Research Progress in Metabolites Produced by Bacillus Against Three Common Plant Pathogenic Fungi

doi:10.13560/j.cnki.biotech.bull.1985.2022-1315

Abstract

Abstract:

Plant pathogenic fungi are one of the major threats to agricultural production. Applying biological agents to control pathogenic fungi is widely considered to be a safer and more sustainable strategy. Bacillus species can produce a variety of antifungal active substances（lipopeptides, bacteriocins and enzymes, etc.）, which is the most widely used biocontrol bacteria at present. Biocontrol agents based on Bacillus spp. and its metabolites can effectively control plant pathogenic fungi and play an important role in agricultural production. This paper focuses on the biological control potential of Bacillus metabolites and their antagonistic properties and mechanisms against three common plant pathogenic fungi（Magnaporthe oryzae, Fusarium oxysporum, and Botrytis cinerea）. Several important Bacillus metabolites were introduced by investigating the related literatures published in recent years on the antifungal activities of Bacillus metabolites, and the antifungal effects and mechanisms of Bacillus metabolites on important plant pathogenic fungi were summarized. And meanwhile the research methods and effects of Bacillus metabolites on the damages of cell wall and cell membrane of pathogenic fungi, inhibition of fungal spore germination and mycelial growth, and competitive binding with fungal DNA were summarized. The aim of this review is to provide guidance for the preparation and application of Bacillus biocontrol agents in the future.

Key words: Bacillus species, metabolite, biological control, plant pathogenic fungi, antifungal mechanism

WANG Wei-chen, ZHAO Jin, HUANG Wei-yi, GUO Xin-zhu, LI Wan-ying, ZHANG Zhuo. Research Progress in Metabolites Produced by Bacillus Against Three Common Plant Pathogenic Fungi[J]. Biotechnology Bulletin, 2023, 39(3): 59-68.

Figures/Tables 2

References 61

[1]	Fira D, Dimkić I, Berić T, et al. Biological control of plant pathogens by Bacillus species[J]. J Biotechnol, 2018, 285: 44-55. doi: 10.1016/j.jbiotec.2018.07.044 URL
[2]	Osaki C, Miyake S, Urakawa S, et al. Growth inhibitory mechanism of contact-independent antifungal TM-I-3 Bacillus sporothermodurans strain against Aspergillus fumigatus and Cladosporium cladosporioides[J]. Biocontrol Sci, 2021, 26(1): 49-53. doi: 10.4265/bio.26.49 URL
[3]	刘翠玲, 张冉, 杨桂玲, 等. 三唑类杀菌剂在蔬菜中的残留分布及对不同人群的累积性膳食摄入风险[J]. 农药学学报, 2021, 23(6): 1194-1204.
	Liu CL, Zhang R, Yang GL, et al. Residues distribution of triazole fungicides in vegetables and cumulative dietary intake risk to different populations[J]. Chin J Pestic Sci, 2021, 23(6): 1194-1204.
[4]	宋桂芳, 张世文, 庄红娟, 等. 农用地大环内酯类抗生素与杀菌剂残留污染评价[J]. 环境化学, 2022, 41(7): 2309-2319.
	Song GF, Zhang SW, Zhuang HJ, et al. Pollution assessment of macrolide antibiotics and fungicides residues in agricultural land[J]. Environ Chem, 2022, 41(7): 2309-2319.
[5]	Flampouri E, Theodosi-Palimeri D, Kintzios S. Strobilurin fungicide kresoxim-methyl effects on a cancerous neural cell line: oxidant/antioxidant responses and in vitro migration[J]. Toxicol Mech Methods, 2018, 28(9): 709-716. doi: 10.1080/15376516.2018.1506848 URL
[6]	沙月霞. 生物农药在稻瘟病防治中的应用及前景分析[J]. 植物保护, 2017, 43(5): 27-34.
	Sha YX. Application of biopesticide against rice blast and analysis of its prospect[J]. Plant Prot, 2017, 43(5): 27-34.
[7]	Abbey JA, Percival D, Abbey L, et al. Biofungicides as alternative to synthetic fungicide control of grey mould(Botrytis cinerea)- prospects and challenges[J]. Biocontrol Sci Technol, 2019, 29(3): 207-228. doi: 10.1080/09583157.2018.1548574 URL
[8]	Caulier S, Nannan C, Gillis A, et al. Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group[J]. Front Microbiol, 2019, 10: 302. doi: 10.3389/fmicb.2019.00302 pmid: 30873135
[9]	Kaspar F, Neubauer P, Gimpel M. Bioactive secondary metabolites from Bacillus subtilis: a comprehensive review[J]. J Nat Prod, 2019, 82(7): 2038-2053. doi: 10.1021/acs.jnatprod.9b00110 pmid: 31287310
[10]	Gomaa EZ, El-Mahdy OM. Improvement of chitinase production by Bacillus thuringiensis NM101-19 for antifungal biocontrol through physical mutation[J]. Microbiology, 2018, 87(4): 472-485. doi: 10.1134/S0026261718040094
[11]	Salazar F, Ortiz A, Sansinenea E. A strong antifungal activity of 7-O-succinyl macrolactin A vs macrolactin A from Bacillus amyloliquefaciens ELI149[J]. Curr Microbiol, 2020, 77(11): 3409-3413. doi: 10.1007/s00284-020-02200-2
[12]	Santos JCP, Sousa RCS, Otoni CG, et al. Nisin and other antimicrobial peptides: Production, mechanisms of action, and application in active food packaging[J]. Innov Food Sci Emerg Technol, 2018, 48: 179-194. doi: 10.1016/j.ifset.2018.06.008 URL
[13]	Calvo H, Mendiara I, Arias E, et al. Antifungal activity of the volatile organic compounds produced by Bacillus velezensis strains against postharvest fungal pathogens[J]. Postharvest Biol Technol, 2020, 166: 111208. doi: 10.1016/j.postharvbio.2020.111208 URL
[14]	Fernandez J, Orth K. Rise of a cereal killer: the biology of Magnaporthe oryzae biotrophic growth[J]. Trends Microbiol, 2018, 26(7): 582-597. doi: S0966-842X(17)30280-9 pmid: 29395728
[15]	Ma ZW, Zhang SY, Sun K, et al. Identification and characterization of a cyclic lipopeptide iturin A from a marine-derived Bacillus velezensis 11-5 as a fungicidal agent to Magnaporthe oryzae in rice[J]. J Plant Dis Prot, 2020, 127(1): 15-24. doi: 10.1007/s41348-019-00282-0
[16]	Zhang LL, Sun CM. Fengycins, cyclic lipopeptides from marine Bacillus subtilis strains, kill the plant-pathogenic fungus Magnaporthe grisea by inducing reactive oxygen species production and chromatin condensation[J]. Appl Environ Microbiol, 2018, 84(18): e00445-e00418.
[17]	Chen Z, Zhao L, Chen WQ, et al. Isolation and evaluation of Bacillus velezensis ZW-10 as a potential biological control agent against Magnaporthe oryzae[J]. Biotechnol Biotechnol Equip, 2020, 34(1): 714-724. doi: 10.1080/13102818.2020.1803766 URL
[18]	Kgosi VT, Bao TT, Ying Z, et al. Anti-fungal analysis of Bacillus subtilis DL76 on conidiation, appressorium formation, growth, multiple stress response, and pathogenicity in Magnaporthe oryzae[J]. Int J Mol Sci, 2022, 23(10): 5314. doi: 10.3390/ijms23105314 URL
[19]	Dong YL, Li H, Rong SH, et al. Isolation and evaluation of Bacillus amyloliquefaciens Rdx5 as a potential biocontrol agent against Magnaporthe oryzae[J]. Biotechnol Biotechnol Equip, 2019, 33(1): 408-418. doi: 10.1080/13102818.2019.1578692 URL
[20]	Li H, Guan Y, Dong YL, et al. Isolation and evaluation of endophytic Bacillus tequilensis GYLH001 with potential application for biological control of Magnaporthe oryzae[J]. PLoS One, 2018, 13(10): e0203505. doi: 10.1371/journal.pone.0203505 URL
[21]	López-Berges MS. ZafA-mediated regulation of zinc homeostasis is required for virulence in the plant pathogen Fusarium oxysporum[J]. Mol Plant Pathol, 2020, 21(2): 244-249. doi: 10.1111/mpp.12891 pmid: 31750619
[22]	Hanif A, Zhang F, Li PP, et al. Fengycin produced by Bacillus amyloliquefaciens FZB42 inhibits Fusarium graminearum growth and mycotoxins biosynthesis[J]. Toxins, 2019, 11(5): 295. doi: 10.3390/toxins11050295 URL
[23]	Montalvão SCL, de Castro MT, Blum LEB, et al. Biocontrol of Fusarium oxysporum f. sp. vasinfectum with Bacillus spp. strains[J]. J Agric Sci, 2021, 13(9): 1.
[24]	Jangir M, Pathak R, Sharma S, et al. Biocontrol mechanisms of Bacillus sp., isolated from tomato rhizosphere, against Fusarium oxysporum f.sp. lycopersici[J]. Biol Control, 2018, 123: 60-70. doi: 10.1016/j.biocontrol.2018.04.018 URL
[25]	Yadav K, Damodaran T, Dutt K, et al. Effective biocontrol of banana Fusarium wilt tropical race 4 by a Bacillus rhizobacteria strain with antagonistic secondary metabolites[J]. Rhizosphere, 2021, 18: 100341. doi: 10.1016/j.rhisph.2021.100341 URL
[26]	Li H, Chen Y, Zhang ZQ, et al. Pathogenic mechanisms and control strategies of Botrytis cinerea causing post-harvest decay in fruits and vegetables[J]. Food Qual Saf, 2018, 2(3): 111-119.
[27]	Petrasch S, Knapp SJ, Kan JALV, et al. Grey mould of strawberry, a devastating disease caused by the ubiquitous necrotrophic fungal pathogen Botrytis cinerea[J]. Mol Plant Pathol, 2019, 20(6): 877-892. doi: 10.1111/mpp.12794 pmid: 30945788
[28]	Salvatierra-Martinez R, Arancibia W, Araya M, et al. Colonization ability as an indicator of enhanced biocontrol capacity—An example using two Bacillus amyloliquefaciens strains and Botrytis cinerea infection of tomatoes[J]. J Phytopathol, 2018, 166(9): 601-612. doi: 10.1111/jph.2018.166.issue-9 URL
[29]	Maung CEH, Lee HG, Cho JY, et al. Antifungal compound, methyl hippurate from Bacillus velezensis CE 100 and its inhibitory effect on growth of Botrytis cinerea[J]. World J Microbiol Biotechnol, 2021, 37(9): 159. doi: 10.1007/s11274-021-03046-x
[30]	Ren L, Zhou JB, Yin H, et al. Antifungal activity and controlefficiency of endophytic Bacillus velezensis ZJ1 strain and its volatile compounds against Alternaria solani and Botrytis cinerea[J]. J Plant Pathol, 2022, 104(2): 575-589. doi: 10.1007/s42161-022-01056-8
[31]	Jiang CH, Liao MJ, Wang HK, et al. Bacillus velezensis, a potential and efficient biocontrol agent in control of pepper gray mold caused by Botrytis cinerea[J]. Biol Control, 2018, 126: 147-157. doi: 10.1016/j.biocontrol.2018.07.017 URL
[32]	Calvo H, Roudet J, Gracia AP, et al. Comparison of efficacy and modes of action of two high-potential biocontrol Bacillus strains and commercial biocontrol products against Botrytis cinerea in table grapes[J]. OENO One, 2021, 55(3): 228-243. doi: 10.20870/oeno-one.2021.55.3.4688 URL
[33]	Chen XM, Wang YJ, Gao Y, et al. Inhibitory abilities of Bacillus isolates and their culture filtrates against the gray mold caused by Botrytis cinerea on postharvest fruit[J]. Plant Pathol J, 2019, 35(5): 425-436. doi: 10.5423/PPJ.OA.03.2019.0064 URL
[34]	Owens RA, Doyle S. Effects of antifungal agents on the fungal proteome: informing on mechanisms of sensitivity and resistance[J]. Expert Rev Proteomics, 2021, 18(3): 185-199. doi: 10.1080/14789450.2021.1912601 URL
[35]	Sui Y, Ma ZX, Meng XH. Proteomic analysis of the inhibitory effect of oligochitosan on the fungal pathogen, Botrytis cinerea[J]. J Sci Food Agric, 2019, 99(5): 2622-2628. doi: 10.1002/jsfa.2019.99.issue-5 URL
[36]	Shi LM, Ge BB, Wang JZ, et al. iTRAQ-based proteomic analysis reveals the mechanisms of Botrytis cinerea controlled with Wuyiencin[J]. BMC Microbiol, 2019, 19(1): 280. doi: 10.1186/s12866-019-1675-4
[37]	Medeot DB, Fernandez M, Morales GM, et al. Fengycins from Bacillus amyloliquefaciens MEP218 exhibit antibacterial activity by producing alterations on the cell surface of the pathogens Xanthomonas axonopodis pv. vesicatoria and Pseudomonas aeruginosa PA01[J]. Front Microbiol, 2020, 10: 3107. doi: 10.3389/fmicb.2019.03107 URL
[38]	Humisto A, Jokela J, Teigen K, et al. Characterization of the interaction of the antifungal and cytotoxic cyclic glycolipopeptide hassallidin with sterol-containing lipid membranes[J]. Biochim Biophys Acta Biomembr, 2019, 1861(8): 1510-1521. doi: 10.1016/j.bbamem.2019.03.010 URL
[39]	Guo HL, Qiao BX, Ji XS, et al. Antifungal activity and possible mechanisms of submicron chitosan dispersions against Alteraria alternata[J]. Postharvest Biol Technol, 2020, 161: 110883. doi: 10.1016/j.postharvbio.2019.04.009 URL
[40]	Jin PF, Wang HN, Tan Z, et al. Antifungal mechanism of bacillomycin D from Bacillus velezensis HN-2 against Colletotrichum gloeosporioides penz[J]. Pestic Biochem Physiol, 2020, 163: 102-107. doi: 10.1016/j.pestbp.2019.11.004 URL
[41]	Xu WH, Wang HX, Lv ZH, et al. Antifungal activity and functional components of cell-free supernatant from Bacillus amyloliquefaciens LZN01 inhibit Fusarium oxysporum f.sp. niveum growth[J]. Biotechnol Biotechnol Equip, 2019, 33(1): 1042-1052. doi: 10.1080/13102818.2019.1637279 URL
[42]	Wu JJ, Chou HP, Huang JW, et al. Genomic and biochemical characterization of antifungal compounds produced by Bacillus subtilis PMB102 against Alternaria brassicicola[J]. Microbiol Res, 2021, 251: 126815. doi: 10.1016/j.micres.2021.126815 URL
[43]	Li YG, Wang RT, Liu JX, et al. Identification of a biocontrol agent Bacillus vallismortis BV23 and assessment of effects of its metabolites on Fusarium graminearum causing corn stalk rot[J]. Biocontrol Sci Technol, 2019, 29(3): 263-275. doi: 10.1080/09583157.2018.1548575 URL
[44]	Rong SH, Xu H, Li LH, et al. Antifungal activity of endophytic Bacillus safensis B21 and its potential application as a biopesticide to control rice blast[J]. Pestic Biochem Physiol, 2020, 162: 69-77. doi: 10.1016/j.pestbp.2019.09.003 URL
[45]	Choub V, Won SJ, Ajuna HB, et al. Antifungal activity of volatile organic compounds from Bacillus velezensis CE 100 against Colletotrichum gloeosporioides[J]. Horticulturae, 2022, 8(6): 557. doi: 10.3390/horticulturae8060557 URL
[46]	Tian DD, Song XP, Li CS, et al. Antifungal mechanism of Bacillus amyloliquefaciens strain GKT04 against Fusarium wilt revealed using genomic and transcriptomic analyses[J]. MicrobiologyOpen, 2021, 10(3): e1192.
[47]	Jiang CM, Li ZZ, Shi YH, et al. Bacillus subtilis inhibits Aspergillus carbonarius by producing iturin A, which disturbs the transport, energy metabolism, and osmotic pressure of fungal cells as revealed by transcriptomics analysis[J]. Int J Food Microbiol, 2020, 330: 108783. doi: 10.1016/j.ijfoodmicro.2020.108783 URL
[48]	Mishra A, Bhattacharya A, Chauhan P, et al. Phenotype microarray analysis reveals the biotransformation of Fusarium oxysporum f.sp. lycopersici influenced by Bacillus subtilis PBE-8 metabolites[J]. FEMS Microbiol Ecol, 2022, 98(10): fiac102. doi: 10.1093/femsec/fiac102 URL
[49]	Liu D, Li KY, Hu JL, et al. Biocontrol and action mechanism of Bacillus amyloliquefaciens and Bacillus subtilis in soybean Phytophthora blight[J]. Int J Mol Sci, 2019, 20(12): 2908. doi: 10.3390/ijms20122908 URL
[50]	沙月霞, 隋书婷, 曾庆超, 等. 贝莱斯芽孢杆菌E69预防稻瘟病等多种真菌病害的潜力[J]. 中国农业科学, 2019, 52(11): 1908-1917. doi: 10.3864/j.issn.0578-1752.2019.11.006
	Sha YX, Sui ST, Zeng QC, et al. Biocontrol potential of Bacillus velezensis strain E69 against rice blast and other fungal diseases[J]. Sci Agric Sin, 2019, 52(11): 1908-1917.
[51]	陈奕鹏, 杨扬, 桑建伟, 等. 拮抗内生芽孢杆菌BEB17分离鉴定及其挥发性物质抑菌活性分析[J]. 植物病理学报, 2018, 48(4): 537-546.
	Chen YP, Yang Y, Sang JW, et al. Isolation and identification of antagonistic endophytic Bacillus BEB17 and analysis of antibacterial activity of volatile organic compounds[J]. Acta Phytopathol Sin, 2018, 48(4): 537-546.
[52]	Kang BR, Song YS, Jung WJ. Differential expression of bio-active metabolites produced by chitosan polymers-based Bacillus amyloliquefaciens fermentation[J]. Carbohydr Polym, 2021, 260: 117799. doi: 10.1016/j.carbpol.2021.117799 URL
[53]	Prakash J, Arora NK. Novel metabolites from Bacillus safensis and their antifungal property against Alternaria alternata[J]. Antonie Van Leeuwenhoek, 2021, 114(8): 1245-1258. doi: 10.1007/s10482-021-01598-4 pmid: 34076810
[54]	Shastri B, Kumar R, Lal RJ. Isolation and Identification of antifungal metabolite producing endophytic Bacillus subtilis(S17)and its in vitro effect on Colletotrichum falcatum causing red rot in sugarcane[J]. Vegetos, 2020, 33(3): 493-503. doi: 10.1007/s42535-020-00133-6
[55]	Nawaz HH, Nelly Rajaofera MJ, He QG, et al. Evaluation of antifungal metabolites activity from Bacillus licheniformis OE-04 against Colletotrichum gossypii[J]. Pestic Biochem Physiol, 2018, 146: 33-42. doi: 10.1016/j.pestbp.2018.02.007 URL
[56]	Liu WX, Sun CM. C17-fengycin B, produced by deep-sea-derived Bacillus subtilis, possessing a strong antifungal activity against Fusarium solani[J]. J Oceanol Limnol, 2021, 39(5): 1938-1947. doi: 10.1007/s00343-020-0215-2
[57]	Zhang YH, Zhao MX, Chen W, et al. Multi-omics techniques for analysis antifungal mechanisms of lipopeptides produced by Bacillus velezensis GS-1 against Magnaporthe oryzae in vitro[J]. Int J Mol Sci, 2022, 23(7): 3762. doi: 10.3390/ijms23073762 URL
[58]	Wang F, Xiao J, Zhang YZ, et al. Biocontrol ability and action mechanism of Bacillus halotolerans against Botrytis cinerea causing grey mould in postharvest strawberry fruit[J]. Postharvest Biol Technol, 2021, 174: 111456. doi: 10.1016/j.postharvbio.2020.111456 URL
[59]	Zhang D, Yu SQ, Zhao DM, et al. Inhibitory effects of non-volatiles lipopeptides and volatiles ketones metabolites secreted by Bacillus velezensis C16 against Alternaria solani[J]. Biol Control, 2021, 152: 104421. doi: 10.1016/j.biocontrol.2020.104421 URL
[60]	Wang DK, Li YC, Yuan Y, et al. Identification of non-volatile and volatile organic compounds produced by Bacillus siamensis LZ88 and their antifungal activity against Alternaria alternata[J]. Biol Control, 2022, 169: 104901. doi: 10.1016/j.biocontrol.2022.104901 URL
[61]	Wu SJ, Yun JM, Wang R, et al. Analysis of the effects of antifungal peptide P-1 from Bacillus pumilus HN-10 on energy metabolism of Trichothecium roseum[J]. Food Biosci, 2022, 47: 101668. doi: 10.1016/j.fbio.2022.101668 URL

病原真菌 Pathogenic fungus	代谢产物Metabolites
病原真菌 Pathogenic fungus	脂肽、细菌素、聚酮 Lipopeptides, bacteriocins, and polyketones	酶 Enzymes	挥发性化合物 Volatile compounds
稻瘟菌Magnaporthe oryzae	Iturin、fengycin等导致细胞渗漏，抑制孢子萌发	纤维素酶、蛋白酶等抑制菌丝生长及孢子萌发	抑制菌丝生长
尖孢镰刀菌 Fusarium oxysporum	Fengycin等导致真菌菌丝结构变形，引起细胞渗漏	β-1, 3-葡聚糖酶、蛋白酶和几丁质酶等水解细胞壁	竞争氧气，减缓真菌生长速度
灰葡萄孢菌 Botrytis cinerea	Fenycin、bacillaene诱导菌丝形态变化，surfactin破坏真菌脂膜	水解酶破坏细胞结构	抑制孢子形成，导致内容物泄露

病原真菌 Pathogenic fungus	代谢产物Metabolites
病原真菌 Pathogenic fungus	脂肽、细菌素、聚酮 Lipopeptides, bacteriocins, and polyketones	酶 Enzymes	挥发性化合物 Volatile compounds
稻瘟菌Magnaporthe oryzae	Iturin、fengycin等导致细胞渗漏，抑制孢子萌发	纤维素酶、蛋白酶等抑制菌丝生长及孢子萌发	抑制菌丝生长
尖孢镰刀菌 Fusarium oxysporum	Fengycin等导致真菌菌丝结构变形，引起细胞渗漏	β-1, 3-葡聚糖酶、蛋白酶和几丁质酶等水解细胞壁	竞争氧气，减缓真菌生长速度
灰葡萄孢菌 Botrytis cinerea	Fenycin、bacillaene诱导菌丝形态变化，surfactin破坏真菌脂膜	水解酶破坏细胞结构	抑制孢子形成，导致内容物泄露

研究方法 Research method	抗菌机制Antifungal mechanisms
研究方法 Research method	壁膜损伤 Damage of cell wall and cell membrane^{[40⇓⇓⇓⇓⇓⇓⇓-48]}	抑制孢子形成和菌丝生长Inhibition of spore formation and mycelial growth^{[49⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓-60]}	竞争性结合DNA Competitive binding with DNA^[61]
显微镜观察 Microscopic observation	扫描/透射电镜可分析细胞形态变化，如孢子表面凹陷、细胞壁和细胞膜的破坏、内容物的泄漏和细胞器的聚集。共聚焦显微镜可观察膜的完整性	扫描/透射电镜观察菌丝和孢子结构；光学显微镜直接观察菌丝结构	透射电镜观察孢子的超微结构
组学方法 Omics method	比较蛋白质组学等可分析膜电位，进一步分析诱导膜损伤的机制	转录组学、代谢组学分析氨基酸代谢、氧化磷酸化等基因的表达；组学结合生物信息学分析抗菌生物合成基因	转录组学、蛋白组学初步分析真菌胞内核酸、蛋白质的变化
其他方法 Other method	结合形态学观察和差异转录组学分析细胞壁、细胞膜形态以及与转运能力相关的基因	色谱/质谱法分析识别脂肽和挥发性化合物等	荧光探针、核酸释放量监测细胞通透性；荧光强度分析与EB竞争性结合DNA

研究方法 Research method	抗菌机制Antifungal mechanisms
研究方法 Research method	壁膜损伤 Damage of cell wall and cell membrane^{[40⇓⇓⇓⇓⇓⇓⇓-48]}	抑制孢子形成和菌丝生长Inhibition of spore formation and mycelial growth^{[49⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓-60]}	竞争性结合DNA Competitive binding with DNA^[61]
显微镜观察 Microscopic observation	扫描/透射电镜可分析细胞形态变化，如孢子表面凹陷、细胞壁和细胞膜的破坏、内容物的泄漏和细胞器的聚集。共聚焦显微镜可观察膜的完整性	扫描/透射电镜观察菌丝和孢子结构；光学显微镜直接观察菌丝结构	透射电镜观察孢子的超微结构
组学方法 Omics method	比较蛋白质组学等可分析膜电位，进一步分析诱导膜损伤的机制	转录组学、代谢组学分析氨基酸代谢、氧化磷酸化等基因的表达；组学结合生物信息学分析抗菌生物合成基因	转录组学、蛋白组学初步分析真菌胞内核酸、蛋白质的变化
其他方法 Other method	结合形态学观察和差异转录组学分析细胞壁、细胞膜形态以及与转运能力相关的基因	色谱/质谱法分析识别脂肽和挥发性化合物等	荧光探针、核酸释放量监测细胞通透性；荧光强度分析与EB竞争性结合DNA