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Nov 2016

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Field Inoculation and Classification of Maize Ear Rot Caused by Fusarium verticillioides
田间接种和轮枝镰孢菌引起的玉米穗粒腐病的分类   

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Abstract

Maize ear rot is a worldwide fungal disease mainly caused by Fusarium verticillioides and Fusarium graminearum. Maize planted in the field was inoculated with Fusarium verticillioides at the filling stage, 15 days after pollination. Two milliliters of spore suspension with a concentration of 5 x 106/ml was injected into the middle of the top ear using pricking ear method to cause maize ear rot. The thirty days after inoculation was the most suitable time for phenotypic evaluation of Fusarium resistance.

Keywords: Maize (玉米), Ear rot (穗粒腐病), Fusarium verticillioides (轮枝镰孢菌), Field (田间), Inoculation (接种), Classification (分类)

Background

Maize ear rot is a fungus-induced ear disease that occurs widely in Europe, America, Africa, Asia and Oceania (Desjardins et al., 2000; Pamphile and Azevedo, 2002; Hussain et al., 2005; Anjorin et al., 2008; Görtz et al., 2008; Rahjoo et al., 2008; Dorn et al., 2009; Eckard et al., 2011; Scauflaire et al., 2011; Madania et al., 2013). In 1977, maize ear rot swept through the state of Meghalaya, India. In 1988, maize ear rot was prevalent in the USA, and became a key biological control factor in maize production of African, resulting in a 48% reduction in maize yield (Vigier et al., 2001). Moreover, the infected maize ears also produced mycotoxins such as fumonisin and deoxynivalenol, which seriously threatened human and animals’ health (Gelderblom et al., 1988; Wild and Turner, 2002; Zhou et al., 2010).

At present, it is generally and internationally considered that Fusarium is the main pathogen causing ear rot in maize. However, different countries or different regions of the same country may have different types of pathogens, and the dominant pathogen may also be different in different regions. The dominant pathogen is Fusarium graminearum in the USA, Austria and Germany (Schaafsma et al., 1997; Goertz et al., 2010; Mesterházy et al., 2012), but Fusarium verticillioides in France, South Africa, Croatia, Iran, Argentina, Brazil and China (Presello et al., 2004; Aliakbari et al., 2007; Ivić et al., 2008; Folcher et al., 2010; Small et al., 2012; Stumpf et al., 2013; Qin et al., 2014). No matter what the pathogen is, it is very important to have an effective artificial inoculating method in order to get the accuracy resistance evaluation.

To date, a total of four inoculation methods were reported in publications, silk channel injection method, silk spray method, prick ear method and toothpick method. A large number of maize germplasms with resistance to maize ear rot were screened by prick ear method and the genetic basis of the resistance were elucidated in our previous study (Chen et al., 2002; Dong et al., 2006; Li et al., 2008; Sun, 2009; Chen, 2012; Li, 2012; Wang et al., 2016). This method that can easily control the inoculation dose together with the suitable inoculation time can accurately reflect the underlying resistance in the maize materials.

Materials and Reagents

  1. Gauze
  2. Mask
  3. Adhesive tape of paper
  4. Newspaper
  5. Inoculating needle
  6. Elastic
  7. Petri dish, 90 x 90 mm (Biosharp, catalog number: BS-90-D)
  8. Latex gloves (Biosharp, catalog number: BS-ST-001)
  9. Parafilm, 100 mm x 38 m (Parafilm, catalog number: PM-996)
  10. Aseptic breathable seal membrane
  11. Cover glass
  12. Scalpel
  13. Newly harvested maize hybrid seeds (Used for the growth of Fusarium verticillioides, Procedure B)
  14. Potato
  15. Fusarium verticillioides (The fungus strain was stored with glycerol in -80 °C refrigerator)
  16. Tween-80 (Solarbio, catalog number: P8360)
  17. Alcohol
  18. Sterile water
  19. D-glucose (Solarbio, catalog number: G8150)
  20. Agar powder (Solarbio, catalog number: A8190)
  21. PDA medium (see Recipes)

Equipment

  1. Glass rod
  2. 10 μl pipette (Eppendorf, 0.5-10 μl)
  3. Alcohol lamp filled with alcohol
  4. 2,000 ml beaker
  5. 500 ml conical flask
  6. 500 ml-1,000 ml water bottle
  7. Plastic bucket
  8. Super clean bench (HDL, model: HD-1360)
  9. Light incubator (Haixiang, model: PGX-400BP)
  10. High-pressure sterilizer (Boxun, model: YXQ-LS-52SII)
  11. Veterinary adjustable continuous syringe (Jiashan, model: 2ml-K, catalog number: JS-003, URL: http://www.jiashanqx.com/cn/products.asp?Bid=51)
  12. Light microscope (Motic, model: BA310)
  13. Hemacytometer (0.1 mm, 1/400 mm2, 16 x 25) (Qiujing, model: XB-K-25)
  14. Refrigerator (Haier, model: BCD-215KS)

Procedure

  1. Propagation of fungi on PDA medium (operation should be carried out in a super clean bench)
    1. Preparation of PDA plate
      The melted PDA is cooled to about 60 °C after sterilization under 121 °C high-temperature steam for half an hour, and then poured into each Petri dish for about 18-20 ml until completely set.
    2. Subculture on PDA media
      Use flame to disinfect the scalpel, and pick a bit of cold stored Fusarium verticillioides onto the plates. Repeat this step for a few other plates. Use parafilm to seal the plates and keep them in the dark at 28 °C for 5 to 7 days.
    3. Mass propagation on PDA plates
      Cut off a block of 1 cm2 from the edge of the PDA plate full of Fusarium verticillioides cultured in Step A2 above with a flame-disinfected scalpel, and put it on a new PDA plate for culture. Repeat this step until enough plates meet your needs. Then culture these plates at the same condition in Step A2 above.
    4. Preserve cultured Fusarium verticillioides at 4 °C refrigerator
      When the PDA plates were filled with Fusarium verticillioides after 5 to 7 days, keep them in 4 °C refrigerator for use.

  2. Propagation of Fusarium verticillioides on maize seeds media (operation should be carried out in super clean bench)
    1. Boil maize seeds
      Prepare 1,000 g newly harvested maize hybrid seeds without impurities, mildew and moth damage. Wash the seeds with tap water for 2-3 times, then use distilled water wash 1 time again to remove the remaining tap water. Add 3 L distilled water to the seed samples, and boil about 2 h or more time until the seeds just crack and the water content is moderate. The proper boiling time can avoid the grain rupture seriously and high moisture content which is not conducive to the growth of Fusarium verticillioides.
    2. Divide boiled seeds into conical flasks
      Remove the remaining water from the boiled maize seeds. And then fill boiled seeds in 500 ml conical flasks to the 200 ml line, and take this as a standard. Normally, 1,000 g seeds can make 10 conical flasks with around 200 ml boiled maize seeds.
    3. Seal the conical flasks
      Use two-layer aseptic breathable seal membrane or one-layer aseptic breathable seal membrane plus two-layer newspaper to seal the conical flasks with rubber band.
    4. Sterilization
      Sterilize the conical flasks with seeds in a high-pressure steam sterilizer at 121 °C for 30 min. Then, keep them at room temperature for 48 h to ensure no microbial contamination (Figure 1A).


      Figure 1. Operation procedure of Fusarium verticillioides propagation on maize medium. A. The maize seeds after sterilization. B. Cut the PDA culture medium with newly cultured Fusarium verticillioides into small pieces. C. Put three pieces into a conical flask. D. The maize culture medium after fully mixing. E. Fusarium verticillioides grow for 3 days on maize medium. F. Fusarium verticillioides grow for 8 days on maize medium.

    5. Inoculate the maize seeds
      In the super clean bench, put three pieces of 1 cm2 PDA medium with newly cultured Fusarium verticillioides in the conical flask and shake fully (Figures 1B-1D). Of course, you can also put more than three pieces for faster propagation. Then, culture them in a constant temperature incubator of 28 °C in the dark. Generally, the best harvest time is on the 7th-10th day because of the large number of spores and strong spore activity (Figures 1E and 1F).
      Note: With time going on, the number of spores does not increase, and some spores begin to deform. So it is best to harvest them in 10 days.

  3. Preparation of spore suspension for inoculation
    1. Collect spores
      One day before inoculation, open the conical flask filled with hypha, stir gently in one direction after add 200 ml sterilized water, then filter through eight layers of gauze into a 2,000 ml beaker. Normally, after repeat it three or four times, the most of spores could be washed off.
    2. Preparation of working solution
      Dilute the spore suspension above and mix well, then count the number of spores using a hemacytometer under a microscope. Ensure the final concentration of spore suspension is 5 x 106/ml. At last, add 2 μl Tween-80 in per ml spore suspension, mix evenly and stay at 4 °C for use. Generally, the spores in each conical flask can be made into 5 L working solution.
      Note: Once the working solution is prepared, use it as soon as possible, because we found that the concentration would change after two days in 4 °C refrigerator. You need to recalculate the number of spore in the working solution before use it after a long time.

  4. Inoculating maize
    1. Rational experimental design and excellent field management play an important role in reducing error
      Plant about 4 rows of border maize around the inoculation test field to ensure that the growth of the inoculation test materials is uniform. Set 2-3 replications to reduce the experimental errors. It is very important to control the pests, such as corn borers, especially.
    2. Inoculating time
      According to our study, the best inoculation period is at milk stage, around 15 days after pollination. At this time, the husk looks still green and the silk has just become brown and dry (Figure 2A). The kernels are full of milky liquid and look big and plump with the water content of about 60% (Figure 2B). So it is very easy to perform inoculation and suitable for growth of Fusarium verticillioides. Neither earlier nor later inoculation is helpful to inoculation and phenotypic evaluation. If earlier, the kernels are too little to carry out inoculation, and the whole ear will be faced with earlier death because of infection of Fusarium verticillioides. If later, the kernels become too hard to penetrate, and the fungus will not develop well because of lack of enough water in kernels. So the epidemic area of the ear is too small to reveal the difference of resistance to maize ear rot. In practice, it is very important to pay much attention to the growth stage of different maize materials.


      Figure 2. The 17-day-old ear of inbred line N6 after pollination. A. The husk and silk of the ear. B. The appearance of kernels.

    3. Inoculating method
      We usually perform the inoculation at 6:30-10:00 and 16:00-18:30 to avoid the high temperature to affect the efficiency of inoculation. The optimum dose, 2 ml, need to be injected into the middle of the ear at two times, 1 ml for one time, to make two inoculating holes that are 1 cm apart. Because the kernels in the middle are often plump and two inoculation holes can increase the success rate of inoculation. It’s better to put the needle into the seeds than between the seeds, since it can increase the rate of successful inoculation. When inoculating, you should limit the extension of the needle to less than 90 degrees at the ear in order to reduce the out-flow of the spore suspension from the inoculating hole (Figure 3A). At last, seal the inoculation point with a piece of paper adhesive tape (Figure 3B).
      Note: For the beginners, it is very necessary to practice a few times before inoculate the experiment in order to reduce experimental error caused by operation.


      Figure 3. Inoculation method in the field. A. The syringe needle penetrates into the kernel but not into the cob. B. Seal the inoculation point with paper adhesive tape.

  5. Scoring disease
    1. The phenotypic evaluation is conducted at the maturity stage. Generally speaking, after 3 days of inoculation, the color around the wound will turn to brown and there will be sparse white mycelium around the wound. At this time, there will also be thin white mycelium on the surface of the kernels near the wound and the space between kernel rows. The infected kernels begin to become yellowish-brow at the top, the junction of the silk and kernel. With time going on, the infected area will expand and the infected kernels gradually turned brown until rot. On the 20th day after inoculation, the diseased grains appear in the softening, which is typical symptom of maize ear rot. The hyphae start to spread slowly after twenty days of inoculation, so the infection areas almost keep stable until maturity.
    2. We find at least 3 types of symptoms caused by Fusarium verticillioides, as summarized below (Sun, 2009), and the resistance evaluation of germplasm resources according to the infected area (the percentage of infected kernels in the whole ear).
      1. Kernel rotten with germination. For example, the seed of maize line 177 under the higher humidity environment will germinate, and there are white mycelium around the germinated seeds in serious condition (Figure 4A).
      2. Kernel rotten within limited area, such as Yu 374 (Figure 4B).
      3. Kernel rotten within large area, such as inbred line N6 (Figure 4C).


        Figure 4. Different ear rot symptoms on different maize inbred lines. A. Maize line 177. B. Yu 374. C. Inbred line N6.

    3. Maize ear rot is classified into seven grades according to the infected area (the percentage of infected kernels in the whole ear) (Table 1). Generally, in order to minimize the error, two experienced researchers are enough to carry out the phenotypic evaluation.

      Table 1. Rating grade of maize ear rot


    4. It is very difficult to finish the large-scale evaluation of maize ear rot in a short time. So, you can harvest them with husk. Dry the ear with husk in the sun for 2-3 days; then phenotypic evaluation can be conducted. If without husk, it is very easy to result in kernel loss because of dry ear. However, it's better to evaluate the phenotype as soon as possible to minimize the experimental error.
    5. In addition, you can organize labor to peel off the husk in the field and break the upper stem of the ear, leaving only ears for evaluation. According to the evaluation of plot area, select a number of evaluators to evaluate. An experienced evaluator can evaluate around 0.1 hectare of inoculated field within 8 h. Note that the selection of evaluators should be consistent with the assessment criteria, and the rank of the same material should be consistent as far as possible.
    6. Generally, we record both the percentage of infected kernels in the whole ear and the grade at the same time.

Data analysis

  1. The method of analysis will depend on the experimental design.
  2. In order to better assess the disease grades, seed setting rate of the materials should be evaluated according to the 1-3 grades at the same time. During your later statistical calculation, you can effectively eliminate the abnormal value caused by the lack of full fruiting. Grade 1 of the seed setting rate indicates that full grain occupies more than 75% of panicle area, and grade 2 indicates that full grain occupies 50%-75% of panicle area. Grade 3 means full grain occupies less than 50% of panicle area.

Notes

In genetic research and breeding, in order to reduce the error of the evaluation of maize ear rot caused by fructifying, you can collect the pollen of other maize plants to increase the seed setting rate by artificial assistant pollination. Our study shows that individual material may have Xenia effect caused by artificial supplementary pollination, but the pollen of other materials has no effect on the contemporary grain resistance to maize ear rot.

Recipes

  1. PDA medium
    200 g/L potato (Boil and filter into beaker)
    20 g/L D-glucose
    15 g-20 g/L agar powder
    Autoclave at 121 °C for 30 min

Acknowledgments

This project was funded by the National Natural Science Foundation of China (NSFC) (Grant No. 31761143009) and Key Scientific Research Projects of Henan Colleges and Universities (Grant No. 18A180014). We are grateful to the graduate students in our team for their efforts on the maize ear rot related research.

Competing interests

The author states that there is no conflict of interest or competing interest.

References

  1. Aliakbari, F., Mirabolfathy, M., Emami, M. and Mazhar, S. F. (2007). Natural occurrence of Fusarium species in maize kernels at gholestan province in northern Iran. Asian J Plant Sci 6(8): 1276-1281.
  2. Anjorin, S., Makun, H., Adesina, T., and Kudu, I. (2008). Effects of Fusarium verticilloides, its metabolites and neem leaf extract on germination and vigour indices of maize (Zea mays L.). Afr J Biotechnol 7(14): 2402-2406.
  3. Chen, J. (2012). Combine association and linkage mapping method to identify the resistance QTL for Fusarium ear rot. Henan Agricultural University. (in Chinese)
  4. Chen, W., Wu, J. and Yuan, H. (2002). Identification of resistance on maize ear rot. J Maize Sci 10(4): 59-60. (in Chinese)
  5. Desjardins, A. E., Manandhar, G., Plattner, R. D., Maragos, C. M., Shrestha, K. and McCormick, S. P. (2000). Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels. J Agric Food Chem 48(4): 1377-1383. 
  6. Dong, H., Song, W., Dai, X., Li, J., liu, C., Wu, J. (2006). Resistance of different tissues of maize ear to Fusarium moniliforme and Fusarium graminearum. J Maize Sci 14(4): 141-144. (in Chinese)
  7. Dorn, B., Forrer, H.-R., Schürch, S. and Vogelgsang, S. (2009). Fusarium species complex on maize in Switzerland: occurrence, prevalence, impact and mycotoxins in commercial hybrids under natural infection. Eur J Plant Pathol 125(1): 51-61.
  8. Eckard, S., Wettstein, F. E., Forrer, H. R. and Vogelgsang, S. (2011). Incidence of Fusarium species and mycotoxins in silage maize. Toxins (Basel) 3(8): 949-967.
  9. Folcher, L., Delos, M., Marengue, E., Jarry, M., Weissenberger, A., Eychenne, N. and Regnault-Roger, C. (2010). Lower mycotoxin levels in Bt maize grain. Agron Sustain Dev 30(4): 711-719.
  10. Gelderblom, W. C., Jaskiewicz, K., Marasas, W. F., Thiel, P. G., Horak, R. M., Vleggaar, R. and Kriek, N. P. (1988). Fumonisins--novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme. Appl Environ Microbiol 54(7): 1806-1811.
  11. Goertz, A., Zuehlke, S., Spiteller, M., Steiner, U., Dehne, H. W., Waalwijk, C., de Vries, I. and Oerke, E. C. (2010). Fusarium species and mycotoxin profiles on commercial maize hybrids in Germany. Eur J Plant Pathol 128(1): 101-111.
  12. Görtz, A., Oerke, E. C., Steiner, U., Waalwijk, C., Vries, I. and Dehne, H. W. (2008). Biodiversity of Fusarium species causing ear rot of maize in Germany. Cereal Res Commun 36(Supplement 6): 617-622.
  13. Hussain, S. M., Hess, K. L., Gearhart, J. M., Geiss, K. T. and Schlager, J. J. (2005). In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol In Vitro 19(7): 975-983.
  14. Ivić, D., Čabrić, M., Palaveršić, B. and Cvjetković, B. (2008). No correlation between pericarp thickness and Fusarium ear rot (Fusarium verticillioides) in croatian maize hybrids and lines. Maydica 53(1): 297-301.
  15. Li, H. (2012). QTL verification and mapping for resistance to Fusarium verticillioides ear rot based on near isogenic lines in maize. Henan Agricultural University. (in Chinese)
  16. Li, J., Wang, R., Han, Y., Chen, J., Sun, X. Liu, C., Ding, J., Wu, J et al. (2008). Near isogenic lines breeding with the resistance gene to maize ear rot by marker assistant selection. J Henan Agric Univ 42(3): 250-254. (in Chinese)
  17. Madania, A., Altawil, M., Naffaa, W., Volker, P. H. and Hawat, M. (2013). Morphological and molecular characterization of Fusarium isolated from maize in Syria. J Phytopathol 161(7-8): 452-458.
  18. Mesterházy, Á., Lemmens, M. and Reid, L. M. (2012). Breeding for resistance to ear rots caused by Fusarium spp. in maize – a review. Plant Breeding 131(1): 1-19.
  19. Pamphile, J. A. and Azevedo, J. L. (2002). Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L). World J Microb Biot 18(5): 391-396.
  20. Presello, D. A., Reid, L. M. and Mather, D. E. (2004). Resistance of argentine maize germplasm to Gibberella and Fusarium ear rots. Maydica 49(49): 73-81.
  21. Qin, Z., Ren, X., Jiang, K., Wu, X., Yang, Z. and Wang, X. (2014). Identification of Fusarium species and F.graminearum species complex causing maize ear rot in China. J Plant Protection 41(5): 589-596. (in Chinese)
  22. Rahjoo, V., Zad, J., Javan-Nikkhah, M., Gohari, A. M., Okhovvat, S. M., Bihamta, M. R., Razzaghian, J. and Klemsdal, S. S. (2008). Morphological and molecular identification of Fusarium isolated from maize ears in Iran. J Plant Pathol 90(3): 463-468.
  23. Scauflaire, J., Mahieu, O., Louvieaux, J., Foucart, G., Renard, F. and Munaut, F. (2011). Biodiversity of Fusarium species in ears and stalks of maize plants in Belgium. Eur J Plant Pathol 131(1): 59.
  24. Schaafsma, A. W., Nicol, R. W. and Reid, L. M. (1997). Evaluating commercial maize hybrids for resistance to gibberella ear rot. Eur J Plant Pathol 103(8): 737-746.
  25. Small, I. M., Flett, B. C., Marasas, W. F. O., McLeod, A., Stander, M. A. and Viljoen, A. (2012). Resistance in maize inbred lines to Fusarium verticillioides and Fumonisin accumulation in South Africa. Plant Disease 96(6): 881-888.
  26. Stumpf, R., Santos, J. d., Gomes, L. B., Silva, C. N., Tessmann, D. J., Ferreira, F. D., Machinski Junior, M. and Del Ponte, E. M. (2013). Fusarium species and fumonisins associated with maize kernels produced in Rio Grande do Sul State for the 2008/09 and 2009/10 growing seasons. Braz Jo Microbiol 44: 89-95.
  27. Sun, X. (2009). Inheritance of resistance to Fusarium moniliforme kernel and cob rot in maize. Henan Agricultural University. (in Chinese)
  28. Vigier, B., Reid, L. M., Dwyer, L. M., Stewart, D. W., Sinha, R. C., Arnason, J. T. and Butler, G. (2001). Maize resistance to gibberella ear rot: symptoms, deoxynivalenol, and yield1. Can J Plant Pathol 23(1): 99-105.
  29. Wang, Y., Zhou, Z., Gao, J., Wu, Y., Xia, Z., Zhang, H. and Wu, J. (2016). The Mechanisms of maize resistance to Fusarium verticillioides by comprehensive analysis of RNA-seq data. Front Plant Sci 7: 1654.
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  31. Zhou, X., Heyer, C., Choi, Y.-E., Mehrabi, R. and Xu, J.-R. (2010). The CID1 cyclin C-like gene is important for plant infection in Fusarium graminearum. Fungal Gene Biol 47(2): 143-151.

简介

玉米穗腐病是一种世界范围的真菌病,主要由 Fusarium verticillioides >和 Fusarium graminearum >引起。 在授粉后15天,在灌浆阶段,在田间种植的玉米用 Fusarium verticillioides >接种。 使用刺耳法将2毫升浓度为5×10 6 p / sup / ml的孢子悬浮液注入顶耳的中部以引起玉米穗腐烂。 接种后30天是最适合表达评估 Fusarium >抗性的时间。

【背景】玉米穗腐病是一种真菌引起的耳病,广泛存在于欧洲,美洲,非洲,亚洲和大洋洲(Desjardins et al。>,2000; Pamphile and Azevedo,2002; Hussain et al 。>,2005; Anjorin et al。>,2008;Görtz et al。>,2008; Rahjoo et al。>,2008; Dorn et al。>,2009; Eckard et al。>,2011; Scauflaire et al。>,2011; Madania et al。,2013)。 1977年,玉米穗腐烂席卷印度梅加拉亚邦。 1988年,玉米穗腐病在美国普遍存在,并成为非洲玉米生产的关键生物控制因子,导致玉米产量减少48%(Vigier 等,>,2001)。此外,受感染的玉米穗还产生霉菌毒素,如伏马菌素和脱氧雪腐镰刀菌烯醇,严重威胁人类和动物的健康(Gelderblom et al。>,1988; Wild and Turner,2002; Zhou et al 。>,2010)。

目前,一般而且国际上认为 Fusarium >是引起玉米穗腐病的主要病原体。然而,同一国家的不同国家或不同地区可能具有不同类型的病原体,并且主要病原体在不同地区也可能不同。在美国,奥地利和德国,主要病原体是 Fusarium graminearum >(Schaafsma et al。>,1997; Goertz et al。>,2010;Mesterházy et al。>,2012),但 Fusarium verticillioides >在法国,南非,克罗地亚,伊朗,阿根廷,巴西和中国(Presello et al。 >,2004; Aliakbari et al。>,2007;Ivić et al。>,2008; Folcher et al。>,2010; Small 等人,>,2012; Stumpf et al。>,2013; Qin et al。>,2014)。无论病原体是什么,具有有效的人工接种方法以获得准确度抗性评估是非常重要的。

迄今为止,在出版物,丝通注射法,丝喷法,刺耳法和牙签法中共报道了四种接种方法。通过刺耳法筛选出大量对玉米穗腐病具有抗性的玉米种质,并在我们之前的研究中阐明了抗性的遗传基础(Chen et al。>,2002; Dong et al。>,2006; Li et al。>,2008; Sun,2009; Chen,2012; Li,2012; Wang et al。>,2016) 。该方法可以容易地控制接种剂量和合适的接种时间,可以准确地反映玉米材料中的潜在抗性。

关键字:玉米, 穗粒腐病, 轮枝镰孢菌, 田间, 接种, 分类

材料和试剂

  1. 纱布
  2. 面具
  3. 胶带纸
  4. 报纸
  5. 接种针
  6. 培养皿,90 x 90 mm(Biosharp,目录号:BS-90-D)
  7. 乳胶手套(Biosharp,目录号:BS-ST-001)
  8. Parafilm,100 mm x 38 m(Parafilm,目录号:PM-996)
  9. 无菌透气密封膜
  10. 盖玻片
  11. 解剖刀
  12. 新收获的玉米杂交种子(用于 Fusarium verticillioides >的生长,程序B)
  13. 土豆
  14. Fusarium verticillioides >(真菌菌株与甘油在-80°C冰箱中保存)
  15. Tween-80(Solarbio,目录号:P8360)
  16. 无菌水
  17. D-葡萄糖(Solarbio,目录号:G8150)
  18. 琼脂粉(Solarbio,目录号:A8190)
  19. PDA介质(见食谱)

设备

  1. 玻璃棒
  2. 10μl移液器(Eppendorf,0.5-10μl)
  3. 酒精灯充满酒精
  4. 2,000毫升烧杯
  5. 500毫升锥形瓶
  6. 500毫升-1000毫升水瓶
  7. 塑料桶
  8. 超洁净工作台(HDL,型号:HD-1360)
  9. 轻型孵化器(海翔,型号:PGX-400BP)
  10. 高压灭菌器(Boxun,型号:YXQ-LS-52SII)
  11. 兽医可调连续注射器(嘉善,型号:2ml-K,目录号:JS-003,URL: http://www.jiashanqx.com/cn/products.asp?Bid=51 )
  12. 光学显微镜(Motic,型号:BA310)
  13. 血细胞计数器(0.1 mm,1/400 mm 2 ,16 x 25)(秋菁,型号:XB-K-25)
  14. 冰箱(海尔,型号:BCD-215KS)

程序

  1. 真菌在PDA培养基上的繁殖(操作应在超洁净的工作台上进行)
    1. PDA板的制备
      在121℃高温蒸汽灭菌半小时后,将熔化的PDA冷却至约60℃,然后倒入每个培养皿中约18-20ml直至完全凝固。
    2. PDA媒体上的亚文化
      使用火焰对手术刀进行消毒,并在平板上挑选一些冷藏的 Fusarium verticillioides >。对其他一些印版重复此步骤。使用封口膜密封板,并在28°C的黑暗中保持5至7天。
    3. PDA平板上的质量传播
      从上面步骤A2中培养的装有 Fusarium verticillioides >的PDA板的边缘用火焰消毒的手术刀切下1cm 2 的块,然后将其放在上面用于培养的新PDA板。重复此步骤,直到足够的板满足您的需求。然后在上述步骤A2中以相同条件培养这些板。
    4. 在4°C冰箱中保存培养的 Fusarium verticillioides >
      当PDA板在5至7天后用 Fusarium verticillioides >填充时,将它们保持在4℃冰箱中使用。

  2. Fusarium verticillioides >在玉米种子培养基上的繁殖(操作应在超洁净工作台上进行)
    1. 煮玉米种子
      准备1,000克新收获的玉米杂交种子,没有杂质,霉菌和蛾的损害。用自来水洗涤种子2-3次,然后再用蒸馏水洗1次,除去剩余的自来水。向种子样品中加入3L蒸馏水,煮沸约2小时或更长时间,直到种子破裂并且含水量适中。适当的煮沸时间可以避免严重的颗粒破裂和高含水量,不利于 Fusarium verticillioides >的生长。
    2. 将煮沸的种子分成锥形烧瓶
      从煮沸的玉米种子中除去剩余的水。然后将煮沸的种子装入500毫升锥形瓶中,加入200毫升生产线,并以此为标准。通常,1,000克种子可以制作10个锥形烧瓶,其中含有约200毫升煮沸的玉米种子。
    3. 密封锥形烧瓶
      使用双层无菌透气密封膜或单层无菌透气密封膜加双层报纸密封带橡皮筋的锥形瓶。
    4. 消毒
      使用高压蒸汽灭菌器在121°C下将带有种子的锥形瓶灭菌30分钟。然后,将它们在室温下保持48小时,以确保没有微生物污染(图1A)。


      图1. Fusarium verticillioides > 在玉米培养基上繁殖的操作程序。 :一种。灭菌后的玉米种子。 B.用新培养的 Fusarium verticillioides >切割PDA培养基成小块。 C.将三片放入锥形瓶中。 D.充分混合后的玉米培养基。 E. Fusarium verticillioides >在玉米培养基上生长3天。 F. Fusarium verticillioides >在玉米培养基上生长8天。

    5. 接种玉米种子
      在超洁净工作台中,将三片1cm 2 PDA培养基与新培养的 Fusarium verticillioides >一起放入锥形瓶中并充分摇动(图1B-1D)。当然,你也可以放三个以上的部分来加快传播速度。然后,在28℃的恒温培养箱中在黑暗中培养它们。一般来说,最佳收获时间是在第7天 -10 th 天,因为孢子数量很大,孢子活性很强(图1E和1F)。 > 注意:随着时间的推移,孢子数量不会增加,一些孢子会开始变形。所以最好在10天内收获它们。>

  3. 接种孢子悬浮液的制备
    1. 收集孢子
      接种前一天,打开装有菌丝的锥形瓶,加入200ml无菌水后,在一个方向轻轻搅拌,然后通过八层纱布过滤到2,000ml烧杯中。通常,重复三到四次后,大部分孢子都会被洗掉。
    2. 制备工作溶液
      稀释上面的孢子悬浮液并充分混合,然后在显微镜下使用血细胞计数器计数孢子数。确保孢子悬浮液的最终浓度为5×10 6 / ml。最后,每ml孢子悬浮液中加入2μlTween-80,混合均匀,保持在4°C使用。通常,每个锥形瓶中的孢子可以制成5L工作溶液。
      注意:一旦准备好工作溶液,请尽快使用,因为我们发现在4°C冰箱中两天后浓度会发生变化。在长时间使用之前,您需要重新计算工作溶液中孢子的数量。>

  4. 接种玉米
    1. 合理的实验设计和优秀的现场管理在减少误差方面发挥着重要作用 在接种试验田周围种植约4行边界玉米,以确保接种试验材料的生长是均匀的。设置2-3次重复以减少实验误差。控制害虫非常重要,例如玉米螟。
    2. 接种时间
      根据我们的研究,最佳接种期是在授粉后约15天的乳汁阶段。此时,稻壳看起来仍然是绿色,丝绸刚刚变成棕色和干燥(图2A)。谷粒充满乳白色液体,看起来大而丰满,含水量约为60%(图2B)。因此,接种非常容易,适合 Fusarium verticillioides >的生长。无论是早期还是晚期接种都不利于接种和表型评估。如果早些时候,核心太少,无法进行接种,并且由于感染 Fusarium verticillioides >,整个耳朵将面临早期死亡。如果以后,内核变得难以穿透,并且由于内核中缺乏足够的水,真菌将不会发育良好。因此,耳朵的流行区域太小,无法揭示对玉米穗腐病的抗性差异。在实践中,非常重视不同玉米材料的生长阶段。


      图2.授粉后的自交系N6的17日龄耳朵。 :一种。耳朵的外壳和丝绸。 B.内核的外观。

    3. 接种方法
      我们通常在6:30-10:00和16:00-18:30进行接种,以避免高温影响接种效率。最佳剂量2毫升,需要两次注入耳中,1毫升,一次,以制作两个相距1厘米的接种孔。因为中间的籽粒经常丰满,两个接种孔可以提高接种成功率。将针头放入种子中比放在种子之间更好,因为它可以提高成功接种的速度。接种时,应将针头的延伸限制在耳朵的90度以下,以减少孢子悬液从接种孔中的流出(图3A)。最后,用一张纸胶带密封接种点(图3B)。
      注意:对于初学者,在接种实验之前练习几次是非常必要的,以减少操作引起的实验误差。>


      图3.现场接种方法。 A.注射器针头穿入内核但不进入穗轴。 B.用纸胶带密封接种点。

  5. 评分疾病
    1. 表型评估在成熟阶段进行。一般来说,接种3天后,伤口周围的颜色会变成棕色,伤口周围会有稀疏的白色菌丝体。此时,伤口附近的核表面和核心行之间的空间也会有薄的白色菌丝体。受感染的谷粒在顶部开始变成黄褐色,这是丝绸和仁的交界处。随着时间的推移,受感染的区域将扩大,受感染的内核逐渐变为褐色直至腐烂。在接种后的第20天天,病害的谷粒出现在软化过程中,这是玉米穗腐病的典型症状。在接种20天后,菌丝开始缓慢扩散,因此感染区域几乎保持稳定直至成熟。
    2. 我们发现至少有3种类型的症状由 Fusarium verticillioides >引起,如下文所述(Sun,2009),以及根据感染区域的种质资源的抗性评估(感染内核的百分比)耳朵)。
      1. 核仁发芽腐烂。例如,在较高湿度环境下的玉米品系177的种子将发芽,并且在发芽种子周围存在严重条件下的白色菌丝体(图4A)。
      2. 核在有限区域内腐烂,例如Yu 374(图4B)。
      3. 核心在大面积内腐烂,如近交系N6(图4C)。


        图4.不同玉米自交系的不同穗腐病症状。 A.玉米品系177. B. Yu 374. C.近交系N6。

    3. 玉米穗腐烂根据感染面积(整个穗中受感染的籽粒的百分比)分为七个等级(表1)。通常,为了最大限度地减少错误,两位经验丰富的研究人员足以进行表型评估。

      表1.玉米穗腐病的评级等级


    4. 在短时间内完成玉米穗腐病的大规模评估是非常困难的。所以,你可以用稻壳收获它们。用稻壳在阳光下干燥耳朵2-3天;然后可以进行表型评估。如果没有外壳,由于耳朵干燥,很容易导致内核损失。但是,最好尽快评估表型,以尽量减少实验误差。
    5. 此外,你可以组织劳动力去除田间的稻壳,打破耳朵的上部茎,只留下耳朵进行评估。根据绘图区域的评估,选择一些评估者进行评估。经验丰富的评估员可在8小时内评估约0.1公顷的接种田地。请注意,评估者的选择应与评估标准一致,并且相同材料的等级应尽可能一致。
    6. 通常,我们同时记录整个耳朵中受感染内核的百分比和等级。

数据分析

  1. 分析方法取决于实验设计。
  2. 为了更好地评估疾病等级,应根据1-3个等级同时评估材料的结实率。在您稍后的统计计算中,您可以有效地消除由于缺乏完整结果而导致的异常值。结实率1级表明全粒占穗区面积的75%以上,2级表示全粒占穗区面积的50%-75%。 3级意味着全谷物占穗区面积的不到50%。

笔记

在遗传研究和育种中,为了减少果实引起的玉米穗腐病评价误差,可以通过人工辅助授粉,收集其他玉米植株的花粉,提高结实率。我们的研究表明,个别材料可能具有人工补充授粉引起的森果效应,但其他材料的花粉对当代玉米穗腐病的抗性没有影响。

食谱

  1. PDA媒体
    200克/升马铃薯(煮沸并过滤入烧杯)
    20克/升D-葡萄糖
    15克-20克/升琼脂粉
    在121°C高压灭菌30分钟

致谢

该项目由国家自然科学基金(NSFC)(批准号:31761143009)和河南省高校重点科研项目(批准号:18A180014)资助。我们感谢团队中的研究生们为玉米穗腐烂相关研究所做的努力。

利益争夺

作者指出,没有利益冲突或竞争利益。

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引用:Dong, C., Wu, Y., Gao, J., Zhou, Z., Mu, C., Ma, P., Chen, J. and Wu, J. (2018). Field Inoculation and Classification of Maize Ear Rot Caused by Fusarium verticillioides. Bio-protocol 8(23): e3099. DOI: 10.21769/BioProtoc.3099.
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