短密木霉新菌株BF06对黄瓜土传病害的防治效果与促生作用.pdf

343449-460 中国生物防治学报 Chinese Journal of Biological Control 2018 年 6 月 收稿日期 2017-10-18 基金项目国家自然科学基金（ 21525625）；北京市科技项目（ D151100003915003） 作者简介李进一，本科生， Email ； *通信作者，苏海佳，博士，教授， E-mail ；刘伟成，博士，研究员， E-mail 。 DOI 10.16409/ki.2095-039x.2018.03.015 Trichoderma brevicompactum BF06, a novel strain with potential for biocontrol of soil-borne diseases and promotion of plant growth of cucumber LI Jinyi1, LU Caige2, LIU Ting2, LIU Weicheng2*, SU Haijia1*1. College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China; 2.Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China Abstract Soil-borne diseases of cucumber have become the most important factor limiting cucumber production in China as a consequence of continuous cropping. These diseases are difficult to control by chemical and agricultural measures, and biocontrol is a desirable alternative strategy. Trichoderma brevicompactum strain BF06 isolated from forest soil exhibited strong antagonistic activity against five soil-borne phytopathogenic pathogens Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, Sclerotinia sclerotiorum, F. s o l a n i f. sp. cucurbitae, and Phytophthora cryptogea that cause cucumber fusarium wilt, basal stem rot, sclerotinia rot, root rot, and phytophthora blight, respectively. Greenhouse experiments indicated that control effects of BF06 on the five cucumber root diseases exceeded 60; notably, up to 90.4 and 88.8 on cucumber fusarium wilt and basal stem rot, respectively. In addition, BF06 improved plant growth by increasing the length of lateral shoots and number of lateral roots. Our results indicated that T. brevicompactum is a novel resource of potential biocontrol agents for effectively controlling cucumber root diseases caused by soil-borne pathogens. Key words Trichoderma brevicompactum; antagonism; cucumber; soil-borne pathogens; plant growth regulation 短密木霉新菌株 BF06对黄瓜土传病害的防治效果与促生作用 李进一1，卢彩鸽2，刘 霆2，刘伟成2*，苏海佳1*（ 1. 北京化工大学生命科学与技术学院，北京 100029； 2. 北京市农林科学院植物保护环境保护研究所，北京 100097） 摘要连作导致枯萎病等土传病害发生日益严重，已成为我国黄瓜生产的重要限制因素。采用化学防治和农业措施防治土传根部病害，操作都较为困难，迫切需要开发环境友好的生物防治技术。本研究从森林土壤分离鉴定一株短密木霉菌株BF06，通过对峙培养发现该菌株可以附着和缠绕病原菌的菌丝，对引起黄瓜枯萎病、茎基腐病、菌核病、根腐病和疫病的病原菌Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, Sclerotinia sclerotiorum, F. s o l a n i f. sp. cucurbitae, and Phytophthora cryptogea具有较强的拮抗作用。温室盆栽试验发现短密木霉菌株BF06可以迅速附着定殖于黄瓜根部表面，对上述5种黄瓜根部病害的防效达60以上，对黄瓜枯萎病和茎基腐病的防效分别为90.4和88.8。此外，利用植物组织培养基培养观察发现BF06显著地促进幼苗黄瓜侧根的形成和生长。本研究的结果表明短密木霉菌株BF06是一种可以有效防治黄瓜根部病害的新生防资源。 关 键 词 Trichoderma brevicompactum；拮抗；黄瓜；土传病菌；植物生长调控 中图分类号 S476 文献标识码 A 文章编号 1005-9261201803-0449-12 450 中 国 生 物 防 治 学 报 第 34 卷 Cucumber Cucumis sativus L. is an important and popular vegetable cash crop that is grown worldwide. Cucumber root diseases caused by soil-borne phytopathogenic fungi, including Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, Sclerotinia sclerotiorum, F. solani f. sp. cucurbitae, and Phytophthora cryptogea, are very destructive to cucumber production in most areas of the world[1-4]. In recent years in China, different soil-borne root diseases of cucumber have occurred simultaneously in greenhouse due to continuous cropping, resulting in frequent severe yield losses[5,6]. Practices that include crop rotations and use of fungicides to reduce the damage of these cucumber diseases have been suggested. However, it is difficult to efficiently prevent and cure soil-borne root diseases since most of these soil-borne pathogens can survive several years in soils without planting cucumber and new populations can be continuously generated by mutation[7]. Chemical fungicides have played important roles in controlling soil-borne root diseases of crops, but their overuse has favored the development of pathogens resistant to fungicides, and has not been friendly to the environment. The use of biological agents against these pathogens is risk-free and can enhance resident antagonists. Many Trichoderma species not only antagonize plant-pathogenic fungi[8, 9], but can even enhance plant growth and elicit plant defense responses to pathogen attack[10, 11]. In this study, we found that Trichoderma brevicompactum is a novel biocontrol agent that can effectively control root diseases caused by soil-borne pathogens and promote plant root growth of cucumber. 1 Materials and s 1.1 Isolation and identification of BF06 The T. brevicompactum BF06 was isolated from forest soil collected from Phoenix Mountain in Haidian District, Beijing. A 10 g soil sample was suspended in 100 mL of sterile-distilled water, serial dilutions of 10−1to 10−5were prepared, and 0.1 mL of each dilution was inoculated onto potato dextrose agar PDA plates. The plates were incubated at 26 C for 48 72 h. After that, typical colonies of Trichoderma were streaked on the same medium to get pure colonies. Preliminary screening for pure isolates with antagonistic activity against F. oxysporum f. sp. cucumerinum was conducted as described by Lin et al[12]. The strain BF06 with distinct antagonistic activity was obtained. The morphological characteristics of BF06 were observed under a microscope after being cultured on PDA plates at 26 C for 5 d, and its identification was further confirmed by partial nucleotide sequencing of internal transcribed spacers ITS using PCR with ITS1/ITS4 primers[13], and partial nucleotide sequencing of RNA polymerase II subunit RPB2 and translation elongation factor 1 alpha TEF1α with species-specific primers RPB2_210up/RPB2_1450low and tef85f/tef954r according to[14,15]. The PCR products were sequenced using an ABI3730XL DNA sequencer ABI Applied Biosystems, Shanghai, China. Publically available gene sequences of Trichoderma sp. strains were retrieved from the NCBI GenBank. All obtained sequences were compared with the NCBI nucleotide sequence database using the blast program. Nucleotide sequences of BF06 and other 25 Trichoderma sp. strains Table S1 were aligned using the ClustalW available in MEGA 5.2 program[16]. A phylogenetic tree was constructed by the neighbor-joining bootstrap analysis with 1000 replicates was conducted based on the partial sequences of ITS, RPB2 and TEF1α in MEGA 5.2. 1.2 Antagonistic activity assay for BF06 against pathogens on the dual culture Strain BF06 with antagonistic effect on F. oxysporum f. sp. cucumerinum was further tested for inhibitory activity against five other phytopathogenic fungi F. oxysporum f. sp. cucumerinum, R. solani, S. sclerotiorum, F. solani f. sp. cucurbitae, and P. cryptogea that cause cucumber fusarium wilt, basal stem rot, sclerotinia rot, root rot, and phytophthora blight, respectively. BF06 and the pathogens were respectively cultured at 26 C for 48 h. After that, 6-mm diameter mycelial pads with medium were excised from the edge of actively growing colonies. The mycelial pad of each pathogen was respectively placed on the surface of PDA plates together with that of 第 3 期 李进一等短密木霉新菌株 BF06 对黄瓜土传病害的防治效果与促生作用 451 BF06 at 3 cm apart from each other. For each pathogen, five plates containing the BF06 and pathogen were used for the experiment, while five plates inoculated with the pathogen alone served as the control. The experiment was repeated three times, and each replicate included five plates. The plates were incubated at 26 C in darkness for 5 d. The inhibitory activity was calculated mean pathogen colony radius of control − mean pathogen colony radius of dual culture/mean pathogen colony radius of control100. 1.3 Biocontrol test for BF06 against cucumber root diseases in greenhouse BF06 and the five pathogens Table 1 were cultured at 26 C for 3 d, and then 6 mm diameter mycelial pads with medium were excised from the edge of actively growing colonies of each fungus. The mycelial pads were placed into flasks containing PDB on a rotary shaker and cultured at 180 r/min for 7 d at 26 C. The harvested hyphae were suspended in sterilized water and shattered using Disperser IKA T18 IKA Works Inc., Guangzhou, China at 5000 r/min for 5 min, and 10 mL aliquots of suspension of propagules 1106CFU/mL were adjusted using a hemocytometer. Table 1 Antagonistic effects of T. brevicompactum BF06 against soil-borne phytopathogenic fungi on the dual culture Radius of pathogenic colony mm Phytopathogenic fungi Strain code Treatment Control Inhibitory activity F. oxysporum f. sp. cucumerinum HG15061107 4.10.0*37.00.0 88.90.3 R. solani HG14072204 8.90.0 42.00. 78.80.4 S. sclerotiorum HG11102015 4.80.1 42.00.0 88.60.2 F. solani f. sp. cucurbitae HG11082218 5.20.2 40.00.2 87.10.5 P. cryptogea HG14082114 8.70.2 40.90.2 78.70.5 * Data presented as meanstandard deviations were calculated from three independent experiments. Seeds of cucumber cv. Zhongnong No. 6 were soaked in 70 vol/vol ethanol for 2 min and washed three times with sterilized water, and the treated seeds were dipped in sterilized water for 1 d and then inoculated on wet filter paper in plates in darkness at 28 C for 3 5 d to germinate. The agricultural soil utilized was autoclaved at 121 C for 1 h in three successive days. The seedling pots containing 30 g of sterilized soil mixed with 10 mL propagule suspension of BF06 and 10 mL propagule suspension of one pathogen, and seedling pots containing soil only with added 10 mL suspension of pathogen used as control, were prepared. Next, germinating cucumber seeds with a 0.5 cm radicle were planted in the pots Table 2. The experiment was conducted with 30 pots per test, and was repeated three times. Table 2 Biocontrol effect of T. brevicompactum BF06 on cucumber root diseases Disease Treatment Disease index Control effect F. oxysporum f. sp. cucumerinumBF06 9.22.9 c*90.4 Cucumber fusarium wilt F. oxysporum f. sp. cucumerinum 95.02.5 a R. solaniBF06 10.80.7 c 88.8 Cucumber basal stem rot R. solani 96.91.0 aS. sclerotiorumBF06 22.80.4 b 73.4 Cucumber sclerotinia rot S. sclerotiorum 85.62.1 a F. solani f. sp. cucurbitaeBF06 30.83.1 c 66.1 Cucumber root rot F. solani f. sp. cucurbitae 90.84.3 aP. cryptogeaBF06 31.10.5 c 64.0 Cucumber phytophthora blight P. cryptogea 86.42.7 a Note Data presented as meanstandard deviations were calculated from three independent experiments. Means followed by the same letters within each column are not significantly different at P0.05 according to Duncan’s multiple range test. 452 中 国 生 物 防 治 学 报 第 34 卷 The pots were kept at 25 C in a greenhouse and watered daily with sterile water. Disease incidence was investigated at 15 d post-sowing. Disease severity was an arbitrary scale of 0 4, based on symptoms of infected plants 0, growth of seedlings was normal, no lesions; 1, light lesions on hypocotyl or cotyledon; 2, obvious necrosis on hypocotyl, cotyledon, or cotyledon etiolation; 3, local wilting of seedling, or cotyledon withered; and 4, whole seedling wilted, lodged, or dead. The disease index was calculated Disease index[∑each scale valuenumber of seedlings infected with the corresponding scale/total number of seedlings tested the highest scale value]100. The control effectdisease index of control disease index of BF06 treatment/disease index of control 100. 1.4 Monitoring for interaction of BF06 with F. oxysporum f. sp. cucumerinum and cucumber Green fluorescent protein GFP and red fluorescent protein RFP were used to label T. brevicompactum strain BF06 and F. oxysporum f. sp. cucumerinum strain HG15061107, respectively. For amplification of GFP region from pMDC43 vector[17]and creation of compatible restriction sites for HindIII and XbaI, forward 5′-GATCTCTAGAATGAGTAAAGGAGAAGAACTTTTCA-3′ and reverse 5′-GATCAAGCTTTTATTTGTA TAGTTCATCCATGCC-3′ primers each containing a 5′-oligonucleotide extension underlined nucleotides were used. For amplification of RFP region from pGWB554[18]and creation of compatible restriction sites for Hind III and Xba I, forward 5′-GATCTCTAGAATGGCCTCCTCCGAGGACG-3′ and reverse 5′-GATCAAGCTTTTA GGCGCCGGTGGAGTG-3′ primers each containing a 5′-oligonucleotide extension were used. The 717 bp GFP and 678 bp RFP fragments were respectively ligated into the vector pCTHyg digested with Hind III and XbaI, and the construction of recombinant pCTHyg vector containing GFP and RFP under the control of Aspergillus nidulans gpd gene promoter and Nos terminator was carried out using standard cloning techniques. The construction of pCTHyg-GFP and pCTHyg-RFP were verified by sequencing of the plasmid in both directions, and pCTHyg-GFP and pCTHyg-RFP were respectively transed into Agrobacterium tumefaciens AGL-1. The T. brevicompactum BF06 was grown on PDA plates at 26 C for 5 d and spores were harvested; F. oxysporum f. sp. cucumerinum strain HG15061107 was grown in flasks containing PDB on a rotary shaker at 180 r/min for 3 d at 26 C and spores were collected. Vectors pCTHyg–GFP and pCTHyg–RFP were transed into BF06 and HG15061107 by A. tumefaciens-mediated transation according to Xu et al[19]. A transant named T. brevicompactum gpdgfp with strong green fluorescence intensity and a transant F. oxysporum f. sp. cucumerinum gpdrfp with strong red fluorescence intensity were obtained. Co-culture studies of interaction between the T. brevicompactum gpdrfp strain and F. oxysporum f. sp. cucumerinum gpdrfp strain were pered on glass slides on which 150 μL of 25 PDA was spread flat onto an area 25 mm15 mm1.5 mm as described by Lu et al[20]. A 6 mm diameter mycelial pad of T. brevicompactum and a mycelial pad of 6 mm diameter F. oxysporum f. sp. cucumerinum from the margin of actively growing colonies were inoculated on the PDA area at opposite ends. The slides were incubated with the PDA-side up at 26 C in darkness in a Petri dish containing a layer of sterilized filter paper and sealed with Parafilm. After 72 h, the slides were viewed with a confocal scanning laser microscope CSLM, Zeiss Microsystems LSM 700. The germinating cucumber seeds with 0.5 cm radicle were planted in seedling pots which contained 30 g of sterilized soil mixed with 10 mL suspension of T. brevicompactum GFPgpd as described in 2.3. The pots were kept at 25 C in a greenhouse, and the colonization of T. brevicompactum GFPgpd on the root surface of cucumber seedlings was viewed with a CSLM at 2 and 4 d after inoculation. 1.5 Effect of BF06 on seedling growth The effect of T. brevicompactum BF06 on growth of cucumber seedlings was determined using the modified s described by Contreras-Cornejo et al[21]. Seeds of cv. Zhongnong No. 6 were sterilized with 95 v/v ethanol for 10 min and 20 v/v NaClO for 5 min. After rinsing in distilled water fi