Induced formation method and germination characteristics of chlamydospores by Fusarium oxysporum f. sp. medicaginis

doi:10.11686/cyxb2023316

Abstract

Abstract:

Chlamydospores are the main survival structure of Fusarium oxysporum in soil， and the number and germination status of chlamydospores in soil directly affects the occurrence and severity of the disease. In this study， we established a system to induce chlamydospore production by F. oxysporum f. sp. medicaginis （Fom） by culturing the pathogen on synthetic low nutrient agar （SNA） medium， synthetic low nutrient agar with filter （SNAF） medium， or in two-salt solution （KH₂PO₄ and MgSO₄·7H₂O） with glucose or magnesium carbonate at a range of concentrations. The chlamydospore induction system was verified， and the effects of different carbon and nitrogen sources on chlamydospore germination were studied. The T6 and T9 strains of Fom produced many chlamydospores after static culture for 7 days in the two-salt solution with glucose at 2 mg·L^–1， producing 4.2×10⁵ and 5.1×10⁵ chlamydospores per mL， respectively. Both T6 and T9 produced more chlamydospores under static culture than under shaking culture （4.2- and 2.8- times， respectively， at 7 days of culture）. All Fom strains produced many chlamydospores after 7 days of culture in the two-salt solution， with a rapid increase of 2.3- times compared with 3 days， followed by slow increases at 14 and 21 days， with an average increase of only 1.2- times from 7 to 21 days of culture. Comparing all the carbon and nitrogen sources， glucose and ammonium chloride had the strongest promoting effects on the germination and germ tube growth of chlamydospores， whereas lactose and urea had the weakest effects. The results show that chlamydospore formation by Fomstrains requires a trace amount of a carbon source and a low-oxygen environment， and the germination and growth of chlamydospores require suitable carbon and nitrogen sources. These findings provide new insights into effective management of soil-borne diseases in alfalfa through controlling the primary infection source， i.e.， the chlamydospores of the pathogen.

Key words: Fusarium oxysporum, soilborne fungal pathogen, survival structure, primary inoculum, chlamydospore

Xiang-ling FANG, Shi-yang XU, Zhi-biao NAN. Induced formation method and germination characteristics of chlamydospores by Fusarium oxysporum f. sp. medicaginis[J]. Acta Prataculturae Sinica, 2024, 33(7): 130-141.

Figures/Tables 7

Fig.1 Spore production of strains T6 and T9 on SNA and SNAF medium

Fig. 2 Chlamydospore production of strains T6 and T9 in two-salt solution with glucose

Fig.3 Chlamydospore production of strains T6 and T9 in two-salt solution with glucose under shaking culture and static culture

Fig.4 Chlamydospore production of different strains in two-salt solution with glucose under static culture at 3 and 7 days

Fig.5 Chlamydospore production of different strains in two-salt solution with glucose under static culture with times

Fig.6 Germination rate and germ tube length of strains T6 and T9 chlamydospores in medium with different carbon source

Fig.7 Germination rate and germ tube length of strains T6 and T9 chlamydospores in medium with different nitrogen source

References 51

1	Berrocal-Lobo M， Molina A. Arabidopsis defense response against Fusarium oxysporum. Trends in Plant Science，2008， 13（3）： 145-150.
2	Dean R， Van Kan J A L， Pretorius Z A， et al. The top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology，2012， 13（4）： 414-430.
3	Fravel D， Olivain C， Alabouvette C. Fusarium oxysporum and its biocontrol. New Phytologist，2003， 157（3）： 493-502.
4	Gordon T R. Fusarium oxysporum and the Fusarium wilt syndrome. Annual Review of Phytopathology，2017， 55： 23-39.
5	Ma L J， Van Der Does H C， Borkovich K A， et al. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature，2010， 464（7287）： 367-373.
6	Michielse C B， Rep M. Pathogen profile update： Fusarium oxysporum. Molecular Plant Pathology，2009， 10（3）： 311-324.
7	Wang Z H， Fang X L. The research on genetic diversity of Fusarium oxysporum. Chinese Journal of Grassland， 2021， 43（5）： 106-114.
	王泽华，方香玲. 尖孢镰刀菌遗传多样性研究进展. 中国草地学报，2021， 43（5）： 106-114.
8	Edel-Hermann V， Lecomte C. Current status of Fusarium oxysporum formae speciales and races. Phytopathology，2019， 109（4）： 512-530.
9	Fang X L， Zhang C X， Wang Z， et al. Co-infection by soil-borne fungal pathogens alters disease responses among diverse alfalfa varieties. Frontiers in Microbiology，2021， 12： 1-15. DOI： 10.3389/fmicb.2021.664385.
10	Ohara T， Tsuge T. FoSTUA， encoding a basic helix-loop-helix protein， differentially regulates development of three kinds of asexual spores， macroconidia， microconidia， and chlamydospores， in the fungal plant pathogen Fusarium oxysporum. Eukaryotic Cell， 2004， 3（6）： 1412-1422.
11	Nan Z B. Establishing sustainable management system for diseases of pasture crops in China. Acta Prataculturae Sinica， 2000， 9（2）： 1-9.
	南志标. 建立中国的牧草病害可持续管理体系. 草业学报， 2000， 9（2）： 1-9.
12	Nan Z B. Alfalfa diseases and their comprehensive control system in China. Animal Science ＆ Veterinary Medicine， 2001， 18（4）： 81-84.
	南志标. 我国的苜蓿病害及其综合防治体系. 动物科学与动物医学，2001， 18（4）： 81-84.
13	Peterson J J， Samac D A， Grau C R. First report of Fusarium wilt of alfalfa caused by Fusarium oxysporum f. sp. medicaginis in Wisconsin. Plant Disease，2018， 102（2）： 447.
14	Rispail N， Rubiales D. Identification of sources of quantitative resistance to Fusarium oxysporum f. sp. medicaginis in Medicago truncatula. Plant Disease，2014， 98（5）： 667-673.
15	Williams A H， Sharma M， Thatcher L F， et al. Comparative genomics and prediction of conditionally dispensable sequences in legume-infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors. BMC Genomics，2016， 17（1）： 1-24. DOI： 10.3389/fmicb.2021.664385.
16	Fang X L， Zhang C X， Nan Z B. Research advances in Fusarium root rot of alfalfa （Medicago sativa）. Acta Prataculturae Sinica， 2019， 28（12）： 169-183.
	方香玲，张彩霞，南志标. 紫花苜蓿镰刀菌根腐病研究进展. 草业学报，2019， 28（12）： 169-183.
17	Wille L， Messmer M M， Studer B， et al. Insights to plant-microbe interactions provide opportunities to improve resistance breeding against root diseases in grain legumes. Plant， Cell & Environment，2019， 42（1）： 20-40.
18	Spraker J E， Sanchez L M， Lowe T M， et al. Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues. The ISME Journal，2016， 10（9）： 2317-2330.
19	Wang F， Sethiya P， Hu X， et al. Transcription in fungal conidia before dormancy produces phenotypically variable conidia that maximize survival in different environments. Nature Microbiology，2021， 6（8）： 1066-1081.
20	Xu S， Liu Y X， Cernava T， et al. Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts. Nature Microbiology， 2022， 7（6）： 831-843.
21	Ma C， Yang X R， Jiang G F， et al. Research progresses on key factors affecting survival of Ralstonia solanacearum in soils. Acta Pedologica Sinica， 2021， 58（6）： 1359-1367.
	马超，杨欣润，江高飞，等. 病原青枯菌土壤存活的影响因素研究进展. 土壤学报，2021， 58（6）： 1359-1367.
22	Fang X L， Barbetti M J. Differential protein accumulations in isolates of the strawberry wilt pathogen Fusarium oxysporum f. sp. fragariae differing in virulence. Journal of Proteomics，2014， 108（2014）： 223-237.
23	Henry P M， Pincot D D， Jenner B N， et al. Horizontal chromosome transfer and independent evolution drive diversification in Fusarium oxysporum f. sp. fragariae. New Phytologist，2021， 230（1）： 327-340.
24	Liu S， Li J， Zhang Y， et al. Fusaric acid instigates the invasion of banana by Fusarium oxysporum f. sp. cubense TR 4. New Phytologist， 2020， 225（2）： 913-929.
25	Masachis S， Segorbe D， Turrà D， et al. A fungal pathogen secretes plant alkalinizing peptides to increase infection. Nature Microbiology，2016， 1（6）： 1-9.
26	Wang C， Guo H， He X， et al. Scaffold protein GhMORG1 enhances the resistance of cotton to Fusarium oxysporum by facilitating the MKK6‐MPK4 cascade. Plant Biotechnology Journal，2020， 18（6）： 1421-1433.
27	Zuriegat Q， Zheng Y， Liu H， et al. Current progress on pathogenicity-related transcription factors in Fusarium oxysporum. Molecular Plant Pathology，2021， 22（7）： 882-895.
28	Akhter A， Hage-Ahmed K， Soja G， et al. Potential of Fusarium wilt-inducing chlamydospores， in vitro behaviour in root exudates and physiology of tomato in biochar and compost amended soil. Plant and Soil，2016， 406： 425-440.
29	De Cal A， Pascual S， Melgarejo P. Infectivity of chlamydospores vs microconidia of Fusarium oxysporum f. sp. lycopersici on tomato. Journal of Phytopathology，1997， 145（5/6）： 231-233.
30	Yun Y， Zhou X， Yang S， et al. Fusarium oxysporum f. sp. lycopersici C₂H₂ transcription factor FolCzf1 is required for conidiation， fusaric acid production， and early host infection. Current Genetics，2019， 65（3）： 773-783.
31	Sun L， Chu X J， Hao Y， et al. FoPLC4， encoding phospholipase C4， is involved in sporulation and pathogenicity in Fusarium oxysporum. Scientia Agricultura Sinica， 2014， 47（12）： 2357-2364.
	孙玲，褚小静，郝宇，等. 尖孢镰刀菌 FoPLC4 参与调控孢子形成和致病性. 中国农业科学，2014， 47（12）： 2357-2364.
32	Qu J X， Fang X L. Research progress on the spore formation and germination mechanism of plant pathogenic fungus Fusarium. Chinese Journal of Grassland， 2021， 43（8）： 106-113.
	屈佳欣，方香玲. 植物病原真菌镰刀菌孢子形成与萌发机理研究进展. 中国草地学报，2021， 43（8）： 106-113.
33	Ding Z J， Qi Y X， Zeng F Y， et al. Amino sugar metabolism pathway involved in chlamydospore formation of Fusarium oxysporum f. sp. cubense. Mycosystema， 2019， 38（4）： 485-493.
	丁兆建，漆艳香，曾凡云，等. 氨基糖代谢通路影响尖孢镰刀菌古巴专化型厚垣孢子的形成. 菌物学报，2019， 38（4）： 485-493.
34	Ding Z J， Qi Y X， Zeng F Y， et al. Amino acid involved in chlamydospore formation of Fusarium oxysporum f. sp. cubense. Mycosystema， 2021， 40（6）： 1413-1426.
	丁兆建，漆艳香，曾凡云，等. 氨基酸影响尖孢镰孢菌古巴专化型厚垣孢子的形成. 菌物学报，2021， 40（6）： 1413-1426.
35	Yang X Y， Li M， Zhang L， et al. Transcriptome analysis of Trichoderma harzianum Th-33 in chlamydospore formation. Chinese Journal of Biological Control， 2015， 31（1）： 85-95.
	杨晓燕，李梅，张林，等. 哈茨木霉 Th-33 厚垣孢子形成过程的转录组变化分析. 中国生物防治学报，2015， 31（1）： 85-95.
36	Qureshi A， Page O. Observations on chlamydospore production by Fusarium in a two-salt solution. Canadian Journal of Microbiology，1970， 16（1）： 29-32.
37	Schippers B， Old K. Factors affecting chlamydospore formation by Fusarium solani f. sp. cucurbitae in pure culture. Soil Biology and Biochemistry，1974， 6（3）： 153-160.
38	Zhang L， Jiang X L， Yang X Y， et al. Inhibition of chlamydospore germination and mycelial growth of Trichoderma spp. by chemical fungicides. Chinese Agricultural Science Bulletin， 2014， 30（33）： 150-155.
	张林，蒋细良，杨晓燕，等. 化学杀菌剂对木霉菌厚垣孢子萌发及菌丝生长的抑制作用. 中国农学通报，2014， 30（33）： 150-155.
39	Du Y X， Shi N N， Ruan H C， et al. Conditions for germination of Mycogone perniciosa chlamydospores. Fujian Journal of Agricultural Sciences， 2020， 35（1）： 67-73.
	杜宜新，石妞妞，阮宏椿，等. 有害疣孢霉厚垣孢子萌发特性研究. 福建农业学报，2020， 35（1）： 67-73.
40	Griffin G J. Roles of low pH， carbon and inorganic nitrogen source use in chlamydospore formation by Fusarium solani. Canadian Journal of Microbiology，1976， 22（9）： 1381-1389.
41	Smith S N， Snyder W. Germination of Fusarium oxysporum chlamydospores in soils favorable and unfavorable to wilt establishment. Phytopathology，1972， 62： 273-277.
42	Leslie J F， Summerell B A. The Fusarium laboratory manual. USA： Blackwell Publishing， 2006.
43	Peng H X， Sivasithamparam K， Turner D W. Chlamydospore germination and Fusarium wilt of banana plantlets in suppressive and conducive soils are affected by physical and chemical factors. Soil Biology and Biochemistry，1999， 31（10）： 1363-1374.
44	Fang X L， Kuo J， Finnegan P M， et al. Comparative root colonisation of strawberry cultivars Camarosa and Festival by Fusarium oxysporum f. sp. fragariae. Plant and Soil，2012， 358（1/2）： 75-89.
45	Steinkellner S， Mammerler R， Vierheilig H. Germination of Fusarium oxysporum in root exudates from tomato plants challenged with different Fusarium oxysporum strains. European Journal of Plant Pathology， 2008， 122（3）： 395-401.
46	Bennett R S， Davis R M. Method for rapid production of Fusarium oxysporum f. sp. vasinfectum chlamydospores. Journal of Cotton Science，2013， 17（1）： 52-59.
47	Goyal J， Maraite H， Meyer J. Abundant production of chlamydospores by Fusarium oxysporum f. sp. melonis in soil extract with glucose. Netherlands Journal of Plant Pathology，1973， 79： 162-164.
48	Zhang D F， Liu M T， Zhu S A. Study on inducing conditions of the chlamydospores from Fusarium oxyporium f. sp. cucumarinum. Journal of Henan Institute of Science and Technology， 2010， 38（1）： 30-32.
	张定法，刘鸣韬，朱盛安. 黄瓜枯萎病菌产生厚垣孢子诱导条件的研究. 河南科技学院学报，2010， 38（1）： 30-32.
49	Böttcher B， Pöllath C， Staib P， et al. Candida species rewired hyphae developmental programs for chlamydospore formation. Frontiers in Microbiology，2016， 7： 1-17. DOI： 10.3389/fmicb.2016.01697.
50	Gordon T， Martyn R. The evolutionary biology of Fusarium oxysporum. Annual Review of Phytopathology，1997， 35（1）： 111-128.
51	Sephton-Clark P C， Voelz K. Spore germination of pathogenic filamentous fungi. Advances in Applied Microbiology， 2018， 102： 117-157.