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Biology

用于确定未知 尖孢镰刀菌 f.sp的种族的三种接种技术的对比。 尼维姆 分离

Published: October 28, 2021
doi:
10.3791/63181
, , , , , , , , ,
1Department of Plant Pathology,University of Florida, 2University of Florida Institute of Food and Agricultural Sciences, 3Gulf Coast Research and Education Center,University of Florida, 4Horticultural Sciences Department,University of Florida, 5Tropical Research and Education Center,University of Florida, 6Department of Horticulture,University of Georgia

Summary

管理西瓜枯萎病需要了解存在的病原体种族。在这里，我们描述了根浸，感染的内核播种和改良的托盘浸渍接种方法，以证明它们在致病真菌 尖孢镰刀菌 f. sp niveum （Fon）的种族分型中的功效。

Abstract

由尖孢镰刀菌 f. sp. niveum （Fon） 引起的西瓜镰刀菌枯萎病 （Citrullus lanatus） 已重新成为美国东南部，特别是佛罗里达州的主要生产制约因素。部署综合虫害管理战略，如针对特定种族的抗性品种，需要有关种植者田间病原体的多样性和种群密度的信息。尽管在开发用于鉴定病原体分离株的分子诊断工具方面取得了一些进展，但种族确定通常需要生物测定方法。

通过根浸接种，侵扰籽粒播种法和改良的托盘浸渍法（黑钻石，查尔斯顿灰，卡尔霍恩灰，植物介绍296341-FR）中的每一种进行种族分型。通过计算接种后五周的疾病发病率，为分离株分配种族名称。如果特定品种中不到33%的植物有症状，则它们被归类为抗性植物。发生率大于33%的品种为易感品种。本文介绍了三种不同的接种方法，以确定种族，根浸，侵染的内核和改良的托盘浸渍接种，其应用因实验设计而异。

Introduction

构成 尖孢镰刀菌 物种复合物（FOSC）的土生真菌是影响性的半生物养植物病原体，可导致多种作物的严重疾病和产量损失1。由 F. oxysporum f. sp. niveum （Fon） 引起的西瓜镰刀菌枯萎病在过去 几十年中在世界各地的范围、发病率和严重程度都在增加2，3.在幼苗中，镰刀菌枯萎病的症状通常类似于阻尼。在较老的植物中，叶子变成灰色，绿化和坏死。最终，植物的枯萎进展为植物完全崩溃和死亡4。直接产量损失是由于植物的症状和死亡而发生的，而间接产量损失可能是由于太阳损害造成的，由叶面冠层5的消除引起的。在 F. oxysporum 中从未观察到有性繁殖和相关生殖结构。然而，病原体产生两种类型的无性孢子，微分生孢子和大分生孢子，以及称为衣原体孢子的更大的长期生存结构，可以在土壤中存活多年6。

FOSC根据观察到的宿主范围被分类为特殊形态，通常限于一个或几个宿主物种1。虽然最近的研究表明，这种物种复合物可能是15种不同物种的复合物，但感染西瓜的特定物种目前尚不清楚7种。 F. oxysporum f. sp. niveum （Fon） 是专门感染 Citrullus lanatus 或驯化西瓜8，9 的菌株组的名称。在大多数致病性特产中的 F. oxysporum 菌株在其遗传成分和对宿主物种的毒力方面显示出一定程度的多样性。例如，一种菌株可能感染宿主的所有栽培品种，而另一种菌株可能只感染更易感的品种。为了解释这种变异，这些群体根据进化关系或共同的表型特征被非正式地分为种族。在Fon中，根据其对一组精选西瓜品种的致病性，对4个种族（0，1，2和3）进行了表征，最近发现了10个种族3。

尽管存在这种明显的多样性，但孢子或菌丝的形态在Fon种族之间无法区分，这意味着需要分子或表型测定来鉴定分离株的独特种族11。分子研究已经确定了一些遗传差异。例如，分泌在木质部（SIX）效应子中的作用已经在 F. oxysporum 中研究了多年，其中一些效应子已经位于水平基因转移过程中交换的染色体上12。例如，SIX6在Fon种族0和1中找到，但在种族213中找不到。六个效应子与 F. oxysporum f. sp . lycopersici 和 F. oxysporum f. sp. cubense的致病性有关，它们分别导致番茄和香蕉上的镰刀菌枯萎病，分别为14，15，16，17。对菠菜上的镰刀菌枯萎病病原体F . oxysporum f. sp. spiniciae菌株的SIX效应谱的分析使得分类能够准确反映遗传和表型多样性18。然而，Fon种族的毒力机制之间的差异目前尚不完全清楚，并且使用它们时开发的分子测定显示出不一致和不准确的结果19。因此，感染检测的表型结果是目前对分离株进行分类的最佳方法。

F. oxysporum 最初通过根部感染宿主，然后向上进入木质部20。这使得直接接种特定宿主品种的根系成为进行种族分型的有效方法，并且是根浸和托盘浸渍接种方法21的基础。当不感染宿主时， F. oxysporum 居住在土壤中，并且可以保持休眠多年。在土壤中从感兴趣的田地种植易感的西瓜品种是测试Fon存在的一种方法。将这种方法扩展到包括故意用Fon侵扰的土壤中具有不同已知抗性水平的品种也是进行种族分型的好方法（表1），并且是受感染的内核播种方法的基础。改进的托盘浸渍方法是原始托盘浸渍方法的变体，该方法允许高通量的竞赛类型，其中可以快速研究许多植物和田间分离株22。快速和成功的种族分型生物测定的重要因素包括使用已记录对不同病原体种族的抗性差异的品种，确保接种物在感染期间具有生物活性和丰富性，保持对病原体和宿主都有利的环境，以及使用一致的疾病严重程度或发病率评级系统。本文描述了基于上述原理的根浸23，24，出没的内核播种25，26和改进的托盘浸渍22 表型种族分型方法。

Protocol

1. 通过浸根法（RDM）确定种族 实验环境的准备 由于症状表达高度依赖于环境条件，因此将植物保持在受控区域。监测相对湿度、温度、光周期和光照强度（图1）。 将温度设置为26-28°C，相对湿度设置为50-75%，并设置16小时的光周期，以确保植物足够的生长和健康。注意：为了防止缺氧，幼苗枯萎和/或种子腐烂，不要在幼苗周围过度浇水?…

Representative Results

这些实验有助于确定通常生长的品种的相对抗性（表1）。然后，这些信息可用于指导基于当地Fon人口的管理建议。换句话说，如果已知种族0或1存在于商业田地中，那么农民可能倾向于种植“抗性”品种，如Calhoun Gray，Sunsugar或同等品种。使用所有方法的生物测定结果表明，当幼苗感染Race 1分离株时，黑钻石和查尔斯顿灰品种死亡或表现出严重症状，而Calhoun Grey和PI品种表现出抗性（<…

Discussion

已经提出了三种种族分型方法。这些方法中的每一种都最适合特定的问题和实验条件。侵扰的籽粒接种方法（土壤侵扰）可能更简单，更直接，使其对于评估致病性30特别有用。使用这种方法进行简单的种族分型是非常有效的。然而，应用该方法来确定特定品种的抗性可能具有挑战性，因为每种植物可能不面临相同程度的感染或暴露，并且可能需要同样高水平的疾病来测试感兴?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢阿里博士和植物分子诊断实验室以及佐治亚大学的平升吉博士，他们的领导和支持帮助建立了我们的Fon计划。

Materials

100% Fuller’s Earth Sigma-Aldrich F200-5KG
1 L glass Erlenmeyer Flask PYREX 4980-1L
15 mL falcon tubes Fisher Scientific 14-959-49B
50 mL graduated cylinder Lab Safety Supply 41121805
50 mL Eppendorf Conical Tubes Fisher Scientific 05-413-921
Aluminum foil wrap Reynolds Wrap 720
Bleach Walmart 587192290
Bunsen burner Fisher Scientific 03-391-301
CaCO3 sigma-Aldrich 239216
cell spreaders Fisher Scientific 08-100-11
Cheesecloth Lions Services, Inc 8305-01-125-0725
Clear plastic dishes Visions Wave 999RP6CLSS ~15 cm diameter
Clear vinyl tubing for mushroom bag clamps Shroom Supply 6" for small bag, 8" for medium bag, 10" for large bag
Cotton Balls Fisherbrand 22-456-885 Sterile
Ethanol Fisher Chemical A4094 100%, then combine with water to make 70% for use
Flourescent Tube Lights MaxLume Model T5 2800 K Color Temperature, 24'' or 48'' long
granulated agar VWR International 90000-786
Hand-held Spray Bottle Ability One 24122002 ~0.95 L
hemacytometer Fisher Scientific 02-671-55A Two chamber hemacytometer
Lab trays Fisher Scientific 15-236-2A
Large, sealable plastic bags Ziploc 430805 38 cm x 38 cm
Mister / watering can Bar5F B10H22
Mushroom Bag Clamp Shroom Supply 6" for small bag, 8" for medium bag, 10" for large bag
Nitrile Gloves Fisher Scientific 19-130-1597D
Organic Rye Berries Shroom Supply 0.5 gallon or 25 lb bags
P1000 pipette and tips Fisher Scientific 14-388-100
Petri dishes Fisherbrand FB0875713 Round, 100 mm diameter, 15 mm height
Planting media Jolly Gardener Pro-Line C/B
Plastic Pitcher BrandTech UX0600850 1 L or larger
Plastic planting pots Neo/SCI 01-1177 ~15 cm diameter and ~10 cm height
Plastic, autoclave-safe bin Thermo Scientific UX0601022 3 L
Quarter-strength potato dextrose agar media Cole-Parmer UX1420028 Use powder in combination with recipe for QPDA
Scientific Balance Scale, measuring in g Ohaus 30208458 Any precise scale that can hold and measure 200g will work
Size #4 cork bore Cole-Parmer NC9585352
Small Mushroom grow bag Shroom Supply 0.5 micron filter, also comes in medium and large sizes
Soil trowel Walmart 563876946
Styrofoam flats (6 x 12 cells) Speedling Model TR72A
Styrofoam flats (8 x 16 cells) Speedling Model TR128A
Syringe (5 or 10 mL) fisher Scientific 14-829-19C
Timer Walmart TM-01
V8 Original 100% Vegetable Juice Walmart 564638212
vortex Fisher Scientific 02-215-418
Watermelon Seed – Black Diamond Willhite Seed Inc 17
Watermelon Seed – Calhoun Gray Holmes Seed Company 4440
Watermelon Seed – Charleston Gray Bonnie Plants 7.15339E+11
Watermelon Seed – PI 296341-FR Contact authors Contact authors
Wheat Kernels (Maxie var.) (optional) Alachua County Feed & Seed
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Tags

Fusarium WiltRace-typingBioassayInoculationWatermelonPathogenDisease ManagementPlant HealthV8 Juice MediumPotato Dextrose AgarConidiaSpore QuantificationBleachPlantletsRoot Rinsing
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Fulton, J. C., Cullen, M. A., Beckham, K., Sanchez, T., Xu, Z., Stern, P., Vallad, G., Meru, G., McGregor, C., Dufault, N. S. A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates. J. Vis. Exp. (176), e63181, doi:10.3791/63181 (2021).
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