苏云金芽胞杆菌防治草地贪夜蛾的研究和应用进展

doi:10.16409/j.cnki.2095-039x.2023.11.013

摘要/Abstract

摘要： 草地贪夜蛾Spodoptera frugiperda是世界性的重大农业害虫，至今已在全世界126个国家和地区传播，对玉米等多种主要粮食和经济作物造成严重为害。草地贪夜蛾于2019年1月入侵我国，并迅速扩散至27个省。2022年草地贪夜蛾发生面积超过3531万hm²。苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt）是一种用于防治农业害虫的革兰氏阳性细菌，具有对害虫高效专一、环境友好、人畜安全等特点。Bt主要依靠其产生的杀虫蛋白发挥杀虫作用。如今，国际上主要依靠转Bt杀虫基因的抗虫作物以及Bt微生物农药对草地贪夜蛾进行绿色防治。我国目前已开发出Bt天然菌株、基因工程菌株和转基因抗虫作物等多种高效Bt产品用于草地贪夜蛾绿色防治。本文将对国内外Bt防治草地贪夜蛾的最新研究进展进行介绍。

关键词: 草地贪夜蛾, 苏云金芽胞杆菌, 生物防治, 微生物农药, 转基因抗虫作物

Abstract: The fall armyworm (Spodoptera frugiperda) is a major agricultural pest in the world. So far, it has spread to126 countries and regions around the world, causing serious damage to many major food and economic crops such as maize. Fall armyworm invaded China in January 2019 and quickly spread to 27 provinces. In 2021, the occurrence area of S. frugiperda has exceeded 1.38 millionha. Bacillus thuringiensis (Bt) is a gram-positive bacterium used to control agricultural pests. It has the characteristics of high efficiency and specificity for pests, environmental friendliness, and safety for humans and animals.Bt mainly relies on the insecticidal protein produced by it to exert its insecticidal effect. The sustainable control of fall armyworm mainly relies on genetically modified crops with Bt insecticidal genes and Bt microbial pesticides now. At present, a variety of high-efficiency Bt products such as Bt natural strains, genetically engineered Bt strains, and genetically modified (GM) crops have been developed in my country for the control of fall armyworm. This article will introduce the latest progress in the research of Bt control of fall armyworm domestic and abroad.

Key words: Spodoptera frugiperda, Bacillus thuringiensis, biological control, microbial pesticides, genetically modified crops

中图分类号:

S476.1

徐国力, 王泽宇, 王奎, 束长龙, 耿丽丽, 廖敏, 张杰. 苏云金芽胞杆菌防治草地贪夜蛾的研究和应用进展[J]. 中国生物防治学报, 2024, 40(5): 1181-1193.

XU Guoli, WANG Zeyu, WANG Kui, SHU Changlong, GENG Lili, LIAO Ming, ZHANG Jie. Advances in Research and Application of Bacillus thuringiensis for Controlling Spodoptera frugiperda[J]. Chinese Journal of Biological Control, 2024, 40(5): 1181-1193.

参考文献

[1] Johnson S. Migration and the life history strategy of the fall armyworm, Spodoptera frugiperda in the western hemisphere[J]. International Journal of Tropical Insect Science, 1987, 8(4-5-6): 543-549.
[2] Todd E, Poole R. Keys and illustrations for the armyworm moths of the noctuid genus Spodoptera Guenée from the Western Hemisphere[J]. Annals of the Entomological Society of America, 1980, 73(6): 722-738.
[3] Murúa G, Molina-Ochoa J, Coviella C. Population dynamics of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) and its parasitoids in northwestern Argentina[J]. Florida Entomologist, 2006, 89(2): 175-182.
[4] Early R, González-Moreno P, Murphy S T, et al. Forecasting the global extent of invasion of the cereal pest Spodoptera frugiperda, the fall armyworm[J]. BioRxiv, 2018: 391847.
[5] Wyckhuys K A, O’Neil R J. Population dynamics of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) and associated arthropod natural enemies in Honduran subsistence maize[J]. Crop Protection, 2006, 25(11): 1180-1190.
[6] Nagoshi R N, Goergen G, Koffi D, et al. Genetic studies of fall armyworm indicate a new introduction into Africa and identify limits to its migratory behavior[J]. Scientific Reports, 2022, 12(1): 1-12.
[7] Day R, Abrahams P, Bateman M, et al. Fall armyworm: impacts and implications for Africa[J]. Outlooks on Pest Management, 2017, 28(5): 196-201.
[8] Van den Berg J, Prasanna B M, Midega C A O, et al. Managing fall armyworm in Africa: can Bt maize sustainably improve control?[J]. Journal of Economic Entomology, 2021, 114(5): 1934-1949.
[9] Tippannavar P S, Talekar S C, Mallapur C P, et al. An outbreak of fall armyworm in Indian subcontinent: a new invasive pest on maize[J]. Maydica, 2019, 64(1).
[10] FAO. FAO Statement on fall armyworm in Sri Lanka. Available online[EB/OL]. (2019-01-24)[2023-01-27]. https://www.fao.org/srilanka/news/detail-events/en/c/1177796/.
[11] FAO. First detection of fall army worm on the border of Thailand[EB/OL]. (2018-12-19)[2023-01-27]. https://www.ippc.int/en/countries/thailand/pestreports/2018/12/first-detection-of-fall-army-worm-on-the-border-of-thailand/.
[12] Alam S N, Sarker D, Pradhan M Z H, et al. First report of occurrence of fall armyworm. Spodoptera frugiperda in Bangladesh[J]. Bangladesh Journal of Entomology, 2018, 28(1): 97-101.
[13] Naeem-Ullah U, Ansari M A, Iqbal N, et al. First authentic report of Spodoptera frugiperda (JE Smith)(Noctuidae: Lepidoptera) an alien invasive species from Pakistan[J]. Applied Sciences and Business Economics, 2019, 6(1): 1-3.
[14] 杨学礼, 刘永昌, 罗茗钟, 等. 云南省江城县首次发现迁入我国西南地区的草地贪夜蛾[J]. 云南农业, 2019(1): 72.
[15] FAO. First detection report of the fall armyworm Spodoptera frugiperda (Lepidoptra: Noctuidae) on maize in Myanmar[EB/OL]. (2019-01-11) [2023-01-27]. https://www.ippc.int/en/countries/myanmar/pestreports/2019/01/first-detection-report-of-the-fall-armyworm-spodoptera-frugiperda-lepidoptranoctuidae-on-maize-in-myanma/.
[16] 全国农业技术推广服务中心. 全国农技推广网[EB/OL]. (2022)[2023-01-11]. https://www.natesc.org.cn/.
[17] FAO. The Occurence of fall armyworm (Spodoptera frugiperda) in Indonesia[EB/OL]. (2019-07-11)[2023-01-27]. https://www.ippc.int/zh/countries/indonesia/pestreports/2019/07/the-occurence-of-fall-armyworm-spodoptera-frugiperda-in-indonesia/.
[18] Ryu M, Lee S J, Lee H S, et al. First report of fall armyworm, Spodoptera frugiperda and its molecular characteristics[C]//2019 Spring International Conference of KSAE. 2019: 66-66.
[19] Jamil S, Mohd Masri S, Hudin L, et al. First incidence of the invasive fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797) attacking maize in Malaysia[J]. BioInvasions Records, 2021, 10: 81-90.
[20] Maino J L, Schouten R, Overton K, et al. Regional and seasonal activity predictions for fall armyworm in Australia[J]. Current Research in Insect Science, 2021, 1: 100010.
[21] EPPO. Spodoptera frugiperda. EPPO datasheets on pests recommended for regulation[EB/OL]. (2022)[2023-01-11]. https://gd.eppo.int.
[22] FAO. FAO Global Action for Fall Armyworm (FAW) Control[EB/OL]. (2022)[2023-01-18]. https://www.ippc.int/en/fallarmyworm/.
[23] Zhou Z S, YANG S J, SHU C L, et al. Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain[J]. Journal of Integrative Agriculture, 2015, 14(8): 1598-1603.
[24] Crickmore N, Berry C, Panneerselvam S. Bacterial pesticidal protein resource center[EB/OL]. (2022)[2023-01-12]. http://www.bpprc.org/.
[25] Dulmage H T. Insecticidal activity of HD-1, a new isolate of Bacillus thuringiensis var. alesti[J]. Journal of Invertebrate Pathology, 1970, 15(2): 232-239.
[26] Arango J A, Romero M, Orduz S. Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae)[J]. Journal of Applied Microbiology, 2002, 92(3): 466-474.
[27] Monnerat R G, Batista A C, de Medeiros P T, et al. Screening of Brazilian Bacillus thuringiensis isolates active against Spodoptera frugiperda, Plutella xylostella and Anticarsia gemmatalis[J]. Biological Control, 2007, 41(3): 291-295.
[28] Park M G, Choi J Y, Kim J H, et al. Isolation and molecular characterization of Bacillus thuringiensis subsp. kurstaki toxic to lepidopteran pests Spodoptera spp. and Plutella xylostella[J]. Pest Management Science, 2022, 78(7): 2976-2984.
[29] Sathyan T, Jayakanthan M, Mohankumar S, et al. Genome profiling of an indigenous Bacillus thuringiensis isolate, T405 toxic against the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)[J]. Microbial Pathogenesis, 2022, 173: 105820.
[30] 姚萌, 王奎, 束长龙, 等. 苏云金芽胞杆菌G03菌株的比较基因组分析[J]. 生物技术通报, 2018, 34(10): 187-193.
[31] 李青, 刘华梅, 丁咏梅, 等. 几株具有杀虫特异性的苏云金芽胞杆菌菌株的生物活性及杀虫蛋白基因型的鉴定[J]. 现代农业科学, 2009, 16(4): 9-11, 18.
[32] 刘华梅, 胡虓, 王应龙, 等. 对草地贪夜蛾高毒力的苏云金杆菌菌株筛选[J]. 中国生物防治学报, 2019, 35(5): 721-728.
[33] 王宇航, 束长龙, 耿丽丽, 等. 苏云金芽胞杆菌G033A产业化现状及应用前景分析[J]. 中国生物防治学报, 2020, 36(6): 837-841.
[34] 王建, 杨小雪, 王丹丹, 等. 对草地贪夜蛾高毒力的苏云金芽胞杆菌菌株筛选与杀虫活性研究[J]. 中国生物防治学报, 2021, 37(4): 660-670.
[35] Hernández-Rodríguez C S, Hernández-Martínez P, Rie J V, et al. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda[J]. PLoS ONE, 2013, 8(7): e68164.
[36] Sena J A, Hernández-Rodríguez C S, Ferré J. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites[J]. Applied and Environmental Microbiology, 2009, 75(7): 2236-2237.
[37] Fang J, Xu X L, Wang P, et al. Characterization of Chimeric Bacillus thuringiensis Vip3 Toxins[J]. Applied and Environmental Microbiology, 2007, 73(3): 956-961.
[38] Caccia S, Chakroun M, Vinokurov K, et al. Proteolytic processing of Bacillus thuringiensis Vip3A proteins by two Spodoptera species[J]. Journal of Insect Physiology, 2014, 67: 76-84.
[39] Banyuls N, Hernández-Rodríguez C S, Van Rie J, et al. Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects[J]. Scientific Reports, 2018, 8(1): 1-14.
[40] de Escudero I R, Banyuls N, Bel Y, et al. A screening of five Bacillus thuringiensis Vip3A proteins for their activity against lepidopteran pests[J]. Journal of Invertebrate Pathology, 2014, 117: 51-55.
[41] Gómez I, Rodríguez-Chamorro D E, Flores-Ramírez G, et al. Spodoptera frugiperda (J. E. Smith) aminopeptidase N1 is a functional receptor of the Bacillus thuringiensis Cry1Ca Toxin[J]. Applied and Environmental Microbiology, 2018, 84(17): e01089-18.
[42] Wang Y fei, Wang J ling, Fu X, et al. Bacillus thuringiensis Cry1Da_7 and Cry1B.868 protein interactions with novel receptors allow control of resistant fall armyworms, Spodoptera frugiperda (J.E. Smith)[J]. Applied and Environmental Microbiology, 2019, 85(16): e00579-19.
[43] Graser G, Walters F S, Burns A, et al. A general approach to test for interaction among mixtures of insecticidal proteins which target different orders of insect pests[J]. Journal of Insect Science, 2017, 17(2): 39.
[44] Soares Figueiredo C, Nunes Lemes A R, Sebastião I, et al. Synergism of the Bacillus thuringiensis Cry1, Cry2, and Vip3 proteins in Spodoptera frugiperda control[J]. Applied Biochemistry and Biotechnology, 2019, 188(3): 798-809.
[45] 贾倩, 郑怀国, 赵静娟. 跨国种企作物育种专利布局及对我国的启示[J]. 中国生物工程杂志, 2022, 42(10): 112-124.
[46] 智慧芽. 智慧芽-中国及多国专利查询_专利检索_中国专利网查询平台[EB/OL]. (2022)[2023-01-11]. https://www.zhihuiya.com/.
[47] Ibrahim M A, Griko N, Junker M, et al. Bacillus thuringiensis: a genomics and proteomics perspective[J]. Bioengineered Bugs, 2010, 1(1): 31-50.
[48] US EPA O. Biopesticide Active Ingredients[EB/OL]. (2022)[2023-01-11]. https://www.epa.gov/ingredients-used-pesticide-products/biopesticide-activeingredients.
[49] National Pesticide Information Center. NPIC Product Research Online(NPRO)[EB/OL]. (2022)[2023-01-11]. http://npic.orst.edu/NPRO/.
[50] Valent BioSciences. DiPel^® Biological Insecticide[EB/OL]. (2023-01-30)[2023-03-24]. https://www.valentbiosciences.com/agriculture/products/dipel/.
[51] Ministério da Agricultura, Pecuária e Abastecimento. Agrofit Consulta Aberta[EB/OL]. (2022)[2023-01-11]. https://agrofit.agricultura.gov.br/agrofit_cons/principal_agrofit_cons.
[52] Wang G J, Zhang J, Song F P, et al. Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests[J]. Applied Microbiology and Biotechnology, 2006, 72(5): 924-930.
[53] 张桂芬, 张毅波, 张杰, 等. 苏云金芽胞杆菌G033A对新发南美番茄潜叶蛾的室内毒力及田间防效[J]. 中国生物防治学报, 2020, 36(2): 175-183.
[54] 胡飞, 徐婷婷, 苏贤岩, 等. 苏云金杆菌微型颗粒剂对玉米鳞翅目害虫的防治效果研究[J]. 中国生物防治学报, 2023, 39(1): 46-53.
[55] 农业农村部农药检定所. 行业数据-中国农药信息网[EB/OL]. (2022)[2023-01-11]. http://www.chinapesticide.org.cn/hysj/index.jhtml.
[56] Briefs I. Global status of commercialized biotech/GM crops in 2017: Biotech crop adoption surges as economic benefits accumulate in 22 years[J]. ISAAA Brief, 2017, 53: 25-26.
[57] ISAAA. GM Approval Database ISAAA.org[EB/OL]. (2022)[2023-01-11]. https://www.isaaa.org/gmapprovaldatabase/default.asp.
[58] US EPA O. Current and previously registered section 3 plant-incorporated protectant (PIP) registrations[EB/OL]. (2022)[2023-01-11]. https://www.epa.gov/ingredients-used-pesticide-products/current-and-previously-registered-section-3-plant-incorporated.
[59] Christou P, Capell T, Kohli A, et al. Recent developments and future prospects in insect pest control in transgenic crops[J]. Trends in Plant Science, 2006, 11(6): 302-308.
[60] Koziel M G, Beland G L, Bowman C, et al. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis[J]. Biotechnology, 1993, 11(2): 194-200.
[61] Baktavachalam G B, Delaney B, Fisher T L, et al. Transgenic maize event TC1507: Global status of food, feed, and environmental safety[J]. GM Crops & Food, 2015, 6(2): 80-102.
[62] Yang F, Huang F, Qureshi J A, et al. Susceptibility of Louisiana and Florida populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) to transgenic Agrisure^®VipteraTM 3111 corn[J]. Crop Protection, 2013, 50: 37-39.
[63] Drury S M, Reynolds T L, Ridley W P, et al. Composition of forage and grain from second-generation insect-protected corn MON 89034 is equivalent to that of conventional corn (Zea mays L.)[J]. Journal of Agricultural and Food Chemistry, 2008, 56(12): 4623-4630.
[64] Siebert M W, Nolting S P, Hendrix W, et al. Evaluation of corn hybrids expressing Cry1F, Cry1A. 105, Cry2Ab2, Cry34Ab1/Cry35Ab1, and Cry3Bb1 against southern United States insect pests[J]. Journal of Economic Entomology, 2012, 105(5): 1825-1834.
[65] Naegeli H, Bresson J L, Dalmay T, et al. Assessment of genetically modified maize MON 87427×MON 87460×MON 89034×1507×MON 87411×59122 and subcombinations, for food and feed uses, under Regulation (EC) No 1829/2003(application EFSA-GMO-NL-2017-139)[J]. EFSA Journal, 2021, 19(1): e06351.
[66] USDA. USDA Economics, Statistics and Market Information System[EB/OL]. (2022)[2023-01-13]. https://usda.library.cornell.edu/concern/publications/ j098zb09z.
[67] Brookes G, Barfoot P. Environmental impacts of genetically modified (GM) crop use 1996–2015: impacts on pesticide use and carbon emissions[J]. GM Crops & Food, 2017, 8(2): 117-147.
[68] 农业农村部科技教育司. 2019年农业转基因生物安全证书批准清单[EB/OL]. (2020)[2023-01-11]. http://www.moa.gov.cn/ztzl/zjyqwgz/spxx/201912/t20191230_6334015.htm.
[69] Yang X M, Zhao S Y, Liu B, et al. Bt maize can provide non‐ chemical pest control and enhance food safety in China[J]. Plant Biotechnology Journal, 2023, 21(2): 391-404.
[70] 农业农村部科技教育司. 关于DBN9501等 2个转基因植物品种命名的公示[EB/OL]. (2021)[2023-01-11]. http://www.kjs.moa.gov.cn/gzdt/202101/t20210113_6359908.htm.
[71] 农业农村部科技教育司. 2021年农业转基因生物安全证书批准清单[EB/OL]. (2021)[2023-01-11]. http://www.moa.gov.cn/ztzl/zjyqwgz/spxx/202104/ t20210407_6365331.htm.
[72] Wang W H, Zhang D D, Zhao S Y, et al. Susceptibilities of the invasive fall armyworm (Spodoptera frugiperda) to the insecticidal proteins of Bt maize in China[J]. Toxins, 2022, 14(8): 507.
[73] 农业农村部科技教育司. 2021年农业转基因生物安全证书批准清单(三)[EB/OL]. (2021)[2023-01-11]. http://www.moa.gov.cn/ztzl/zjyqwgz/spxx/202112/t20211227_6385638.htm.
[74] 农业农村部再批3 个转基因玉米安全证书[J]. 农药, 2022, 61(2): 86.
[75] Zhao S Y, Yang X M, Liu D Z, et al. Performance of the domestic Bt corn event expressing pyramided Cry1Ab and Vip3Aa19 against the invasive Spodoptera frugiperda (JE Smith) in China[J]. Pest Management Science, 2023, 79(3): 1018-1029.
[76] 农业农村部科技教育司. 2022年农业转基因生物安全证书批准清单(一)[EB/OL]. (2022)[2023-01-11]. https://www.moa.gov.cn/ztzl/zjyqwgz/spxx/202204/t20220429_6398211.htm.
[77] Li G P, Ji T J, Zhao S Y, et al. High-dose assessment of transgenic insect-resistant maize events against major lepidopteran pests in China[J]. Plants, 2022, 11(22): 3125.
[78] 张丹丹, 吴孔明. 国产Bt-Cry1Ab和Bt-(Cry1Ab+Vip3Aa)玉米对草地贪夜蛾的抗性测定[J]. 植物保护, 2019, 45(4): 54-60.
[79] 金文涌, 叶凤林, 刘定富, 等. 中美转基因作物产业化最新进展[J]. 中国种业, 2022(9): 1-6.
[80] Storer N P, Babcock J M, Schlenz M, et al. Discovery and characterization of field resistance to Bt maize: Spodoptera frugiperda (Lepidoptera: Noctuidae) in Puerto Rico[J]. Journal of Economic Entomology, 2010, 103(4): 1031-1038.
[81] Omoto C, Bernardi O, Salmeron E, et al. Field-evolved resistance to Cry1Ab maize by Spodoptera frugiperda in Brazil[J]. Pest Management Science, 2016, 72(9): 1727-1736.
[82] Farias J R, Andow D A, Horikoshi R J, et al. Dominance of Cry1F resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) on TC1507 Bt maize in Brazil[J]. Pest Management Science, 2016, 72(5): 974-979.
[83] Fatoretto J C, Michel A P, Silva Filho M C, et al. Adaptive potential of fall armyworm (Lepidoptera: Noctuidae) limits Bt trait durability in Brazil[J]. Journal of Integrated Pest Management, 2017, 8(1): 17.
[84] Hamadou-Charfi D B, Boukedi H, Abdelkefi-Mesrati L, et al. Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin[J]. Journal of Invertebrate Pathology, 2013, 114(2): 139-143.
[85] Gouffon C V, Van Vliet A, Van Rie J, et al. Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin[J]. Applied and Environmental Microbiology, 2011, 77(10): 3182-3188.
[86] Jackson R E, Marcus M A, Gould F, et al. Cross-resistance responses of Cry1Ac-selected Heliothis virescens (Lepidoptera: Noctuidae) to the Bacillus thuringiensis protein Vip3A[J]. Journal of Economic Entomology, 2007, 100(1): 180-186.
[87] 何康来, 王振营. 草地贪夜蛾对Bt玉米的抗性与治理对策思考[J]. 植物保护, 2020, 46(3): 1-15.
[87] Tabashnik B E, Brévault T, Carrière Y. Insect resistance to Bt crops: lessons from the first billion acres[J]. Nature Biotechnology, 2013, 31(6): 510-521.
[89] Hutchison W D, Burkness E C, Mitchell P D, et al. Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers[J]. Science, 2010, 330(6001): 222-225.
[90] Zhao J Z, Cao J, Li Y X, et al. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution[J]. Nature Biotechnology, 2003, 21(12): 1493-1497.
[91] Chitkowski R L, Turnipseed S G, Sullivan M J, et al. Field and laboratory evaluations of transgenic cottons expressing one or two Bacillus thuringiensis var. kurstaki Berliner proteins for management of noctuid (Lepidoptera) pests[J]. Journal of Economic Entomology, 2003, 96(3): 755-762.
[92] 吴孔明. 中国草地贪夜蛾的防控策略[J]. 植物保护, 2020, 46(2): 1-5.
[93] 王文荟, 黄运新, 万鹏, 等. 草地贪夜蛾对国产Bt玉米的抗性风险评估[J]. 植物保护学报, 2022, 50(2): 1-14.
[94] 梁晋刚, 张旭冬, 毕研哲, 等. 转基因抗虫玉米发展现状与展望[J]. 中国生物工程杂志, 2021, 41(6): 98-104.