Research progress on occurrence and control of litchi anthracnose
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摘要:
荔枝Litchi chinensis Sonn.是中国重要的热带亚热带水果之一，然而刺盘孢属真菌Colletotrichum spp.侵染所致的荔枝炭疽病严重影响了荔枝的生产和果实采后贮运。荔枝炭疽病为害荔枝叶、枝梢、花穗和果实，症状复杂，病原种类多样，迄今已报道了5个复合种共22个种，其中，盘长孢状刺盘孢复合种C. gloeosporioides species complex为优势种群。高温、高湿的气候条件适合病害的发生，每年4—6月为我国荔枝炭疽病的爆发期。本文从荔枝炭疽病病害症状、病原、发生规律、致病机制以及防治技术等方面进行了全面综述，根据其侵染过程对已报道的刺盘孢属真菌的致病因子进行了归纳，对荔枝炭疽病的防控技术进行了总结分析，并对今后的荔枝炭疽致病机制研究和绿色防控技术进行了展望。
Abstract:Litchi (Litchi chinensis Sonn.) is one of the important tropical and subtropical fruits in China. However, anthracnose caused by Colletotrichum spp. severely impacts the yield and quality of litchi, posing a major threat during both production and postharvest storage. Litchi anthracnose infects leaves, flower clusters, shoots and fruits, exhibiting diverse and complex symptoms. To date, total 22 species belonging to five Colletotrichum species complex have been reported worldwide to infect litchi, with the C. gloeosporioides species complex being the dominant group. High temperature and humidity are conducive to the occurrence of litchi anthracnose, and the outbreak period of litchi anthracnose is from April to June every year in China. This paper reviewed comprehensively the symptoms, pathogens, occurrence patterns, pathogenic mechanisms, and control techniques of litchi anthracnose. The reported infection process and virulent factors of Colletotrichum spp. were categorized, the control technologies for litchi anthracnose were summarized and analyzed. Finally, the future research on pathogenic mechanisms and green prevention and control strategies for litchi anthracnose were prospected.
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草地贪夜蛾Spodoptera frugiperda为鳞翅目夜蛾科灰翅夜蛾属，该虫源自北美，2019年1月入侵我国云南省，并迅速扩展到全国26个省份[1-3]。作为联合国粮农组织全球预警的跨国界迁飞性重大害虫，草地贪夜蛾具有寄主范围宽、适生区域广、增殖能力强、扩散速度快、突发危害重等特点[4-5]。
与其他鳞翅目昆虫一样，草地贪夜蛾主要在幼虫时期为害。目前草地贪夜蛾雌、雄幼虫为害行为性别差异的研究较少，因为缺乏幼虫性别鉴定的快速简便的手段。与许多鳞翅目昆虫一样，草地贪夜蛾在蛹和成虫时期不再取食，因此幼虫时期的取食量对其化蛹、羽化、产卵、迁飞等行为具有重要影响，不同性别的幼虫取食量存在差异。林玉英等[5]对椰子织蛾Opisina arenosella 1龄幼虫取食量的研究表明，雌虫取食量显著大于雄虫，结合幼虫取食量可作为其龄期的判断依据之一，从而为制定椰子织蛾防控措施奠定基础；同时，大量研究表明，昆虫幼虫在抵抗高温、抗核型多角体病毒等方面有性别差异[6-8]，成虫在感光、触角结构等方面也存在显著的性别差异[9]，昆虫在取食、感光、抗病等行为上的性别差异研究，可为农业害虫的精准防控提供理论支持。因此，性别鉴定可以作为研究昆虫雌、雄行为差异的一种便捷有效的工具，有助于制定更加精准高效的农业害虫防控治理策略。
目前，草地贪夜蛾的性别主要是通过蛹期和成虫时期的外露生殖器及翅上的斑纹差异进行区分[10-11]。草地贪夜蛾入侵中国后，性信息素诱捕、高空灯诱捕在虫情预测预报中发挥了非常重要的作用。由于缺乏对幼虫形态学有效的判断标准，而田间捕捉的草地贪夜蛾成虫非常活跃，鳞羽容易掉落，给性别鉴定造成了困难，影响了测报结果的准确性。对于鳞羽掉落的草地贪夜蛾样本和未经过性别鉴定的DNA样本，也缺乏有效的性别鉴定手段。因此，根据雌、雄虫性信息素结合蛋白(Pheromone-binding protein, PBP)基因的序列差异，开发简便、准确的功能性分子标记，对鉴定幼虫期乃至成虫期的草地贪夜蛾的性别具有理论和实际应用意义。
1. 材料与方法
1.1 供试材料
草地贪夜蛾为实验室饲养种群，饲养条件参考王世英等[12]方法，温度为(26.0±0.5) ℃；相对湿度为 65%±5%；光周期为16 h光∶8 h暗。
1.2 试验方法
1.2.1 草地贪夜蛾雌、雄虫分子标记引物的设计
通过在线网站( https://pfam.xfam.org)寻找并下载PBP隐马尔科夫模型，使用Bio-Linux软件进行生物信息学分析得到草地贪夜蛾PBP基因家族的氨基酸序列，通过在线网站( http://www.omicsclass.com/article/681)手动确认每个蛋白的结构域，总共筛选得到21个PBP，使用Bio-Linux软件进行生物信息学分析获得对应蛋白的CDS序列等相关信息，所得序列与NCBI上已发表的PBP基因序列进行比对，比对结果为本研究的PBP基因的CDS序列与已发表的4个PBP基因(SfruPBP1、SfruPBP2、SfruPBP3、SfruPBP4)[13]的CDS序列不存在相似性(结果未显示)。对获得的各基因片段进行PCR测序，结果发现Sf-10911基因序列在雌、雄个体中存在较大差异。通过多个已知雌、雄样本检测后，确认该基因为性别差异基因，针对草地贪夜蛾雌、雄虫Sf-10911基因的差异区段设计了3对引物(表1)，开发雌、雄性别鉴定的特异标记，引物设计见图1。利用设计合成的引物，对鉴别过已知性别的草地贪夜蛾虫蛹样本进行PCR扩增，筛选得到分子标记。
表 1 引物序列表Table 1. List of primer sequence引物名称1) Primer name 引物序列(5′→3′) Primer sequence Sf-F TAGCCGTGAGTTTGAATAGGGT Sf-female-R-1 CCTGCCAGTGCCTTATTAATTAA Sf-male-R-1 TTTTGGCAGTGCCTTATTGATTA Sf-female-R-2 CTCAGAGGTTTTTGATATGGTTT Sf-male-R-2 TGTATTCTTCTCAGTGCGAAGAC Sf-female-R-3 TTAACAACGCTCCATAATAACCT Sf-male-R-3 TAAGAACCAGTTCTTATAAACAC 1) F、R分别表示正、反向引物
1) F and R respectively represents forward and reverse primers![]()
图 1 草地贪夜蛾性别鉴定引物设计深蓝色表示相同的核苷酸序列，浅蓝色表示差异位点，黑点表示缺失位点；Sf-male-R：雄虫基因差异区段；Sf-female-R：雌虫基因差异区段；Sf-F：正向引物；Sf-R：反向引物Figure 1. Primers design for sexual identification of Spodoptera frugiperdaDark blue represents the same nucleotide sequence, light blue represents the differencial sites and black dots represent the missing sites; SF-male-R: Differential gene segment of male; Sf-female-R: Differential gene segment of female; Sf-F: Forward primer; Sf-R: Reverse primer1.2.2 草地贪夜蛾蛹期DNA提取及PCR扩增
根据草地贪夜蛾蛹期雌、雄虫形态差异区分出雌、雄后(图2)，利用微量样品基因组DNA 提取试剂盒进行DNA的提取。采用雌、雄特异性引物对提取的DNA样本进行PCR扩增。扩增产物用琼脂糖凝胶电泳检测，筛选分子标记。PCR 扩增的体系为：PrimerSTAR Max 6.25 μL，上游和下游引物(10 μmol/L)各0.5 μL，模板0.5 μL，加 ddH2O至15 μL。PCR 扩增的反应程序为：98 ℃ 预变性2 min；98 ℃变性 10 s，58 ℃退火 30 s，72 ℃延伸 30 s，35 个循环；72 ℃延伸5 min。
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图 2 草地贪夜蛾蛹期雌、雄虫腹部末端差异对比a：臀刺；b：肛门；c：第10腹节；d：第9腹节e：半圆形瘤状突起；f：第8腹节；g：产卵孔；h：生殖孔Figure 2. Distinction between abdomen ends of male and female of Spodoptera frugiperda at pupal stagea: Buttocks stab; b: Anus; c: The 10th abdominal segment; d: The 9th abdominal segment; e: Semicircular tumor-like protrusion; f: The 8th abdominal segment; g: Spawning hole; h: Genital hole2. 结果与分析
2.1 草地贪夜蛾性别鉴定引物的开发及筛选
针对草地贪夜蛾雌、雄虫Sf-10911基因的性别差异区段设计了3对引物，开发性别鉴定的特异标记。利用设计合成的3对引物，对已知性别的草地贪夜蛾样本进行PCR扩增，筛选得到分子标记，该分子标记可以扩增出450 bp左右的条带。之后，利用筛选出的分子标记对经过形态鉴定的雌、雄虫样本再次进行PCR扩增。
首先，利用3对标记引物扩增草地贪夜蛾的雌、雄虫DNA样本，所用样本为经过测序鉴定的雌、雄虫DNA样本；图3表明，引物Sf-female-R-1、Sf-male-R-3搭配Sf-F均不能扩增出特异条带；搭配引物Sf-F扩增时，其中雄性样本可以用雄性特异性引物Sf-male-R-2扩增得到特异条带，而雌性样本只有雌性特异性引物Sf-female-R-2可以扩增得到特异条带，与测序结果一致。因此，选择Sf-female-R-2和Sf-male-R-2作为草地贪夜蛾雌、雄虫特异性引物。
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图 3 3对引物对草地贪夜蛾雌、雄虫样本的扩增M：2000 DNA marker; m1: Sf-F/ Sf-male-R-1; m2: Sf-F/Sf-male-R-2; m3: Sf-F/Sf-male-R-3; f1: Sf-F/Sf-female-R-1; f2: Sf-F/Sf-female-R-2; f3: Sf-F/Sf-female-R-3Figure 3. Amplification of different sexual samples of Spodoptera frugiperdaby three pairs of primers2.2 性别分子标记引物对草地贪夜蛾的鉴定
为进一步验证筛选出的标记引物的准确性，对经过形态鉴定的雌、雄虫蛹进行PCR检测(图4)。从图4可以看出，利用雌虫标记引物Sf-female-R-2扩增雌、雄虫DNA样本时，只有雌虫才能扩增出450 bp左右的特异性条带；用雄虫标记引物Sf-male-R-2扩增雌、雄虫DNA样本时，只有雄虫才能扩增出450 bp左右的特异性条带。检测结果与形态鉴定结果一致，说明筛选出的引物适用于草地贪夜蛾的性别鉴定。
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图 4 基于PCR扩增对草地贪夜蛾雌、雄虫蛹性别鉴定F1~F5：雌虫蛹DNA；M1~M5：雄虫蛹DNA；a、c：雌虫标记引物对 Sf-F/Sf-female-R-2；b、d：雄虫标记引物对Sf- F/Sf-male-R-2Figure 4. Sex identification of male and female pupae of Spodoptera frugiperdabased on PCR amplificationF1−F5: DNA of female pupae; M1−M5: DNA of male pupae; a and c: Pair of female marker primers of Sf-F/Sf-female-R-2; b and d: Pair of male marker primers of Sf-F/Sf-male-R-23. 讨论与结论
农业害虫的性别鉴定对于害虫的有效防治和农业生产具有重要意义。不同性别的昆虫在虫体形态上往往存在差异，甜菜夜蛾Spodoptera exigua Hübner、桉袋蛾Acanthopsyche subferalbata Hampson以及凤凰木夜蛾Pericyma cruegri在其蛹及成虫时期的形态存在明显的性别差异[14-16]，利用这种形态上的差异，研究人员可以快速简便地鉴定雌、雄虫，及时为田间种群动态的监测和预测预报提供数据。
利用雌、雄虫形态差异鉴定性别的方法虽然简单快捷，但却无法对一些不存在性别形态差异或是生长发育早期无形态差异的昆虫进行鉴定。牛宝龙等[17]以棉铃虫Helicoverpa armigera雌、雄虫基因组DNA为模板，筛选了1条雌特异随机扩增多态性DNA(Random amplified polymorphic DNA，RAPD)，根据该特异性分子标记的核苷酸序列设计雌性特异引物，并对棉铃虫基因组DNA进行PCR扩增，雌性棉铃虫可以扩增出目的条带，可将此标记用于棉铃虫幼虫乃至胚胎的性别鉴定；王慧超等[18]也早在2004年运用RADP技术对家蚕Bombyx mori Linnaeus上得到的雌特异性片段设计引物并进行了PCR验证。此外，张利娜[19]从外部形态学、血清生化指标建立了鳗鲡Anguilla japonica的性别判定函数，用SRAP分子标记获得F5R2雌性特异DNA序列，根据测序结果设计序列特定扩增区域(Sequence characterized amplified regions，SCAR)特异引物并进行性别鉴定；Masaru等[20]用日本青鳉Oryzias latipes的雄性Y特异性DM结构域基因开发引物鉴定了弓背青鳉Oryzias curvinotus的遗传性别；中国大鲵Andrias davidianus、双须骨舌鱼Osteoglossum bicirrhosum的性别鉴定也利用雌、雄虫基因差异序列开发分子标记引物并进行了有效的验证[21-22]。
PBP在草地贪夜蛾的信息素识别过程中发挥着重要作用，雄虫通过触角感受雌虫性腺释放的性信息素，寻找合适的交配对象。PBP的功能特征决定了其基因序列以及表达模式在雌、雄虫之间必然存在差异，具有明显的性二型性[23]。牛小慧[24]对甜菜夜蛾的不同PBP进行RT-PCR检测发现，PBP在雌、雄虫之间的表达量存在显著差异；刘苏等[13]通过对草地贪夜蛾4个PBP基因的克隆及表达模式分析发现，定位于成虫触角上的SfruPBP1和SfruPBP2蛋白在雄虫中具有更高的表达量。本研究发现草地贪夜蛾雌、雄虫中的PBP基因Sf-10911存在核苷酸序列差异，进而根据该差异设计了针对雌、雄虫扩增的引物对，通过琼脂糖凝胶电泳检测出450 bp左右的特异条带，作为其性别鉴定的分子标记，以期为研究草地贪夜蛾某些性状可能存在的性别差异提供快速有效的手段。
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图 1 荔枝炭疽病症状
A：褐色斑状病叶；B、D：病果；C：麻点状病叶；E：感染炭疽病的花穗。
Figure 1. Symptoms of litchi anthracnose
A: Symptoms of brown lesions associated with leaves; B, D: Diseased fruit; C: Symptoms of pepper spots associated with leaves; E: Symptoms of anthracnose associated with inflorences.
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图 2 荔枝炭疽病菌发现时间轴线
图中不同背景颜色的荔枝炭疽病菌表示归属于不同的刺盘孢复合种；括号里面的内容表示报道的作者及其报道年份；CN、AU和JA分别表示该病菌的地理分布为中国、澳大利亚和日本。
Figure 2. Timeline for discovering litchi anthracnose pathogenic species
Pathogens of litchi anthracnose with different background colors represented different Colletotrichum species complex; The details in parentheses indicated the reporter and the reported year of the pathogens; CN, AU and JA indicated the distribution of the pathogens in China, Australia and Japan respectively .
表 1 已报道的引起炭疽病的致病因子
Table 1 The reported pathogenic factors of Colletotrichum
侵染过程
Infection process致病因子
Pathogenic factor基因
Gene种类
Species参考文献
Reference穿透寄主表面前
Pre-penetration of
host surface附着胞细胞壁蛋白 Cap20 盘长孢状刺盘孢 C. gloeosporioides [27] 附着胞细胞壁蛋白 Cap22 盘长孢状刺盘孢 [28] 金属硫蛋白 Cap3 盘长孢状刺盘孢 [28] 金属硫蛋白 Cap5 盘长孢状刺盘孢 [28] 异柠檬酸裂合酶 ICL1 瓜类刺盘孢 C. lagenarium [29] 蛋白激酶 RPK1 瓜类刺盘孢 [30] 甾醇糖基转移酶 Chip6 盘长孢状刺盘孢 [31] 穿透寄主表面
Penetration of host
surface角质酶 CutA 盘长孢状刺盘孢 [32] 聚酮合成酶 Pks1 瓜类刺盘孢 [33] 铜离子转运ATP酶 Clap1 豆类刺盘孢 C. lindemuthianum [34] 小柱孢酮脱水酶 Scd1 瓜类刺盘孢 [35] 单羧酸转运蛋白 CgMCT1 盘长孢状刺盘孢 [36] 寡肽转运因子 CgOPT2 盘长孢状刺盘孢 [36] 1,3,8−二羟基萘还原酶 Thr1 瓜类刺盘孢 [37] 泛素结合酶蛋白 Chip1 盘长孢状刺盘孢 [38] 漆酶 Lac2 圆孢刺盘孢 C. orbiculare [39] 假定钙调素激酶 CaMK 盘长孢状刺盘孢 [40] 四跨膜区蛋白 Pls1 豆类刺盘孢 [41] 自噬相关蛋白 Atg26 瓜类刺盘孢 [42] 穿透寄主表面相关的
信号转导途径
Signal transduction of
penetrating host
surface假定的丝氨酸/苏氨酸激酶 Clk1 豆类刺盘孢 [43] 过氧化物酶体蛋白 ClaPEX6 瓜类刺盘孢 [44] 丝裂原活化蛋白激酶 Cmk1 瓜类刺盘孢 [45] MAPK激酶 CgMEK 盘长孢状刺盘孢 [46] cAMP依赖性蛋白激酶 CtPKAC 苜蓿刺盘孢 C. trifolli [47] cAMP依赖性蛋白激酶 CgPKAC 盘长孢状刺盘孢 [48] 穿透寄主表面相关
的转录因子
Transcriptional
factors of penetrating host
surface转运因子 CLTA1 豆类刺盘孢 [49] Ste12 CST1 瓜类刺盘孢 [50] Ste12-like CLSTE12 豆类刺盘孢 [51] 转录因子 CMR1 葫芦刺盘孢 C. lagenaria [52] 肌动相关蛋白 ArpA 盘长孢状刺盘孢 [53] pH响应转录因子 PacC 尖孢刺盘孢 C. acutatum [54] 穿透寄主表面相关
的水解酶
Hydrolytic enzymes of
penetrating host
surface果胶酸裂解酶 CgPel 盘长孢状刺盘孢 [55] 果胶酸裂解酶 CgPel 盘长孢状刺盘孢 [56] 果胶酸裂解酶 Pel 壮丽刺盘孢 C. magna [57] 果胶酸裂解酶 PelB 盘长孢状刺盘孢 [58] 果胶酸裂解酶 PecCl1 豆类刺盘孢 [59] 果胶酸裂解酶 CcpelA 球刺盘孢 C. coccodes [60] 果胶酸酯裂解酶 PnlA 盘长孢状刺盘孢 [61] 
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