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Bacteria-fungal Confrontation and Fungal Growth Prevention Assay
细菌 - 真菌对抗和真菌生长预防试验   

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参见作者原研究论文

本实验方案简略版
Nature Communications
Sep 2017

Abstract

There are some bacteria which can grow and multiply at the cost of living fungal biomass. They can potentially utilize fungi as a source of nutrients to forage over them. Such phenomenon is known as bacterial mycophagy, however, its mechanistic insights need to be explored to identify the molecules involved in mycophagy for potential utilization in controlling various fungal diseases. Recently we have demonstrated that a rice-associated bacteria Burkholderia gladioli strain NGJ1 exhibits mycophagous ability on several fungi, including Rhizoctonia solani, the necrotrophic fungal pathogen causing sheath blight disease in rice. We hereby describe our validated and efficient methods used to study B. gladioli strain NGJ1-R. solani interactions. These methodologies would be useful for designing assays to study the confrontation between bacteria and fungi which in turn enable discovery of novel antifungal molecules from such bacteria.

Keywords: Burkholderia gladioli (唐菖蒲伯克霍尔德菌), Bacterial mycophagy (细菌真菌吞噬), Bacterial-fungal interaction (细菌 - 真菌相互作用), Rhizoctonia solani (立枯丝核菌), Sheath blight disease (纹枯病)

Background

Rhizoctonia solani is an important plant pathogenic fungi with a wide range of hosts. It causes sheath blight disease in rice; the second most devastating fungal disease of rice after Blast disease (Fisher et al., 2012; Ghosh et al., 2014; Ghosh et al., 2017). In wake of developing control methods of sheath blight disease of rice, we isolated a bacterium with a broad spectrum antifungal activity from rice seedling. Based upon the rDNA and draft genome sequencing, the bacterium had been identified as Burkholderia gladioli strain NGJ1 (Jha et al., 2015). Beside antifungal property, we observed that the NGJ1 has the ability to forage over fungi and exhibit mycophagous ability on R. solani as well as various other tested fungi. Upon interaction with R. solani mycelia, NGJ1 growth was drastically enhanced. The NGJ1 imparted cell death response in R. solani and caused disintegration of fungal mycelia. We further established that the bacteria utilize type III secretion system to deliver a prophage tail-like protein (Bg_9562) to feed on R. solani (Swain et al., 2017).

Materials and Reagents

  1. 1.5 ml and 2.0 ml microcentrifuge tubes (Tarson, catalog numbers: 500010 and 500020 )
  2. Pipette tips (10 μl) (Tarson, catalog number: 521000 )
  3. Pipette tips (200 μl) (Tarson, catalog number: 521010 )
  4. Pipette tips (1,000 μl) (Tarson, catalog number: 521020 )
  5. 15 ml culture tubes (Riviera, catalog number: 71200155 )
  6. 30 ml culture tubes (Riviera, catalog number: 71200305 )
  7. Microscopic glass slides (GEM, catalog number: 051 )
  8. Toothpicks
  9. Petri dishes (90 mm) (Tarson, catalog number: 460090 )
  10. Rhizoctonia solani AG1-IA strain (Lab collection)
  11. Burkholderia gladioli strain NGJ1 (Lab collection)
  12. Milli-Q water
  13. Ethanol (Merck)
  14. Potato dextrose agar (PDA) (HiMedia Laboratories, catalog number: GM096-500G )
  15. Potato dextrose broth (PDB) (HiMedia Laboratories, catalog number: GM403-500G )
  16. Sodium nitrate (NaNO3) (Fisher Scientific)
  17. Dipotassium phosphate (HiMedia Laboratories, catalog number: MB044-500G )
  18. Magnesium sulfate heptahydrate ( MgSO4·7H2O) (HiMedia Laboratories, catalog number: GRM684 )
  19. Potassium chloride (KCl) (HiMedia Laboratories, catalog number: MB043 )
  20. Ferrous sulfate heptahydrate ( FeSO4·7H2O) (HiMedia Laboratories, catalog number: GRM1377 )
  21. Dimethyl sulfoxide (MP Biomedicals, catalog number: 02196055 )
  22. Czapek dox broth (CDB) (HiMedia Laboratories, catalog number: M076-500G )
  23. Czapek dox agar (CDA) (HiMedia Laboratories)
  24. (Optional) KBA (HiMedia Laboratories, catalog number: M1544-500G )
  25. Potato Dextrose Agar (PDA) (see Recipes)
  26. Potato Dextrose Broth (PDB) (see Recipes)
  27. Czapek dox agar (CDA) (see Recipes)
  28. Czapek dox Broth (CDB) (see Recipes)

Equipment

  1. Forceps
  2. Stainless steel forceps (HiMedia Laboratories, catalog number: LA710 )
  3. Pipettes (Eppendorf-that can accommodate pipette tips of 10 μl, 200 μl, 1,000 μl)
  4. Milli-Q water purification system (Merck, model: Milli-Q® Advantage A10,catalog number: Z00Q0V0WW )
  5. Autoclave (INDFOS-110-PB)
  6. Incubator and shaker (28 °C) (mrc, catalog number: LOM-65 )
  7. Microcentrifuge (TOMY DIGITAL BIOLOGY, model: MX-301 )
  8. Laminar Air Flow (Azbil Telstar, model: Telstar Bio II Advance, catalog number: EN 12469 )
  9. Light microscope (Nikon, model: Eclipse E100 )

Procedure

  1. Bacterial-fungal confrontation assay on solid media
    Note: All steps should be carried out in sterile conditions.
    1. Pick a Rhizoctonia solani AG1-IA strain BRS1 sclerotium from approx. 2-week-old PDA plate using sterile toothpick/forceps, spot it at the center of fresh PDA plate (see Recipes) and incubate at 28 °C for a week. The R. solani sclerotium germinates to form mycelia which upon incubation produce plenty of black colored secondary sclerotia, the spore-like resting structures.
    2. Pick a single colony of B. gladioli strainNGJ1 from PDA plate and inoculate it in 10 ml PDB broth (see Recipes) and incubate it for 48 h at 28 °C at 200 rpm. The bacterial number is quantified using serial dilution plating and colony counting.
    3. Using sterile toothpick/forceps, place R. solani sclerotium at the center of PDA or CDA plate (see Recipes) and spot the B. gladioli strain NGJ1 culture (20 µl of 109 cells/ml) at four corners of the plate, equidistant from the center.
    4. Further, incubate the plate at 28 °C and routinely monitor for bacterial spread over fungal mycelia (Figure 1; Please note the NGJ1 spreads over fungal mycelia, induces cell death response and feed on them).


      Figure 1. B. gladioli strain NGJ1 demonstrates mycophagy on R. solani. A. Growth pattern of R. solani sclerotia on PDA plates. During 3 and 5 dpi of growth, the fungal mycelia spread over petri-plate and by 7 dpi, plenty of secondary sclerotia are produced. B. Growth pattern of B. gladioli strain NGJ1 on PDA plates. Even during 7 dpi of growth, the bacteria failed to cover the entire plate (C) Confrontation of NGJ1 with R. solani on PDA plates. Initially by 3 dpi, the bacteria show limited antifungal activity but during 5 dpi, the bacteria started spreading over fungal biomass and by 7 dpi, the bacteria spread over entire fungal biomass.

  2. Bacterial-fungal confrontation assay in liquid media
    1. Collect fresh sclerotia from one-week-old plate of R. solani AG1-IA strain BRS1 using sterile toothpick/forceps. Inoculate 5-10 sclerotia in 10 ml PDB/CDB broth (see Recipes) and allow them to grow for 48 h at 28 °C with constant shaking (200 rpm) to obtain mycelial mass.
    2. Pick a single colony of B. gladioli strain NGJ1 and inoculate it in 10 ml PDB/CDB broth at 28 °C for 48 h at 200 rpm.
    3. 100 µl of 109 cells/ml culture of B. gladioli strain NGJ1 was inoculated into 10 ml PDB/CDB media containing 48 h pre-grown fungal mycelial mass.
    4. Collect 100 µl of the bacterial solution at three different time points (24 h, 48 h and 72 h) of co-cultivation with pre-grown R. solani mycelia and upon serial dilution (105, 106, 107) plate 100 µl of bacterial solution on PDB/CDB plates. Use NGJ1 grown in absence of R. solani mycelia in PDB/CDB broth as a control.
    5. Incubate plates at 28 °C for 24 h and count the bacterial colonies appeared on the plate.

  3. Sclerotial growth prevention assay
    1. Pick a single colony of B. gladioli strain NGJ1 and inoculate in 10 ml PDB broth and incubate at 28 °C for 48 h at 200 rpm.
    2. Prepare 10 ml of 109, 107, 105, and 103 cells/ml dilutions of bacterial culture using sterile PDB.
    3. Add 5-10 R. solani sclerotia in each of the diluted bacterial culture.
    4. Incubate for 4 h at 28 °C with constant shaking at 200 rpm.
    5. After 4 h, take out the treated sclerotia and individually place each sclerotium on fresh PDA plates.
    6. For control, incubate the sclerotia for 4 h in PDB broth (without NGJ1).
    7. Further, incubate plates at 28 °C and routinely monitor for mycelial growth.

  4. Agar slide confrontation assay
    1. Clean the microscopic glass slides with ethanol and autoclave them.
    2. Prepare 1% agar solution (autoclaved) and gently pour 4 ml of it drop wise on the sterile glass slide to obtain thin agar layered slides. Let the agar solidify for 30 min in laminar air flow.
    3. Place 2 freshly collected sclerotia on both ends of the thin agar layered slides, incubate the slides in a closed sterile Petri-plate having moist tissue paper (to maintain humid condition) and allow the sclerotia to germinate at 28 °C for 24 h.
    4. Using a pipette, spot 20 µl of pre-grown culture (109 cells/ml) of B. gladioli strain NGJ1 at the center of the agar slides containing pre-grown R. solani mycelia.
    5. Incubate at 28 °C under aseptic condition and using a light microscope, observe the slides (for bacterial spread and damage of mycelia) after 24 h, 48 h and 72 h of confrontation.

Data analysis

All the information about data processing, statistical tests, details of replicates and independent experiments was already included in original research paper (Swain et al., [2017]. A prophage tail-like protein is deployed by Burkholderia bacteria to feed on fungi. Nature Communications. 404(8). doi:10.1038/s41467-017-00529-0).

Notes

  1. The R. solani sclerotial germination rate is not 100%, and this should be kept in mind while designing experiment.
  2. Apart from PDA and CDA; the bacterial-fungal confrontation can also be studied on King’s Medium B base (KBA, HiMedia).

Recipes

  1. Potato dextrose agar (PDA) for 400 ml
    40 g potato infusion form
    4 g dextrose
    6 g agar
    Add Milli-Q water up to 400 ml and autoclave the media
    Pour into sterile Petri plates, let the plates completely solidify and store them at 4 °C
  2. Potato dextrose broth (PDB) for 400 ml
    40 g potato infusion form
    4 g dextrose
    Add Milli-Q water up to 400 ml, autoclave the media
  3. Czapek doxagar (CDA) 400 ml
    12 g sucrose
    1.2 g sodium nitrate
    0.4 g dipotassium phosphate
    0.2 g magnesium sulfate
    0.2 g potassium chloride
    0.004 g ferrous sulfate
    6 g agar
    Add Milli-Q water up to 400 ml and autoclave the media
    Pour into sterile Petri plates, let the plates completely solidify and store them at 4 °C
  4. Czapek dox broth (CDB) 400 ml
    12 g sucrose
    1.2 g sodium nitrate
    0.4 g dipotassium phosphate
    0.2 g magnesium sulfate
    0.2 g potassium chloride
    0.004 g ferrous sulfate
    Add Milli-Q water up to 400 ml and autoclave the media

Acknowledgments

The research funding from DBT, Government of India and core research grant from National Institute of Plant Genome Research, India to support the GJ lab are gratefully acknowledged. RK and SG acknowledge fellowship from CSIR and IT, SKY, JD acknowledges fellowship from DBT, Govt. of India. The authors declare no conflict of interest.

References

  1. Fisher, M. C., Henk, D. A., Briggs, C. J., Brownstein, J. S., Madoff, L. C., McCraw, S. L. and Gurr, S. J. (2012). Emerging fungal threats to animal, plant and ecosystem health. Nature 484(7393): 186-194.
  2. Ghosh, S., Gupta, S. K. and Jha, G. (2014). Identification and functional analysis of AG1-IA specific genes of Rhizoctonia solani. Curr Genet 60(4): 327-341.
  3. Ghosh, S., Kanwar, P. and Jha, G. (2017). Alterations in rice chloroplast integrity, photosynthesis and metabolome associated with pathogenesis of Rhizoctoniasolani. Sci Rep 7: 41610.
  4. Jha, G., Tyagi, I., Kumar, R. and Ghosh, S. (2015).Draft genome sequence of broad-spectrum antifungal bacterium Burkholderia gladiolistrain NGJ1, isolated from healthy rice seeds. Genome Announc 3: 803-815.
  5. Swain, D. M., Yadav, S. K., Tyagi, I., Kumar, R., Kumar, R., Ghosh, S., Das, J. and Jha, G. (2017). A prophage tail-like protein is deployed by Burkholderia bacteria to feed on fungi. Nat Commun 8(1): 404.

简介

有一些细菌可以生长和繁殖以牺牲真菌生物量为代价。 他们可能潜在地利用真菌作为营养素的来源来搜寻它们。 这种现象被称为细菌真菌病,然而,其机理性的见解需要被探索,以确定参与mycophagy分子潜在的利用控制各种真菌病害。 最近我们已经证明,稻米相关的细菌伯克霍尔德氏唐菖蒲菌株NGJ1在几种真菌上展现出真菌的能力,包括水稻纹枯病真菌病原体,即立枯丝核菌(Rhizoctonia solani)。 我们在此描述我们用于研究B的已验证的和有效的方法。 剑兰菌株NGJ1- R。 solani 互动。 这些方法学可用于设计分析来研究细菌和真菌之间的对抗,从而能够从这些细菌中发现新的抗真菌分子。

【背景】立枯丝核菌(Rhizoctonia solani)是具有多种宿主的重要的植物病原真菌。导致稻纹枯病;在爆发性疾病之后,稻米的第二大破坏性真菌病(Fisher等人,2012; Ghosh等人,2014; Ghosh等人, >,2017)。在发展水稻纹枯病防治方法之后,我们从水稻幼苗中分离出一株具有广谱抗真菌活性的细菌。基于rDNA和基因组测序,该细菌已经被鉴定为唐菖蒲伯克霍尔德氏菌菌株NGJ1(Jha等人,2015)。除了抗真菌特性外,我们观察到NGJ1具有对真菌进行觅食的能力,并且在禾本科植物上表现出食菌能力。 solani 以及其他各种真菌。在与 R交互之后。 solani菌丝体,NGJ1的生长得到大幅度的提高。 NGJ1在 R中传递了细胞死亡反应。 solani 引起真菌菌丝体解体。我们进一步确定,细菌利用III型分泌系统来递送原噬菌体尾样蛋白(Bg_9562)以在R上饲喂。 solani (Swain et。,2017)。

关键字:唐菖蒲伯克霍尔德菌, 细菌真菌吞噬, 细菌 - 真菌相互作用, 立枯丝核菌, 纹枯病

材料和试剂

  1. 1.5 ml和2.0 ml微量离心管(Tarson,目录号:500010和500020)
  2. 移液器吸头(10μl)(Tarson,目录号:521000)
  3. 移液器吸头(200μl)(Tarson,目录号:521010)
  4. 移液器吸头(1,000μl)(Tarson,目录号:521020)
  5. 15毫升培养管(里维埃拉,目录号:71200155)
  6. 30毫升培养管(里维埃拉,目录号:71200305)
  7. 显微镜载玻片(GEM,目录号:051)
  8. 牙签
  9. 培养皿(90毫米)(Tarson,目录号:460090)
  10. 立枯丝核菌(Rhizoctonia solani)AG1-IA株(Lab collection)
  11. 剑杆菌(Burkholderia gladioli)菌株NGJ1(Lab collection)
  12. Milli-Q水
  13. 乙醇(Merck)
  14. 马铃薯葡萄糖琼脂(PDA)(HiMedia实验室,目录号:GM096-500G)
  15. 马铃薯葡萄糖肉汤(PDB)(HiMedia Laboratories,目录号:GM403-500G)
  16. 硝酸钠(NaNO 3)(Fisher Scientific)
  17. 磷酸二钾(HiMedia Laboratories,目录号:MB044-500G)
  18. 硫酸镁七水合物(MgSO 4•7H 2 O)(HiMedia Laboratories,目录号:GRM684)
  19. 氯化钾(KCl)(HiMedia Laboratories,目录号:MB043)
  20. 硫酸亚铁七水合物(FeSO 4•7H 2 O)(HiMedia Laboratories,目录号:GRM1377)
  21. 二甲基亚砜(MP Biomedicals,目录号:02196055)
  22. Czapek dox肉汤(CDB)(HiMedia Laboratories,目录号:M076-500G)
  23. Czapek dox琼脂(CDA)(HiMedia实验室)
  24. (可选)KBA(HiMedia实验室,目录号:M1544-500G)
  25. 马铃薯葡萄糖琼脂(PDA)(见食谱)
  26. 马铃薯葡萄糖肉汤(PDB)(见食谱)
  27. Czapek dox琼脂(CDA)(见食谱)
  28. Czapek dox肉汤(CDB)(见食谱)

设备

  1. 镊子
  2. 不锈钢钳(HiMedia实验室,目录号:LA710)
  3. 移液器(Eppendorf-可以容纳10μl,200μl,1000μl移液器吸头)
  4. Milli-Q水纯化系统(Merck,型号:Milli-Q Advantage A10,目录号:Z00Q0V0WW)
  5. 高压灭菌器(INDFOS-110-PB)
  6. 培养箱和摇床(28°C)(mrc,目录号:LOM-65)
  7. 微量离心机(TOMY DIGITAL BIOLOGY,型号:MX-301)
  8. 层流空气流动(Azbil Telstar,型号:Telstar Bio II Advance,目录号:EN 12469)
  9. 光学显微镜(尼康,型号:Eclipse E100)

程序

  1. 固体培养基上的细菌 - 真菌对抗试验
    注意:所有步骤应在无菌条件下进行。
    1. 从大约一个挑选一个立枯丝核菌AG1-IA株BRS1菌核。 2周龄的PDA平板,使用无菌牙签/镊子,将其置于新鲜PDA平板的中心(参见食谱),并在28℃下孵育一周。 R。因此,菌核会萌发形成菌丝体,经过孵化后产生大量黑色的继发性菌核,孢子状的休息结构。
    2. 选择一个B的单个殖民地。来自PDA平板的唐菖蒲菌株GNJ1接种于10ml PDB肉汤中(见配方)并在28℃以200rpm将其温育48小时。使用系列稀释平板和菌落计数对细菌数量进行定量。
    3. 使用无菌牙签/镊子,将R.solani菌核置于PDA或CDA平板的中心(参见食谱),并将B.randoli菌株NGJ1培养物(20μl的10 9细胞/ ml),在距离中心等距离的四个角落处。
    4. 进一步,在28°C孵育板和常规监测细菌蔓延真菌菌丝体(图1;请注意NGJ1蔓延真菌菌丝体,诱导细胞死亡反应和饲料)。


      图1. B。剑兰菌株NGJ1在 R上展示mycophagy。菌。 :一种。 R的增长模式solani在PDA平板上的菌核。在3和5dpi生长期间,真菌菌丝体在培养皿上扩散,并且通过7dpi,产生大量的继发性菌核。 B. B的增长模式剑杆菌菌株NGJ1在PDA平板上。即使在7dpi的生长期间,细菌也不能覆盖整个平板(C)NGJ1与R的对抗。 solani 在PDA平板上。最初由3dpi,细菌显示有限的抗真菌活性,但在5dpi,细菌开始蔓延真菌生物量和7dpi,细菌蔓延到整个真菌生物量。

  2. 液体培养基中的细菌 - 真菌对抗试验
    1. 从一周龄的板块R收集新鲜的菌核。 solani AG1-IA菌株BRS1使用无菌牙签/镊子。在10ml PDB / CDB培养液中接种5-10个菌核(参见食谱),让它们在28℃下持续摇动(200rpm)培养48小时以获得菌丝体。
    2. 选择一个B的单个殖民地。唐菖蒲菌株NGJ1并在28℃以200rpm接种于10ml PDB / CDB肉汤中48小时。
    3. 100μl的10 9细胞/ ml的B培养物。唐菖蒲菌株NGJ1接种到含有48小时预生长的真菌菌丝体的10ml PDB / CDB培养基中。
    4. 在三个不同时间点(24小时,48小时和72小时)收集100μL的细菌溶液与预先生长的R株共培养。 (10 5,10 6,10 7)平板稀释后,将100μl细菌溶液在PDB / CDB板。在没有R的情况下使用NGJ1。以PDB / CDB培养液中的solani菌丝体作为对照。

    5. 在28°C孵育板24小时,并计数出现在板上的细菌菌落。

  3. 菌核生长预防试验
    1. 选择一个B的单个殖民地。唐菖蒲菌株NGJ1并接种于10ml PDB肉汤中,并在28℃以200rpm孵育48小时。
    2. 准备10ml的10 9,10 7,10 5,和10 3细胞/ ml的细菌稀释液使用无菌PDB培养。
    3. 添加5-10 R。每个稀释的细菌培养物中都含有solani菌核。

    4. 在28°C恒温振荡培养4小时
    5. 4小时后,取出处理后的菌核,并将每个菌核分别放在新鲜的PDA平板上。
    6. 为了控制,将菌核在PDB肉汤(不含NGJ1)中孵育4小时。
    7. 此外,培养板在28°C和常规监测菌丝生长。

  4. 琼脂片对抗试验

    1. 用乙醇清洁显微镜载玻片,高压灭菌
    2. 准备1%的琼脂溶液(高压灭菌),并轻轻倒在无菌载玻片4毫升,以获得薄琼脂分层的幻灯片。让琼脂在层流空气中固化30分钟。
    3. 将2个新收集的菌核在薄琼脂层状载玻片的两端放置,在具有湿纸巾的封闭的无菌培养皿(保持潮湿的条件)中孵育载玻片,并使菌核在28℃发芽24小时。 br />
    4. 使用移液管,将20μl预先生长的培养物(10 9细胞/ ml)的斑点B.在含有预先生长的R.solani菌丝体的琼脂载玻片中心的唐菖蒲菌株NGJ1。
    5. 在无菌条件下于28°C孵育,并使用光学显微镜观察对抗24小时,48小时和72小时后的载玻片(细菌传播和菌丝体损伤)。

数据分析

所有关于数据处理,统计检验,重复的细节和独立实验的信息都已经包含在原始的研究论文中(Swain等人,[2017]。 Nature Communications 404(8)。doi:10.1038 / s41467-017-00529-0)。

笔记

  1. R. solani菌核的发芽率不是100%,在设计实验时应该牢记这一点。
  2. 除了PDA和CDA;细菌 - 真菌对抗也可以在King's B的基础上进行研究(KBA,HiMedia)。

食谱

  1. 马铃薯葡萄糖琼脂(PDA)400毫升
    40克土豆注入形式
    4克葡萄糖
    6克琼脂
    加入Milli-Q水至400毫升,并高压灭菌媒介
    倒入无菌培养皿中,让培养皿完全固化,并将其储存在4°C
  2. 马铃薯葡萄糖肉汤(PDB)400毫升
    40克土豆注入形式
    4克葡萄糖
    加Milli-Q水至400毫升,高压灭菌介质
  3. Czapek doxagar(CDA)400毫升
    12克蔗糖
    1.2克硝酸钠
    0.4克磷酸二钾
    0.2克硫酸镁
    0.2克氯化钾
    0.004克硫酸亚铁
    6克琼脂
    加入Milli-Q水至400毫升,并高压灭菌媒介
    倒入无菌培养皿中,让培养皿完全固化,并将其储存在4°C
  4. Czapek dox肉汤(CDB)400毫升
    12克蔗糖
    1.2克硝酸钠
    0.4克磷酸二钾
    0.2克硫酸镁
    0.2克氯化钾
    0.004克硫酸亚铁
    加入Milli-Q水至400毫升,并高压灭菌媒介

致谢

感谢来自印度政府DBT,印度国家植物基因组研究所核心研究基金的支持GJ实验室的研究经费。 RK和SG承认CSIR和IT,SKY,JD的奖学金,并承认DBT,Govt的奖学金。的印度。作者宣称没有利益冲突。

参考

  1. Fisher,M.C.,Henk,D.A.,Briggs,C.J.,Brownstein,J.S.,Madoff,L.C.,McCraw,S.L。和Gurr,S.J。(2012)。 新出现的对动植物和生态系统健康的真菌威胁 484(7393):186-194。
  2. Ghosh,S.,Gupta,S.K。和Jha,G。(2014)。 Rhizoctonia solani的AG1-IA特异性基因的鉴定和功能分析。 Curr Genet 60(4):327-341。
  3. Ghosh,S.,Kanwar,P.和Jha,G。(2017)。 与Rhizoctoniasolani发病相关的水稻叶绿体完整性,光合作用和代谢组的变化。 Sci Rep 7:41610.
  4. Jha,G.,Tyagi,I.,Kumar,R.和Ghosh,S。(2015)。
  5. Swain,D.M.,Yadav,S.K。,Tyagi,I.,Kumar,R.,Kumar,R.,Ghosh,S.,Das,J。和Jha,G。(2017)。 噬菌体类似于尾巴的蛋白质被布鲁克菌(Burkholderia)细菌所部署真菌。 Nat Commun 8(1):404.
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引用:Kumar, R., SWAIN, D. M., YADAV, S. K., Tyagi, I., Kumar, R., Das, J., Ghosh, S. and Jha, G. (2018). Bacteria-fungal Confrontation and Fungal Growth Prevention Assay. Bio-protocol 8(2): e2694. DOI: 10.21769/BioProtoc.2694.
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