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Large Scale Field Inoculation and Scoring of Maize Southern Leaf Blight and Other Maize Foliar Fungal Diseases
玉米南方叶斑病及其他叶片真菌病的大面积野外接种与评分   

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Nature Genetics
Feb 2011

Abstract

Field-grown maize is inoculated with Cochliobolus heterostrophus, causal agent of southern leaf blight disease, by dropping sorghum grains infested with the fungus into the whorl of each maize plant at an early stage of growth. The initial lesions produce secondary inoculum that is dispersed by wind and rain, causing multiple cycles of infection that assures a high uniform disease pressure over the entire field by the time of disease scoring, which occurs after anthesis. This method, with slight modifications, can also be used to study the maize fungal diseases northern leaf blight (caused by Exserohilum turcicum) and gray leaf spot (Cercospora zeae-maydis).

Keywords: Maize (玉米), Cochliobolus heterostrophus (异旋孢腔菌), Inoculation (接种), Fungal disease (真菌病)

Background

Southern leaf blight (SLB), caused by Cochliobolus heterostrophus (Drechs.) Drechs. [anamorph = Bipolaris maydis (Nisikado) Shoemaker], is a widespread maize disease which causes significant yield losses in hot, humid tropical and sub-tropical regions, such as the southeastern USA, parts of India, Africa, Latin America and Southern Europe. In 1970-71 an SLB epidemic caused by C. heterostrophus race T infecting hybrids carrying Texas male-sterile cytoplasm (cms-T) caused an estimated 15% loss in total maize production in the US (Ullstrup, 1972). After the 1970 epidemic, cms-T maize was replaced by race T-resistant, normal cytoplasm maize.

Currently, race O is the predominant cause of SLB in the US and worldwide (Wang et al., 2017). SLB resistance to C. heterostrophus race O is quantitatively inherited with primarily additive or partially dominant gene action (Holley and Goodman, 1989). Under experimental conditions, yield losses due to infection with C. heterostrophus race O as high as 46% have been observed (Fisher et al., 1976; Byrnes and Pataky, 1989). However, losses in commercial production are generally much less severe (Mueller et al., 2016).

This approach to inoculation and rating is based on methodology developed by Carson et al. (2004), though similar methods had been used in numerous previous studies (e.g., Fisher et al., 1976). We have used it in a number of studies to screen germplasm for SLB resistance and to elucidate its genetic basis (Balint-Kurti et al., 2006; 2007 and 2008b; Zwonitzer et al., 2009 and 2010; Kump et al., 2011; Negeri et al., 2011; Belcher et al., 2012; Santa-Cruz et al., 2014; Yang et al., 2017). We have also used an essentially identical method to assess resistance to two other foliar fungal diseases; Gray leaf spot caused by Cercospora zeae-maydis (e.g., Balint-Kurti et al., 2008a) and northern leaf blight (NLB) caused by Exserohilum turcicum (e.g., Balint-Kurti et al., 2010; Chung et al., 2010; Zwonitzer et al., 2010). This method has provided reliable data with high correlations between replications and environments.

Materials and Reagents

  1. Petri dishes, 100 x 15 mm (Genesee Scientific, catalog number: 32-107G )
  2. Micro-spatula (VWR, catalog number: 82027-518 )
  3. Parafilm M (Bemis, catalog number: PM999 )
  4. 50-ml conical tubes (Corning, Falcon®, catalog number: 352070 )
  5. 15-ml sterile tube
  6. Garbage bag large enough to line cooler
  7. Newspapers
  8. Gloves
  9. Identi-plug foam plugs (Jaece Industries, catalog number: L800-E )
  10. Aluminum foil
  11. Small metal beads (Ballistic Products, #4 shot SHZ04 or similar)
  12. Isolates of Cochliobolus heterostrophus frozen in 50% glycerol
  13. Sorghum grain (wheat or barley may also be used)
    Note: The sorghum should not be treated with any chemicals or fungicides. Sorghum intended for birdseed, also called milo, is ideal.
  14. Difco Potato Dextrose Agar (PDA) media (BD, DifcoTM, catalog number: 213400 )
  15. A small quantity of 70% ethanol in a glass container with a lid
  16. Tween-20
  17. V8 juice
  18. Agar
  19. CaCO3
  20. V8-agar medium (see Recipes)

Equipment

  1. Laminar flow workbench (NuAire, model: AireGardTM ES NU-301 , catalog number: 301-630)
  2. Incubator (Percival Scientific, model: I-35LL )
  3. Tongs or large tweezers
  4. Scalpel (EISCO, catalog number: BIO182A )
  5. 1 L Erlenmeyer flasks (Corning, PYREX®, catalog number: 5100-1L )
  6. Autoclave
  7. Alcohol lamp (such as C&A Scientific, catalog number: 97-5313 ) filled with ethanol
  8. Plastic buckets
  9. Ventilated trays (Buckhorn, catalog number: BT28220522 )
  10. Oscillating fan (Air King, catalog number: 9119 )
  11. Cooler
  12. Pails

Procedure

  1. Propagating fungal cultures on potato dextrose agar (the work should be performed inside a laminar flow hood)
    1. Make PDA plates, preparing media and autoclaving according to package instructions. After autoclaving, the molten PDA should be allowed to cool to about 60 °C before pouring approximately 18 to 20 ml into each Petri dish.
    2. Flame-sterilize the micro-spatula and use it to scoop a small chunk–50 to 100 µl–of frozen C. heterostrophus fungal stock onto a plate; repeat for each desired C. heterostrophus isolate. Seal the plates with Parafilm and place them into an incubator at constant 25 °C, 12-h light/dark. Allow to grow for 10 to 14 days.
    3. Propagate the fungal cultures: To produce several plates from the initial plate, use a flame-sterilized scalpel to cut a 1-cm square from the leading edge of a colony and dab it over the surface of a fresh plate. Incubate as before (Step A2). These secondary plates will be used to inoculate the grain.
    4. If desired, cultures can be transferred again onto fresh PDA plates for several more generations. Plates should be used at least 10 to 14 days after inoculation, but older plates can be used. We have used plates as old as 3 months. Fungal pathogens are known to sometimes become less aggressive pathogens after prolonged growth and repeated transfer on plates. While we have not observed this phenomenon with C. heterostrophus, we start a fresh culture from the frozen stock to produce inoculum each season.

  2. Propagating fungal cultures on grain
    1. Begin preparation of the grain three or four days before inoculating with fungal cultures. Fill plastic buckets about half full with sorghum (or barley or wheat), then add lukewarm water to three-quarters full. Allow the grain to soak for three days.
    2. Drain the water away by holding a screen against the edge of the bucket to catch debris and pouring the water off. Scoop the grain into 1-L flasks, filling to 700 ml. As each flask is filled with grain, add water to the flask, swirl to rinse the grain, and strain the water away through a screen.
    3. Cap each flask with an Identi-plug and cover the top with a square of foil. Autoclave for 1 h at 121 °C. Allow to cool.
    4. Prepare sterile tubes for production of fungal suspension: Into each 50-ml conical tube, add about 4 metal beads and 35 ml water. Autoclave for 15 min at 121 °C. Allow to cool.
    5. Prepare fungal suspension using the sterile tubes and fungal cultures grown on PDA plates: In laminar flow hood using a flame-sterilized scalpel, cut the fungal mat and underlying media into thin strips. Divide the material from one plate into two tubes (Figure 1). Cap the tubes tightly and shake vigorously to further break up the fungal culture. This will produce a slurry containing a suspension of fungal spores and mycelial fragments.


      Figure 1. Making fungal suspension

    6. Inoculate the grain: In the laminar flow hood, one tube of fungal suspension can be dispersed into three or four flasks of sterilized grain. Collect the flasks and the tube of fungal suspension. Shake the flasks to loosen grain. Remove the foil caps. Flame-sterilize the large forceps, shake the fungal suspension and remove the cap, then work quickly to remove a foam stopper with the forceps, pour in 10-15 ml of fungal suspension, and replace the foam stopper for each flask (Figure 2). Replace the foil caps. Gently shake each flask to distribute the fungus.


      Figure 2. Pouring the fungal suspension into sterile sorghum grain

    7. Incubate on a lab bench at room temperature (Figure 3) for about 10 days or until grains are evenly covered in fungus (Figure 4), shaking the flasks every 2 to 3 days to distribute the fungus and allow for even growth. Vigorous shaking is sometimes required to break up clumps.


      Figure 3. Hyphae growing from PDA fragment 1 day post-inoculation


      Figure 4. A mature sorghum grain culture

    8. Dry the grain inoculum: Empty the inoculum from the flasks into buckets lined with plastic bags. A sterile spatula can be used to break up large clumps. Transport to a dry, covered location such as a garage or barn. Line ventilated trays with newspaper and spread the grain in a layer no deeper than 3 cm. Allow the trays of inoculum to dry, using a fan for air circulation; stir and break up clumps with gloved hands every few days. It is important that the grain inoculum is dried rapidly as it is susceptible to molding. The drying process usually takes about two weeks.
    9. When the inoculum is completely dry, store in closed plastic bags at 2-7 °C and < 50% relative humidity. The inoculum may remain viable for several years if well-dried and kept cool.

  3. Inoculating maize
    1. The field experiment should be set up with one or two rows of border corn on all sides of the experimental block so that experimental plots do not experience ‘edge effects’. Disease symptoms are often slightly lower on the very edges of a field due, presumably, to lower disease pressure and humidity. We generally set up two or three replications of each experiment and try to physically separate the replications as much as possible so that if one replication is planted in a ‘bad’ part of the field, the other replication is unlikely to be in that same area. In general, most experiments are performed over two or three years or environments with two or three replications per environment/year.
    2. Field-grown maize plants should ideally be inoculated as early as possible after most plants in the field have formed a whorl. This is usually after about 5 to 6 weeks growth when the plants are at the 7 to 9 leaf stage, although when working with diverse lines, some will likely be larger and smaller. Some variation within the field is acceptable. If some plants are too small to inoculate that is also OK as they will be infected by the secondary inoculum that is generated.
    3. On the day of inoculation, combine the dry sorghum grain inoculum of the desired fungal isolates in a cooler. About 10 gallons of inoculum is used to inoculate about 3 acres of experiments or approximately 40,000 plants.
    4. If a large experiment (say more than 2 acres) is to be inoculated, it is best to assemble a team of several volunteers, say 1-2 people per acre, to inoculate it. This is because inoculation can take some time and is strenuous since there is a lot of bending involved. Give each volunteer a pail of inoculum and have them drop about ten to twenty grains of inoculum into the whorl of each maize plant (Figure 5, Video 1). Move across the field row by row until every row is inoculated. N.B. While the goal is to inoculate every plant with an equal amount of inoculum, this is not realistically achievable, nor is it essential. The goal of the initial inoculation is to generate secondary inoculum which will spread over the field creating a high, uniform disease pressure. For the same reason, it is not important that the same person inoculate a whole experiment.


      Figure 5. Details of field inoculation. A. Applying inoculum in the field; B. Maize plants immediately after inoculation.

      Video 1. Video of field inoculation

    5. The inoculum is ‘activated’ by moisture which initiates fungal growth. If overhead irrigation is available, it should be applied briefly. If not, rain or dew will collect in the whorl, allowing the fungus to grow and proliferate. Initial SLB symptoms should be apparent in 4-7 days (Figure 6), but disease severity should not be rated based on this early infection.


      Figure 6. Maize plants 2 weeks after inoculation, showing disease symptoms where leaves were directly inoculated. Subsequent growth is initially free of symptoms, though symptoms will develop on these leaves later in the season.

  4. Scoring disease
    1. In general, the initial symptoms of the disease arising from the primary inoculum are quite severe but the corn quickly outgrows these and much milder (or no) symptoms are apparent initially on subsequent growth. Symptoms then become more severe after flowering. Plants should be rated for disease soon after anthesis when an ear is apparent, and then one to three times more at approximately 10-14 day intervals. Disease is rated by observing the ear leaf and the leaf above, then rating the symptoms on a 1 to 9 scale where the maximum score of nine indicates no disease symptoms and the minimum score of one indicates complete death of the plant (Figure 7, reproduced from Kump et al. (2011)). Generally, several plants in each plot are inspected briefly and one representative score is recorded for each plot unless disease severity obviously differs from plant to plant. In general an experienced scorer can rate a plot in about 15 to 20 sec.


      Figure 7. SLB Disease symptom scoring guide. SLB scoring rubric: 9-No evidence of leaf blight; 8-A few spots on the lower leaves; 7-A few spots on the ear leaf; 6-More spots on the ear leaf but the lesions don’t coalesce; 5-Lesions on the ear leaf have grown together, particularly at the tip of the leaf to give quite large necrotic areas; 4-Lesions on the leaf above the ear leaf have grown together too; 3-Leaf above the ear leaf almost completely dead; 2-Almost all tissue on the plant dead; 1-Everything brown (reproduced from Kump et al., 2011).

    2. Once the plant has started to senesce such that necrosis due to senescence is visible on the ear leaf, then it is no longer possible to score disease accurately. In general, scoring is possible for about five weeks after the start of anthesis but unusually hot dry weather can decrease this period.
    3. In hotter environments, for instance in the southern US, leaves will curl up in the afternoons, making scoring difficult. In these cases, it is advantageous to try to start to score as early as possible in the morning before curling occurs.
    4. The Field Book app (Rife and Poland, 2014) on a tablet computer is useful for recording scores of large populations in the field.
    5. It is important that an entire experimental replication is rated on the same day by the same person since the disease progresses rapidly and scores may change from day to day. We have found that scores between different scorers are somewhat variable but that relative score differentials between plots do not vary significantly. This has been noted elsewhere (Poland and Nelson, 2011). 
    6. In some cases, for various reasons, individual plants or rows or sets of rows may not grow as they should or may be damaged by insect or animal pests. In our experience, it is preferable to record the scores of such rows as missing data rather than to try to ascribe a score to a row or plant that may not be accurate. We have found that plants that do not produce a healthy ear with normal developing seed may give inaccurate scores since source-sink relationships in the plant significantly affect the development of symptoms. While in general, the disease pressure is uniform across the field, various factors such as soil type and local topography may cause the disease pressure to vary somewhat. Rows that are to be directly compared should therefore be planted as close to each other as possible.

Data analysis

The analytical approach will depend on experimental design. Typically, when three or more sets of disease ratings have been recorded over the season, the disease rating for each plot is calculated as an area under disease progress curve (AUDPC) or a standardized AUDPC (sAUDPC). sAUDPC is calculated by taking the average value of two consecutive ratings and multiplying by the number of days between the ratings. Values are then summed over all the intervals and then adjusted by dividing by the number of days of evaluation, so that the sAUDPC scores are on a similar one to nine scale as the initial ratings (Campbell and Madden, 1990). In other cases, the scores from the different scoring dates can be analyzed separately to identify resistance that is apparent at different times of the season (e.g., Balint-Kurti et al., 2006).

Notes

  1. Northern Leaf Blight inoculation and rating. Exserohilum turcicum (causal agent of Northern Leaf Blight) grows more slowly than C. heterostrophus, so its inoculum can be produced by following the same protocol but allowing two to four extra days for the fungus to grow at each stage (on the PDA plates and on the sterile sorghum). We have found that scoring NLB infection based on a percentage necrotic leaf area over the whole plant is more effective than using a 1 to 9 scale as described for SLB above.
  2. Gray Leaf Spot inoculation and rating. Cercospora zeae-maydis (causal agent of Gray Leaf Spot) grows more slowly than C. heterostrophus and produces a leathery, dense fungal mat when grown on PDA, which is not easily distributed in the sterile sorghum. To produce inoculum of C. zeae-maydis, following the initial growth on PDA, prepare a suspension by shaking several slices of the fungal culture in a 15-ml sterile tube containing 7.5 ml of 0.05% Tween-20 and several metal balls. Pipet 1 to 2 ml of the suspension onto V8-agar (10% V8 juice, 15 g/L agar, 1g/L CaCO3, autoclave for 30 min) plates, tilt to spread evenly, allow to dry slightly, then Parafilm and incubate for about 2 weeks. Use the V8-agar cultures to inoculate the sterile sorghum. GLS is scored in an essentially identical way to SLB.

Recipes

  1. V8-agar medium
    10% V8 juice
    15 g/L agar
    1g/L CaCO3
    Autoclave at 121 °C for 30 min

Acknowledgments

We would like to thank all our jolly bands of inoculators over the years for sacrificing their backs for the sake of science, especially Greg Marshall. We thank Cathy Herring and her staff at Central Crops Research Station in Clayton NC, where most of these inoculations and ratings were performed. Dr. Martin Carson provided critical guidance and advice in setting up this protocol. We appreciate Brent McCraven’s help with video editing. Luis Lopez-Zuniga took some of the pictures shown in the figures. This work has been funded by USDA-ARS and by NSF grant # 1127076. The authors declare no conflicts of interest or competing interests.

References

  1. Balint-Kurti, P. J., Krakowsky, M. D., Jines, M. P., Robertson, L. A., Molnar, T. L., Goodman, M. M. and Holland, J. B. (2006). Identification of quantitative trait Loci for resistance to southern leaf blight and days to anthesis in a maize recombinant inbred line population. Phytopathology 96(10): 1067-1071.
  2. Balint-Kurti, P. J., Wisser, R. and Zwonitzer, J. C. (2008a). Use of an advanced intercross line population for precise mapping of quantitative trait loci for gray leaf spot resistance in maize. Crop Sci 48: 1696-1704.
  3. Balint-Kurti, P. J., Yang, J., Van Esbroeck, G., Jung, J. and Smith, M. E. (2010). Use of a maize advanced intercross line for mapping of QTL for northern leaf blight resistance and multiple disease resistance. Crop Sci 50: 458-466.
  4. Balint-Kurti, P. J., Zwonitzer, J. C., Pe, M. E., Pea, G., Lee, M. and Cardinal, A. J. (2008b). Identification of quantitative trait Loci for resistance to southern leaf blight and days to anthesis in two maize recombinant inbred line populations. Phytopathology 98(3): 315-320.
  5. Balint-Kurti, P. J., Zwonitzer, J. C., Wisser, R. J., Carson, M. L., Oropeza-Rosas, M. A., Holland, J. B. and Szalma, S. J. (2007). Precise mapping of quantitative trait loci for resistance to southern leaf blight, caused by Cochliobolus heterostrophus race O, and flowering time using advanced intercross maize lines. Genetics 176(1): 645-657.
  6. Belcher, A. R., Zwonitzer, J. C., Santa Cruz, J., Krakowsky, M. D., Chung, C. L., Nelson, R., Arellano, C. and Balint-Kurti, P. J. (2012). Analysis of quantitative disease resistance to southern leaf blight and of multiple disease resistance in maize, using near-isogenic lines. Theor Appl Genet 124(3): 433-445.
  7. Byrnes, K.J. and Pataky, J. K. (1989). Relationships between yield of three maize hybrids and severity of southern leaf blight caused by race O of Bipolaris maydis. Plant Disease 73: 834-840.
  8. Campbell, C. L. and Madden, L. V. (1990). Introduction to plant disease epidemiology. John Wiley and Sons pp: P192-194.
  9. Carson, M. L., Stuber, C. W. and Senior, M. L. (2004). Identification and mapping of quantitative trait Loci conditioning resistance to southern leaf blight of maize caused by Cochliobolus heterostrophus race O. Phytopathology 94(8): 862-867.
  10. Chung, C. L., Longfellow, J. M., Walsh, E. K., Kerdieh, Z., Van Esbroeck, G., Balint-Kurti, P. and Nelson, R. J. (2010). Resistance loci affecting distinct stages of fungal pathogenesis: use of introgression lines for QTL mapping and characterization in the maize--Setosphaeria turcica pathosystem. BMC Plant Biol 10: 103.
  11. Fisher, D. E., Hooker, A. L., Lim, S. M. and Smith, D. R. (1976). Leaf infection and yield loss caused by four Helminthosporium leaf diseases of corn. Phytopathology 66: 942-944.
  12. Holley, R. N. and Goodman, M. M. (1989). New sources of resistance to southern corn leaf blight from tropical hybrid maize derivatives. Plant Dis 73: 562-564.
  13. Kump, K. L., Bradbury, P. J., Wisser, R. J., Buckler, E. S., Belcher, A. R., Oropeza-Rosas, M. A., Zwonitzer, J. C., Kresovich, S., McMullen, M. D., Ware, D., Balint-Kurti, P. J. and Holland, J. B. (2011). Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population. Nat Genet 43(2): 163-168.
  14. Mueller, D. S., Wise, K. A., Sisson, A. J., Allen, T. W., Bergstrom, G. C., Bosley, B., Bradley, C. A., Byamukama, E. C., Chilvers, M. I., Collins, A., Faske, T., Friskop, A. J., Hollier, C. A., Isakeit, T., Jackson-Ziems, T. A., Jardine, D. J., Kinzer, K., Koenning, S. R., Malvick, D. K., Meyer, R. F., McMullen, M., Mostrom, M. S., Paul, P., Robertson, A. E., Roth, G. W., Smith, D. L., Tande, C. A., Tenuta, A., Vincelli, P. and Warner, F. (2016). Corn disease loss estimates from the United States and Ontario, Canada from 2012 to 2015. Plant Health Progress.
  15. Negeri, A. T., Coles, N. D., Holland, J. B. and Balint-Kurti, P. J. (2011). Mapping QTL controlling southern leaf blight resistance by joint analysis of three related recombinant inbred line populations. Crop Sci 51: 1571-1579.
  16. Poland, J. A. and Nelson, R. J. (2011). In the eye of the beholder: the effect of rater variability and different rating scales on QTL mapping. Phytopathology 101(2): 290-298.
  17. Rife, T. W. and Poland, J. A. (2014). Field book: an open-source application for field data collection on android. Crop Sci 54: 1624-1627.
  18. Santa-Cruz, J. H., Kump, K. L., Arellano, C., Goodman, M. M., Krakowsky, M. D., Holland, J. B. and Balint-Kurti, P. J. (2014). Yield effects of two southern leaf blight resistance Loci in maize hybrids. Crop Sci 54: 882-894.
  19. Ullstrup, A. J. (1972). The impacts of the southern corn leaf blight epidemics of 1970-1971. Annu Revi Phytopathol 10: 37-50.
  20. Wang, M., Wang, S., Ma, J., Yu, C., Gao, J. and Chen, J. (2017). Detection of Cochliobolus heterostrophus races in South China. J Phytopathology 165: 681-691.
  21. Yang, Q., He, Y., Kabahuma, M., Chaya, T., Kelly, A., Borrego, E., Bian, Y., El Kasmi, F., Yang, L., Teixeira, P., Kolkman, J., Nelson, R., Kolomiets, M., J, L. D., Wisser, R., Caplan, J., Li, X., Lauter, N. and Balint-Kurti, P. (2017). A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens. Nat Genet 49(9): 1364-1372.
  22. Zwonitzer, J. C., Bubeck, D. M., Bhattramakki, D., Goodman, M. M., Arellano, C. and Balint-Kurti, P. J. (2009). Use of selection with recurrent backcrossing and QTL mapping to identify loci contributing to southern leaf blight resistance in a highly resistant maize line. Theor Appl Genet 118(5): 911-925.
  23. Zwonitzer, J. C., Coles, N. D., Krakowsky, M. D., Arellano, C., Holland, J. B., McMullen, M. D., Pratt, R. C. and Balint-Kurti, P. J. (2010). Mapping resistance quantitative trait Loci for three foliar diseases in a maize recombinant inbred line population-evidence for multiple disease resistance? Phytopathology 100(1): 72-79.

简介

田间种植的玉米接种南方叶枯病的致病剂异叶蜗杆菌,通过在生长的早期阶段,将侵染了真菌的高粱谷物滴入每个玉米植株的螺纹中。 最初的病变产生由风和雨分散的二次接种物,引起多次感染循环,从而在开花后发生疾病评分时在整个区域确保高度均匀的疾病压力。 这种方法只需稍作修改,也可用于研究北方叶枯病(由Exserohilum turcicum引起)和灰叶斑点(Emesospora zeae-maydis)引起的玉米真菌病害。


【背景】南方叶枯病(SLB),由蜗杆异轴旋回(Drechs。)Drechs引起。 [anamorph = Bipolaris maydis (Nisikado)Shoemaker]是一种普遍的玉米病害,在热带,潮湿的热带和亚热带地区,如美国东南部,印度部分地区,非洲,拉丁美洲和南欧。在1970 - 71年,由C引起的SLB流行。异质性种群T感染了携带德克萨斯雄性不育细胞质(cms-T)的杂交种,导致美国总玉米产量估计损失15%(Ullstrup,1972)。在1970年流行后,cms-T玉米被抗T种质的正常细胞质玉米所取代。

目前,O型是美国和全球SLB的主要原因(Wang等人,2017)。 SLB抵抗 C。 heterostrophus race O是主要由加性或部分显性基因作用定量遗传的(Holley and Goodman,1989)。在实验条件下,由于感染C而导致产量损失。已经观察到异质体动物种族O高达46%(Fisher等人,1976; Byrnes和Pataky,1989)。然而,商业生产中的损失通常不那么严重(Mueller等人,2016年)。

接种和评级的这种方法基于Carson等人(2004)开发的方法学,尽管类似的方法已经用于许多先前的研究(例如,Fisher >等人,1976年)。我们已将其用于许多研究中以筛选SLB抗性的种质并阐明其遗传基础(Balint-Kurti等人,2006; 2007和2008b; Zwonitzer等人, 2009年和2010年; Kump等人,2011年; Negeri等人,2011年; Belcher等人,2012年; Santa-Cruz等人,2014; Yang等人,2017)。我们还使用了基本相同的方法来评估对另外两种叶面真菌病害的抗性;例如,由Cercospora zeae-maydis (例如,Balint-Kurti等人,2008a)和北方叶枯病(NLB)引起的灰叶斑病( eg,eg ,Balint-Kurti et al。,2010; Chung et al。,2010; Zwonitzer等人,2010)。该方法提供了复制和环境之间高度相关的可靠数据。

关键字:玉米, 异旋孢腔菌, 接种, 真菌病

材料和试剂

  1. 培养皿,100×15毫米(Genesee Scientific,目录号:32-107G)
  2. 微铲(VWR,目录号:82027-518)
  3. Parafilm M(Bemis,目录号:PM999)
  4. 50毫升锥形管(Corning,Falcon ,目录号:352070)
  5. 15毫升无菌管
  6. 垃圾袋足够大,可放置冷却器
  7. 报纸
  8. 手套
  9. 标识泡沫塞(Jaece Industries,目录号:L800-E)
  10. 铝箔
  11. 小金属珠(弹道产品,#4拍摄SHZ04或类似)
  12. 冷冻在50%甘油中的 Cochliobolus heterostrophus 的分离株
  13. 高粱谷物(小麦或大麦也可以使用)
    注意:高粱不应该用任何化学物质或杀真菌剂处理。高粱意欲用于鸟类,也称为米罗,是理想的。
  14. Difco马铃薯葡萄糖琼脂(PDA)培养基(BD,Difco TM,目录号:213400)
  15. 少量的70%乙醇在带盖玻璃容器中
  16. Tween-20
  17. V8果汁
  18. 琼脂
  19. CaCO 3
  20. V8-琼脂培养基(见食谱)

设备

  1. 层流工作台(NuAire,型号:AireGard TM ES NU-301,目录号:301-630)
  2. 孵化器(Percival Scientific,型号:I-35LL)
  3. 夹钳或大镊子
  4. 手术刀(EISCO,目录号:BIO182A)
  5. 1升锥形瓶(Corning,PYREX ®,目录号:5100-1L)
  6. 高压灭菌器
  7. 酒精灯(如C&amp; A Scientific,目录号:97-5313)填充乙醇
  8. 塑料桶
  9. 通风盘(Buckhorn,目录号:BT28220522)
  10. 摆动风扇(Air King,目录号:9119)
  11. 冷却器

程序

  1. 在马铃薯葡萄糖琼脂上繁殖真菌培养物(工作应在层流罩内进行)
    1. 根据包装说明制作PDA平板,准备培养基和高压灭菌。高压灭菌后,应将熔化的PDA冷却至约60℃,然后向每个培养皿中倒入约18至20ml。
    2. 对微刮刀进行火焰消毒,并用它来舀取一小块50至100μl的冷冻的C。 heterostrophus 霉菌原液加入平板;重复每个期望的C. heterostrophus分离物。用Parafilm密封平板并置于恒温25°C,12小时光照/黑暗的培养箱中。允许增长10到14天。
    3. 传播真菌培养物:为了从最初的平板上产生几块平板,使用火焰消毒的手术刀从殖民地的前缘切下1厘米的正方形,并将其贴在新鲜平板的表面上。像以前一样孵育(步骤A2)。这些辅助板将用于接种谷物。
    4. 如果需要,可以再次将培养物转移到新鲜的PDA平板上几代。接种后至少10至14天应使用平板,但可使用较旧的平板。我们已经使用了3个月的旧版。已知真菌病原体在长时间生长和在平板上重复转移后有时变得不那么侵略性的病原体。虽然我们没有观察到这种现象,但是我们从冷冻原料开始新鲜培养,每个季节都能生产接种物。

  2. 在谷物上传播真菌培养物
    1. 在接种真菌培养物之前三或四天开始准备谷物。用高粱(或大麦或小麦)填满大约一半的塑料桶,然后加入温水至四分之三满。让谷物浸泡三天。
    2. 通过在水桶边缘放置一个筛子来清除水分,以清除水中的杂物并将水倒掉。将谷物铲入1升烧瓶中,装入700毫升。当每个烧瓶装满谷物时,向烧瓶中加水,旋转冲洗谷物,并通过筛网将水分滤出。
    3. 用Identi-plug盖住每个烧瓶并用一个方形箔覆盖顶部。在121℃高压灭菌1小时。允许冷却。
    4. 准备用于生产真菌悬浮液的无菌试管:在每个50ml锥形管中加入约4个金属珠和35ml水。在121℃高压灭菌15分钟。允许冷却。
    5. 使用生长在PDA平板上的无菌管和真菌培养物制备真菌悬浮液:在使用火焰消毒的手术刀的层流罩中,将真菌垫和下面的介质切成细条。将材料从一块板分成两个管(图1)。紧紧盖住试管并大力摇动以进一步分解真菌培养物。这将产生含有真菌孢子和菌丝碎片悬浮液的浆液。

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      图1.制造真菌悬浮

    6. 接种谷物:在层流罩中,可将一管真菌悬浮液分散到三或四个灭菌谷物瓶中。收集烧瓶和真菌悬液管。摇动烧瓶以放松粮食。取下铝箔盖。对大镊子进行火焰消毒,摇动真菌悬液并取下盖子,然后迅速用钳子取出泡沫塞,倒入10-15ml真菌悬液,并替换每个瓶子的泡沫塞(图2) 。更换箔盖。轻轻摇动每个烧瓶分发真菌。


      图2.将真菌悬浮液倒入无菌高粱谷物

    7. 在室温下(图3)在实验台上孵育约10天,或者直到谷物被真菌均匀覆盖(图4),每2至3天摇动培养瓶以分布真菌并允许均匀生长。
      有时需要剧烈晃动才能分解团块。


      图3.接种后1天PDA菌体生长的菌丝


      图4.成熟的高粱谷物培养

    8. 干燥谷物接种物:将烧瓶中的接种物倒入装有塑料袋的桶中。无菌铲可以用来分解大块。运输到干燥,有盖的位置,如车库或谷仓。用报纸将通风的托盘放在一起,并将其撒在不超过3厘米的层中。使用风扇进行空气循环,让接种物盘子干燥;每隔几天用手套手搅动并打碎丛块。由于易于成型,因此谷物接种物很快干燥很重要。
      干燥过程通常需要两周左右
    9. 当接种物完全干燥时,将其储存在2-7℃的封闭塑料袋中, 50%的相对湿度。
      如果干燥并保持凉爽,接种物可保持数年
  3. 接种玉米
    1. 田间试验应在试验区块的两侧设置一排或两排边界玉米,以便实验地块不会遇到“边缘效应”。可能是由于疾病压力和湿度降低,疾病的症状通常在野外的边缘稍低。我们通常为每个实验设置两到三个重复,并尝试尽可能多地物理分离复制,以便如果一个复制在种子的“坏”部分种植,另一个复制不太可能在同一个区域。一般来说,大多数实验是在两年或三年内进行的,或者是每个环境/年有两到三次重复的环境。
    2. 理想情况下,田间种植的玉米植株应在田间大部分植株形成螺纹后尽早接种。这通常在植物处于7至9叶阶段后约5至6周的生长之后,但是当处理不同的系时,一些可能会越来越小。该领域内的一些变化是可以接受的。如果一些植物太小而不能接种,那也是可以的,因为它们会被所产生的二次接种物感染。
    3. 在接种当天,将所需真菌分离物的干燥高粱粒接种物在冷却器中结合。
      约10加仑的接种物用于接种约3英亩的实验或约40,000株植物。
    4. 如果要接种一个大型实验(比方说2英亩以上),最好组装一个由几个志愿者组成的团队,每英亩1-2人接种。这是因为接种需要一些时间并且很费劲,因为涉及很多弯曲。给每个志愿者一桶接种物,并让他们将大约10到20粒接种物滴入每个玉米植株的螺纹中(图5,视频1)。逐行移动场,直到每行被接种。注:虽然目标是接种每个植物等量的接种物,但这不是现实可行的,也不是必需的。初始接种的目标是产生二次接种物,该接种物将在整个田间扩散,产生高度均匀的疾病压力。出于同样的原因,同一个人接种整个实验并不重要。


      图5.现场接种的细节。 :一种。在田间施用接种物; B.接种后马上接种玉米。

      视频1

    5. 接种物由引发真菌生长的水分“活化”。如果可以使用高架灌溉,则应该简单应用。否则,雨水或露水会积聚在螺纹中,使真菌生长繁殖。初始SLB症状应在4-7天内明显(图6),但不应根据此早期感染评估疾病严重程度。


      图6.接种2周后的玉米植株,显示直接接种叶子的疾病症状。 随后的生长最初没有任何症状,尽管季节后期会在这些叶子上出现症状。

  4. 评分疾病
    1. 一般来说,由主要接种物引起的疾病的最初症状非常严重,但玉米迅速长大,最初在随后的生长中明显更温和(或没有)症状。开花后症状会变得更加严重。开花后不久,当耳朵明显时,植物应评为疾病,然后以约10-14天的间隔进行1-3次。通过观察上面的耳叶和叶子来评级疾病,然后以1至9的等级评分症状,其中最高评分9表示无疾病症状,最低评分1表示植物完全死亡(图7,转载来自Kump 等人(2011))。一般情况下,每个地块的几个植物进行简单的检查,每个地块记录一个代表性得分,除非植物对植物的疾病严重程度明显不同。一般来说,经验丰富的得分手可以在大约15至20秒内对情节进行评分。


      图8. SLB疾病症状评分指南。 SLB评分标准:9-没有叶枯病的证据; 8-下部叶子上的几个点; 7 - 耳叶上的几个点; 6-叶上有更多的斑点,但病斑不结合;耳叶上的5-病斑已经一起生长,特别是在叶尖处,以产生相当大的坏死区域;穗上叶上的4个病斑也一起生长;耳叶上方的3叶几乎完全死亡; 2-几乎植物上的所有组织都死亡; 1-Everything brown(转载自Kump et al。,2011)。

    2. 一旦植物开始衰老,使得由于衰老的坏死在耳叶上可见,则不再可能准确评分疾病。一般来说,在开花后五周左右可以进行评分,但异常炎热干燥的天气可能会缩短这段时间。
    3. 在比较炎热的环境中,例如在美国南部,树叶会在下午卷曲,使得评分困难。在这些情况下,尽早在冰壶发生前尽早开始评分是有利的。
    4. 平板电脑上的Field Book应用程序(Rife and Poland,2014)对于录制现场大量人群的分数非常有用。
    5. 由于疾病进展迅速,并且分数可能每天都在变化,所以整个实验复制在同一天由同一个人评定是很重要的。我们发现不同得分者之间的得分有些变化,但是情节之间的相对得分差异并没有显着变化。这已在其他地方提到过(波兰和尼尔森,2011)。&nbsp;
    6. 在某些情况下,由于各种原因,单个植物或行或一组行可能不会生长,因为它们应该或可能被昆虫或动物害虫破坏。根据我们的经验,最好将这些行的分数记录为缺失数据,而不是试图将分数归于可能不准确的行或植物。我们发现,由于植物源 - 汇关系显着影响症状的发展,因此不会产生具有正常发育种子的健康耳朵的植物可能得不准确的分数。一般来说,整个地区的疾病压力是均匀的,因此土壤类型和局部地形等多种因素可能会导致疾病压力有所变化。因此,应该直接比较的行应尽可能地彼此靠近。

数据分析

分析方法将取决于实验设计。通常情况下,当本季节记录了三组或更多组疾病等级时,每个小区的疾病等级计算为疾病进展曲线下面积(AUDPC)或标准化AUDPC(sAUDPC)。 sAUDPC是通过取两个连续评级的平均值并乘以评级之间的天数来计算的。然后将所有间隔的数值相加,然后除以评估的天数进行调整,以便sAUDPC评分与初始评分相似(Campbell和Madden,1990)。在其他情况下,可以分别分析不同评分日期的得分,以确定在季节不同时间显而易见的抗性(例如Balint-Kurti等人 ,2006)。

笔记

  1. 北方叶枯病菌的接种和评级
  2. 灰叶斑点接种和评级。 Cercospora zeae-maydis (灰叶斑病的致病因子)比生长得更慢。在PDA上生长时,它会产生一种皮革状的,致密的真菌垫,这种垫不容易分布在无菌高粱中。为了产生 C的接种物。在PDA上初始生长后,通过在含有7.5ml 0.05%Tween-20和几个金属球的15ml无菌试管中摇动几片真菌培养物来制备悬浮液。吸取1到2毫升悬浮液到V8-琼脂(10%V8汁,15克/升琼脂,1克/升碳酸钙,高压灭菌30分钟)上,倾斜以均匀分布,稍微干燥,然后封口膜并孵育约2周。使用V8-琼脂培养物接种无菌高粱。 GLS的得分方式与SLB基本相同。

食谱

  1. V8-琼脂培养基
    10%V8果汁

    15克/升琼脂 1g / L CaCO 3
    在121°C高压灭菌30分钟

致谢

我们要感谢多年来我们所有的乐队为了科学而牺牲自己的背部,特别是Greg Marshall。我们感谢Cathy Herring和她在克莱顿北卡罗莱纳州中央作物研究站的工作人员,他们在那里进行了大部分接种和评级。 Martin Carson博士提供了建立该协议的重要指导和建议。我们非常感谢Brent McCraven在视频编辑方面的帮助。 Luis Lopez-Zuniga拍摄了一些图中所示的照片。这项工作由USDA-ARS和NSF资助#1127076资助。作者声明没有利益冲突或利益冲突。

参考

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引用:Sermons, S. M. and Balint-Kurti, P. J. (2018). Large Scale Field Inoculation and Scoring of Maize Southern Leaf Blight and Other Maize Foliar Fungal Diseases. Bio-protocol 8(5): e2745. DOI: 10.21769/BioProtoc.2745.
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