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目 录

1. OsABT 基因在水稻苗期根系耐盐进程中的作用机制 .............................................................................. 1

2. 菠萝全基因组 SNP 标记的开发及指纹图谱构建.................................................................................... 2

3. 基于 PCR 检测黄龙病菌在寄主体内含量动态变化研究 ........................................................................ 3

4. 芒果病程相关蛋白 MiPR1 基因家族鉴定和 MiPR1A 基因功能分析 ................................................... 14

5. A sugarcane smut fungus effector simulates the host endogenous elicitor peptide to suppress plant

immunity ...................................................................................................................................................... 15

6. 油莎豆油体蛋白基因的全基因组鉴定与功能解析 ............................................................................... 30

7. 热区植原体遗传多样性与其病害传播流行关系研究 ........................................................................... 31

8. 热区棕榈作物植原体病害检测技术研发与应用 ................................................................................... 32

9. 孕穗期喷施外源硒对水稻产量和糙米硒含量的影响 ........................................................................... 33

10. 广藿香组织培养工厂化育苗技术研究................................................................................................. 34

11. 旋切式电动割胶刀在茂名植胶区试验初报 ......................................................................................... 35

12. The rotary cutting electric rubber tapping knife was first reported in the rubber planting area of Maoming

..................................................................................................................................................................... 35

13. 乙烯利对五个菠萝品种成花及品质的影响 ......................................................................................... 37

14. 生物有机配方肥连续替代化肥对剑麻生长、土壤质量的影响 .......................................................... 38

15. 基于 2021-2022 年粤西蔗区甘蔗主要病虫害监测数据的分析及综合防治探索................................ 46

16. Analysis and Integrated Pest Management Exploration Based on the Monitoring Data of Major Pests and

Diseases in the Western Guangdong Sugarcane Region from 2021 to 2022 .................................................. 46

17. 不同光质 LED 补光对火龙果激素含量的影响 ................................................................................... 47

18. 湛江蔗区螟虫动态监测........................................................................................................................ 48

19. 基于国内剑麻产业形势视角下分析探讨广东垦区剑麻产业发展新路径........................................... 49

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20. 火炬农场剑麻产业发展问题与对策探讨............................................................................................. 55

21. 湛江农垦品牌现状分析与建设思考..................................................................................................... 59

22. 湛江农垦橡胶产业现状及思考 ............................................................................................................ 67

23. 发展橡胶产业助力乡村振兴的有效举措............................................................................................. 73

24. 剑麻化肥农药减施增效生产技术应用报告 ......................................................................................... 76

25. 生物有机配方肥连续替代化肥对剑麻生长、土壤质量的影响 .......................................................... 79

26. 红江橙黄化树种类调查及防控措施..................................................................................................... 80

27. 探讨广东农垦培育发展甘薯产业的意义及建议 ................................................................................. 85

28. 胶园套种岗梅高效栽培技术研究 ........................................................................................................ 87

29. 云南德宏石斛产业发展现状调查报告................................................................................................. 90

30. 孕穗期喷施外源硒对水稻产量和糙米硒含量的影响.......................................................................... 97

31. 农作物重要农业性状表型鉴定无损检测方法 ....................................................................................104

32. 广东农垦剑麻产业发展历程、瓶颈与建议 ........................................................................................105

33. 50 份芒果材料对细菌性黑斑病和坏死病的抗性评价.........................................................................110

34. 核磁共振技术在油棕的研究应用 .......................................................................................................112

35. 柑橘黑点病研究进展...........................................................................................................................113

36. 广西芒果主要细菌性病害病原菌鉴定、药剂敏感性测定及 抗病材料筛选....................................125

37. 广东农垦培育发展甘薯产业的意义及建议 ........................................................................................140

38. 李树叶部真菌性病害病原鉴定及快速检测技术研究.........................................................................142

39. 热区菠萝施肥管理研究及其生态效益评价 ........................................................................................143

40. 热区植原体遗传多样性与其病害传播流行关系研究.........................................................................144

41. 香蕉枯萎病菌的遗传进化及分子检测研究 ........................................................................................145

42. 湛江蔗区螟虫动态监测.......................................................................................................................146

43. 应用 SSR 和 InDel 标记鉴定槟榔杂交子代的真实性 ........................................................................147

44. 蔬菜害虫轻简化绿色防控技术 ...........................................................................................................148

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OsABT 基因在水稻苗期根系耐盐进程中的作用机制

闻丹妮，鲍聆然，沈 怡，沈 波*

杭州师范大学生命与环境科学学院，浙江杭州 311121

E-mail:bshen65@163.com

摘要：水稻是重要的粮食作物，提高水稻产量和扩大水稻种植面积对确保世界粮食安全具有举足轻重的作用。盐胁迫

是影响水稻产量的主要因素之一，因此，开展水稻耐盐机制的基础研究具有重要理论和应用意义。含 WD40 重复序列

的蛋白在真核生物的发育和环境适应中起着重要作用，课题组前期运用生物信息学和同源克隆的方法，在水稻中找到

了一个编码 WD40 重复蛋白的基因 OsABT(Oryza sativa L.ABA Signaling Terminator) ，过表达 OsABT 的水稻在幼苗发

育过程中对盐胁迫的耐受性显著增强，但 OsABT 如何参与水稻耐盐的尚未可知。本试验以日本晴和 OsABT 过表达水稻

为材料，对盐处理的根系进行根系活力、相对电导率、丙二醛（MDA）、H2O2、O2

*- 和钠钾离子含量等相关生理指标

的测定；利用转录组分析以及 qRT-PCR 分析，找到 OsABT 基因可能参与的代谢及信号转导途径；利用酵母双杂交等实

验查找可能与 OsABT 互作的蛋白，探究 OsABT 基因在水稻苗期耐盐中的作用机理。研究结果显示，与日本晴相比，

OsABT 过表达水稻表现出更强的耐盐性，OsABT 过表达水稻株系根系中的相对电导率、MDA 含量、H2O2 含量、O2

*- 含

量均显著或极显著低于日本晴，根系活力和 K+

/Na+

显著高于日本晴；转录组数据显示，日本晴和 OsABT 过表达水稻在

未盐胁迫和盐胁迫 12h、24h、48h 分别有 1950、1646、3499 和 1522 个差异表达基因。这些差异表达基因主要富集在

盐胁迫响应、脱落酸响应和转录调控等 GO 条目中，富集的重要代谢通路主要是植物激素信号转导、植物 MAPK 信号

传导途径和苯丙烷生物合成、类黄酮生物合成相关的次生代谢途径等；通过 qRT-PCR 结果发现，与日本晴相比，OsABT

过表达水稻根系中 ABA 合成相关基因 OsNCED3、OsNCED4 的表达量显著下调，而 ABA 分解代谢基因 OsABA8ox2 的

表达量显著升高，同时 ABA 信号转导相关基因 OsPYLs、OsABIL2，盐胁迫响应相关基因 OsRab16A、OsLEA3、OsHAK5

和 OsSOS1 等均有显著差异表达；此外，酵母双杂实验发现 OsABT 与 OsABIL2、OsPYL4 和 OsPYL10 存在相互作用。

综上，OsABT 可以通过 SOS 通路减少根系 Na+

内流，减轻盐胁迫对水稻产生的离子毒害；OsABT 蛋白与 OsABIL2、

OsPYL4 和 OsPYL10 蛋白相互作用，说明 OsABT 通过影响 ABA 信号途径参与调控 OsRab16A、OsLEA3 等逆境相关基

因的表达来提高苗期水稻的耐盐性。

关键词：水稻；根系；盐胁迫；ABA；转录组分析

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菠萝全基因组 SNP 标记的开发及指纹图谱构建

贾盼盼 1,2，林文秋 1

，刘胜辉 1

，孙伟生 1

，吴青松 1*

1.中国热带农业科学院南亚热带作物研究所，广东湛江 524088；2.华中农业大学园艺林学学院，湖北武汉 430070

摘要：针对菠萝种质资源存在本底不清、同名异物或同物异名等问题，本研究以 179 份菠萝种质为材料，根据重测序

数据的 SNP 位点信息设计引物，开发分子标记，构建菠萝种质的指纹图谱。结果表明在菠萝 25 个连锁群上共有 171

个 SNP 标记分型成功，转化率为 34.2%，缺失率为 0.05，平均 MAF 为 0.38，平均基因多样性为 0.45，平均 PIC 为 0.35。

进一步基于次要等位基因频率大于 0.40，多态性信息含量大于 0.37，缺失率小于 0.03，杂合率小于 0.5 等参数，筛选

出 23 个具有代表性的核心 SNP 分子标记，构建菠萝种质资源的指纹图谱。聚类分析结果显示，179 份种质可分为 4 大

类，并能将同名异物或同物异名的种质区分。SNP 标记的开发为菠萝种质资源的鉴定和鉴别，遗传图谱构建以及基因

定位及分子标记辅助育种等提供研究基础。

关键词：菠萝；种质资源；SNP；遗传多样性；指纹图谱

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基于 PCR 检测黄龙病菌在寄主体内含量动态变化研究

王盼 1,1

1. 广东农垦热带作物科学研究所，广东茂名 525000

摘 要：柑橘黄龙病（Citrus Huanglongbing, HLB）是危害柑橘最严重的一种病害，严重影响了柑橘产业的发展。砂糖

橘（Citrus reticulata cv Shatangju）作为广东柑橘主要栽培品种之一，长期受到黄龙病为害，造成了巨大的经济损失，

砂糖橘黄龙病病原菌为亚洲韧皮杆菌（Candidatus Liberibacter asiaticus，CLas）。本研究应用实时荧光定量 TaqMan qPCR

技术跟踪检测黄龙病砂糖橘叶内病菌含量的周年动态变化，明确黄龙病菌的消长规律，比较分析砂糖橘病株梢期和果

实成熟期 5 个不同发育阶段黄龙病菌含量的变化情况，结果发现：一年内不同月份平均黄龙病菌含量：4 月<5 月<6 月

<3 月<2 月<7 月<8 月<1 月<9 月<10 月<11 月<12 月；全年每克组织黄龙病菌平均含量在 2.28×107

~4.85×108 拷贝之间，

4-6 月病菌量出现低峰，7 月-12 月病菌量呈现增长趋势，并在 12 月份达到增长高峰。不同发育时期发病株组织内的平

均黄龙病菌含量：春梢期<夏梢期<果实成熟期<秋梢期<冬梢期。

关键词：砂糖橘；黄龙病菌；TaqMan qPCR；动态变化

Study on the dynamics of Huanglongbing bacterial content in

hosts based on PCR detection

WANG Pan1,1

1. Guangdong Nongken Tropical Crops Scientific Research Institute, Maoming, Guangdong 525000, China

Abstract: The citrus Huanglongbing (HLB), caused by phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), is

one of the most devastating diseases that seriously jeopardizes the development of citrus industry. On Citrus reticulata cv.

Shatangju, a main cultivar of citrus in Guangdong province, China, the long-term damaging by HLB has resulted in huge

economic losses. In this study, the annual dynamics of CLas content in the leaves of HLB-affected Shatangju was explored via

TaqMan qPCR-based analyses, where the variation of pathogen contents in five growing periods of the diseased plants was

studied. The results obtained in this study are as follows:The average CLas content in leaf tissues of infected Shatangju, ranging from 2.28×107

to 4.85×108

copies per gram of tissue, varied in different months during a year, in a pattern of April < May

< June < March < February < July < August < January < September < October < November < December, i.e., a low peak in

April followed by a steady rising which lasted through December to the climax. Besides, the average CLas content in infected

plants exhibited an increasing trend in the five sequential growing stages: spring sprouting, summer sprouting, fruit ripening,

autumn sprouting, and winter sprouting periods.

Keywords: Shatangju; CLas; TaqMan qPCR; dynamic change

柑橘是世界上种植十分广泛的一种作物，我国的柑橘属（Citrus L.）品种大概有 20 种左右，我国

是世界上第一柑橘生产大国，全国大约有 1000 个市县区有种植柑橘的记录，其中主要集中种植区是广

东和广西等地区。柑橘是主要分布在热带、亚热带地区的多年生常绿果树，是一种非常重要的商品水

果。柑橘果实汁液中含有丰富的具有抗氧化活性的类黄酮和可作为芳香剂使用的单萜类化合物，具有

巨大的经济和药用价值[1-2]。世界上有 140 个国家和地区生产柑橘，中国的柑橘种植面积和产量在 200

但柑橘种植地多数处于亚热带气候，受病虫害影响较为严重，其中柑橘黄龙病（Citrus Huanglongbing，

作者简介 王盼（1998—），女，硕士研究生，研究方向：植物病害综合防控。

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简称 HLB）是迄今为止整个柑橘产业所面临的最为复杂严重的一种灾害。此病害传播速度快，且不易

预防，一旦被感染，整个果园都会受到影响。柑橘黄龙病在我国最早于 20 世纪 90 年代初期在广东潮

汕发现，柑橘叶片出现典型斑驳、黄化等，症状与黄龙病类似[3]。柑橘属植物的所有栽培品种几乎都

能够感染黄龙病，例如常见的桔类、橙类和柚类等，很少有品种能表现出对黄龙病的耐受性，且在整

个生长发育周期植株均能够被黄龙病为害[4]。

柑橘一年中随气候周期性变化，可抽生 3-4 次枝梢，即春梢、夏梢、秋梢和冬梢。春梢 2-4 月抽生，

是一年中数量最多的枝梢，春梢发育好坏，影响着当年产量。夏梢 5-7 月陆续零星抽生，长势不一，

抽梢时与幼果争夺养分，常加剧生理落果，故除幼树利用扩大、填补树冠外，生产上多抹除。秋梢 8-10

月分批抽生，幼龄树及初结果树，秋梢抽发数量较多。栽培中常采用抹除夏芽，促放秋梢的措施，以

增加翌年开花结果数量。在冬季温暖的地区和年份，11 月前后能抽生冬梢，数量少，较零星，不易老

熟，低温时常受冻害[5]。本研究利用实时荧光定量 TaqMan qPCR 技术跟踪检测同一地理区域内感病砂

糖橘叶内黄龙病菌含量的周年动态变化，明确黄龙病菌的消长规律，比较分析砂糖橘病株梢期和果实

成熟期 5 个不同发育阶段黄龙病菌含量的变化情况，以期为微生物源农药防控砂糖橘黄龙病指导施药

最佳时间，为确定砂糖橘果园田间黄龙病普查最佳时期提供参考依据。

1 材料与方法

1.1 实验材料

1.1.1 供试植株

供试品种为砂糖橘，分别来自 4 个果园，每个果园按五点取样法选取 10 株带菌植株作为供试植株，

病株均至少感染黄龙病 3 年。

试验地点：2020 年 1 月至 2020 年 12 月在广东省广州市白云区雄伟村进行。

1.1.3 主要试剂和试剂盒

EasyPure® Plant Genomic DNA Kit：北京全式金生物技术有限公司；PerfectStart® II Probe qPCR

SuperMix UDG：北京全式金生物技术有限公司；特异性引物：生工生物工程股份有限公司合成；2×Taq

PCR Master Mix：生工生物工程股份有限公司；D2000 DNA Marker：生工生物工程股份有限公司；

FastPure® Gel DNA Extraction Mini Kit：诺唯赞生物科技有限公司；pMD™18-T Vector Cloning Kit：

TAKARA；快速感受态细胞制备试剂盒（一步法）：生工生物工程股份有限公司；SanPrep 柱式质粒 DNA

小量抽提试剂盒：生工生物工程股份有限公司。

1.1.4 主要试验仪器

qTOWER3 qPCR 仪：德国 Jena 公司；移液枪：德国 Eppendorf 公司；PowerPac Basic 电泳仪：

美国 Bio-rad 公司；T100 Thermal Cycler PCR 仪：美国 Bio-rad 公司；Centrifuge 5425 离心机：德国

Eppendorf 公司；Gel Doc XR+自动凝胶成像系统：美国 Bio-rad 公司；IMPLEN 微量分光光度计：德

国 IMPLEN 公司；FastPrep-24 组织匀浆器：美国 MP；Accu Therm 恒温金属水浴锅：莱伯特公司。

1.2 方法

1.2.1 试验设计

针对柑橘上的重要病害柑橘黄龙病，选定广东省广州市白云区钟落潭镇雄伟村的 4 个果园作为试

验地，果园种植品种为 5 年生砂糖橘。采集每个果园疑似感病植株的叶片，通过 PCR 对采集的柑橘叶

片样本进行定性检测，将健康与感病样品分类后，每个果园按五点采样法选择 10 株感病砂糖橘植株确

定为试验株，本次试验植株总计 40 株。本实验于 2020 年度果实成熟期（1 月 16 日）、春梢期（2 月

15 日、3 月 14 日、4 月 12 日），夏梢期（5 月 13 日、6 月 14 日、7 月 15 日），秋梢期（8 月 13 日、9

月 14 日、10 月 16 日）以及冬梢期（11 月 15 日、12 月 16 日）采集植株病样。提取叶片总 DNA，用

5

TaqMan qPCR 荧光定量检测技术进行检测，根据得到的 CT 值绝对定量标曲，计算样品黄龙病菌含量

的动态变化情况，各样品数据为 3 个重复的平均值，探索感病砂糖橘黄龙病菌在梢期和果实成熟期 5

个不同发育阶段的动态变化规律。

1.2.2 砂糖橘黄龙病与病菌含量的检测

1.2.2.1 采样与预处理

田间采样部位及方法：每株树采样时先把树冠分为东南西北中 5 个方位，每个方位分别随机采集

3-4 片老叶和嫩叶，每棵树总共采集 15-20 片叶子混合作为一个样本。采完的样品运回实验室立即处理

或则将样品放入 4℃冰箱，为了保证叶片的湿度，在自封袋内放入加灭菌水的纸团，可延长其保存时间。

实验室处理：实验样品的处理在洁净无污染的环境下进行，将单株植株所有叶片样品用无菌水洗

净并擦拭干净，之后用剪刀切取叶中脉偏上部位[6]，处理前后剪刀应用酒精消毒，之后将样品剪至 1 cm

左右，混合，装入研磨管中进行后续的 DNA 提取，处理好的叶片研磨样品可以立即进入后续实验，剩

余叶片放入-20℃冰箱，冷冻保存，待需要时可重新研磨取样。

1.2.2.2 叶样品的 DNA 提取

对叶样品的 DNA 提取，使用 Plant Genomic DNA Kit 试剂盒，操作如下：

（1）将预处理中提取出的叶片中脉 100 mg 进行碾磨；

（2）将磨碎的样品放入容量为 2 mL 试管中，加入 500 µL 重悬缓冲液（Resuspension Buffer, RB1)

后，加入 15 µL 核糖核酸酶 A（RNase A)；

（3）55℃水浴 15 min；

（4）12000 rpm，5 min，上清液移入 2 mL 试管中；

（5）加入 100 µL 的沉淀缓冲液（Precipitation Buffer, PB1），放入冰盒中冰浴 5 min 后，12000 rpm，

5 min；

（6）将上清液移至 2 mL 试管中，加入 375 µL 结合缓冲液（Binding Buffer, BB1）；

（7）吸取全部液体至 1 mL 离心柱管中后，12000 rpm，30 s，去除缓冲液；

（8）加入 500 µL 清除缓冲液（Clean Buffer，CB1），12000 rpm，30 s，去除缓冲液；

（9）加入 500 µL 漂洗缓冲液（Wash Buffer，WB1），12000 rpm，30 s，去除缓冲液；

（10）重复加入 WB1 并离心；

（11）12000 rpm，5 min，去除残留液；

（12）离心柱放入新的 2 mL 试管后，加入 100 µL 预热好的洗脱液（Elution Buffer, EB）60-70℃，

12000 rpm，1 min，洗脱 DNA。

1.2.2.3 PCR 扩增

表 1.1 和表 1.2 为 PCR 扩增体系和条件，根据文献[7]，选用 OI1/OI2c 引物判断样品感病与否，特

异性引物序列为：

OI1：5’-GCGCGTATCCAATACGAGCGGCA-3’

OI2c：5’-GCCTCGCGACTTCGCAACCCAT-3’

表 1.1 PCR 扩增体系

Table 1.1 The PCR amplification system

反应成分 Composition of reaction 体积(µL) Volume (µL)

OI1 1

OI2c 1

ddH2O 9.5

2×Taq PCR Master Mix 12.5

样品 DNA 1

6

PCR 扩增完成后，将扩增产物加注 1%琼脂糖凝胶进行电泳（30 min，120 V），完成后使用凝胶成

像仪观察，并对健康与感病样品分类。

表 1.2 PCR 扩增条件

Table 1.2 The condition of PCR amplification reaction

循环数

cycles

步骤

step

温度(℃)

Temperature ( ) ℃

时间(s)

Time (s)

1 预变性 96 60

35 变性 94 30

35 退火 55 30

35 延伸 72 60

1 修复延伸 72 240

1.2.2.4 质粒标准品制备

本试验选择 Li 等（2006）[8]设计的黄龙病菌亚洲种检测体系对采集的样品中黄龙病菌含量进行检

测，以黄龙病病原菌基因组 CLas 基因片段（5'-GTCGAGCGCGTATGCAATACGAGCGGCAGACGGGTGAGTAACGCGTAGGAATCTACCTTTTTCTACGGGATAACGCA-3'）为模板扩增目的片段，所用引物序

列[9-10]如表 1.3 所示。

表 1.3 黄龙病检测探针序列

Table 1.3 Probe for HLB quantitative detection

名称

name

目的基因引物序列（5'-3'）

Target gene primer sequence (5'-3')

扩增片段大小

Amplified fragment size

HLB asf TCGAGCGCGTATGCAATACG 75 bp

HLB asr GCGTTATCCCGTAGAAAAAGGTAG

HLB Probe FAM-AGACGGGTGAGTAACGCG-BHQ1

首先以提取的阳性基因组总 DNA 为模版，进行普通 PCR 扩增，PCR 反应条件：96℃预变性 4 min；

94℃变性 30 s，55℃退火 30 s，72℃延伸 1 min，35 个循环；72℃修复延伸 4 min；PCR 反应体系（25

µL）。反应体系如表 1.4 所示：

表 1.4 PCR 扩增体系

Table 1.4 The PCR amplification system

反应成分

Composition of reaction

体积(µL)

Volume (µL)

HLB asf(10 µM) 1

HLB asr(10 µM) 1

ddH2O 9.5

2×Taq PCR Master Mix 12.5

样品 DNA 1

PCR 扩增反应结束后，将产物进行 1.5%琼脂糖凝胶电泳（20 min，150 V），观察是否扩增出目的

条带。获得目的条带后，进行胶回收，使用 FastPure® Gel DNA Extraction Mini Kit 试剂盒，操作如下：

（1）DNA 电泳结束后，在紫外灯下快速切下含有目的 DNA 片段的凝胶。

（2）加入等倍体积的 Buffer GDP，56℃水浴 10 min，确保凝胶块完全溶解。水浴期间颠倒混匀 2

次加速溶胶。

7

（3）短暂离心。将≤700 µL 胶溶液转移至吸附柱中，12000 rpm，30 s。若大于 700 µL，将吸附柱

置回收集管，剩余溶液转移至吸附柱中，12000 rpm，30 s。

（4）弃滤液。加入 300 µL Buffer GDP 至吸附柱中。静置 1 min。12000 rpm，30 s。

（5）弃滤液。加入 700 µL Buffer GW （已加入无水乙醇）至吸附柱中。12000 rpm, 30 s。（沿吸

附柱壁四周加入 Buffer GW，或加入 Buffer GW 后盖盖颠倒混匀 3 次有助于冲洗粘附在壁上的盐分）

（6）重复第 5 步。

（7）弃滤液。12000 rpm，2 min。

（8）将吸附柱置于 1.5 mL 灭菌的离心管中，加入 30 µL Elution Buffer 或已灭菌去离子水至吸附

柱中央，放置 2 min。12000 rpm，1 min。

（9）将得到的溶液重新加入离心吸附柱中，12000 rpm，1 min（二次洗脱）。

（10）弃去吸附柱，把 DNA 保存于-20℃。

将纯化后的 DNA 进行克隆测序，使用 pMD™18-T Vector Cloning Kit 试剂盒，在 PCR 管中配制下

列反应体系，16℃连接 1 h，将目的基因片段连接到 pMD18-T Vector 载体上，反应体系如表 1.5 所示。

表 1.5 T 载反应体系

Table 1.5 The pMD™18-T reaction system

反应成分

Composition of reaction

体积(µL)

Volume (µL)

Solution I 5

pMD® 18-T Vector 0.2

PCR Product 4.8

Total 10

连接成功后，进行连接产物转化，使用生工一步法快速感受态细胞制备试剂盒（产品编号：SK9307）。

第二天挑选出白色菌落，通过菌落 PCR 扩增确认载体中插入片段的长度。根据 pMD™18-T Vector

Cloning Kit 试剂盒，使用 T 载的引物序列为：

M13F：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’

M13R：5’-GAGCGGATAACAATTTCACACAGG-3’

PCR 反应条件：96℃预变性 60 s；94 ℃变性 30 s，55℃退火 30 s，72℃延伸 1 min，35 个循环；

72℃修复延伸 4 min；PCR 反应体系（25 µL）。

PCR 反应体系如表 1.6 所示。

表 1.6 PCR 扩增体系

Table 1.6 The PCR amplification system

反应成分

Composition of reaction

体积(µL)

Volume (µL)

2×Taq PCR Master Mix 12.5

M13F(10 µM) 1

M13R(10 µM) 1

Template（菌液） 1

ddH20 9.5

Total 25

PCR 扩增反应结束后，将产物进行 2%琼脂糖凝胶电泳（25 min，90 V），观察是否扩增出目的条

8

带。条带大小比对正确后，从 10 µL 菌液中吸取 1 µL 菌液到 LB 液体培养基中，于恒温摇床中过夜培

养（37℃，200 rpm），次日吸取 600 µL 菌液加入 600 µL 甘油，将菌液保存到 1.5 mL 离心管中。将阳

性菌液送至生工生物工程（上海）股份有限公司进行测序，测序结果比对正确后，使用生工质粒提取

试剂盒 B518191 SanPrep 柱式质粒 DNA 小量抽提试剂盒提取质粒。

1.2.2.5 TaqMan qPCR 检测分析

提取成功的质粒标准品以双蒸水（ddH2O）10 倍梯度稀释之后作为模板，90 µL 稀释液+10 µL 质

粒进行 6 个浓度梯度稀释，进行 TaqMan qPCR 检测，每个浓度梯度做三个技术重复，通过预实验选取

合适标准品用于制备标准曲线，20 µL 的 TaqMan qPCR 反应体系如表 1.7 所示：

表 1.7 TaqMan qPCR 扩增体系

Table 1.7 The TaqMan qPCR amplification system

反应成分

Composition of reaction

浓度

concentration

体积(µL)

Volume (µL)

TaqMan Fast qPCR Master Mix 2× 10

HLB asf(10 µM) 10 µM 0.4

HLB asr(10 µM) 10 µM 0.4

HLB Probe(10 µM) 10 µM 0.4

ddH20 7.8

Template(DNA) 1

Total 20

PCR 循环条件：95℃，3 min；95℃，10 s，57℃，15 s，72℃，30s，45 个循环周期。

完成上述步骤后，把加好样品的 96 孔板放在 qTOWER3 qPCR 仪中进行反应，得出标准曲线，以

质粒制作标准曲线来计算样品中 CLas 基因组拷贝数（copy number，CN）。

2 结果与分析

2.1 砂糖橘果园采集情况

根据 PCR 扩增结果，确定感病样品，每个果园按五点（东、西、南、北和中方位）采样法选择 10

株黄龙病植株作为试验样品，本次试验植株总计 40 株，以感病砂糖橘叶片标准品作为阳性对照，以

健康砂糖橘叶片标准品作为阴性对照，扩增结果如图 1.1 所示。

注：M 为 DL2000 DNA marker，由 6 条特定分子量的双链 DNA 片段组成，标注为图中左侧数字；

+为阳性对照，-为阴性对照，A1~D10 为样品植株编号。

图 1.1 CLas 的 PCR 扩增产物 1%琼脂糖电泳图

Figure 1.1 1% agarose electrophoresis of PCR amplification products of CLas

9

本试验样品植株共 40 株，周年共获得叶样品 480 份，周年 DNA 提取样本共计 1440 份，PCR 检测

结果发现，果园中采集的疑似患病样品都确诊感染柑橘黄龙病，该果园黄龙病扩散程度较大，病情较

为严重。选定 40 株感病植株测定病树内黄龙病菌含量的周年动态变化，明确黄龙病菌的消长规律，为

黄龙病的综合防控提供参考依据。

2.2 质粒的构建

以提取的柑橘叶片阳性基因组总 DNA 为模版，使用 HLB asf/HLB asr 引物进行普通 PCR 扩增，扩

增出目的条带，如图 1.2 所示。

注：M 为 DL10000 DNA marker，片段标注为图中左侧数字。

图 1.2 CLas 的 PCR 扩增产物 1%琼脂糖电泳图

Figure 1.2 1% agarose electrophoresis of PCR amplification products of CLas

验证目标片段载体，将 HLBas/HLBr 扩增片段连接到上 pMD 18-T Vector 得到 pMD 18-T 载体，使

用引物 M13F/M13R 扩增得到 211 bp 特异条带。测序后使用 DNAMAN 进行序列比对，与 CLas 基因组

中相应序列相似性为 100%，验证结果正确，如图 1.3 所示。

注：M 为 DL2000 DNA marker，由 6 条特定分子量的双链 DNA 片段组成，标注为图中左侧数字；

+为阳性对照，-为阴性对照，数字为样品编号。

图 1.3 pMD 18-T 载体的验证

Figure 1.3 Validation of pMD 18-T vector

表 1.8 HLB 质粒相关数值

Table 1.8 HLB plasmid correlation valuesn

名称 name 质粒 plasmid

扩增子长度 75(bp)

质粒大小 2767(bp)

浓度 86.35(ng/µL)

拷贝数 2.89E+10(copies/µL)

标曲最高拷贝数 2.89E+07(copies/µL)

注：[1]质粒浓度换算公式(copies/µL)=(mol 数/µL×6.02×1023=[质量(g)/分子量]/µL×6.02×1023=[质量(ng)×10-9/

10

分量]/µL×6.02×1023=浓度(ng/µL)×6.02×1014/分子量；[2]分子量=(载体片段碱基对+PCR 产物碱基对)×650；[3]双链

DNA 分子的分子量(道尔顿)=碱基对数目×650；[4]一个 DNA 碱基对(钠盐)的平均分子量=650 道尔顿。

2.3 标准曲线的制备

目的基因 HLB 梯度稀释样品扩增和标准曲线结果如图 1.4 和图 1.5 所示。

图 1.4 目的基因梯度稀释样品 TaqMan qPCR 扩增曲线

Figure 1.4 TaqMan qPCR amplification curve of gradient dilution sample of target gene

注：纵坐标是 CT 值；横坐标 LogCO，LogCO 是指 Log 浓度即取浓度的对数。Slope=-3.078；

扩增效率：E=10-1/斜率-1=10-1/-3.078-1=111.283%；相关系数：R2

=0.999；Y-inter=40.63。

图 1.5 CLas 检测的荧光定量 TaqMan qPCR 标准曲线

Figure 1.5 TaqMan qPCR standard curve for Las detection

标准曲线 y=-3.078x+40.63(R2

=0.999, E=111.283%)。y 为 CT 值，x 为 DNA 浓度的对数，DNA 浓度

转化为模板拷贝数，单位 copies/µL。得到公式 Y=-3.078lg(CN)+40.63（Y 为 CT 值，CN 为模板拷贝数）。

2.4 梢期及果实成熟期 5 个不同发育阶段砂糖橘 CLas 含量的变化规律

利用实时荧光定量 TaqMan qPCR 检测 40 株砂糖橘的病原菌含量周年变化，周年中 1 月为果实成

熟期，2-4 月为春梢期，5-7 月为夏梢期，8-10 月为秋梢期，11-12 月为冬梢期，进而分析梢期和果实

成熟期 5 个不同发育阶段的感病砂糖橘黄龙病菌含量变化规律。根据上一章制备标曲得出的公式

Y=-3.078lg(CN)+40.63 来计算黄龙病菌的含量，Y 为样品进行 TaqMan qPCR 检测得出的 CT 值，CN 为

DNA 浓度，单位为 copies/µL，qPCR 体系中的样品 DNA 量为 1 µL，用于提取全基因组 DNA 的样品

叶片质量为 0.1 g，根据线性回归方程计算每克病样组织内黄龙病菌量=10^[(Y-40.63)/-3.078]×103

，单位

为 copies/g。

利用 Excel 2021 进行数据记录与处理，用 IBM SPSS statistics 25 对检测数据做单因素方差分析，

显著性 P<0.05，使用 GraphPad Prism 8.0 作图，将不同月份及 4 个梢期和果实成熟期的砂糖橘样品 DNA

11

的 CT 值和样品叶片病原菌含量的变化数据进行相关性分析，探索 HLB 的流行规律。结果如下所示：

表 1.9 一年内不同月份样品含菌 CT 值变化

Table 1.9 Changes of CT values of bacteria in different months of the year

栽培品种

cultivated variety

一年内不同月份样品病菌含量 CT 平均值

CT mean value of pathogen content in samples in different months within a year

1 月 2 月 3 月

(24.65±0.12)d (27.37±0.18)b (27.35±0.18)b

4 月 5 月 6 月

(28.74±0.14)a (28.59±0.18)a (27.03±0.13)b

7 月 8 月 9 月

(26.41±0.18)c (25.06±0.12)d (27.12±0.18)b

10 月 11 月 12 月

砂糖橘

(25.07±0.13)d (26.06±0.16)c (24.19±0.16)e

注：数据表示平均值±标准误(n=120)，同行数据后不同小写字母表示在 0.05 水平差异显著(P<0.05)。

利用实时荧光定量 TaqMan qPCR 每个月对 40 株感病砂糖橘内含菌量变化进行连续定量检测，结

果表明，所有被检测样品均显阳性（CT 值小于 35）；黄龙病菌 CT 值在不同月份有明显的变化，感病

砂糖橘树体含菌量 CT 平均值的趋势为：4 月份>5 月份>2 月份>3 月份>9 月份>6 月份>7 月份>11 月份

>10 月份>8 月份>1 月份>12 月份。其中 4 月和 5 月病菌含量 CT 值无显著差异，2 月、3 月、6 月和 9

月病菌含量 CT 值无显著差异，7 月和 11 月病菌含量 CT 值无显著差异，1 月、8 月和 10 月病菌含量

CT 值无显著差异（P>0.05）；12 月份病菌含量 CT 值显著低于其他月份；4 月和 5 月病菌含量 CT 值显

著高于其他月份（P<0.05）。

表 1.10 不同梢期和果实成熟期样品含菌 CT 值变化

Table 1.10 Changes of CT values of bacteria in samples at different shoot and fruit ripening stages

栽培品种

cultivated variety

不同梢期和果实成熟期样品含菌 CT 值变化

Changes of CT of bacteria in samples at different shoot and fruit ripening stages

春梢期 夏梢期 秋梢期

(27.82±1.94)e (27.35±2.01)a (25.75±1.87)b

冬梢期 果实成熟期

砂糖橘

(25.13±2.02)c (24.65±1.29)d

注：数据表示平均值±标准误，同行数据后不同小写字母表示在 0.05 水平差异显著(P<0.05)。

结果表明：黄龙病菌 CT 值在不同梢期及果实成熟期内有明显的变化，感病砂糖橘树体含菌量平均

CT 值的趋势：春梢期>夏梢期>秋梢期>冬梢期>果实成熟期，春梢期、夏梢期、秋梢期、冬梢期和果

实成熟期感病砂糖橘树体含菌量平均 CT 值差异显著(P<0.05)。

表 1.11 样品黄龙病菌含量在一年内不同月份的动态变化

Table 1.11 Dynamic changes of pathogen content in samples in different months in a year

栽培品种

cultivated variety

样品黄龙病菌含量在一年内不同月份的动态变化

Dynamic change of Pathogen content of HLB in different months of a year (copies/g)

1 月 2 月

(24.51±2.47)×107

bcd (10.24±2.84)×107

cde

砂糖橘

3 月 4 月

12

(6.71±1.28)×107

de (22.82±6.61)×106

e

5 月 6 月

(3.88±1.05)×107

e (42.95±4.14)×106

e

7 月 8 月

(16.47±3.95)×107

cde (20.73±3.23)×107

bcde

9 月 10 月

(2.56±1.02)×108

bcd (29.77±9.06)×107

bc

11 月 12 月

(3.79±1.39)×108

ab (48.45±7.16)×107

a

注：数据表示平均值±标准误(n=120)，同行数据后不同小写字母表示在 0.05 水平差异显著(P<0.05)。

结果表明：根据砂糖橘黄龙病菌 16S rDNA 基因的 qPCR 标准曲线线性回归方程，把获得的黄龙病

菌菌量 CT 值转化为每克砂糖橘组织内的含菌量。一年内不同月份平均感病砂糖橘的黄龙病菌含量：4

月<5 月<6 月<3 月<2 月<7 月<8 月<1 月<9 月<10 月<11 月<12 月；全年砂糖橘每克组织黄龙病菌平均

含量在 2.28×107

~4.85×108 拷贝之间，4-6 月病菌量出现低峰，7-12 月病菌量呈现增长趋势，并在 12 月

份达到增长高峰。其中 11 月和 12 月病菌含量无显著差异，4 月、5 月和 6 月病菌含量显著低于其他月

份，11 月和 12 月病菌含量显著高于其他月份(P<0.05)。

表 1.12 不同梢期和果实成熟期的黄龙病菌含量变化

Table 1.12 Changes of pathogen contents in different shoot stages and fruit ripening stages

栽培品种

cultivated variety

不同梢期和果实成熟期的黄龙病菌含量变化

Changes of Pathogen content in Different shoot and fruit maturity stages (copies/g)

春梢期 夏梢期

(6.41±1.07)×107

c (8.21±1.40)×107

c

秋梢期 冬梢期

(25.37±4.65)×107

b (43.17±7.80)×107

a

果实成熟期

砂糖橘

(24.51±2.47)×107

b

注：数据表示平均值±标准误，同行数据后不同小写字母表示在 0.05 水平差异显著(P<0.05)。

结果表明：不同梢期和果实成熟期的感病砂糖橘的平均黄龙病菌含量：春梢期＜夏梢期＜果实成

熟期＜秋梢期＜冬梢期；全年砂糖橘每克组织黄龙病菌平均含量在 2.28×107

~4.85×108 拷贝之间。其中

秋梢期和果实成熟期黄龙病菌含量无显著差异，春梢期和夏梢期黄龙病菌含量无显著差异(P>0.05)；冬

梢期黄龙病菌含量显著高于春梢期、夏梢期、秋梢期和果实成熟期，春梢期和夏梢期黄龙病菌含量显

著低于果实成熟期、秋梢期和冬梢期，果实成熟期和秋梢期的黄龙病菌含量显著低于冬梢期(P<0.05)。

3 讨论

本研究明确了感病砂糖橘体内黄龙病菌的年动态变化规律，比较分析了砂糖橘病株梢期和果实成

熟期 5 个不同发育阶段黄龙病菌含量的变化情况，为确定果园田间普查病情和检测植株是否带菌最佳

时期和黄龙病的最佳防控时期提供参考依据。胡浩研究结果显示在柑橘黄龙病菌含量在 10 月时含量最

高，3-5 月时植株病菌含量比较少[11]。研究发现黄龙病菌含量的最高值出现在 10 月和 12 月，3 月和 5

月病菌含量较少[12]。程保平研究发现柑橘病树内的黄龙病菌含量在春梢期最低，夏梢、秋梢期次之，

冬梢期及果实成熟期最高[13]。李敏在感病砂糖橘根和叶的菌含量周年动态研究中，发现叶的菌含量始

终显著高于根的菌含量，根和叶的菌含量均在秋季最高，夏季最低。叶的菌含量变化相对稳定，但是

13

10-12 月份的菌含量迅速增长且显著高于其他月份[14]。李智鹏等研究发现果园中柑橘黄龙病亚洲种含

量变化呈现一定规律消涨趋势，与以往的研究结果有相同之处，在 10 月存在高峰，柑橘体内亚洲种含

量变化与温度和下雨天数这 2 个气候因子无相关性[15]。上述学者的研究结果都说明病树体内黄龙病菌

含量在 10-12 月份较其他月份会升高，与本研究的结果大致相似。

本研究发现柑橘黄龙病菌一年内在 4-6 月份呈现低谷的变化趋势，从 7 月份开始逐步增长，春梢

期和夏梢期病菌含量低于其他时期，差异显著，果实成熟期和秋梢期的黄龙病菌含量显著低于冬梢期，

这说明感病砂糖橘体内的黄龙病菌含量变化与不同的砂糖橘生长时期可能有着联系。春、夏梢期的时

候气温比较高，环境气候比较适宜柑橘的生长发育，树体强健，从而能够抑制黄龙病菌的生命活动；

而在冬梢、秋梢期及果实成熟期的时候，天气开始变冷，气候适宜度降低，而随着果实的成熟，柑橘

树体的营养会向果实集中运输，冬天的时候植物的根系吸收能力会有所减弱，这些原因可能会导致感

病砂糖橘树的营养失衡、树的长势变弱，黄龙病菌的活动加强，从而难以抑制。此外，因为果园的地

理位置差异，不同的地区气候以及不同品种的柑橘植株都会给实验结果带来一些影响。综上，黄龙病

的防控可以通过培养健壮发达的苗木根系，从而提高感病柑橘植株的营养吸收和传导的能力，促进树

体强健，增强植株抗逆性，可以依据消长规律，做好果园管理，抑制黄龙病菌。本试验结果可为黄龙

病果园监测、产量损失和综合治理模型的建立提供一定参考依据，对柑橘黄龙病的防控具有一定指导

意义。本试验的结果可以为后续柑橘黄龙病的田间防控的最佳用药时间提供指导依据，建议于 7 月份

黄龙病高发期之前进行药剂处理。

参考文献

[1] 陈源, 张迪, 潘鹤立, 等. 不同品种柑橘果皮的抗氧化能力研究[J]. 热带作物学报, 2019, 40(8): 1633-1637.

[2] 唐传核, 彭志英. 柑橘类的功能性成分研究概况[J]. 四川食品与发酵, 2000, 1(4): 1-7.

[3] Reinking O A. Host index of diseases of economic plants in the Philippines[J]. Philippine Agriculturist, 1919, 8(91): 38-54.

[4] Bové J M, Barros A P D. Huanglongbing: A destructive, newly emerging, century-old disease of citrus[J]. Journal of Plant

Pathology, 2006, 88(1): 7-37.

[5] 佚名. 柑橘各梢段特点与春梢管理[J]. 农化市场十日讯, 2019, 2(5): 2-4.

[6] Teixeira D C, Saillard C, Couture C, et al. Distribution and quantification of Candidatus Liberibacter americanus, agent of

huanglongbing disease of citrus in so Paulo State, Brasil, in leaves of an affected sweet orange tree as determined by PCR[J].

Molecular & Cellular Probes, 2008, 22(3): 139-150.

[7] 肖婉钰, 黄江华. 砂糖桔黄龙病 3 种检测引物灵敏性比较研究[J]. 仲恺农业工程学院学报, 2019, 32(3): 14-17.

[8] Li W, Hartung J S, Levy L. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species

associate with citrus huanglongbing[J]. Journal of Microbiological Methods, 2006, 66(1): 104-11.

[9] 王华堂, 曾鑫年, 薛培培, 等. Direct-PCR 检测柑橘黄龙病的快速制样方法研究[J]. 果树学报, 2014, 31(4): 733-738.

[10] 陈冬梅. 柑桔黄龙病叶近红外光谱特征及药效评价应用[D]. 华南农业大学, 2016.

[11] 胡浩. 应用荧光定量 PCR 技术研究亚洲韧皮部杆菌在寄主体内的动态变化及分布[D]. 重庆大学, 2007.

[12] Wang Z, Yin Y, Hu H, et al. Development and application of molecular-based diagnosis for 'Candidatus Liberibacter asiaticus', the causal pathogen of citrus huanglongbing[J]. Plant Pathology, 2010, 55(5): 630-638.

[13] 程保平, 彭埃天, 宋晓兵, 等. 广东三个柑橘品种不同生育期的黄龙病菌含量动态变化研究[J]. 植物保护, 2017,

43(4): 208-212.

[14] 李敏. 黄龙病菌在柑橘根部的动态消长及其对丛枝菌根侵染的影响[D]. 华南农业大学, 2018.

[15] 李智鹏, 关巍, 黄洋, 等. 柑橘黄龙病菌在寄主体内含量动态变化研究[J]. 果树学报, 2019, 36(11): 1540-1548.

14

芒果病程相关蛋白 MiPR1 基因家族鉴定和 MiPR1A 基因功能分析

李佳俊，罗聪，杨小州，彭龙辉，陆婷婷，何新华*

*为通讯作者

广西大学农学院，亚热带农业生物资源保护与利用国家重点实验室，广西农业环境与

农产品安全重点实验室，广西南宁 530004

摘 要：病程相关蛋白 PR1 是植物防御机制中抵抗各种生物和非生物胁迫的重要组成部分。在本研究中，通过对四季

蜜芒基因组进行生物信息学分析，鉴定到 10 个 PR1 基因家族成员，系统分析了这些基因的结构、理化性质、染色体定

位、启动子顺势原件分析和进化关系。MiPR1 基因不均匀的分布在芒果的二十条染色体中，对其进行组织、生物胁迫

与非生物胁迫表达分析，发现 MiPR1 基因家族在不同组织中表达并不相同，生物和非生物胁迫在不同程度上诱导了

MiPR1 基因的表达。在这些基因中，MiPR1A 基因在芒果的芽和胚中略高表达，在盐、干旱胁迫和接种胶孢炭疽菌的处

理中显著上调。进一步对 MiPR1A 基因转拟南芥进行功能研究。与野生型拟南芥相比，MiPR1A 基因在拟南芥中过表达

增加了转基因拟南芥的耐盐性和抗旱性，超氧化物歧化酶（SOD）活性和脯氨酸含量显著升高，丙二醛（MDA）含量

显著下降。转 MiPR1A 基因，能增强转基因拟南芥对胶孢炭疽菌的抗性。

关键词 芒果；全基因组鉴定；PR1 基因家族；表达分析；功能验证

基金资助 广西创新驱动发展专项资金资助项目(桂科 AA22068098-2), 国家现代农业产业技术体系广西芒果创新团队

栽培与病虫害防治岗位项目(nycytxgxcxtd-2021-06-2)

A sugarcane smut fungus effector simulates the host

endogenous elicitor peptide to suppress plant immunity

Hui Ling1,2* , Xueqin Fu1

*, Ning Huang2

*, Zaofa Zhong1

, Weihua Su1

, Wenxiong Lin1

, Haitao Cui1 and

Youxiong Que1

1

Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Plant

Immunity Center, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2

College of Agriculture, Yulin Normal University, Yulin 537000, China

Authors for correspondence:

Youxiong Que

Email: queyouxiong@126.com

Haitao Cui

Email: cui@fafu.edu.cn

Received: 6 July 2021

Accepted: 22 October 2021

New Phytologist (2021)

doi: 10.1111/nph.17835

Key words: effector, PEPR1, plant immunity,

smut fungus, sugarcane.

Summary

The smut fungus Sporisorium scitamineum causes the most prevalent disease on sugarcane. The mechanism of its pathogenesis, especially the functions and host targets of its effector proteins, are unknown.

In order to identify putative effectors involving in S. scitamineum infection, a weighted

gene co-expression network analysis was conducted based on the transcriptome profiles of

both smut fungus and sugarcane using a customized microarray. A smut effector gene, termed SsPele1, showed strong co-expression with sugarcane PLANT ELICITOR PEPTIDE

RECEPTOR1 (ScPEPR1), which encodes a receptor like kinase for perception of plant elicitor

peptide1 (ScPep1). The relationship between SsPele1 and ScPEPR1, and the biological function of SsPele1 were characterized in this study.

The SsPele1 C-terminus contains a plant elicitor peptide-like motif, by which SsPele1 interacts strongly with ScPEPR1. Strikingly, the perception of ScPep1 on ScPEPR1 is competed by

SsPele1 association, leading to the suppression of ScPEPR1-mediated immune responses.

Moreover, the Ustilago maydis effector UmPele1, an ortholog of SsPele1, promotes fungal

virulence using the same strategy.

This study reveals a novel strategy by which a fungal effector can mimic the plant elicitor

peptide to complete its perception and attenuate receptor-activated immunity.

Introduction

Sugarcane (Saccharum spp. hybrids) is a multifunctional crop

especially for sugar production (Marques et al., 2017). It is

impacted by various diseases, including sugarcane smut, caused

by Sporisorium scitamineum (Ustilaginomycetes), which occur in

the growing areas all over the world (Marques et al., 2017). Smut

fungi colonize the apical meristematic tissue of the germinating

lateral bud or stem apex, resulting in the degradation of plant cell

wall, enlargement of the intercellular space, hormonal imbalance

and the development of a whip-like sorus in sugarcane (Marques

et al., 2017, 2018). The sorus is an elongated internode whose

growth is mediated by high mitotic activity of the intercalary

meristem at the base (Marques et al., 2018). The underlying

mechanisms driving the cellular changes in the host tissue remain

to be elucidated.

Smut fungi are facultative biotrophs, as they can grow saprotrophically as yeast-like cells on culture media but require the

biotrophic infection of host cells to complete their life cycle (Sundar et al., 2012). To establish a biotrophic parasite, these fungi

deliver large amounts of effectors to counteract host defenses.

Ustilago maydis and Sporisorium reilianum, which cause smut disease on maize, both produce more than four hundred putative effectors (Schuster et al., 2018). Many smut effectors have enzymatic

activities, such as mutase, peroxidase or protease (Doehlemann et

al., 2009, 2011; Djamei et al., 2011; Hemetsberger et al., 2012;

Mueller et al., 2013; Ma et al., 2018; Schweizer et al., 2018). However, many smut-secreted effectors, accounting for nearly half of the

secretome, lack known functional domains (Schuster et al., 2018).

The S. scitamineum genome encodes 622 proteins with signal peptides, among which 537 were predicted as candidate-secreted effector proteins (Que et al., 2014b; Dutheil et al., 2016), including the

orthologs of well-studied effectors in U. maydis, such as Cmu1,

Pep1, Pit2, Stp1 and Tin2 (Tumor inducing2) (Tanaka et al.,

2014). The transcription of these effector genes was significantly

upregulated in the infected tissue (Barnabas et al., 2017). However,

none of the putative effectors in S. scitamineum has been functionally

characterized.

Different technologies, including metabolomics (Sanchez- ´

Elordi et al., 2019), proteomics (Barnabas et al., 2017) and

DNA/RNA related-omics (Que et al., 2014a,b; Su et al., 2019),

have been used in identifying S. scitamineum factors that were

involved in sugarcane–S. scitamineum interactions. These studies

*These authors contributed equally to this work. usually focused on either sugarcane or smut fungus, but not on

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This is an open access article under the terms of the Creative Commons Attribution License, which permits use,

distribution and reproduction in any medium, provided the original work is properly cited.

15

Research

both. Furthermore, most S. scitamineum transcripts or proteins

are missing from the published sugarcane–S. scitamineum mixed

transcriptome or proteome data probably due to the low biomass

of S. scitamineum in the mixed samples. Weighted gene coexpression network analysis (WGCNA) is a widely used systemic

biology method to construct gene networks, detect gene modules

and identify the central players within modules (Langfelder &

Horvath, 2008). In a co-expression network between the biomass

and gene expression levels in U. maydis using WGCNA, three

well-known effector genes Pep1, Pit2 and Stp1, and 25 uncharacterized core effector genes were clustered into the same module

and were speculated to be important for establishing biotrophy

(Lanver et al., 2018). An Arabidopsis thaliana–Botrytis cinerea

gene co-expression network generated using WGCNA revealed

that fungal phytotoxins, such as sesquiterpene botrydial and

polyketide botcinic acid, likely targeted host defense/camalexin

related components to inhibit host immunity (Zhang et al.,

2019). To date, WGCNA has not yet been used to construct sugarcane–S. scitamineum gene co-expression networks and to identify key players in this pathosystem.

The first layer of the plant immune system consists of

PAMP-triggered immunity (PTI) that is activated by cellsurface-resident pattern recognition receptors (PRRs), perceiving

pathogen/microbe associated-molecular patterns (PAMPs/

MAMPs) or damage-associated molecular patterns (DAMPs).

PTI activates a series of immune responses, including production of reactive oxygen species (ROS) and nitric oxide, phosphorylation of mitogen-activated protein kinase (MAPK) cascades,

transcriptional reprograming, changing of hormone homeostasis, and callose deposition (Cui et al., 2015; Liang & Zhou,

2018; Wang et al., 2020). PRRs consist primarily of receptorlike kinases (RLKs) and receptor-like proteins (RLPs). There are

approximately 610 and approximately. 1100 RLKs accounting

for c. 2% of the coding genes in the Arabidopsis and rice

genomes, respectively (Liang & Zhou, 2018). In sugarcane, 427

RLKs and 157 RLPs are coded in the genome of the ancestral

sugarcane genotype Saccharum spontaneum (Rody et al., 2019).

RNA-seq detected 290 RLK and 194 RLP transcripts in modern

sugarcane varieties (Rody et al., 2019). Among them, 18 were

significantly upregulated in a smut-resistant variety (SP80–

3280), whereas only six were upregulated in the smutsusceptible variety (IAC66–6) (Rody et al., 2019), indicating

their involvement in resistance to smut disease. However, none

of these RLKs or RLPs have been functionally characterized in

the sugarcane–S. scitamineum interaction thus far.

In this study, using a customized Agilent microarray combined

with WGCNA, we discovered S. scitamineum putative effectors

that exhibit strong co-expression with a sugarcane PLANT

ELICITOR PEPTIDE RECEPTOR1 (ScPEPR1) gene. Plant

PEPRs are PRRs recognizing plant elicitor peptides (Peps) that

are DAMPs being produced primarily after wounding (Tang &

Zhou, 2016). We found that an apoplastic effector, termed

SsPele1, interacts with ScPEPR1. SsPele1 has a Peps-like motif

on the C-terminus and could bind to the ScPEPR1 extracellular

LRR domain to compete ligand binding, leading to the suppression of ScPEPR1-mediated immune responses. The works reveal

a novel virulence mechanism of fungal apoplastic effector to suppress host defenses by competing perception of Peps.

Materials and Methods

Plant materials, growth conditions and pathogen infection

Sugarcane genotypes, NCo376 (highly resistant to Sporisorium scitamineum), YC71-374 (highly susceptible to S. scitamineum) and

ROC22 (the most prevalent variety in China) were used. Robust

sugarcane plants with uniform growth from NCo376 and YC71-

374 were collected from the field and cut into single-bud stalks.

These stalks were grown in an incubator at 28  0.5°C 3000 lx,

with a 16 h : 8 h, light : dark photoperiod, until the buds sprouted

and the seedlings emerged, then three biological replicates were

injected with S. scitamineum at 5 × 106 spores ml−1 or water (the

control) (Huang et al., 2018), respectively.

For testing the biological relevance of the 25-amino-acid peptide

of the C-terminal of SsPele1 (SsPel25) in sugarcane-S. scitamineum

pathosystem, three biological replicates each containing green

sheaths from 10 ROC22 plants were surface-sterilized in 3%

NaClO (v/v, contained 0.02% Tween20) for c. 20 min and washed

with sterilized water. The outermost leaf sheath was discarded and

40 inner leaf sheaths were cut into 80 slices at about 0.5 cm × 2 cm.

These slices were divided into two equal parts and immersed into

the smut fungus solution (diploid-type Ss17-18 at OD600 = 0.008

in water plus 0.02% Tween20) only or containing the SsPel25 peptide (5 μM), respectively, and then vacuumized (0.4 kg cm−2

) for

10 min. They were placed on the filter paper covering solid

Murashige & Skoog medium, and grown in an incubator (28 +

0.5°C, 3000 lx, 16 h : 8 h, light : dark). About 10 slices were

pooling-sampled at 0, 12 and 24 h for DNA isolation, respectively.

Arabidopsis thaliana Col-0 was used for sugarcane and S. scitamineum gene transformation and protoplast isolation. Seeds were

germinated in soil, and plants were grown in an incubator at

22°C, 60% relative humidity with a 16 h : 8 h, light : dark photoperiod.

RNA isolation, cDNA amplification and quantitative realtime (qRT)-PCR

The sugarcane buds and Arabidopsis leaves were collected for

RNA isolation using a TRIzol kit (#10296028; Invitrogen).

RNA samples from NCo376 and YC71-374 sugarcane genotypes

infected with S. scitamineum at 0 d postinoculation (dpi), 3 dpi

(Peters et al., 2017), 5 dpi (Schaker et al., 2016) and 7 dpi (Singh

et al., 2004) were used for microarray hybridization and qRTPCR validation. Total RNA (1 μg) was used for cDNA synthesis

and qRT-PCR using ChamQTM Universal SYBR qPCR Master

Mix (#Q331-02; Vazyme, Nanjing, China) on a QuantStudio 3

machine (Applied Biosystems, Foster City, CA, USA). In qRTPCR analysis, the relative expression level of sugarcane genes was

normalized to the reference genes acyl-CoA dehydrogenase and serine/arginine repetitive matrix protein 1 (Livak & Schmittgen,

2001; Huang et al., 2018), and that of S. scitamineum genes was

normalized to the reference genes inosine 50

-monophosphate

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dehydrogenase and SEC65-signal recognition particle subunit (Livak

& Schmittgen, 2001; Huang et al., 2018), whereas that of Arabidopsis genes was normalized to Actin with 2ΔΔCq method

(Livak & Schmittgen, 2001; Wang et al., 2017).

TaqMan based detection of S. scitamineum

The fungal biomass of S. scitamineum in the infected sugarcane

buds and sheath slices were quantified using a TaqMan-based

qPCR method as described by Su et al. (2013). The cycle amplification of bE, a S. scitamineum gene related to mating (Albert &

Schenck, 1996), in DNA samples and the plasmid pMD19-bE

was compared (Su et al., 2013). The primer pairs and the probes

are listed in the Supporting Information Table S1.

Microarray design, hybridization, validation and data analysis

In order to identify the differentially expressed genes (DEGs)

during the S. scitamineum–sugarcane interaction, a customized 8

× 60 K Agilent microarray (Agilent Technologies, Santa Clara,

CA, USA), targeting nonredundant sugarcane genes (20 392,

Table S2) (Que et al., 2014a) and S. scitamineum genes (6621

coding sequences) (Que et al., 2014a), was designed. The probes

against the sugarcane genes had two technical replicates, whereas

those against the S. scitamineum genes had three technical replicates. The microarray hybridization and the systemic normalization of gene expression, general data analysis of sample groups

and calculation of differential gene expression level were processed by Shanghai Biotechnology Co. Ltd (SBC, Shanghai,

China). R software was used for normalization per chip and systemic normalization of sugarcane and smut gene differential

expression level with the QUANTILE algorithm separately. The

DEGs with expression fold-change ≥ 2 or ≤ 0.5 (P < 0.05) were

identified using the LIMMA package (www.bioconductor.org)

(Bolstad et al., 2003). All of these data were deposited in the

Gene Expression Omnibus (GEO, GSE140801). Data analysis

and figure illustration were performed using toolkit TBTOOLS

(Chen et al., 2020). For identifying the effector genes, the coding

DNA sequences (CDSs) of S. scitamineum genes were aligned to

the effector sequences of other gramineous smut fungi (Laurie et

al., 2012; Ali et al., 2014; Brefort et al., 2014; Que et al., 2014a;

Dutheil et al., 2016), using TBTOOLS (Chen et al., 2020).

Construction of a sugarcane–S. scitamineum co-expression

network and screening of key genes

The R package based WGCNA (http://labs.genetics.ucla.edu/

horvath/CoexpressionNetwork/Rpackages/WGCNA/) was used

for identifying the DEG matrix (module) that was significantly

associated with incubation time and fungal biomass. The parameters (soft threshold, 20; minimum module size, 30; merge cut

height, 0.20) were fitted, and the modules, including DEGs with

similar expression tendencies, were generated statistically and

equitably with a one-step automatic construction method and

indicated by different colors according to the correlation patterns.

The DEGs from those modules significantly associated with the

increase of the fungal biomass and incubation period were chosen. The connectivity value between genes was obtained from the

WGCNA. The top DEGs with the highest connectivity value were

used to generate the visible co-expression network in CYTOSCAPE

(v.3.5.0) software (Shannon et al., 2002; Cline et al., 2007).

Gene cloning, plasmid construction and sequence analysis

Sugarcane PLANT ELICITOR PEPTIDE RECEPTOR1 (ScPEPR1)

and four S. scitamineum putative effectors, SsPE1, SsPE4, SsPE14

(SsPele1) and SsPE15, were cloned from genotype ROC22 (Table

S3). The open reading frames (ORFs) of these genes were cloned

into destination vectors, including pGBKT7, pGADT7,

pSUC2T7M13ori, pFastR06, pXCSG and pCMABIA1306

(Table S1), using a cloning Kit (#C112-01; Vazyme). Likewise,

the ORFs of ScPEPR1 and SsPE14 were introduced into

pCMABIA1300S-nYFP and pCMABIA2300S-cYFP, respectively, to generate N-terminal YFP (nYFP) fused to ScPEPR1 by

its N-terminus (nYFP-ScPEPR1) and C-terminal (cYFP) fused

to SsPE14 or SsPE14-Δsp (without signal peptide) by its Cterminus (SsPE14-cYFP and SsPE14-Δsp-cYFP) (YFP, yellow

fluorescent protein). The ORF of AtPEPR1 without a stop codon

also was introduced into pCAMBIA1306-FLAG. The primer

pairs used for plasmid construction are listed in Table S1.

The sequences of the PEPR and peptide1 (Pep1) were downloaded from Sequence Read Archive database (SRP192749) and

NCBI database (S. spontaneum genomic data: GCA_003544955.1;

Table S3). The alignment of PEPR, Pep1 and the effectors was performed with DNAMAN (v.7.0.2.176) and GENEDOC (http://www.flu.

org.cn/en/download-47.html). A phylogenetic tree was generated

using the maximum-likelihood method with 1000 bootstrap replicates in MEGA7 software (Institute for Genomics and Evolutionary

Medicine, Temple University, Philadelphia, PA, USA).

Yeast two-hybrid (Y2H) and glutathione S-transferase

(GST)-pulldown experiments

For validating the protein interactions, prey and bait vectors containing genes as indicated in the figures were co-transformed into Y-2-

HGold chemically competent cells. Positive yeast clones containing

two plasmids were selected from SD/-Trp-Leu medium (#630494;

Clontech, Terra Bella Avenue Mountain View, CA, USA) and were

re-plated on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade medium

(#630494; Clontech).

After codon optimization and synthesis, the CDSs of

ScPEPR1-N and SsPE14 were ligated into plasmids, generating

pCzn1_ScPEPR1-N and pGEX-4T-1_SsPE14, which were transformed into Escherichia coli strain BL21. The histidine (HIS)-

and GST-tagged proteins were purified and the in vitro GST

pulldown experiments were performed according to the method

of Tarun & Sachs (1996).

Secretory function assay of signal peptide in yeast

The assay was performed mainly based on Xu et al. (2019). The

sequence of the signal peptide was inserted into the vector

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pSUC2T7M13ori and transformed into yeast strain YTK121

(Jacobs et al., 1997). The transformed yeast was plated on the

CMD-W (minus tryptophane plates) (Xu et al., 2019), and incubated at 30°C for 3 d in darkness. For invertase secretion assay,

transformers were replica plated on YPRAA plates (1% yeast

extract, 2% peptone, 2% raffinose and 2 μg ml−1 antimicyn A)

lacking glucose. The activity of invertase also was determined by

reducing 2,3,5-triphenyltetrazolium chloride (TTC) to insoluble

red 1,3,5-triphenylmethyl nitrogen (TPF) (Xu et al., 2019).

Confocal images, protein extraction, immunoprecipitation

and immunoblotting assay

The transformed Agrobacterium tumefaciens (GV3101)

cells with plasmids pFastR06-SsPele1-eGFP, pXCSG-SsPele1-

mYFP, pXCSG-SsPele1-ΔC-mYFP, pCMABIA1306-ScPEPR1-

Flag, pCMABIA1306-ZmPEPR1-Flag, pXCSG-UmPele1-mYFP,

pCMABIA1306-ScPEPR1-N-Flag, pCMABIA1300S-ScPEPR1-

nYFP, pCMABIA2300S-SsPE14-cYFP, or pCMABIA2300SSsPE14-Δsp-cYFP were grown at 28°C/200 rpm in lysogeny broth

medium supplemented with kanamycin/spectinomycin (50 μg

ml−1

) and rifampicin (35 μg ml−1

). Agrobacterial cells were collected and re-suspended in MS salt buffer (MS-salt, plus 200 mM

acetosyringone) and injected into Nicotiana benthamiana leaves.

Confocal images of the fluorescent signal in the N. benthamiana

leaves were pictured on a laser confocal microscope Leica TCS SP8

(Leica, Wetzlar, Germany) after 48 h of agroinfiltration.

The N. benthamiana leaves were collected 48 h postagroinfiltration, ground in liquid nitrogen and lysed in extraction buffer EXB

(50 mM Tris pH7.5, 150 mM NaCl, 10% (v/v) glycerol, 2 mM

EDTA, 5 mM DTT, protease inhibitor (Roche), 0.1% Triton).

Lysates were centrifuged for 15 min at 20 000 g at 4°C. Aliquots of

supernatants were used as input samples. Immunoprecipitations

(IPs) were conducted by incubating supernatants with 15 μl GFPTrap beads (#gta-10; ChromoTek, Planegg-Martinsried, Germany) in 1.5 ml tubes for 2 h at 4°C. Beads then were collected by

centrifugation at 1000 g and washed four times with extraction

buffer. Beads then were heated in 2×Laemmli loading buffer, and

the proteins were separated by SDS-PAGE and analyzed by

immunoblotting. The antibodies used included anti-GFP

(#HT801; Transgen, Beijing, China), anti-HA (#11867423001;

Roche), anti-FLAG (#ab1162; Abcam, Cambridge, UK), anti-His

(#ab15149; Abcam) and anti-GST (#ab19256; Abcam).

Modified immunoprecipitation for detecting the

associations of peptides with ScPEPR1

After 48 h of transient expression in N. benthamiana leaves,

ScPEPR1-FLAG was extracted in extraction buffer EXB (50

mM Tris pH7.5, 150 mM NaCl, 10% glycerol, 2 mM ETDA,

0.1% Triton X-100, 0.5% DTT and 1% protein inhibitors

cocktail) and purified by incubating the supernatants with 15 μl

anti-FLAG agarose (#A4596; Sigma) in 1.5 ml tubes for 1 h at

4°C. After centrifugation at 1000 g, the agarose gels were

washed four times in extraction buffer and then incubated in

1 μg horseradish peroxidase (HRP)-conjugated anti-His

antibodies (#ab1187; Abcam) and 10 mM his-tagged peptides

(his-ScPep1, his-SsPel25, or both of his-ScPep1 and SsPel25)

for 1 h at 4°C. The agarose gels then were washed three times

in extraction buffer and transferred into a 96-well plate for

detecting HRP activity using chemiluminescence substrate

(#37069; ThermoFisher, Waltham, MA, USA).

Mitogen-activated protein kinase (MAPK) assays on

sugarcane, maize and Arabidopsis

For MAPK activation assays, 1 μM ScPep1 or ZmPep1 with or

without 5 μM SsPel25 was added into sugarcane (Wang et al.,

2020), maize (Cao et al., 2014) and Arabidopsis (Yoo et al., 2007)

protoplasts, respectively. Then 1 μM AtPep1 peptide plus 0.02%

silwet-77 was sprayed onto the leaves of 4-wk-old Arabidopsis

plants. Total protein samples were collected at the indicated time

points and used for immunoblotting with anti-p44/42 MAPK antibody (#4370; Cell Signaling, Danvers, MA, USA) to detect phosphorylation of MAPKs (Suarez-Rodriguez et al., 2007).

Transient expression and reporter assay in protoplasts

The mesophyll tissues of 4-wk-old Arabidopsis col-0 plants were

used for protoplast isolation and the transfection with DNA plasmids were performed according to Yoo et al. (2007). After that,

protoplasts were incubated at room temperature under weak light

for 16 h, and then used for protein immune-binding and

luciferase assays.

Protoplasts isolated from 4-wk-old Arabidopsis plants were cotransfected with proFRK1-LUC along with the indicated constructs

as described (Li et al., 2005). At 16 h after transfection, the protoplasts were treated with 1 μM peptides as indicated. The luciferase

(LUC) activity was determined at 2 h after the peptide treatments

using the luciferase reporter system (#E1500; Promega).

Generation of transgenic A. thaliana plants and powdery

mildew infection

The A. tumefaciens cells (GV3101) containing plasmid

pCAMBIA1306_ScPEPR1-FLAG or pCAMBIA1301_SsPE14

were used for floral-dipping to generate the transgenic Arabidopsis lines. For powdery mildew infection, the spores of Golovinomyces cichoracearum were blown onto the leaves of the ScPEPR1-

and SsPE14-overexpression lines. At 0, 3, and 5 dpi, leaves were

collected for RNA isolation. The leaves at 5 dpi were collected

for visualizing fungal structures using trypan blue staining (Frye

& Innes, 1998).

Statistical analysis

Statistical analysis of the qRT-PCR data from three biological

replicates was done by SPSS STATISTICS (v.22.0.0.0; IBM,

Armonk, NY, USA). Using two-tailed Student’s t-tests, SEs were

calculated using the variance and covariance values obtained from

the linear model fitting. The expression level was shown as the

mean  SD.

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Results

Gene co-expression network during sugarcane–S.

scitamineum interaction

We used a customized microarray targeting both the representative sugarcane genes and S. scitamineum genes to identify the

DEGs in the host and the fungus along with the progress of the

infections (0, 3, 5 and 7 dpi). The smut fungus grew faster and

was more abundant in the susceptible sugarcane cultivar YC71-

374 than in the resistant NCo376 (Fig. S1a), confirming the successful S. scitamineum infection. There was a uniform gene

expression distribution as revealed by a boxplot (Fig. S1b), and

all biological replicates had a strong correlation and good repeatable performance (R2 ≥ 0.90) (Fig. S1c). Subsequently, compared with the samples at 0 dpi, we identified 3110 sugarcane

DEGs in YC71-374 and 2383 DEGs in NCo376 under the smut

infection (Fig. 1a). Meanwhile 1491 and 1110 smut DEGs,

including 94 putative effector genes, were identified respectively,

after inoculation on YC71-374 and NCo376 (Fig. 1b). As shown

in Fig. S2, the expression tendency of 13 DEGs in qRT-PCR

analysis was consistent with the microarray results, indicating the

high reliability of the microarray data.

We then performed a WGCNA analysis on the microarray

data to construct gene co-expression network. It resulted in six

and eight co-expression modules in the susceptible YC71-374

(Fig. 1c) and the resistant NCo376 (Fig. 1d), respectively.

Notably, both the blue module in YC71-374 and the turquoise

module in NCo376 with the lowest P-value (P = 5 × 10–8 and P

= 9 × 10–6

, respectively) were highly and negatively correlated

with incubation period (r = −0.98 and r = −0.93 respectively)

and fungal biomass (r = −0.62, P = 0.03 and r = −0.75, P =

0.005, respectively) (Fig. 1c,d). We speculated that sugarcane

genes in these two modules were likely involved in defense

responses, and that their expression was suppressed by S. scitamineum. Then, 2959 sugarcane genes and 751 S. scitamineum

genes from these two modules were grouped together to calculate

the connectivity value between genes. The top sugarcane kinase

Fig. 1 Gene co-expression network in

sugarcane–Sporisorium scitamineum

interaction. (a) The total numbers of

differentially expressed sugarcane genes in

sugarcane susceptible genotype YC71-374

and resistant genotype NCo376 at 3, 5, 7 d

postinfection with S. scitamineum. DEG,

differentially expressed gene; nDEG, notdifferentially expressed gene. (b) The total

numbers of S. scitamineum DEGs at 3, 5, 7 d

postinfection in sugarcane YC71-374 and

NCo376. (c, d) Module (DEG matrix)-trait

associated analysis bases on the correlation

of the fungal biomass of S. scitamineum and

incubation period with the expression level of

the DEGs (from both sugarcane and smut) in

YC71-374 (c) and in NCo376 (d). The table

exhibits modules in rows and traits in

columns (fungal biomass and incubation

period). The corresponding correlation value

r and P-value (in closing bracket) between

row and column was shown in the color cell.

The cell color was correlated to r-value

indicated by the color legend on the right,

while the color legend on the left represented

the row name. ME, module eigengenes. (e)

Weighted gene co-expression network

between the top sugarcane kinase DEGs and

smut secreted protein genes. The four

biggest dots in the network represent

sugarcane mitogen-activated protein kinase

kinase kinase A (MAPK, BMK.45382),

mitogen-activated protein kinase

(BMK.69601), receptor-like serine/

threonine-protein kinase (BMK.73537) and

plant elicitor peptide receptor1 (PEPR1)

gene. The red line indicates the positive

correlation between genes, whereas the grey

line indicates the negative correlation. The

size of node reflects the number of

connections.

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genes and smut effector genes with the highest connectivity value

subsequently were selected to construct the gene co-expression

network, where the genes with a WGCNA edge weight > 0.15

were connected with lines (Fig. 1e). The four biggest dots represented sugarcane genes: mitogen-activated protein kinase kinase

kinase A (BMK.45382), mitogen-activated protein kinase

(BMK.69601), receptor-like serine/threonine-protein kinase

(BMK.73537) and plant elicitor peptide receptor1 (ScPEPR1,

BMK.75743). As PEPR1-signaling is an important component

in plant immune system (Tang & Zhou, 2016), we focused on

the ScPEPR1 gene for further analysis here (studies on other three

genes will be reported elsewhere). The expression of ScPEPR1

was decreased in either NCo376 or YC71-374 after S. scitamineum infection, which accorded with the results of our

microarray data (Fig. S2).

ScPEPR1 is a phylogenetical ortholog to Arabidopsis PEPR1

The protein sequence analysis showed that ScPEPR1 contains Nterminal extracellular LRR domain (a LRRNT_2, a LRR1 and a

LRR8), a transmembrane domain and a C-terminal cytoplasmic

kinase domain, sharing the closest relationship to PEPR1s from

Sorghum bicolor and Zea mays (Fig. S3a). The alignment of protein sequence showed that PEPR1 and its ortholog protein share

68.78% identity in amino acid sequence (Fig. S3b). And we confirmed that as a PRR receptor, YFP fused ScPEPR1 had a membrane localization in the leaf cells when transiently expressed in

N. benthamiana (Fig. S3c).

We expressed ScPEPR1 under the control of the constitutive

35S promoter (35S-ScPEPR1) in Arabidopsis. Compared to the

expression of endogenous AtPEPR1 in the control transgenic

line (transformed with empty vector), ScPEPR1 were highly

expressed in the 35S-ScPEPR1 lines (Fig. S4a). We found that

the 35S-ScPEPR1 lines showed fewer fungi and significantly

fewer conidiophores on the leaves infected with powdery

mildew G. cichoracearum at 5 dpi than the control line (Fig.

S4b,c). The fungal-induced expression of the defense-related

gene AtWRKY33 (Gravino et al., 2017) (Fig. S4d) and the SAinduced gene AtPR5 (Sun et al., 2018) (Fig. S4e) were higher

in 35S-ScPEPR1 lines than in the control line at 3 and 5 dpi.

We concluded that overexpression of the sugarcane ScPEPR1

gene in Arabidopsis enhances plant resistance to powdery

mildew. Thus, ScPEPR1 gene is a structural, phylogenetic

orthologous to Arabidopsis PEPR1 and is functional in plant

immunity.

ScPEPR1 interacts with S. scitamineum effector SsPE14

We then tested the possible interactions between ScPEPR1 and

the S. scitamineum putative effectors (SsPEs) in the WGCNA coexpression network (Fig. 1e; Table S4). In the Y2H assay,

ScPEPR1 specifically interacted with SsPE14 (smut.10005024)

(Fig. 2a). In the following protein truncation tests, we found that

the N-terminal extracellular LRR domain (ScPEPR1-N) but not

the C-terminal cytoplasmic kinase domain (ScPEPR1-C) of

ScPEPR1 interacted with SsPE14 (Fig. 2b).

In order to further examine the protein interactions in vivo,

co-immunoprecipitation (co-IP) assays were performed after

transient expression of YFP-tagged SsPE14 and FLAG-tagged

ScPEPR1 or ScPEPR1-N in N. benthamiana. As shown in Fig.

2(c,d), ScPEPR1-FLAG or ScPEPR1-N-FLAG was co-purified

with SsPE14-YFP, rather than with the YFP control. Furthermore, in an in vitro pull-down assay, the direct interaction

between SsPE14-GST and ScPEPR1-N-His also was observed

(Fig. 2e). Together, these results demonstrated that SsPE14 interacts with ScPEPR1 by its N-terminal LRR domain.

As SsPE14 interacts with extracellular LRR domain of

ScPEPR1, we intended to visualize the subcellular location of the

protein complexes formed by the two proteins using bimolecular

fluorescence complementation (BiFC) assay. For this, N-terminal

YFP (nYFP) was fused to ScPEPR1 by its N-terminus (nYFPScPEPR1) and C-terminal YFP (cYFP) was fused to SsPE14 by

its C-terminus (SsPE14-cYFP). Yellow fluorescence was observed

on the cytoplasmic membrane of the leaf cells upon transient coexpression of nYFP-ScPEPR1 and SsPE14-cYFP in N. benthamiana, suggesting that ectopically expressed SsPE14 could be localized to the apoplastic space, where it interacts with extracellular

LRR domain of ScPEPR1 (Fig. 2f).

SsPE14 is a plant elicitor peptide-like effector

The expression of SsPE14 is strongly induced during smut infection (Fig. 3a), suggesting that it might play an important role in

promoting virulence of S. scitamineum. In the NCBI database,

two orthologs of SsPE14 in Sporisorium species, four in Ustilago

species, one in Pseudozyma species and two in Moesziomyces

species were found through BLASTP tool. Phylogenetic tree analysis showed that SsPE14 was closely related to two orthologs from

Sporisorium (Fig. 3b). SsPE14 is likely a secreted protein without

a conserved domain in the NCBI database (https://www.ncbi.

nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Que et al., 2014a). It also

is predicted to be an effector protein (Ratio 0.739) by EFFECTORP

(http://effectorp.csiro.au/). Thus, SsPE14 might be a conserved

effector protein in smut fungi species.

SsPE14 has a predicted signal peptide on its N-terminus. To

functionally validate it, we used a genetic assay based on the

requirement of invertase secretion for yeast cells to grow on

media containing raffinose as sole carbon source. The predicted

signal peptide sequence of SsPE14 was fused in-frame to the yeast

invertase gene in the vector pSUC2T7M13ori (Xu et al., 2019).

The invertase mutant yeast strain YTK121 transformed with

pSUC2T7M13ori-SsPE14 construct grew on the YPRAA

medium (sucrose was replaced by raffinose, the YTK121 can

grow only when invertase is secreted) (Fig. 3c). The invertase

secretion was further confirmed using an enzymatic activity assay

based on invertase-mediated conversion of the colorless dye TTC

into the insoluble red colored triphenylformazan (Fig. 3c). These

results demonstrated that the signal peptide of SsPE14 is functional and SsPE14 is a secreted protein.

Next, a BiFC experiment was performed to test interaction

between nYFP-ScPEPR1 and SsPE14-Δsp-cYFP whose secretion

signal peptide has been deleted. We found that SsPE14-ΔspNew Phytologist (2021)

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cYFP did not interact with nYFP-ScPEPR1 (Fig. S5). This result

indicates that SsPE14 must be secreted into apoplastic space for

interaction with ScPEPR1 LRR domain.

The C-terminal Pep1 like domain is required for SsPele1 to

interact with ScPEPR1

The interaction between the SsPE14 and the extracellular LRR

domain of ScPEPR1 (Fig. 2d–f) is reminiscent of the perception of

Arabidopsis AtPep1 by AtPEPR1 (Yamaguchi et al., 2006; Tang et

al., 2015). We wondered whether there was a sequence similarity

between SsPE14 and plant elicitor peptides. Hence, the conserved

amino acid residues from the plant elicitor peptides and fungal

orthologs of SsPE14 were aligned (Figs 3d, S6). We found that the

C-terminal 26 amino acids of SsPE14 (149–174 aa) and its

orthologs had several conserved sites compared with the plant elicitor peptides (Peps) (Fig. 3d). Hereafter, the SsPE14 was renamed

as SsPele1 (S. scitamineum plant elicitor peptide-like effector 1).

Fig. 2 Sugarcane PLANT ELICITOR PEPTIDE RECEPTOR1 (ScPEPR1) interacts with the smut effector SsPE14. (a) ScPEPR1 interacts with SsPE14 in yeast.

The activation domain (AD) and DNA binding domain (BD) plasmids containing the indicated genes were co-transformed into yeast strain Y-2-Hgold and

screened on synthetic dextrose dropout (SD) media lacking Leu and Trp (SD/-Leu-Trp). The single colonies were serially diluted and spotted onto SD/-LeuTrp and SD/-Leu-Trp-His-Ade to observe the yeast cell growth. Yeast co-transformed with AD-largeT + BD-p53 or AD-largeT + BD-laminC served as a

positive control and negative control, respectively. EV, empty vector. SsPE1/4/14/15, Sporisorium scitamineum putative effector1/4/14/15. (b) The

interaction between ScPEPR1 and SsPE14 in Nicotiana benthamiana, revealed by bimolecular fluorescence complementation assay. The N. benthamiana

leaves were co-infiltrated with 35S:nYFP-ScPEPR1 and 35S:SsPE14-cYFP. Images were captured by a confocal microscope 2 d after Agrobacterium

tumefaciens transformation. Bar, 10 μm. (c) Co-immunoprecipitation (co-IP) analysis of interactions between ScPEPR1-FLAG and SsPE14-YFP in N.

benthamiana leaves. Proteins in total extracts (Input) and after IP with GFP-trap beads (IP (YFP)) were detected on immunoblots using α-FLAG or α-GFP

antibodies. GFP/YFP, green/yellow fluorescent protein. (d) SsPE14 interacts with N-terminal LRR domain of ScPEPR1 (ScPEPR1-N), but not with the Cterminal of ScPEPR1 (ScPEPR1-C) in yeast. The experiment was performed according to the same procedure in (a). (e) SsPE14 interacts with the ScPEPR1-

N, revealed using glutathione S-transferase (GST) pull-down assays. The recombinant ScPEPR1-N-his and SsPE14-GST proteins purified from Escherichia

coli BL21 strain were subjected to a GST pull-down analysis. Proteins in input and pull-down were detected on immunoblots using α-his or α-GST

antibodies. (f) Co-IP analysis of interactions between SsPE14-YFP and ScPEPR1-N-FLAG in N. benthamiana leaves. Proteins in total extracts (Input) and

after IP with GFP-trap beads (IP (YFP)) were detected on immunoblots using α-FLAG or α-GFP antibodies. These experiments were repeated at least three

times with similar results.

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We then tested whether SsPele1 interacts with ScPEPR1

through its C-terminal Pep1 like domain using Y-2-H and Co-IP

assays. We found that deletion of Pep1 like motif (SsPele1-ΔC)

in SsPele1 abolished its association with ScPEPR1 in Y-2-H

experiments (Fig. 3e). Consistently, SsPele1-ΔC-YFP did not

interact with ScPEPR1-FLAG in Co-IP experiments (Fig. 3f).

These data demonstrate that Pep1 like domain of SsPele1 is

required for its association with ScPEPR1.

ScPep1-, but not SsPele1-, perception by ScPEPR1 induces

immune responses

Three orthologs of AtPep1 (NP_569001.1) in sugarcane were

identified by protein blast against our RNA-seq data

(SRP192749) and the published genomic data (S. spontaneum,

GCA_003544955.1). The sugarcane Peps, namely ScPep-#1,

ScPep-#2 (ScPep1) and ScPep-#3 (Table S3), were aligned with

the plant elicitor peptides (AtPep1 from Arabidopsis, ZmPep2

from maize and SvPep from Setaria viridis, Table S3) to assess

their similarity. Clearly, the sugarcane Peps contain the conserved

Fig. 3 The characterization of the smut fungal effector SsPE14. (a)

Quantitative real-time PCR analysis of expression of Sporisorium

scitamineum effector SsPE14 in sugarcane genotypes YC71-374 and

NCo376 at indicated time points. The bars indicate relative fold-change 

SD (n = 3) compared to 0 dpi. *indicates a significant difference between

the 0 dpi, and 3, 5 or 7 dpi (P < 0.05) determined by Student’s t-test. dpi, d

postinoculation. (b) Phylogenetic analysis of SsPE14 and its orthologs from

Sporisorium graminicola, Sporisorium reilianum, Ustilago maydis,

Ustilago trichophora, Ustilago bromivora, Ustilago hordei, Pseudozyma

hubeiensis, Moesziomyces antarcticus, Moesziomyces aphidis and

Kalmanozyma brasiliensis; Numbers at the branches indicate bootstrap

values. The accession number of the sequences used in the present study

can be found in Supporting Information Table S3. (c) Experimental

validation of the signal peptide of SsPE14 using the yeast invertase

secretion assay. Yeast YTK121 strains carrying the SsPele1 fused in frame

to the invertase gene in the pSUC2 vector can grow in the CMD-W media

(with sucrose) and YPRAA media (with raffinose instead of sucrose,

growth only when invertase is secreted). The secreted invertase also can

reduce triphenyltetrazolium chloride (TTC) to red formazan. The negative

control was YTK121 strains carrying the pSUC2 vector. (d) The alignment

of the conserved amino acid sequences of plant elicitor peptides

(Arabidopsis AtPep1, maize ZmPep2, green foxtail SvPep and sugarcane

ScPep-#1/#2/#3) and the fungal homologs of SsPele1 (from S. reilianum,

S. graminicola, U. maydis, U. hordei, U. bromivora, P. hubeiensis, and K.

brasiliensis) obtained from the NCBI database (Table S3). The highly

conserved sites are colored and marked with red triangle symbol on the

top row. SsPele1 and UmPele1 (Um01690) marked with purple triangle

symbol. The accession number of the sequences used in the present study

can be found in Table S3. (e) ScPEPR1 interacts with SsPele1 and SsPele1-

ΔC in yeast. SsPele1-ΔC is the fragment of SsPele1 with deletion of 68

residues in its C-terminus. The activation domain (AD) and binding domain

(BD) plasmids containing the indicated genes were co-transformed into

yeast strain Y-2-Hgold and screened on synthetic dextrose dropout media

lacking Leu and Trp (SD/-Leu-Trp). The single colonies were serially

diluted and spotted onto SD/-Leu-Trp and SD/-Leu-Trp-His-Ade to

observe the yeast cell growth. Yeast co-transformed with AD-largeT + BDp53 or AD-largeT + BD-laminC served as a positive control and negative

control, respectively. EV, empty vector. Three independent experiments

gave consistent results. Pele1, plant Pep1-like effector 1. (f) Coimmunoprecipitation (co-IP) analysis of interactions between ScPEPR1-

FLAG and SsPele1-ΔC-YFP in Nicotiana benthamiana leaves. Proteins in

total extracts (Input) and after IP with GFP-trap beads (IP (YFP)) were

detected on immunoblots using α-FLAG or α-GFP antibodies. These

experiments were repeated at least three times with similar results. GFP/

YFP, green/yellow fluorescent protein.

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amino acids in their C-terminus compared with known plant

Peps (Figs 3d, S6).

In order to test activity of the putative sugarcane Pep1 candidates, we synthesized three peptides of ScPeps, ScPep-#1, ScPep-

#2 and ScPep-#3, and monitored their ability for induction of

proFRK1-LUC in an Arabidopsis protoplast, which is a transient

reporter system widely used in studying PTI signaling (Asai et al.,

2002). FRK1 encodes a receptor-like kinase that is rapidly

induced by PAMPs (Asai et al., 2002). When co-expressing with

ScPEPR1, ScPeps, especially ScPep-#2, strongly induced the

expression of proFRK1-LUC (Fig. 4a). Thus, we named it as

ScPep1. ScPep1 could not induce proFRK1-LUC expression in

the absence of ScPEPR1 (Fig. 4b), indicating that ScPep1 is

specifically perceived by ScPERP1, but not by AtPEPR1 in Arabidopsis protoplasts. Strikingly, the 25-amino-acid-peptide from

SsPele1 C-terminal Pep1 like domain (SsPel25) (Fig. 3d) did not

induce proFRK1-LUC reporter (Fig. 4a), despite its sequence similarity to plant Peps.

SsPel25 inhibits ScPep1-induced immune responses

The interaction of SsPele1 with ScPEPR1 (Fig. 2) without activating the ScPEPR1-mediated FRK1 expression (Fig. 4a) promoted us to test whether it suppresses ScPEPR1-signaling. We

found that the co-application of SsPel25 peptide significantly

suppressed the ScPep1-induced expression of the proFRK1-LUC

Fig. 4 The smut effector SsPele1 suppresses sugarcane peptide1 (ScPep1)-induced immune responses. (a) Sugarcane ScPep1 candidates induce proFRK1-

LUC expression in Arabidopsis protoplasts. The Arabidopsis ecotype Col-0 protoplasts were transfected with proFRK1-LUC along with ScPEPR1. Sixteen

hours later, the protoplasts were treated with 0.2 μM ScPep1-#1, #2, #3, or the 25-amino-acid peptide from SsPele1 (SsPel25), then the LUC reporter

activity was determined 2 h later (LUC, luciferase). Error bars indicate the SD. Different letters indicate statistical significance (P ≤ 0.01) determined by oneway ANOVA followed by Tukey’s honestly significant difference (HSD) tests. (b) ScPep1-induced proFRK1-LUC expression is dependent on sugarcane

PLANT ELICITOR PEPTIDE RECEPTOR1 (ScPEPR1). Col-0 protoplasts were transfected with proFRK1-LUC along with ScPEPR1or the empty vector (EV).

Sixteen hours later, the protoplasts were treated with 0.2 μM ScPep1 for another 2 h, and the LUC reporter activity was determined. * indicate statistical

significance (P ≤ 0.01) to the mock determined by one-way ANOVA followed by Tukey’s HSD tests. (c) The SsPel25 peptide inhibits ScPep1-induced

proFRK1-LUC expression. Col-0 protoplasts were transfected with proFRK1-LUC along with ScPEPR1. The assays were done as in (a). (d, e) SsPel25

suppresses ScPep1-induced phosphorylation of MAPKs (pMPKs). Col-0 (d) and sugarcane (e) protoplasts expressing ScPEPR1 were treated with 1 μM

ScPep1 or ScPep1 plus 2 μM SsPel25, and total protein extracts were prepared at the indicated time points. The phosphorylation of MAPKs was detected

on an immunoblot probed with anti-p44/42 MAPK antibody. (f, g) His tagged ScPep1 and SsPel25 peptides associate with ScPEPR1-FLAG (f), and SsPel25

competes the interaction between His-ScPep1 and ScPEPR1-FLAG (g). The transient expression of ScPEPR1-FLAG samples extracted and purified from

Nicotiana benthamiana leaves with anti-FLAG agarose, and then then incubated in 1 μg horseradish peroxidase (HRP)-conjugated anti-His antibodies

(Abcam; #ab1187) and 10 mM his-tagged peptides or no tagged peptides (his-ScPep1, his-SsPel25, or both of his-ScPe1 and SsPel25). The agarose gels

then were washed three times in extraction buffer and transferred into a 96-well plate for detecting HRP activity using chemiluminescence substrate

(ThermoFisher; #37069). The dot intensity indicates the interactions between ScPEPR1-FLAG and His-tagged peptides. These experiments were repeated

at least three times with similar results.

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reporter in the Arabidopsis protoplasts expressing ScPEPR1 (Fig.

c). In the same protoplasts, we examined the ScPep1-induced

phosphorylation of MAPKs, an early event in PTI responses,

with or without SsPel25. As shown in Fig. 4(d), the ScPep1-

induced transient phosphorylation of MAPKs was reduced in the

presence of SsPel25. Furthermore, we examined whether SsPel25

inhibits ScPep1-induced phosphorylation of MAPKs on sugarcane. Consistently, we found co-application of SsPel25 significantly suppressed ScPep1-induced phosphorylation of MAPK in

sugarcane protoplasts (Fig. 4e). Moreover, application of SsPel25

promoted S. scitamineum propagation on sugarcane sheath tissue

(Fig. S7). Together, these results indicated that SsPel25 inhibits

ScPep1-induced immune responses.

SsPel25 competes with ScPep1 to bind to ScPEPR1

We then sought to examine whether SsPel25 competes with

ScPep1 for perception by ScPEPR1. First, the FLAG-tagged protein ScPEPR1-FLAG or an empty vector as control was transiently expressed in N. benthamiana and total protein extracts

were incubated with anti-FLAG agarose. After washing, the antiFLAG agarose binding ScPEPR1-FLAG were incubated with

mock, the 6× his-tagged peptides His-ScPep1 or His-SsPel25,

respectively, plus anti-His-HRP antibody for detecting the presence of his-tagged peptides in the immune-complexes in 96-well

plates (details in the Materials and Methods section). Both HisScPep1 and His-SsPel25 were detected in the ScPEPR1-FLAG

immune-complexes (Fig. 4f), indicating that both peptides

bound with ScPEPR1. To check for competition between

ScPep1 and SsPel25 for binding to ScPEPR1, the ScPEPR1-

FLAG-binded anti-FLAG agarose was incubated with HisScPep1 in the absence or presence of un-tagged SsPel25. As

shown in Fig. 4(g), the presence of SsPel25 greatly reduced the

signal of His-ScPep1 in the immune-complexes, suggesting that

SsPel25 indeed competed the association between ScPep1 and

ScPEPR1.

Overexpression of SsPele1 in Arabidopsis suppresses

AtPep1-induced immunity

We generated transgenic Arabidopsis lines expressing SsPele1

driven by 35S promoter (35S-SsPele1). As AtPep1 is perceived by

AtPEPR1 in Arabidopsis, we questioned whether SsPele1 interacts with and interferes with AtPEPR1 activation. Co-IP experiments in N. benthamiana showed that SsPele1 also interacted

with AtPEPR1 in planta (Fig. 5a). We then evaluated the plant

immune responses to AtPep1 in the transgenic lines. First,

AtPep1-induced activation of MAPKs was examined. The leaves

of 4-wk-old plants were treated with 1 μM AtPep1, and the total

protein was collected at the indicated time points (Fig. 5b). The

immunoblots with an anti-pERK antibody showed that the

AtPep1-induced transient phosphorylation of MAPKs was

reduced in the 35S-SsPele1 lines than in the control line (Fig. 5b).

We next examined the AtPep1-induced production of ROS,

Fig. 5 Overexpression of smut effector gene SsPele1 in Arabidopsis

suppresses peptide1 (AtPep1)-induced immunity. (a) Coimmunoprecipitation (co-IP) analysis of the interactions between SsPele1-

YFP and AtPEPR1-FLAG in Nicotiana benthamiana). Proteins in total

extracts (Input) and after IP with GFP-trap beads (IP (YFP)) were detected

on immunoblots using anti-FLAG or anti-GFP antibodies (GFP/YFP, green/

yellow fluorescent protein). (b) AtPep1-induced phosphorylation of

mitogen-activated protein kinases (MAPKs) on ScPele1 transgenic line1/2

and empty-vector (EV) transgenic line. Total protein extracts were

prepared from leaves of 4-wk-old transgenic Arabidopsis lines expressing

SsPele1 or an EV at the indicated time points after 1 μM AtPep1

treatment. The phosphorylation of MAPKs was detected on an

immunoblot probed with anti-p44/42 MAPK antibodies. Ponceau staining

of the blot shows equal sample loading. (c) AtPep1-induced H2O2

production was reduced in the SsPele1 transgenic lines. The results shown

are representative of three independent experiments. Each data point

consists of six to eight replicates. Values are means  SD. Different letters

indicate statistical significance (P < 0.01) determined by one-way ANOVA

followed by Tukey’s honestly significant difference. These experiments

were repeated three times with consistent results.

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another typical PTI response, in the 35S-SsPele1 lines. As shown

in Fig. 5(c), the AtPep1-induced ROS were compromised in the

35S-SsPele1 lines compared with the control line. Together, these

results indicated that overexpression of SsPele1 in Arabidopsis

suppresses AtPep1-induced immune responses.

Ustilago maydis effector UmPele1 interacts with ZmPEPR1

and suppresses ZmPEPR1-mediated immunity

Ustilago maydis has an SsPele1 ortholog effector gene, Um01690

(Schilling et al., 2014) (Fig. 3d). The deletion of Um01690

impaired fungal tumor induction on maize seedling leaves

(Schilling et al., 2014), indicating Um01690 is an important

effector for fungal pathogenicity. Um01690 protein sequence

shows high similarity to SsPele1 with conserved secretion signal

peptide and C-terminal Pep1 like domain (Fig. 6a). Thus, we

renamed Um01690 to UmPele1. We then tested whether

UmPele1 interacts with ZmPEPR1 using Co-IP assay in N. benthamiana. As shown in Fig. 6(b), ZmPEPR1-FLAG was copurified with YFP-UmPele1 but not with YFP control, showing

that UmPele1 interacts with ZmPEPR1 in planta.

We then assessed whether UmPele1 suppresses ZmPEPR1-

mediated immunity. ZmPep1 was synthesized and used to activate immunity in maize protoplasts. We also synthesized the 25

amino acid peptide of the UmPele1 C-terminal Pep1 like

domain (highlighted by red triangle in Fig. 6(a), named as

UmPel25). In maize protoplasts, co-application of UmPel25 partially suppressed ZmPep1-induced phosphorylation of

ZmMAPK (Fig. 6c). We conclude that like the S. scitamineum

effector SsPele1, U. maydis effector UmPele1 promotes fungal

virulence at least partly by interacting with ZmPEPR1 and

inhibiting activation of ZmPEPR1.

Discussion

Here we show that the smut fungal effector SsPele1 and its

ortholog UmPele1 contain a plant elicitor peptide-like motif in

its C-terminus (Fig. 3e), by which SsPele1 interacts with the

extracellular leucine-rich repeat (LRR) domain of sugarcane

PLANT ELICITOR PEPTIDE RECEPTOR1 (ScPEPR1), and

completes ScPep1 perception, resulting in the inhibition of

ScPEPR1-mediated defense responses (Fig. 7). This reveals a

novel mechanism whereby a pathogenic fungal effector simulates

a nonfunctional host-endogenous signal peptide to suppress plant

defense responses. Our work also contributes a first mechanistically study on a sugarcane smut fungal effector.

Plant PEPRs are LRR kinases and receptors for endogenous

peptides (Peps) (Yamaguchi et al., 2006, 2010; Ross et al., 2014;

Tang & Zhou, 2016; Xu et al., 2018). The PEPR immune signaling is engaged in PTI and is required for systemic acquired

resistance in Arabidopsis and tomato (Huffaker et al., 2006;

Yamaguchi et al., 2010; Ross et al., 2014; Yamada et al., 2016;

Xu et al., 2018). In tomato, a PEPR1 ortholog is required for

systemin-mediated resistance to the necrotrophic fungus Botrytis

cinerea (Xu et al., 2018). In maize (Zea Mays), ZmPep1 induces

the expression of defense-related genes, the accumulation of plant

defense related hormones, and resistance to pathogens (Huffaker

et al., 2011). We found that the overexpression of ScPEPR1 in

Arabidopsis enhanced resistance to the biotrophic fungal

pathogen and promoted the expression of the defense-related

genes AtWRKY33 and AtPR5 (Fig. S3), indicating that ScPEPR1

is a positive regulator in plant immunity. The targeting of

ScPEPR1 by the effector SsPele1 indicates its importance in

Fig. 6 Smut effector SsPele1’s ortholog, UmPele1, interacts with maize

PLANT ELICITOR PEPTIDE RECEPTOR1 (ZmPEPR1) and suppresses

immune responses. (a) The alignment of Ustilago maydis ortholog

(um01690, Supporting Information Table S3) and SsPele1. The N-terminal

amino acids representing the signal peptide and the C-terminal conserved

residues were marked in blue triangle symbol and red triangle symbol,

respectively. The accession number of the sequences used in the present

study can be found in Table S3. (b) Co-immunoprecipitation (co-IP)

analysis of interactions between (Zea mays) ZmPEPR1-FLAG and

UmPele1-YFP in Nicotiana benthamiana leaves. Proteins in total extracts

(Input) and after IP with GFP-trap beads (IP (YFP)) were detected on

immunoblots using α-FLAG or α-GFP antibodies (GFP/YFP, green/yellow

fluorescent protein). (c) UmPel25 suppresses ZmPep1 induced

phosphorylation of mitogen-activated protein kinases (MAPKs). Maize

protoplasts expressing ZmPEPR1 were treated with 1 μM ZmPep1 or 1 μM

ZmPep1 plus 2 μM UmPel25, and total protein extracts were prepared at

the indicated time points. The phosphorylation of MAPKs was detected on

an immunoblot probed with anti-p44/42 MAPK antibody. These

experiments were repeated at least three times with similar results.

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resistance to smut fungi. Further genetic studies, such as the overexpression or knock-down of ScPEPR1 in sugarcane, would illustrate the biological importance of ScPEPR1 in resistance to smut

disease.

Ustilago maydis effector UmPele1, an ortholog of SsPele1, has

been shown to be required for U. maydis infection in maize

(Schilling et al., 2014). Here, we found that SsPele1 directly

interacted with ScPEPR1 (Figs 2, 3), and UmPele1 interacted

with ZmPEPR1 (Fig. 6). With a functional secretion signal,

SsPele1 is a secreted protein and interacts with the extracellular

LRR domain of ScPEPR1 to interfere with ScPep1-mediated

defense in apoplasts (Figs 3, S5). Moreover, SsPel25 could promote growth of Sporisorium scitamineum on sugarcane sheath

(Fig. S7). Thus, as a conserved smut fungal effector, SsPele1 contributes to the virulence of S. scitamineum.

Interestingly, the C-terminus of SsPele1 and UmPele1 show a

certain sequence similarity to several known plant Peps (Fig. 3d).

As the extracellular domains of cell surface receptors function as

interaction platforms and regulatory modules of receptor activation (Jaillais et al., 2011; Belkhadir et al., 2014), we investigated

the biological relevance between ScPEPR1LRR and SsPele1 using

a well-established reporter system in Arabidopsis protoplasts (Yoo

et al., 2007). We found that ScPep1-, but not SsPele1-, was perceived by ScPEPR1 to induce immune responses (Figs 3e,f, 4a,

b). Besides, SsPel25 competes with ScPep1 to bind to ScPEPR1

and inhibits ScPep1-induced immune responses (Fig. 4). Furthermore, transgenic Arabidopsis overexpressing SsPele1 exhibited reduced AtPep1-induced early immune responses (Fig. 5).

Taken together, these results allow us to present a working model

for SsPele1 as shown in Fig. 7: During S. scitamineum infection,

SsPele1 is induced and delivered to sugarcane apoplasts, where it

competes with endogenous ScPep1 to interact with ScPEPR1,

inhibiting ScPEPR1-mediated immune responses.

It is interesting that both ScPep1 and SsPele1 interact with

ScPEPR1; however, ScPep1 triggers the activation of ScPEPR1

signaling, whereas SsPele1 does not. The underlying mechanisms

remain elusive. In Arabidopsis, AtPep1 perception by AtPEPR1

leads to the stable association of AtPEPR1 with the co-receptor

AtBAK1 (Bri1-associated kinase 1), eliciting immune responses.

Biochemical assays showed that AtPep1 induces the heterodimerization of the extracellular domains of PEPR1LRR-BAK1LRR

(Tang et al., 2015). In addition to AtBAK1, another small LRRreceptor kinase, AtAPEX interacts with AtPEPR1/2LRR in a

ligand-independent manner and is required for appropriate

Pep2-induced responses (Smakowska-Luzan et al., 2018).

Whether SsPele1 affects the heterodimerization of ScPEPR1-

ScBAK1 or ScPEPR1-ScAPEX is worth testing in future studies.

The extracellular space (apoplast) of plant tissue is an important battleground between plants and pathogens. Pathogenic

microbes secrete apoplastic (extracellular) as well as cytoplasmic

(intracellular) effectors to alter host-cell structure and function,

thereby enhancing plant susceptibility (Wawra et al., 2012).

Apoplastic effectors mostly have been reported to function as

inhibitors of proteases, chitinases, or glucanases to prevent the

release of fungal elicitors (Lanver et al., 2017). LysM effectors are

widely used by pathogenic fungi to bind to soluble chitin

oligomers that could be recognized by plant immune receptors,

to prevent the enzymatic hydrolyzation of host chitinase (Mentlak et al., 2012; Zeng et al., 2020). The maize smut fungus U.

maydis secretes the effector Pit2, which inhibits a set of apoplastic

papain-like cysteine proteases by its conserved 14-aa motif and

prevents the release of endogenous SA-associated plant defense

(Mueller et al., 2013; Misas Villamil et al., 2019). The work

described here show that the S. scitamineum apoplastic effector

SsPele1, and its orthologs UmPele1 from U. maydis, prevent the

activation of the host plant DAMP receptor ScPEPR1/

ZmPEPR1 by mimicking its ligand. Besides UmPele1, the

orthologs of SsPele1 were found in related smut species (Fig. 3b).

It is reasonable to speculate that such a virulent strategy used by

SsPele1 might be common for smut pathogens. The intracellular

kinase domain of PRRs has been shown to be targeted and suppressed by pathogen effectors (Heese et al., 2007; Shan et al.,

2008; Zhou et al., 2014). Here, our findings illustrate that the

extracellular ligand-binding domain of ScPEPR1 is targeted by

SsPele1, uncovering a novel virulence mechanism for fungal

apoplastic effectors.

Acknowledgements

We thank Katayoon Dehesh from Botany and Plant Sciences at

the University of California, Riverside for sharing plasmid

Fig. 7 A model for the suppression of the PLANT ELICITOR PEPTIDE

RECEPTOR1 (PEPR1)-signaling by the smut effector SsPele1. The plant

peptide1 (Pep1) perception induces heterodimerization and

transphosphorylation of receptor kinases PEPR1 and the co-receptor BAK1

(Bri1-associated kinase 1). Then the activated PEPR1–BAK1 complexes

induce the activation of mitogen-activated protein kinase (MAPK)

cascades through a series of phosphorylation events, resulting in the

activation of PAMP-triggered-immunity (PTI). During the infection, the

smut fungus Sporisorium scitamineum delivers effector SsPele1 to host

apoplast, where the SsPele1 competitively binds to the extracellular

domain of PEPR1 to suppress PEPR1-mediated immune responses.

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pFAST-R06, Lei Su for sharing pCAMBIA serial vectors, and

Dingzhong Tang, Jie Zhang and Li Xue for critical reading and

suggestions. We thank Yachun Su for help us on sugarcane treatment, Meixin Yan for sharing the smut wild-type haploid Ss17

and Ss18. We also thank SHbio and Zoonbio Biotechnology Co.

Ltd for technical support. This work has been jointly supported

by the following grants: National Key R&D Program of China

(2019YFD1000500), National Natural Science Foundation of

China (31671752, 31901592 and 31770277), and Natural

Science Foundation of Fujian Province for Distinguished Young

Scholars (2015J06006), Natural Science Foundation of Fujian

Province (2018J01609), the Sugar Crop Research System of

China (CARS-17) and the Funding of Yulin Normal University

(GZ2020ZK03). All authors had no conflict of interest to

declare.

Author contributions

HC and YQ designed and supervised the research; HL and NH

carried out the pathogen treatment, analysis of microarray data,

gene cloning, Y2H, sequence alignment, qRT-PCR, BiFC, Arabidopisis transformation, and vector construction; XF performed

the protein blot, ROS, Luciferase and CoIP assays; ZZ performed the yeast secretion trial; WS performed picture capture

on the laser scanning confocal microscope; and HL, HC, NH,

WL and YQ wrote the manuscript. HL, XF and NH contributed

equally to this work.

ORCID

Haitao Cui https://orcid.org/0000-0002-6343-1014

Hui Ling https://orcid.org/0000-0003-0873-7878

Youxiong Que https://orcid.org/0000-0003-1111-5834

Data availability

All the generated and analyzed data from this study are included

in the published article and its Supporting Information.

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Supporting Information

Additional Supporting Information may be found online in the

Supporting Information section at the end of the article.

Fig. S1 Fungal biomass accumulation and microarray hybridization in the sugarcane-S. scitamineum interaction.

Fig. S2 The expression of 14 selected differentially expressed

genes revealed by microarray hybridization and qRT-PCR.

Fig. S3 The phylogenetic analysis of PEPR1 and the expression

of ScPEPR1 in sugarcane.

Fig. S4 Overexpression of ScPEPR1 in Arabidopsis enhances

plant resistance to powdery mildew.

Fig. S5 SsPE14-Δsp lacking signal peptide does not interact with

ScPEPR1 in bimolecular fluorescence complementation assay.

Fig. S6 The alignment of the amino acid sequences of plant elicitor peptides and the fungal homologs of SsPele1.

Fig. S7 SsPel25 promotes the propagation of smut fungus on the

sugarcane sheath tissue.

Table S1 The primers and constructs used in the present study.

Table S2 Screening genes as the detection targets of sugarcaneS. scitamineum customization microarray.

Table S3 The accession number of genes and proteins used in

the present study.

Table S4 Twenty-night candidate secreted effector protein genes

coexpressed with ScPEPR1 gene.

Please note: Wiley Blackwell are not responsible for the content

or functionality of any Supporting Information supplied by the

authors. Any queries (other than missing material) should be

directed to the New Phytologist Central Office.

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30

油莎豆油体蛋白基因的全基因组鉴定与功能解析

邹 智*

，郑玉皎，肖艳华，张中甜，赵永国

海南省南繁生物安全与分子育种重点实验室/中国热带农业科学院热带生物技术研究所/

三亚研究院，海南海口 571101

Abstract

Oleosins (OLEs) are abundant structural proteins of lipid droplets (LDs) that function in LD formation and stabilization

in seeds of oil crops. However, little information is available on their roles in vegetative tissues. In this study, we present

the first genome-wide characterization of the oleosin family in tigernut (Cyperus esculentus L., Cyperaceae), a rare

example accumulating high amounts of oil in underground tubers. Six members identified represent three previously

defined clades (i.e. U, SL and SH) or six out of seven proposed orthogroups (i.e. U, SL1, SL2, and SH1–3). Comparative

genomics analysis reveals that lineage-specific expansion of Clades SL and SH was contributed by whole-genome duplication and dispersed duplication, respectively. Moreover, presence of SL2 and SH3 in Juncus effuses implies their appearance sometime before Cyperaceae-Juncaceae divergence, whereas SH2 appears to be Cyperaceae-specific. Expression analysis showed that CeOLE genes exhibit a tuber-predominant expression pattern and transcript levels are considerably more abundant than homologs in the close relative Cyperus rotundus. Moreover, CeOLE mRNA and protein

abundances were shown to positively correlate with oil accumulation during tuber development. Additionally, two dominant isoforms (i.e. CeOLE2 and -5) were shown to locate in LDs as well as the endoplasmic reticulum of Nicotiana

benthamiana leaves, and are more likely to function in homo and heteromultimers. Furthermore, overexpression of

CeOLE2 and -5 in N. benthamiana leaves could significantly increase the oil content, supporting their roles in oil accumulation. These findings provide insights into lineage-specific family evolution and putative roles of CeOLE genes in

oil accumulation of vegetative tissues, which facilitate further genetic improvement for tigernut.

Keywords: oil crop, oil accumulation, expression divergence, orthogroup, protein interaction, synteny analysis

31

热区植原体遗传多样性与其病害传播流行关系研究

于少帅 1*, 车海彦 2

, 宋薇薇 1

1. 中国热带农业科学院椰子研究所, 海南文昌 571339; 2. 中国热带农业科学院环境与

植物保护研究所, 海南海口 571101

摘 要: 植原体于 1967 年首次报道，基于植原体保守基因及国际植原体分类鉴定标准，目前植原体已鉴定 37 个 16Sr

组和 48 个植原体候选种，引起全球 1000 多种植原体病害，其中我国已报道植原体病害约 200 种。热带地区生物多样

性丰富，海南岛是我国典型的热带岛屿省份，植原体病害约占我国植原体病害的四分之一。目前在海南岛已鉴定、报

道引起植物病害的植原体有 4 个组，分别为 16SrI、16SrII、16SrV、16SrXXXII，其中 16SrI、16SrII 所占比例较高。本

团队前期在海南岛已鉴定植原体病害 20 余种，其中 10 余种植原体病害为首次鉴定报道。经同源比对、变异位点分析

等表明不同植物寄主中的植原休同源性极高，有的株系间甚至高达 100%。植原体不能分离培养，目前研究表明其传播

主要通过寄主植物与媒介昆虫。植原体不同寄主间植原体高同源性，可能导致该类病原在不同寄主间植原体存在互传

现象。国内外已有多项研究表明，植原体可能不同寄主植物中相互传播，导致不同寄主植物植原体病害的发生流行。

本团队前期在槟榔黄化病发病园内及周围，已鉴定苦楝等 7 种寄主植物的植原体与海南槟榔黄化植原体保守基因序列

同源性为 100%，结合槟榔病园发病历史、病情发展等调查，推测植原体可能在其高同源性植物寄主间相互传播，因此，

在相关经济作物植原体等难培养微生物病害的防控过程中，中间寄主是一个不可忽视的问题。植原体寄主的多样性，

导致其传播流行的复杂性与检测防控的困难性。国外一些致死性棕榈作物病害，如椰子、油棕等，国内虽未见发病报

道，但经基因数据比对分析发现，高同源性（甚至 100%）的植原体株系国内已存在，传播风险极大，因此，定期开展

岛内关键作物植原体等病原物“项目”的定期“体检”，明确植原体等在自然条件下的寄主多样性及特征，对于其相关

病害的精准监测与有效防控具有重要意义。

关键词: 植原体; 系统分类; 遗传多样性; 中间寄主; 病害传播流行

基金项目 海南省自然科学基金高层次人才项目（No. 320RC743）；中国热带农业科学院中央级公益性科研院所基本科研业务

费专项（No. 1630152021005，1630152022004）。

作者简介 *通信作者（Corresponding author）：于少帅（YU Shaoshuai），E-mail：hzuyss@163.com。

32

热区棕榈作物植原体病害检测技术研发与应用

于少帅 1*, 覃伟权 1

, 潘英文 2

, 宋薇薇 1

, 车海彦 3

, 朱 辉 1

1. 中国热带农业科学院椰子研究所, 海南文昌 571339; 2. 海口海关热带植物隔离检疫中心, 海南海口 570311;

3. 中国热带农业科学院环境与植物保护研究所, 海南海口 571101

摘 要: 植原体是一类尚难分离培养的原核致病菌，由植原体引起的植物病害被称为植物“癌症”。槟榔、椰子是海南

“六棵树”之一，重要的热带经济作物，国内外研究报道表明由植原体引起的槟榔、椰子病害是致死性的，给海南乃

至全球热区相关产业造成沉重打击。在国家“一带一路”、海南自由贸易港、全球动植物种质资源引进中转基地等建设

的背景下，植原体等外来有害生物入侵与国内有害生物扩散的风险极大增加，一旦入侵，会像槟榔黄化病一样，给海

南椰子产业带来灭顶之灾。快速高效的检测诊断技术对于预防相关病害的发生流行、降低经济损失等具有重要意义。

因此，针对槟榔、椰子等棕榈作物植原体病害研究难点及其产业卡脖子问题，研发其病原快速、高效检测技术体系。

针对我国槟榔黄化植原体序列特征，建立了其 LAMP 快速可视化检测技术，该技术操作简便、检测时间短（40 min 内

可完成），检测结果肉眼可视，适用于田间诊断与推广应用；全球率先将微滴式数字 PCR 技术应用于槟榔黄化病研究

中，建立了槟榔黄化植原体高灵敏检测技术，灵敏度数量级达 10-2 copies/μL，与已报道的检测技术相比，灵敏度提高

了约 1000 倍；建立了全球椰子致死性植原体通用型 LAMP 快速可视化检测技术，该技术一个反应体系可同时检测全球

已知的 6 类椰子致死性植原体，且检测反应仅需 40 min，极大提高了这一全球重大检疫性病害的检测效率。相关技术

已在棕榈作物体内植原体分布规律、海南不同地区槟榔黄化病定期检测监测等方面进行广泛应用，在棕榈作物种苗和

新种植区的检测监测及口岸检疫等方面具有重要的应用前景，对有效降低外来有害生物入侵风险、防止国内相关病害

的扩散流行、精准监测预警相关病害等至关重要。

关键词: 植原体; 棕榈作物; 检测鉴定; 监测预警; 口岸检疫

基金项目 海南省自然科学基金高层次人才项目（No. 320RC743）；中国热带农业科学院中央级公益性科研院所基本科研业务

费专项（No. 1630152021005，1630152022004）。

作者简介 *通信作者（Corresponding author）：于少帅（YU Shaoshuai），E-mail：hzuyss@163.com。

33

孕穗期喷施外源硒对水稻产量和糙米硒含量的影响

李许明，李福燕

广西民族师范学院，广西崇左 532200

摘 要：为了探索桂西南地区氨基酸硒肥对水稻产量及糙米中硒含量的影响，为形成区域安全、低成本的富硒水稻种

植技术，以桂西南六种常见水稻为供试材料，在水稻孕穗期喷施氨基酸有机硒肥，探究喷施硒肥对不同水稻株高、穗

长、产量及糙米硒含量的影响。结果表明：叶面喷施硒肥对水稻株高、穗长、千粒重和产量无显著差异，但不同水稻

品种间存在显著差异，其中表现较好的是中浙优 15 号、昌两优馥香占、耕香优荔丝苗 3 个品种。叶面喷施硒肥对水稻

产量无显著差异，但不同水稻品种间存在显著差异，其中表现较好的是昌两优馥香占、中浙优 15 号、野香优明月丝苗

3 个品种。在硒含量方面，叶面喷施硒肥对水稻糙米硒含量具有显著影响且不同品种对硒的富集能力存在显著差异，其

中野香优莉丝、中浙优 15 号以及野香优明月丝苗 3 个品种富硒能力较强且均符合广西富硒水稻标准。综合分析，6 个

参试水稻品种以昌两优馥香占和中浙优 15 号在株高、穗长、结实率和产量上的表现较佳且富硒能力较强，可为当地种

植优质富硒水稻提供参考。

关键词：水稻; 氨基酸硒肥; 产量; 硒含量; 糙米

34

广藿香组织培养工厂化育苗技术研究

倪燕妹 1,2，陈叶海 1,2*，林杰泽 1,2，黄梅花 3

广东农垦热带农业研究院有限公司，广东广州 511365；2. 广东省农垦南亚热带作物科技中心，

广东广州 510507；3. 广东农垦热带作物科学研究所，广东茂名 525100

摘 要：从广藿香组织培养工厂化育苗的生产流程入手，系统地介绍了其组织培养生产和快速栽培技术，总结出组织培

养工厂化育苗的技术要点。试验结果表明，最优诱导愈伤组织培养基：MS +2，4-D 0.2 mg/L 十 6-BA 1.0mg/L 十 NAA

0.5mg/L；最优诱导从生芽培养基：MS 十 6-BA 1.0mg/L；最优壮苗培养基：MS 十 6-BA 0.1mg/L；最优生根培养基：

1/2 MS。以上培养基每升均加入蔗糖 30g、卡拉胶 6.5g，pH 5.8。培养温度为（25±2）℃，光照度 1500lx，每日光照

10h。最适移植基质为泥炭土与珍珠岩（5∶1）的混合基质。移栽后遮荫保湿，成活率≥95%，按常规组培苗做好水肥

管理和病虫害防治，50d 后苗高约 20cm，叶片在 4 对以上，即可出圃进行大田种植。

关键词：广藿香；组织培养；工厂化育苗；快速栽培

Mass Propagation of Pogostemon Cablin( Blanco)

Benth via Tissue Culture

Ni Yanmei 1,2, Chen Yehai 1,2 *, Lin Jieze 1,2, Huang Meihua 3

1. Guangdong Agribusiness Tropical Agriculture Institute, Guangzhou,Guangdong 511365, China;

2. Guangdong Agribusiness South Subtropical Crops Science and technology Center,

Guangzhou, Guangdong 510507, China;

3. Guangdong Agricultural Reclamation Tropical Crop Science Research Institute,

Maoming, Guangdong 525100, China

Abstract: This study presents a comprehensive exploration of tissue culture production and rapid cultivation techniques

for achieving mass propagation of P. cablin. The paper systematically introduces the production process, highlighting

key technical considerations for successful mass propagation via tissue culture. Experimental findings reveal optimal

conditions for various stages of the propagation process. The induced callus medium demonstrated superior performance

when formulated with MS supplemented with 2,4-D (0.2 mg·L-1), 6-BA (1.0 mg·L-1), and NAA (0.5 mg·L-1).

Similarly, the optimal medium for induction from budding consisted of MS supplemented with 6-BA (1.0 mg·L-1).

Robust seedling growth was achieved using MS supplemented with 6-BA (0.1 mg·L-1). Successful rooting was observed with a medium composition of 1/2 MS, incorporating 30g of sucrose, 6.5g of carrageenan, and adjusted to a pH

of 5.8 per liter. Incubation conditions involved maintaining a temperature of (25±2) ℃ and a light intensity of 1500 lx

for 10 hours. The optimal graft substrate was identified as a peat soil and perlite mixture in a 5:1 ratio.

Post-transplantation, shade and moisture were provided, resulting in an impressive survival rate of 95%. Water and fertilizer management, along with pest control, adhered to conventional tissue culture seedling practices. After 50 days, the

seedlings attained a height of approximately 20cm, accompanied by the emergence of more than 4 pairs of leaves, signifying their readiness for field planting. This study provides valuable insights for the large-scale propagation of P. cablin through tissue culture, enabling enhanced cultivation practices and potential applications in the industry.

Keywords: Pogostemon cablin( Blanco) Benth; Tissue culture; mass propagation; Rapid cultivation

基金项目 广东省农业农村厅 2023 年乡村振兴战略专项“大宗与道地南药广藿香的生态种植关键技术示范及产业化应用”

作者简介 倪燕妹（1984—），女，硕士研究生，科研管理部副部长，高级农艺师，研究方向：科研项目管理、农业技术推广

及植物组织培养研究。*通信作者（Corresponding author）：陈叶海（CHEN Yehai），E-mail：chenyehai@126.com

35

旋切式电动割胶刀在茂名植胶区试验初报

李富存 1

，谢黎黎 1

，潘媛 1

，马启铭 1

，杨江波 2

1. 广东农垦热带作物科学研究所，广东化州 525100；2.广东农垦热带农业研究院有限公司，广东广州 510000

摘 要：茂名植胶区由于胶价低迷，胶工短缺，面临橡胶大面积弃割风险。如何提高现有胶工的潜力，快速培养一批

合格胶工，会影响到天然橡胶发展速度、质量和效益。传统推式割胶方式，是一种精细且技术性很强的手工操作，茂

名植胶区大部分都是使用这种方式进行采胶，采用此方法存在胶工劳动强度大、培训时间长、橡胶树死皮率高、割胶

速度难以再进一步提高等缺点。机械化、智能化割胶工具与技术研究在国内外都备受重视，良具良法的升级换代更是

离不开新的核心资源引进或者创制，本研究通过利用茂名植胶区比较有代表性的品种‘热研 73397’为试验材料，进行

4CJX 型旋切式电动割胶刀和传统推式割胶刀对比分析试验，结果发现：耗皮量明显更低（p<0.01），割胶深度明显更

浅（p<0.05），割胶速度明显更快（p<0.01），劳动强度明显更低（p<0.01），同时培训 7 天就可以进行试验割胶，对缩

短胶工培训时间、降低培训成本有积极作用。割胶方式多样化发展，有利于割胶技术的进一步提升，旋切式电动割胶

刀通过机械结构控制耗皮量、控制割胶深度，能避免胶工超深割胶，不易伤树，对保护橡胶树有积极作用。橡胶树保

持健康，是保持高产、稳产的保障。旋切式电动割胶刀和传统推式割胶刀使用差异比较大，有必要通过加强胶工针对

性培训，来解决减产问题。同时，机械还有不少改进空间，来进行更有针对性的设计。相信随着机械的进一步发展，

技术的进一步成熟，旋切式电动割胶刀会成为解决茂名植胶区割胶难题的重要工具。

关键词：橡胶树；茂名植胶区；旋切式；电动割胶

The rotary cutting electric rubber tapping knife was first

reported in the rubber planting area of Maoming

LI Fucun1

, XIE Lili1

, PAN Yuan1

,MA Qiming1

,YANG Jiangbo2

1. Guangdong Agricultural Reclamation Institute of Tropical Crops Science, Huazhou, Guangdong 525100, China;

2. Guangdong Ag-ricultural Reclamation Academy of Tropical Agriculture Co., Ltd., Guangzhou,

Guangdong 510000, China

Abstract: Due to the low price of rubber and the shortage of glue workers, the rubber planting area in Maoming is facing the risk of large-scale abandonment of rubber. How to improve the potential of existing glue workers and quickly

train a batch of qualified glue workers will affect the development speed, quality and efficiency of natural rubber. The

traditional push rubber tapping method is a fine and highly technical manual operation, most of the Maoming rubber

planting area is used in this way for rubber picking, the use of this method has the shortcomings of high labor intensity,

long training time, high dead skin rate of rubber trees, and it is difficult to further improve the rubber tapping speed.

Mechanized, intelligent rubber tapping tools and technology research are highly valued at home and abroad, the upgrading of good methods is inseparable from the introduction or creation of new core resources, this study by using the

more representative variety of Maoming rubber planting area 'Hot Research 73397' as the test material, 4CJX type rotary

cutting electric rubber tapping knife and traditional push rubber tapping knife comparative analysis test, the results

found: skin consumption is significantly lower (p<0.01), rubber tapping depth is significantly shallower (p<0.05), rubber tapping speed is significantly faster (p<0.01), labor intensity is significantly lower (p<0.01), test tapping can be

carried out after 7 days of training at the same time, which has a positive effect on shortening the training time of glue

workers and reducing training costs. The diversified development of rubber tapping methods is conducive to the further

improvement of rubber tapping technology, and the rotary cutting electric rubber tapping knife controls the skin consumption and the depth of rubber tapping through the mechanical structure, which can avoid the glue worker's ultra-deep

rubber tapping, not easy to damage the tree, and has a positive effect on the protection of rubber trees. Rubber trees

36

maintain health and are the guarantee for maintaining high and stable yields. The use of rotary cutting electric rubber

tapping knives and traditional push rubber tapping knives is quite different, and it is necessary to strengthen the targeted

training of glue workers to solve the problem of reducing production. At the same time, there is still a lot of room for

improvement in the machinery to carry out more targeted design. It is believed that with the further development of

machinery and the further maturity of technology, rotary cutting electric rubber tapping knife will become an important

tool to solve the problem of rubber tapping in Maoming rubber planting area.

Keywords: Rubber tree; Maoming rubber planting area; Rotary cut; Electric tapping

作者简介 李富存（1994—），男，大学本科，研究方向：农业新技术试验、示范及推广，E-mail：171823762@qq.com。

37

乙烯利对五个菠萝品种成花及品质的影响

普跃 1,2，林文秋 1*

，刘朝阳 2

，刘胜辉 1

，吴青松 1

，孙伟生 1

，

1.中国热带农业科学院南亚热带作物研究所，广东湛江 524088；2.华南农业大学园艺学院，广东广州 510642

摘 要 为了筛选出不同菠萝品种适宜的乙烯利催花浓度，本研究利用 25-1000mg/L 乙烯利对‘Josapine’、‘台农 4 号’、

‘MD-2’、‘台农 21 号’、‘台农 22 号’进行灌心处理，探究不同乙烯利浓度对各菠萝品种成花率、现红期、果实内外

品质及畸形率的影响。结果表明，除‘台农 22 号’外，各菠萝品种随着乙烯利浓度的增加成花率显著提升。其中，

‘Josapine’、‘台农 4 号’诱导成花的最佳浓度为 400 mg/L，‘MD-2’、‘台农 21 号’诱导成花的最佳浓度为 800 mg/L；

当处理浓度大于 400mg/L 时，‘Josapine’、‘台农 4 号’、‘台农 21 号’现红时间进一步缩短，‘MD-2’现红时间则逐渐

延长；‘Josapine’、‘MD-2’、‘台农 22 号’纵横径、单果重等形态指标在处理浓度超 200mg/L 时呈下降趋势；随着乙

烯利浓度的增大，各品种可滴定酸含量呈下降的趋势，而其可溶性固形物含量变化趋势则与之相反；此外，五个菠萝

品种在乙烯利诱导下均有畸形果产生，其中‘Josapine’对高浓度乙烯利较为敏感，畸形率最高达到了 65.5%，而‘MD-2’

畸形率仅为 6.7%。综合各项指标，‘Josapine’、‘台农 4 号’最适乙烯利催花浓度为 400mg/L；‘MD-2’、‘台农 21 号’

最适乙烯利催花浓度为 800mg/L；而单一乙烯利不能诱导‘台农 22 号’成花。

关键词：菠萝；成花；现红期；品质；畸形率

38

生物有机配方肥连续替代化肥对剑麻生长、土壤质量的影响

经福林 1

，冯学娟 1

，毛丽君 2

，郭继阳 2

，吴刃 1

，

刘志强 1

，张曼其 1

*

1 广东省湛江农垦科学研究所 广东湛江 524000；2 农业农村部剑麻及制品质量

监督检验测试中心 广东湛江 524000

摘 要 本文通过在剑麻地连续两年施放生物有机配方肥替代传统化肥施放，设置有三个处理：常规施肥（对照 CK），

施放 1/2 常规施肥+剑麻生物有机配方肥 125 kg/hm2

（T1）和施放剑麻生物有机配方肥 250 kg/hm2

（T2），研究剑麻连

续生物有机配方肥对剑麻地土壤养分、微生物量碳氮、酶活以及剑麻叶片生长状况与收益的影响。结果表明：连续施

放生物有机配方肥（T2）促进了剑麻植株对钙的吸收利用，麻片全钙含量高传统化肥施放 0.67 mg/kg，麻片纤维含量

高传统化肥施放 0.35mg/kg；同时，剑麻施放生物有机配方肥（T2）的麻地相对常规施肥（CK），有效提高了剑麻地土

壤微生物量碳氮含量、蔗糖酶活性、脲酶活性、过氧化氢酶活性及土壤质量，减少了化学肥料投入成本，避免化肥过

量施放，通过成本核算，两年内 T2 比 CK 增加收入 2400.5 yuan/hm2

，T1 比 CK 增加收入 663 yuan/hm2

，为促进剑麻产

业的健康持续发展提供了一种有效方法。

关键词 剑麻；生物有机配方肥；生长；土壤；收益

Effect on Sisal Growth and Soil Quality by Bio-organic Formulated Fertilizer Consecutively Replaced Chemical Fertilizer

JING Fulin1

, FENG Xuejuan1

, MAO Lijun2

, GUO Jiyang2

, WU Ren1

,

LIU Zhiqiang1

, ZHANG Manqi1,2*

1.Guangdong Zhanjiang State Farms Research Institute, Zhanjiang, Guangdong 524000, China）;

2.Quality and Safety Inspection Testing Center for Sisal Products, MARA, Zhanjiang, Guangdong 524000, China

Abstract: In this paper, biological organic formula fertilizer was applied in sisal field for two consecutive years instead

of traditional chemical fertilizer. Three treatments were set up: Conventional fertilization (CK), application of 1/2 conventional fertilization + sisal bio-organic formula fertilizer 125 kg/hm2 (T1) and sisal bio-organic formula fertilizer 250

kg/hm2 (T2) were used to study the effects of continuous bio-organic formula fertilizer on soil nutrients, microbial

biomass carbon and nitrogen, enzyme activity, leaf growth and yield of sisal. The results showed that continuous application of bioorganic formula fertilizer (T2) promoted the absorption and utilization of calcium in sisal plants, the total

calcium content of sisal leaves was 0.67 mg / kg higher than that of traditional chemical fertilizer, and the fiber content

of sisal leaves was 0.35 mg / kg higher than that of traditional chemical fertilizer. At the same time, compared with

conventional fertilization (CK), sisal applied bioorganic formula fertilizer (T2), effectively improved soil microbial

biomass carbon and nitrogen content, sucrase activity, urease activity, catalase activity and soil quality, reduced the input cost of chemical fertilizer, and avoided excessive application of fertilizer. In two years, T2 increased its income by

2400.5 yuan/hm2 compared with CK, and T1 increased its income by 663 yuan/hm2 compared with CK, providing an

effective way to promote the healthy and sustainable development of sisal industry.

Key words: Sisal; Bio-organic Formula Fertilizer;Grow; Soil; Earnings

剑麻（学名：Agave sisalana Perr. ex Engelm.）又名菠萝麻，龙舌兰科龙舌兰属，是一种多年生热

带硬质叶纤维作物，原产于墨西哥，中国 1963 年从东非引进优良剑麻品种，经改良后命名“东 1 号”，

资助项目 中央财政资金“耕地建设与利用资金项目”(2130122)。

作者简介 经福林(1984—)，男，高级农艺师；研究方向：土壤与肥料研究。通讯作者(Corresponding author)：张曼其

(ZHANG Manqi)，E-mail：1004596264@qq.com。

39

现已成为国内剑麻行业的当家品种[1]。剑麻喜高温多湿和雨量均匀的高坡环境，尤其日间高温、干燥、

充分日照，夜间多雾露的气候最为理想。适宜生长的气温为 27～30℃，上限温 40℃，下限温 16℃，

昼夜温差不宜超过 7～10℃，适宜的年雨量为 1200～1800mm。其适应性较强，耐瘠、耐旱、怕涝，但

生长力强，适应范围很广，宜种植于疏松、排水良好、地下水位低而肥沃的砂质壤土，排水不良、经

常潮湿的地方则不宜种植。耐寒力较低，易发生生理性叶斑病。近来因受政治动乱、自然灾害、剑麻

病虫害、劳力缺乏、经济萧条等因素影响，加上化学肥料价格上涨及剑麻种植户对土壤耕地保护意识

薄弱，导致剑麻种植生产过程中肥料成本越来越高，收益越来越低，为此，黄标[2]等为解决传统施有

机肥及化肥工效差，成本高问题，结合剑麻田间肥效试验，研制了成功配制了有机与无机肥相结合兼

加有益功能菌的配方颗粒肥，并进行推广应用。但是连续施放剑麻生物有机配方肥替代传统化肥施放

对剑麻叶片纤维含量、麻地土壤质量效果如何，尚未见报道，因此，本文通过连续施放剑麻生物有机

配方肥替代传统化肥施肥模式，分析麻地土壤养分、微生物量碳氮、酶活及剑麻叶片生长、纤维含量

等变化，为施生物有机配方肥提供理论，也为促进剑麻产业的健康持续发展提供了一种有效方法。

1 材料和方法

1.1 材料

供试剑麻品种：H·11648 麻；供试肥料：剑麻生物有机配方肥[含量标准：氮磷钾≥20%（7-4-9），

有机质≥25%，活的有益微生物≥0.2 亿个/g，水分≤20%，PH 值 6.5，主要有羊粪配制，颗粒状]，参

考黄标[2]等生物有机配方肥。试验有机配方肥是由广东省丰收糖业发展有限公司复肥厂加工生产。

1.2 方法

1.2.1 试验地点

试验地设在广东农垦东方红农场有限公司 3 队 59 号地二刀麻，面积 1.79 hm2

。依据全国第二次土

壤普查养分分级标准 [3]，土壤有机质含量 27.90 g/kg，处于中水平；全氮含量 1.35 g/kg，处于中水平；

碱解氮含量 121.41 mg/kg，处于中上水平；速效磷含量 18.78 mg/kg，处于中上水平；速效钾含量 150.08

mg/kg，处于的中上水平；交换性钙 439.54 mg/kg，处于中下水平，交换性镁 31.68 mg/kg，处于缺乏；

土壤类型为砖红壤，旱坡地，地势平整，土壤 PH 值 5.25，处于强酸性。

1.2.2 试验方法及田间设置

采用随机区组设计，3 次重复，施用肥料配方设计如表 1：CK 为对照组，常规施肥；T1 为 1/2 常

规施肥+125 公斤剑麻有机配方肥；T2 为施用剑麻有机配方肥 250 公斤。各小区面积约 0.16 hm2。试验

剑麻 2021 年割第三刀麻，2022 年割第四刀麻。

表 1 生物有机配方肥配方设计

Tab1 Formulation design of bio-organic formula fertilizer

施肥配

方

fertilizer

formula

尿素

Urea

磷肥

Phosphate fertilizer

氯化钾

potassium chloride

有机肥料

organic fertilizer

生物有机配方肥

bio-organic formula fertilizer

肥料成本

fertilizer cost

（kg/hm2） （kg/hm2

） （kg/hm2

） （kg/hm2

） （kg/hm2

） （yuan/hm2

）

CK 450 1500 450 7500 — 9900

T1 225 750 225 3750 1875 8981.25

T2 — — — — 3750 8062.5

注：肥料价格：尿素 3000 元/t，磷肥 1000 元/t，氯化钾 4000 元/t，有机肥料 700 元/t，有机配方肥 2150 元/t。

Note : Fertilizer price : urea 3000 yuan / t, phosphate fertilizer 1000 yuan / t, potassium chloride 4000 yuan / t, organic fertilizer 700 yuan / t,

organic formula fertilizer 2150 yuan / t.

1.2.3 试验经过及田间管理

对照 CK：采用本地区常规田管施肥方法，剑麻采割叶片后，一次性机械培土施下。T1、T2：按

40

试验设计配好肥，参照常规施肥，一次性机械培土施下。田间防虫除草管理相同。

1.2.4 数据分析

剑麻地土壤样品采集参照鲍士旦《土壤农化分析》第三版[4]，在剑麻收割叶片前 1 个月内，各处

理分别采取五个点 0-20cm 表层土壤混合均匀四分法后，装入自封袋低温冷藏运回实验室，并取一半土

样用自封袋密封储存于低温冰箱（-20℃）用于测定土壤微生物量碳、氮，其余土壤样品风干、研磨，

分别过筛（2mm）后用于土壤酶和土壤理化性质的测定。在施肥前，每个小区随机挑选 10 株剑麻调查

增叶数，并在收割叶片前 1 个月内，每株采集 1 片最长叶片用于调查剑麻叶片生长和测定叶片纤维率。

剑麻叶片产量按实际收获统计。

土壤样品测定方法：土壤有机质测定采用高温外热重络酸钾容量法，全氮测定采用凯氏蒸馏法法，

碱解氮测定采用减解扩散中和滴定法，有效磷采用盐酸-1/2 硫酸浸提钼锑抗比色法[5]，速效钾测定采用

乙酸交换 1∶10 原子吸收分光光度法，交换性钙和交换性镁测定采用原子吸收分光光度计法，pH 测定

采用水（去 CO2）浸提 1∶2.5 点位法；土壤微生物量碳、氮采用氯仿熏蒸-K2SO4 浸提法[6]；土壤酶活

性指标包括蔗糖酶（采用 3,5-二硝基水杨酸比色法测定）、脲酶（采用苯酚钠-次氯酸钠比色法测定）、

磷酸酶（采用的是对硝基苯磷酸二钠比色法）、过氧化氢酶活性（采用高锰酸钾滴定法测定），测定方

法参照关松荫[7]的《土壤酶及其研究法》。剑麻叶片样品全氮、磷、钾、钙、镁测定方法：全氮测定采

用 H2 SO4-H2O2 消煮凯氏定氮法[8]；全磷测定采用 H2 SO4-H2O2 消煮，钒钼黄比色法[9]；全钾测定采用

H2SO4-H2O2 消煮，火焰光度计法测定[10]；全钙和全镁测定采用干灰化盐酸溶解，原子吸收分光光度

法[11]。

采用 Microsoft Excel2010 软件完成试验数据统计分析，并用统计软件 SAS 9.0 duncan 新复极差法

对调查结果平均值进行方差分析 Fisher’s-test (LSD)。

2 结果与分析

2.1 生物有机配方肥连续替代化肥对剑麻地土壤养分的影响

连续两年开展有机配方肥替代化肥试验，由表 2 结果可发现：剑麻地土壤大、中元素存在一定差

异，且主要表现在土壤元素有效态方面存在差异，而有机质和全氮三个处理在两年中变化不大，有机

质含量主要在 29.04～30.14 g/kg，土壤全氮含量主要在 1.39～1.42 g/kg。土壤元素有效态方面差异主要

表现在：2021 年三处理土壤碱解氮含量没有显著差异，2022 年 T1 和 T2 显著高于 CK，T1 和 T2 之间

没有明显差异，但 T1 碱解氮含量最高，为 148.59 mg/kg；土壤中有效磷含量，2021 年 CK 和 T1 显著

高于 T2，CK 和 T1 之间没有显著差异，但最高是 CK，为 109.35 mg/kg，而 2022 年，CK 和 T1 还是

显著高于 T2，CK 和 T1 之间没有显著差异，但最高是 T1，为 94.05 mg/kg；土壤中速效钾含量，2021

年 CK 显著高于 T1 和 T2，T1 和 T2 之间没有显著差异，而最高的 CK，是 190.04 mg/kg，而 2022 年

和 2021 年差异性相似，也是 CK 和 T1 显著高于 T2，CK 和 T1 之间没有显著差异，但最高是 T1，为

259.24mg/kg；土壤中交换性钙含量，2021 年 CK 显著高于 T2，与 T1 之间没有显著差异，而 T1 和 T2

之间也没有显著差异，最高是 CK，为 676.10 mg/kg，而 2022 年，最高是 T2，显著高于 CK 和 T1，而

CK 又显著高于 T1，T2、CK、T1、分别为 492.12 mg/kg，428.70 mg/kg、308.84 mg/kg；土壤中交换性

镁含量，2021 年 CK 最高，显著高于 T1 和 T2，T2 显著高于 T1，而 2022 年，最高是 T2，显著高于

CK 和 T1，而 CK 又显著高于 T1，T2、CK、T1、分别为 34.14 mg/kg，27.34 mg/kg、17.39 mg/kg。土

壤 PH 值：2021 年三个处理存在差异，T2 显著高于 T1 和 CK，而 CK 和 T1 没有明显差异，2022 年土

壤 PH 值不存在明显差异，但是 2021 年和 2022 年，T2 处理土壤 PH 值最高，分别为 6.15 和 5.62。因

此，施放化肥（CK）能提高土壤有效态元素含量，而施放有机配方肥可以一定程度提高土壤 PH。

41

表 2 生物有机配方肥替代化肥对土壤养分和 PH 的影响生物

Tab2 Effect on soil nutrients and Ph by bio-organic formulated fertilizer consecutively replaced chemical fertilizer

年份

year

处理

Treatment

有机质

The organic

matter

(g/kg)

(g/kg)

全氮 Total

nitrogen

(g/kg)

碱解氮

Alkaline

hydrolysis

nitrogen

(mg/kg)

有效磷 The

effective

phosphorus

(mg/kg)

速效钾

Rapidly

available potassium

(mg/kg)

交换性钙

Exchangeable

calcium

（mg/kg）

交换性镁

Exchangeable

magnesium

（mg/kg）

PH

2021 CK 29.86±1.12a 1.39±0.04a 149.04±2.38a 109.35±3.52a 190.04±10.22a 676.10±28.12a 71.84±10.01a 5.96±0.05b

T1 30.14±1.57a 1.41±0.05a 150.09±3.59a 106.10±4.66a 164.29±8.92b 644.41±39.23ab 37.75±5.69c 5.95±0.07b

T2 29.52±1.02a 1.40±0.04a 154.90±4.59a 90.10±5.79b 155.56±11.02b 613.06±26.30b 50.63±9.98b 6.15±0.06a

2022 CK 29.88±1.10a 1.39±0.03a 125.14±3.96b 90.74±6.88a 259.24±14.09a 428.70±28.23b 27.34±5.55b 5.54±0.04a

T1 29.04±1.23a 1.39±0.02a 148.59±4.97a 94.05±4.92a 232.92±13.07a 308.84±26.98c 17.39±6.68c 5.57±0.05a

T2 29.23±1.03a 1.42±0.04a 146.63±5.61a 59.53±7.77b 163.44±8.55b 492.12±34.15a 34.14±7.89a 5.62±0.05a

注：同列数字后的不同字母表示处理间差异显著性(p＜0.05， LSD)。下同。

Note Different letters after numbers in the same column indicate significant difference between treatments (P < 0.05,

LSD).

2.2 生物有机配方肥连续替代化肥对剑麻地土壤微生物氮碳的影响

土壤微生物生物量是土壤中的活性营养库，调控着土壤养分的转化循环及有效性。土壤微生物生

物量碳、氮含量与环境、施肥及作物生长有密切关系。大量研究表明不同有机肥和化肥配合施用对土

壤微生物生物量氮影响不同。微生物对施入氮素的固持与释放主要受施入的碳（如有机肥、秸秆等）

和氮的种类和数量等因素支配。施入肥料的碳、氮比越高，土壤微生物对肥料氮的固持量越大，释放

率降低[12]。由表 3 可知，2021 年，土壤微生物量碳含量和氮含量都是 T2 最高，分别为 33.87 mg/kg 和

16.81mg/kg，都显著高于 CK，其中土壤微生物量碳含量和氮含量，T1 和 CK 没有明显差异，但 T1 大

于 CK。2022 年 T2 土壤微生物量碳含量显著高于 T1，T1 显著高于 CK，分别为 384.80 mg/kg 、189.40

mg/kg 、21.40 mg/kg，但是土壤微生物量氮含量 T2 显著高于 CK 和 T1，CK 和 T1 之间没有显著差异。

从碳氮比方面来看，T2>T1>CK，2021 年分别为 2.01、1.57 和 1.52,2022 年 6.06、6.55 和 0.69。因此，

有机配方肥能提高土壤微生物生物量碳、氮含量。

表 3 生物有机配方肥替代化肥对剑麻地土壤微生物碳氮的影响

Tab3 Effect on Soil microbial nitrogen and carbon in sisal field by bio-organic formulated fertilizer consecutively replaced

chemical fertilizer

年份

Year

处理

Treatment

土壤微生物量碳

Soil microbial biomass C mg/kg

土壤微生物量氮

Soil microbial biomass N mg/kg

C/N

2021 CK 23.62±1.22b 15.57±0.64b 1.52

T1 25.75±1.57b 16.45±0.53ab 1.57

T2 33.87±1.85a 16.81±0.71a 2.01

2022 CK 21.40±1.02c 30.90± 1.23b 0.69

T1 189.40± 1.23b 28.90 ±0.98b 6.55

T2 384.80± 1.38a 63.50±2.11a 6.06

2.2 生物有机配方肥连续替代化肥对剑麻地土壤酶活的影响

土壤酶活性是表征土壤质量和土壤肥力的重要指标[13-14]，在土壤生态系统的物质循环和能量流动

方面具有主要的作用[15]，也是衡量土壤质量变化的预警和敏感指标[16]。它能表征土壤的综合肥力特征

及土壤养分转化进程，预测土壤中各种生物化学过程的强度和方向。酸性磷酸酶、脲酶、蔗糖酶和过

氧化氢酶是最常用的土壤酶活性指标，分别是氧化氢和蔗糖的代谢专一酶，对分解土壤过氧化氢，促

42

进活性碳转化有着重要的作用[17]。土壤酸性磷酸酶是土壤中的主要水解酶类， 是土壤组分中最活跃的

有机成分， 其活性与碳、氮、磷、硫等营养元素循环及植物营养状况的关系密切。在多数生态系统中

土壤酸性磷酸酶活性将会影响碳、氮、磷的迁移转化。已有研究结果表明，土壤酸性磷酸酶能够催化

土壤有机磷矿化， 提高土壤磷的有效性，由表 4 可知，酸性磷酸酶活性属 CK 最高，2021 年三个处理

差异不明显，分别为 719.04 U/g.h、709.90 U/g.h 和 705.76 U/g.h，到了 2022 年 CK 明显高于 T1 和 T2，

而 T2 又高于 T1。土壤蔗糖酶是一种水解酶，能够催化土壤中的蔗糖水解为单糖，从而被机体吸收。

土壤蔗糖酶的酶促作用产物与土壤中营养元素（如有机质、氮、磷）含量，微生物数量及土壤呼吸强

度密切相关，可作为评价土壤肥力的重要指标之一。2021 年和 2022 年，T2 都明显高于 CK 和 T1，而

2021 年 T1 显著高于 CK，其中 2021 年和 2022 年 T2 蔗糖酶活性分别为 10.53 U/g 和 8.91 U/g。土壤脲

酶活性，与土壤的微生物数量、有机物质含量、全氮和速效磷含量呈正相关。T2 和 T1 在两年内检测

活性都显著高于 CK，但他们之间差异不明显。过氧化氢酶活性对分解土壤过氧化氢，促进活性碳转化

有着重要的作用[18]。T2 在两年内检测活性都显著高于 CK，T1 在 2022 年显著高于 CK。因此，施放有

机配方肥一定程度提高了土壤蔗糖酶、脲酶和过氧化氢酶活性，改善了土壤地力。

表 4 生物有机配方肥替代化肥对剑麻地土壤酶活的影响

Tab4 Effect on soil enzyme activity by bio-organic formulated fertilizer consecutively replaced chemical fertilizer

年份

Year

处理

Treatment

酸性磷酸酶活性

S-ACP（U/g.h）

蔗糖酶活性

SC（U/g）

脲酶活性

S-UE（U/g）

过氧化氢酶活性

S-CAT（U/g ）

2021 CK 719.04±23.15a 1.47±0.85c 807.26±59.13b 11.78±0.85b

T1 709.90±17.12a 4.51±0.74b 916.86±45.16a 12.49±0.79b

T2 705.76±20.66a 10.53±1.76a 958.91±64.01a 14.90±1.03a

2022 CK 1137.11±50.18a 2.03±0.56b

702.56±46.12b 10.59±0.74b

T1 892.69±68.14c 2.13±0.29b

936.91±52.29a 12.72±0.81a

T2 922.02±45.12b 8.91±1.02a 969.51±56.22a 12.88±1.12a

2.4 生物有机配方肥连续替代化肥对剑麻叶片大、中元素和纤维含量的影响

土壤地力的改善一定程度上反映到剑麻叶片元素含量上，由表 5 可知，有机配方肥替代化肥对剑

麻叶片部分大、中元素和纤维含量存在明显影响，三个处理的剑麻叶片全氮、全磷含量没有显著差异。

剑麻叶片全钾含量在 2021 年 T1 最高，显著高于 CK 和 T2，CK 和 T2 没有显著差异，2022 年 T2 显著

高于 CK 和 T1，达到 2.55%。在 2021 年和 2022 年剑麻叶片全钙含量 T2 显著高于 T1 和 CK，而 T1 又

显著高于 CK，分别为 2021 年 3.22%、2.87%和 2.76%，2022 年 3.62%、3.48%和 2.95%。剑麻叶片全

镁含量在 2021 年和 2022 年都没有明显差异。在 2021 年和 2022 年剑麻叶片纤维含量 T2 显著高于 T1

和 CK，而 T1 又显著高于 CK，分别为 2021 年 3.87%、3.75%和 3.62%，2022 年 4.03%、3.78%和 3.68%。

因此，有机配方肥连续替代化肥可以提高剑麻叶片钙和纤维含量。

表 5 生物有机配方肥替代化肥对剑麻叶片大、中元素和纤维含量的影响

Tab5 Effect on Contents of large, medium elements and fiber in sisal leaves by bio-organic formulated fertilizer consecutively replaced chemical fertilizer

年份

Year

处理

Treatment

氮含量 Nitrogen content(%)

磷含量

phosphorous

content(%)

钾含量 potassium content(%)

钙含量

calcium content(%)

镁含量

magnesium

content (%)

纤维含量

fiber content

（％）

2021 CK 1.16±0.04a 0.08±0.01a 2.49±0.04b 2.76±0.05c 0.46±0.01a 3.62±0.04c

T1 1.10±0.03a 0.08±0.02a 2.57±0.03a 2.87±0.04b 0.45±0.02a 3.75±0.05b

T2 1.12±0.04a 0.08±0.02a 2.42±0.05b 3.22±0.06a 0.48±0.02a 3.87±0.06a

43

2022 CK 1.13±0.05a

0.08±0.02a 2.49±0.03b 2.95±0.05c 0.47±0.01a 3.68±0.05c

T1 1.14±0.02a 0.08±0.03a 2.49±0.04b 3.48±0.05b 0.46±0.02a 3.78±0.04b

T2 1.11±0.03a 0.09±0.02a 2.55±0.05a 3.62±0.06a 0.45±0.03a 4.03±0.05a

2.5 生物有机配方肥连续替代化肥对剑麻叶片生长性状和产量的影响

连续施放有机配方肥后，对剑麻叶片生长存在一定影响，主要表现在：如表 6 可知，剑麻叶长、

叶宽及增叶影响较小，没有明显差异，而差异主要表现在单叶重和叶厚，在 2021 年和 2022 年剑麻叶

片单叶重 T2 显著高于 CK，而 T1 与 CK 没有明显差异，2022 年 T2 显著比 T1 重；在 2021 年和 2022

年剑麻叶片厚 T2 显著厚于 CK，而 T1 与 CK 没有明显差异，2022 年 T2 和 T1 叶厚没有明显差异，T2、

T1 和 CK 在 2021 年和 2022 年叶厚分别为 8.01mm、7.32mm、7.04mm 和 10.70mm、10.25mm、9.89mm。

剑麻叶片产量 T2 、T1 与 CK 没有明显差异，T2、T1 和 CK 在 2021 年和 2022 年产量分别为 6.51 t/667m2

、

6.45 t/667m2

、6.57 t/667m2 和 6.11 t/667m2

、6.09 t/667m2

、6.16 t/667m2

。

表 6 生物有机配方肥替代化肥对剑麻叶片生长性状和产量的影响

Tab6 Effect on Growth traits and yield of sisal leaves by bio-organic formulated fertilizer consecutively replaced chemical fertilizer

年份

Year

处理

Treat

ment

单叶重

weight per leaf

（g)

叶长

leaf length

（cm)

叶宽 leaf width

（mm)

叶厚 thick leaves

（mm)

增叶

Increasing

leaves（片）

产量

Yield

（t/667m2

）

2021 CK 423.26±13.12b 111.98±4.56a 106.55±2.56a 7.04±0.51b 14.2±0.3a 6.57±0.14a

T1 418.98±14.15b 113.54±3.69a 105.82±3.41a 7.32±0.32b 13.8±0.4a 6.45±0.21a

T2 442.32±10.13a 116.58±3.72a 109.95±3.11a 8.01±0.41a 14.1±0.5a 6.41±0.32a

2022 CK 490.02±10.11b 115.56±2.45a 125.21±3.56a 9.89±0.52b 14.0±0.4a 6.16±0.22a

T1 507.38±15.19ab 118.52±3.68a 126.48±3.54a 10.25±0.41ab 13.3±0.6a 6.09±0.24a

T2 512.78±19.58a 117.84±3.55a 126.12±3.01a 10.70±0.36a 13.7±0.5a 6.11±0.13a

2.6 有机配方肥连续替代化肥对剑麻产量效益的影响

通过施放连续有机配方肥可以节省投入成本，增加效益， 由表 7 可知，T2、T1 和 CK 两年投入

的成本分别为 16125 yuan/hm2

、17962.5 yuan/hm2和 19800 yuan/hm2

，而两年收入分别为 78741 yuan/hm2

、

78840 yuan/hm2 和 80014.5 yuan/hm2

，最后两年的利润分别为 62615 yuan/hm2

、60877.5 yuan/hm2 和

60214.5 yuan/hm2

，因此两年内 T2 比 CK 增加收入 2400.5 yuan/hm2

，T1 比 CK 增加收入 663 yuan/hm2

。

表 7 生物有机配方肥连续替代化肥对剑麻产量效益的影响

Tab7 Effect on sisal yield profit by bio-organic formulated fertilizer consecutively replaced chemical fertilizer

年份

Year

处理

Treatment

产量

Yield

（t/hm2）

投入成本

input cost

(yuan/hm2)

麻片收入

income

(yuan/hm2 )

利润

Profit

(yuan/hm2 )

2021 CK 98.55 9900 38434.5 28534.50

T1 96.75 8981.25 37732.5 29126.25

T2 96.15 8062.5 37498.5 28517.25

2022 CK 92.4 9900 41580 31680.00

T1 91.35 8981.25 41107.5 32501.25

T2 91.65 8062.5 41242.5 33180.00

注：剑麻叶片收购价：三刀麻叶片 390yuan/t，四刀麻叶片 450yuan/t。

Note : The purchase price of sisal leaves is 390 yuan / t for three-bladed sisal leaves and 450 yuan / t for four-bladed sisal

leaves.

44

3 讨论

剑麻生物有机配方肥是将有机与无机肥相结合兼加有益功能菌按照一定比例加工造粒，有利于机

械施放，肥料养分可以慢慢释放，并通过适当补充 N， 以延长旺长期，利用 K 与 Ca 拮抗作用，适当

减少钾肥施用量，促进对钙的吸收量， 提高抗剑麻茎腐病能力， 以增加剑麻叶片产量。本文连续两

年施放剑麻生物配方肥的麻地土壤速效钾和交换性钙与传统化肥施放是存在明显差异这种差异表现在

2022 年全施放配方肥土壤速效钾（163.44 mg/kg）显著低于传统化肥施放 CK（259.24 mg/kg），但全施

放配方肥土壤交换性钙（492.12 mg/kg））却显著高于传统化肥施放 CK（428.70 mg/kg），而通过检测

麻片全钾和全钙含量，也发现全施放配方肥剑麻叶片全钙含量显著高于传统化肥施放 CK，第一年高对

照 CK 0.46 mg/kg，第二年全施放配方肥剑麻叶片全钙含量与传统化肥施放 CK 两者差距到 0.67 mg/kg。

由于叶片全钙含量的提升，叶片纤维含量也表现出显著差异，第一年全施放配方肥剑麻叶片全钙含量

高传统化肥施放 CK 0.25 %，到了第二年这个差距达到了 0.35%，因此，全施生物有机配方肥是有利于

促进剑麻对钙的吸收，以致增加了麻片纤维含量。

剑麻全施生物有机配方肥第一年土壤微生物量碳含量 33.87mg/kg，到了第二年再施，麻地土壤微

生物量碳含量达到了 384.80 mg/kg，提高了 11.36 倍，相反传统化肥施放 CK 第二年是 21.40mg/kg，仅

为第一年的 0.91 倍。土壤微生物量氮含量，施放生物有机配方肥的麻地从第一年 16.81 mg/kg，到第二

年的 63.50 mg/kg，增长了 3.78 倍，而传统化肥施放麻地第一年是 15.57mg/kg，第二年是 30.90 mg/kg，

只增长了 1.98 倍。说明连续施放生物有机配方肥有能显著提高土壤微生物量碳氮含量。

施放生物有机肥是有利于增强土壤酶活活性，而连续施放生物有机配方肥是否可以持续增强土壤

酶活，通过文中试验结果分析，施放生物有机配方肥一定程度可以提高土壤蔗糖酶、脲酶和过氧化氢

酶活性，改善了土壤地力。

剑麻生物有机配方肥是通过优化传统剑麻化肥施放量，并配合机械化作业，而研制的一种颗粒生

物有机配方肥，具有机械方便、改良土壤、价格低等优点，1 公顷投入 3750Kg，成本仅为 8062.5 元，

而传统化肥施放 1 公顷需投入 9900 元，通过试验，两者产量没有显著差异，相反剑施放麻生物有机配

方肥比传统化肥施放两年时间增加了收入 2400.5 yuan/hm2，这和黄标等研究是一致的。

4、结论

剑麻连续施放生物有机配方肥促进了剑麻对钙吸收利用，麻片全钙含量高出传统化肥施放达 0.67

mg/kg，有效提高了麻片纤维含量，麻片纤维含量高出传统化肥施放达 0.35mg/kg， 同时，剑麻连续施

放生物有机配方肥相对常规施肥（CK）还能有效提高剑麻地土壤微生物量碳氮含量、蔗糖酶活性、脲

酶活性、过氧化氢酶活性，改良土壤性状，减少了化学肥料投入成本，避免化肥过量施放造成流失。

通过成本核算，发现两年连续施放剑麻颗粒有机配方肥的剑麻比常规施肥，最高增加了收入 2400.5

yuan/hm2。

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46

基于 2021-2022 年粤西蔗区甘蔗主要病虫害监测数据的

分析及综合防治探索

陈士伟 1,2,吴如慧 1,2,廖锡华 3

，徐福生 5

，徐杨玉 1,2，郑志坤 4

，

易建学 5

，谢永议 6

，欧阳司 7

，林明江 8*

（1 广东农垦热带农业研究院有限公司，广东广州 511365；2 广东省农垦南亚热带作物科技中心，广东广州 510507；3

广东广垦糖业集团有限公司，广东湛江 524002；4 广东省广前糖业发展有限公司，湛江遂溪 524348；5 广东省华海糖

业发展有限公司，湛江徐闻 524132；6 广东省丰收糖业发展有限公司，湛江雷州 524200；7 广东农垦火炬农场有限公

司，湛江雷州 524272；8 广东省科学院南繁种业研究所,广东广州 510310）

摘 要：基于 2021-2022 年粤西蔗区甘蔗条螟及其它主要病虫害监测数据，进行了较系统的整理、分析和比较, 总结了

粤西蔗区近年来条螟的发生规律，并建立了监测预警技术体系。同时，通过探索综合防治措施，为甘蔗病虫害的防治

提供了科学依据和实践经验，对促进粤西蔗区甘蔗产业的可持续发展具有重要意义。

关键词：粤西地区；甘蔗；条螟；病虫害；监测；综合防治

Analysis and Integrated Pest Management Exploration Based on the

Monitoring Data of Major Pests and Diseases in the Western

Guangdong Sugarcane Region from 2021 to 2022

CHEN Shiwei1,2,WU Ruhui1,2,LIAO Xihua3

,XU Fusheng5

,XU Yangyu1,2,ZHENG Zhikun4

, YI Jianxue5

,

XIE Yongyi6

,OUYANG Si7

,LIN Mingjiang8*

(1Guangdong Agribusiness Tropical Agriculture Institute, Guangzhou, Guangdong 511365;2Guangdong State Farms

South Subtropical Crop Science & Technology Center, Guangzhou, Guangdong 510507;3Guangdong Guangken Sugar

Group Co.Ltd, Zhanjiang, Guangdong 524002; 4Guangqian Sugar Industry Development Co. Ltd., Suixi, Guangdong

524348;5Guangdong Huahai Sugar Industry Development Co.Ltd, Xuwen, Zhanjiang 524132;6Guangdong Province

Harvest Sugar Industry Development Co.Ltd, Leizhou, Zhanjiang 524200;7Guangdong Nongken Torch Farm Co.Ltd,

Leizhou, Zhanjiang 524272;8Institute of Nanfan & Seed Industry, Guangdong Academy of Sciences,Guangzhou,

Guangdong 510310)

Abstract : Based on the monitoring data of Proceras venosatum Walker and other major pests and diseases in

the western Guangdong sugarcane region from 2021 to 2022, a systematic organization, analysis, and comparison were conducted. The study summarized the occurrence patterns of Proceras venosatum Walker in the

western Guangdong sugarcane region in recent years, and a monitoring and warning technology system was

established. At the same time, exploring comprehensive prevention and control measures provides scientific

basis and practical experience for the prevention and control of sugarcane diseases and pests, which is of

great significance for promoting the sustainable development of the sugarcane industry in the western

Guangdong sugarcane region.

Key words:Western Guangdong region;Sugarcane;Proceras venosatum Walker; Diseases and insect pests;

Monitoring; Integrated pest management

第一作者 陈士伟（1980—），男，硕士，热作高级工程师，主要研究方向为植物保护、农业管理与推广，E-mail：

94169837@qq.com。

*通讯作者：林明江(1972-)，男，硕士，研究员，主要研究方向为从事昆虫信息素与生物防治技术，E-mail:lmjjiang@163.com。

47

不同光质 LED 补光对火龙果激素含量的影响

陈士伟 1,2，徐杨玉 1,2，吴如慧 1,2，张曼其 3

，姚雷业 3

，伍兆诚 1,2，

段门俊 1,2，袁志能 1,2，李栋宇 4**

1.广东省农垦南亚热带作物科技中心，广东广州 510506；2.广东农垦热带农业研究院有限公司，广东广州 511365；

3.广东省湛江农垦科学研究所，广东湛江 524000；4.岭南师范学院 LED 前沿技术与利用研究所，广东湛江 524048

摘要：选用金都一号火龙果为试验材料，设红光、绿光、蓝光比例分别为 23:75:2、19:79:2、18:78:4、24:73:3、16:80:4

的 LED 灯作为补光处理，以不采取补光措施 CK 作为对照，同时设定最佳补光时间段（22:30-02:30）。通过比较不同光

质 LED 补光对火龙果激素（IAA、ABA、CZR、TZR、GA3）含量的变化规律，进一步分析花芽分化的调控机制。结

果表明：不同光质补光处理下，火龙果五种内源激素含量变化规律是：IAA>ABA>TZR>CZR>GA3，其中激素（IAA、

ABA、CZR、TZR）与火龙果花芽分化有正相关性、激素（GA3）与火龙果花芽分化有负相关性。火龙果五种激素间

平衡互作的影响：补光组 CZR/IAA 与 TZR/IAA 的比值均高于对照组。结论：补光可调控火龙果植株激素含量，促进

IAA、ABA、CZR、TZR 合成，抑制 GA3 合成，从而促进火龙果成花，且红光：绿光：蓝光比例为 23:75:2 时，促进

花芽分化的效果最佳。

关键词：火龙果；LED 灯；激素；花芽分化

Effects of LEDs Spectra on the Hormone Content of Pitaya

CHEN Shiwei1,2, XU Yangyu1,2, WU Ruhui1,2, ZHANG Manqi3

, YAO Leiye3

, WU Zhaocheng1,2, DUAN Menjun1,2, YUAN Zhineng1,2, LI Dongyu4**

Guangdong Agricultural Reclamation South Asian Tropical Crop Science and Technology Center, Guangzhou, Guangdong

510506, China;2. Guangdong Agribusiness Tropical Agriculture Institute Co.,Ltd. Guangzhou, Guangdong 511365, China;3.

Zhanjiang Agricultural Reclamation Science Research Institute, Zhanjiang, Guangdong 524000, China; 4. Lingnan Normal

University LED Frontier Technology and Utilization Research Institute, Zhanjiang, Guangdong 524048, China

Abstract: Jindu No.1 pitaya was selected as the experimental material, and LEDs with red, green, and blue light ratios of

23:75:2, 19:79:2, 18:78:4, 24:73:3, and 16:80:4 were used as supplementary lighting treatment. CK without supplementary

lighting measures was used as the control, and the optimal supplementary lighting time period (22:30-02:30) was set. By

comparing the changes of the contents of IAA, ABA, CZR, TZR, GA3 in pitaya fruit under different light quality LED light,

the regulation mechanism of flower bud differentiation was further analyzed.The results showed that under different light

quality supplementary treatments, the changes in the content of five hormones in pitaya were as follows: IAA>ABA>TZR>

CZR>GA3. Among them, IAA, ABA, CZR, TZR were positively correlated with pitaya flower bud differentiation, while GA3

was negatively correlated with pitaya flower bud differentiation. The effect of balance interaction among the five hormones in

pitaya: The ratio of CZR/IAA and TZR/IAA in the light supplement group was higher than that in the control group. Conclusion: Supplementing light can regulate the hormones content of pitaya, promote the synthesis of IAA, ABA, CZR, TZR, inhibit

GA3 synthesis, and promote the flower bud differentiation of pitaya. The best effect is to promote flower bud differentiation

when the ratio of red , green and blue light is 23:75:2.

Keywords: Pitaya; LED lights; Hormones; Flower bud differentiation

基金项目 2020 年农业农村部农业生产发展资金项目（No.2130122）；2020 年度湛江市科技发展专项资金竞争性分配

项目 （No.2020A03003）；2019 年度广东省普通高校“服务乡村振兴计划”重点领域专项（No.2019KZDZX2008）。

第一作者 陈士伟（1980—），男，硕士，热作高级工程师，主要研究方向为植物保护、农业管理与推广，E-mail：

94169837@qq.com。

通讯作者 李栋宇（ 1984— ），男，博士，教授，主要研究方向为光谱农业和发光材料与器件， E-mail：

nanorainbows@163.com。