus resistance in a greenhouse test. DI can be calculated as: DI = n (3)+ n (5)+ n (7)+ n (9) tn Where n (3), n (5), n (7), and n (9) = number of plants showing a reaction in scale; tn = total number of plants scored
  68. 68. The resulting DI can be classified as:The resulting DI can be classified as: DI Reaction 0-3 Resistant/Tolerant 4-6 Moderate 7-9 Susceptible For further confirmation, test materials with DI rating of 0-3 may be tested by forced inoculation using different number of vectors, at different plant growth stages, and may be assayed serologically to differentiate between virus resistance/tolerance or insect (vector) resistance.
  69. 69. Rice Tungro Disease (RTD)Rice Tungro Disease (RTD) Causal agents: Rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV) Symptoms: Yellow to yellow orange leaves, stunting, and slightly reduced tillering. At growth stages (IRRI): 2 (for the GH) 3-5 (for the field)
  70. 70. Scale of RTDScale of RTD 1 no symptom observed 3 1-10% height reduction, no distinct yellow to yellow orange leaf discoloration 5 11-30% height reduction, no distinct yellow to yellow orange leaf discoloration 7 31-50% height reduction, with distinct yellow to yellow orange leaf discoloration 9 More than 50% height reduction, with distinct yellow to yellow orange leaf discoloration
  71. 71. Discussion on genetic studies of host plant-pathogen interactions
  72. 72. Disease reactions of the parents, IR64, Teqing and their introgression lines (ILs) and 75 Teqing ILs to 10 Philippine Xanthomonas oryzae pv oryzae (Xoo) races that cause bacterial leaf blight (BLB) in rice Xoo races IR64 Teqing Binam IR64 ILs Teqing ILs Race 1 R a MS S R S Race 2 S S S S S Race 3 S S S R SRace 3 S S S R S Race 4 MS S S R MS Race 5 MR MR S MR MS Race 6 S S S R/S b S Race 7 MR R S MR S Race 8 R R MS R S Race 9 S R MR R/S b R Race 10 R R S R MS
  73. 73. R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R RM600(1.5) RM525(2.8) RM50(6.3) RM6(2.9) RM10916(?) RM55(3.9) RM411(3.8) IR64 Binam S S S S S S 33 35 36 50 66 5 7 1 78 34 37 77 38 13 46 73 2 15 R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R S S S R MS S S S S S S S S S S S Race3 Race4 Race6 ILs 95 90 89 R R R R R R S S S R R R R R R R R R R R R R R R R R R R R 17 39 93 84 18 96 8 68 4 42 25 21 45 23 55 R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R S S S S S S S S S S S S S S S R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R IR64 Binam S R S R S S R R R R R R R R R R R R R R RM55(3.9) RM600(1.5) RM525(2.8) RM50(6.3) RM6(2.9) RM10916(?) RM411(3.8) Race3 Race4 Race6 ILs Graphic presentation of the genotypes of 99 IR64 ILs at 7 loci associated with resistance to Xoo races 3, 4, and 6. R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R 16 31 15 19 53 24 20 57 62 72 82 44 54 59 67 75 70 63 74 43 83 26 61 71 80 9 51 27 64 65 87 97 88 R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S 76 55 81 91 99 47 32 69 85 94 56 10 58 6 22 52 86 12 92 49 11 60 40 28 29 3 14 98 79 48 30 40 R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R
  74. 74. Conclusion remarksConclusion remarks Appropriate screening methods are essential for identifying resistant plants from a segregating population; Screening resistance to airborne fungal diseases and/or race specific bacterialdiseases and/or race specific bacterial diseases requires use of multiple races/isolates and/or evaluation in multiple sites under natural disease hotspots; Scoring for reactions to race-specific diseases should include both qualitative and quantitative measurements
  75. 75. Screening for BPH Resistance IR64
  76. 76. *A total of 193 BC2F2 populations was screened for BPH resistance using the BPH screening facility at IRRI. *One hundred pre-germinated seeds per BC2F2 bulk were sown in plastic trays (40 cm 60 cm) half-filled with garden soil along with TN1 (susceptible check) and RPs. *Ten days after sowing, each seedling was infested with two 2nd to PROTOCOL *Ten days after sowing, each seedling was infested with two 2nd to 3rd instar nymphs using the local BPH population. *The plastic insect cage was installed on each tray to contain the insect population. *When TN1 and RPs were killed, surviving plants from each BC2F2 population were counted and transferred to the field.
  77. 77. Summary results of BC populations screened for brown planthopper resistance Details Brown planthopper resistance IR64 TQ NPT Total Total BC2F2 populations 64 67 62 193 Single plant selections per BC population 0 - 22 0 - 22 0 - 2 Total selected BC2F3 lines 652 255 2 909 Selection intensity (%) 10.19 3.81 0.03 4.71 Number of indica donors 49 60 49 158 Selected lines 565 221 2 788 Selection intensity (%) 11.53 3.68 0.04 4.99 Number of japonica donors 9 11 7 27 Selected lines 54 21 0 75 Selection intensity (%) 6.00 1.91 0.00 2.78 Number of intermediate donors 4 3 3 10 Selected lines 33 2 0 35 Selection intensity (%) 8.25 0.67 0.00 3.50 For individual BC populations of 100 plants, a difference of 2.5% between two populations in selection intensity (survival rate) is statistically significant at P < 0.05 when the selection intensity is < 0.1.
  78. 78. # of BC2F2 pop. 64 74 62 200 BHP resistance IR64 Teqing NPT Total Summary results of BC populations for screening for BPH resistance # of selections per pop. 0 - 22 0 - 22 0 - 2 Total selected lines 652 255 2 909 # of contributing donors 62 67 59 189 SI (%) 10.19 3.81 0.03 4.71 For individual BC populations of 200 plants, a difference of 4% between two populations in selection intensity (survival rate) is statistically significant at P < 0.05 when the selection intensity is between 0.1 and 0.5.
  79. 79. Ragged stunt virus-BPH outbreak in Sukamandi, Indonesia Ragged stunt virus-BPH –Tolerant inbred lines Promising Multiple disease resistant GSR hybrids at Jakenan rainfed conditions DS2010
  80. 80. Zhongzu14-ski-4-1
  81. 81. BPH and Virus Resistance Screening IRRI-ICRR joint project collaborators: Prof.Baehaki/Drs Muhsin,Untung • 30 BC3F2 and BC2F3 population (CS 3) • 39 BC3F3 and BC2F4 population (CS 4; 3rd year)ongoing BC2 F3 HHZ populations screened against virulent BPH strain that caused outbreak in Sukamandi in 2010 Several populations showed ILs with comparable resistance with the checks in second round of screening. ICRR 8.2011

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