陈敬师, 黄玉洋, 向杰, 郭清华, 李世贵, 顾金刚^,中国农业科学院农业资源与农业区划研究所农业农村部农业微生物资源收集保藏重点实验室,北京100081

Carbon Source Metabolism of Trichoderma afroharzianum with High-Yield of Antifungal Volatile Organic Compounds

CHEN JingShi, HUANG YuYang, XIANG Jie, GUO QingHua, LI ShiGui, GU JinGang^,Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture and Rural Affairs, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081

通讯作者: 顾金刚,E-mail： gujingang@caas.cn

责任编辑: 岳梅
收稿日期:2020-03-7接受日期:2020-04-11网络出版日期:2020-11-16

基金资助:

国家重点研发计划.2017YFD0200604

Received:2020-03-7Accepted:2020-04-11Online:2020-11-16
作者简介 About authors
陈敬师,E-mail： 13126830210@163.com。








摘要
【目的】 获得生防菌非洲哈茨木霉（Trichoderma afroharzianum）ACCC 33109高产抑菌挥发性有机物的突变菌株,并分析其碳源代谢以挖掘高产抑菌挥发性有机物的机制。【方法】 通过原生质体紫外诱变野生株ACCC 33109获得突变株,以对扣法筛选挥发性有机物抑制尖镰孢（Fusarium oxysporum）活性差异大的突变菌株,利用Omnilog表型芯片技术比较野生株ACCC 33109和突变株MU153、MU792对不同种类碳源代谢的差异特征。【结果】 野生株ACCC 33109经紫外线诱变2.0 min（致死率为76.63%）共获得828个突变株,对扣法筛选获得30个挥发性有机物对尖镰孢抑制率高于野生株的突变株,其中突变株MU153抑菌率高达53.86%,较野生株提高了16.68%,而突变株MU792的抑菌率降至15.83%。盆栽试验表明非洲哈茨木霉ACCC 33109和MU153菌株均对黄瓜具有促生作用,对黄瓜枯萎病有防治作用,与野生株ACCC 33109相比,突变株MU153对黄瓜枯萎病的相对防治效果提高了15.88%,相对于清水对照,防病效果高达89.69%。另外,紫外诱变导致突变株的菌落、菌丝、分生孢子梗和孢子形态发生了变化,相对于ACCC 33109,MU153菌丝变为絮状、生长速度快、密度大、并有色素产生,而MU792菌丝生长速度慢、密度低、菌落后期由绿色变为白色;突变株MU153和MU792分生孢子梗增大、孢子变大,且分生孢子梗基部宽度降低。紫外诱变引起突变株对碳源代谢能力的改变,相对于ACCC 33109,MU153对FF板中46种物质的代谢能力较高,包括D-阿拉伯醇、二胺乙醇、麦芽糖、熊果苷、纤维二糖和α-D-葡萄糖等,对其他50种物质的代谢能力低于ACCC 33109,包括对羟基苯乙酸、琥珀酸、琥珀酰胺酸、糖原、溴代丁二酸和L-苏氨酸等。MU792对FF板中27种物质的代谢能力较高,包括γ-羟丁酸、葡萄糖-1磷酸、β-羟丁酸、D-乳酸甲酯、D-山梨醇和丙酰胺等,对其他69种物质的代谢能力低于ACCC 33109,包括琥珀酰胺酸、N-乙酰-D-葡萄糖胺、对羟基苯乙酸、葵二酸、吐温-80和D-糖质酸。α-D-葡萄糖最利于抑菌挥发性有机物的产生,以α-D-葡萄糖为碳源时,ACCC 33109、MU153和MU792的挥发性有机物对尖镰孢的抑菌率依次为48.08%、56.17%和40.94%。【结论】 非洲哈茨木霉突变株MU153具有高产抑菌挥发性有机物的能力,以α-D-葡萄糖为碳源时抑菌效果更佳,是一株具有应用潜力的生防菌。
关键词： 非洲哈茨木霉;挥发性有机物;紫外诱变;表型分析

Abstract
【Objective】The objective of this study is to obtain mutant strains of Trichoderma afroharzianum ACCC 33109 with high yield of inhibitory volatile organic compounds (VOCs) and analyze the carbon utilization mechanism.【Method】Mutant strains were obtained through protoplast ultraviolet mutagenesis of wild-type ACCC 33109 and screened by sandwiched Petri plate method. The wild type and mutant strains MU153, MU792 were then subjected to carbon utilization analysis using Omnilog phenotype microassays.【Result】A total of 828 mutant strains were obtained by 2.0 min ultraviolet mutagenesis, with the lethality rate of 76.63%. Among them, 30 mutants showed higher inhibitory activities against Fusarium oxysporum than the wild type. MU153 showed the highest inhibitory rate (53.86%), which was 16.68% higher than that of the wild type, while the inhibitory rate of MU792 was as low as 15.83%. The pot experiments showed that both ACCC 33109 and MU153 had the effects of promoting cucumber growth and preventing cucumber fusarium wilt. Compared with ACCC 33109, the relative control effect of MU153 on cucumber fusarium wilt increased by 15.88%, which was as high as 89.69%. UV mutagenesis caused changes in the morphologies of colony, hypha, and conidiogenous structures and metabolic capacity to utilize carbon sources of the mutants. Compared with ACCC 33109, the mycelia of MU153 grew rapidly and densely, became flocculent, and pigment was produced, while the mycelia of MU792 grew slowly and loosely, and the colony color changed from green to white at the late stage. The sizes of conidia and pedicels of MU153 and MU792 increased, and the base width of the conidia decreased. Moreover, MU153 had a higher metabolic capacity for 46 substances in FF plate, including D-arabinol, diethanolamine, maltose, arbutin, cellobiose and α-D-glucose, but less active on the other 50 substances, such as 4-hydroxyphenylacetic acid, succinic acid, succinamic acid, glucogen, bromosuccinic acid and L-threonine. MU792 had a higher metabolic capacity for 27 substances in FF plate, including γ-hydroxybutyric acid, glucose-1-phosphate, β-hydroxybutyric acid, D-methyl lactate, D-sorbitol and propanamide, and lower metabolic capacity on the other 69 substances, including succinamic acid, N-acetyl-D-glucosamine, 4-hydroxyphenylacetic acid, sebacic acid, Tween-80, D-saccharic acid. α-D-Glucose was the most favorable substrate for antifungal VOCs production. With α-D-glucose as the carbon source, the inhibitory rates of VOCs of ACCC 33109, MU153 and MU792 to F. oxysporum were 48.08%, 56.17% and 40.94%, respectively. 【Conclusion】T. afroharzianum MU153 has the capability of producing a large amount of inhibitory VOCs, favors α-D-glucose for the highest yield of VOCs, and thus represents a great candidate of biocontrol agent.
Keywords：Trichoderma afroharzianum;volatile organic compounds (VOCs);ultraviolet mutagenesis;phenotypic analysis


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本文引用格式
陈敬师, 黄玉洋, 向杰, 郭清华, 李世贵, 顾金刚. 非洲哈茨木霉产抑菌挥发性有机物碳源代谢机制[J]. 中国农业科学, 2020, 53(22): 4601-4612 doi:10.3864/j.issn.0578-1752.2020.22.007
CHEN JingShi, HUANG YuYang, XIANG Jie, GUO QingHua, LI ShiGui, GU JinGang. Carbon Source Metabolism of Trichoderma afroharzianum with High-Yield of Antifungal Volatile Organic Compounds[J]. Scientia Agricultura Sinica, 2020, 53(22): 4601-4612 doi:10.3864/j.issn.0578-1752.2020.22.007


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0 引言

【研究意义】木霉具有生长快、适应性强和抑菌谱广等特点,对辣椒炭疽病菌（Colletotrichum capsici）、灰霉菌（Botrytis cinerea）、镰孢菌（Fusarium spp.）、疫霉（Phytophthora spp.）和葡萄钩丝壳菌（Uncinula necator）等病菌引起的病害均具有很好的防治效果^[1,2,3,4]。木霉的生防机制包括营养和生态位竞争、重寄生、拮抗、促生、诱导植物产生系统抗性和产生抑菌性次级代谢产物等。木霉产生的抑菌性次级代谢产物主要分为3类：一是挥发性有机物（volatile organic compounds,VOCs）,二是水溶性化合物（water-soluble compounds）,三是哌珀霉素类（peptaibols）。挥发性有机化合物在水、空气和土壤中传播,并且传播距离较远,因此较其他次级代谢产物在防治土传病害方面有更大的优势^[5,6,7]。碳源和氮源是微生物生长的必需物质,不同碳源和氮源对其生长、产孢和抑菌性次级代谢物质的产生影响较大^[8,9],YONG等^[10]研究了碳源对绿色木霉（Trichoderma viride）产抑菌性气体6-戊基-吡喃-2酮的影响,发现甘油和葡萄糖均能促进绿色木霉产生6-戊基-吡喃-2酮,而山梨醇和酸类物质不利于绿色木霉产生6-戊基-吡喃-2酮。因此,研究木霉对碳源的代谢机制,对提高木霉生物防治的应用潜力具有重要意义。【前人研究进展】目前,已被鉴定的具有生物活性的挥发性有机化合物有1 006种^[11],其中约390种由木霉产生^[12];具有广谱抑菌性的哌珀霉素类物质有220种^[13,14,15],至少12种来自木霉,这些物质在诱导植物抗性和抑制土壤和根际微生物生长方面具有重要作用^[16,17,18]。不同木霉菌株产生的挥发性有机物组分存在明显差异,如木霉T36和T50菌株产生的挥发性有机物均可抑制禾谷镰孢（Fusarium graminearum）、立枯丝核菌（Rhizoctonia solani）和终极腐霉（Pythium ultimum）的生长,但T36菌株对3种病原菌的抑制率均高于T50^[19]。BELéN等^[20]研究发现,Thmbf 1与哈茨木霉（Trichoderma harzianum）抑菌挥发性有机物的产生密切相关,过表达Thmbf 1会降低哈茨木霉挥发性有机物对尖镰孢和灰霉菌的抑制率;PACHAURI等^[21]研究发现,木霉挥发性有机物中包含大量的萜类物质,其形成与3-磷酸甘油醛脱氢酶息息相关。【本研究切入点】针对非洲哈茨木霉（Trichoderma afroharzianum）ACCC 33109及其突变株产生的挥发性有机物对多种病原菌具有抑制作用,且不同碳源条件下菌株抑菌率存在差异的现象,采用Omnilog表型芯片技术分析菌株高产抑菌挥发性有机物与碳源代谢种类之间的关系。【拟解决的关键问题】通过原生质体紫外诱变获得高产抑菌挥发性有机物的突变株,明确与抑菌挥发性有机物产生相关碳源种类,为促进木霉菌株高产挥发性代谢物并高效应用于生物防治提供理论依据。

1 材料与方法

试验于2018年9月至2019年12月在中国农业科学院农业资源与农业区划研究所完成。

1.1 材料

1.1.1 菌种 非洲哈茨木霉ACCC 33109和尖镰孢（黄瓜枯萎病菌,Fusarium oxysporum sp. cucumebrium）ACCC 37438,均保藏于中国农业微生物菌种保藏管理中心（ACCC）。

1.1.2 培养基 PDA、CMA和SNA培养基配方详见文献[22]。原生质体再生培养基：葡萄糖 20 g,琼脂20 g,酵母膏1 g,(NH₄)₂SO₄ 1.4 g,KH₂PO₄ 2 g,MgSO₄·7H₂O 0.3 g,FeSO₄·7H₂O 0.05 g,MnSO₄ 0.01 g,ZnSO₄·7H₂O 0.01 g,以0.6 mol·L^-1的NaCl渗透压稳定剂定容至1 L。合成培养基：果糖20 g,琼脂20 g,酵母膏3 g,苏氨酸3 g,K₂HPO₄ 1 g,MgSO₄·4H₂O 0.5 g,KCl 0.5 g,ZnSO₄·7H₂O 0.1 g,FeSO₄ 0.01 g,以蒸馏水定容至1 L。

1.1.3 主要试剂与仪器 蜗牛酶、纤维素酶、培养基（CMA、PDA、PDB和SNA）均购自北京百乐欣生物科技有限公司。紫外光催化/反应箱（CBIO21）购自北京赛百奥科技有限公司。Omnilog微生物表型分析系统（71000）购自美国Biolog有限公司。

1.2 方法

1.2.1 原生质体制备 ACCC 33109的新鲜菌丝转接入含50 mL PDB培养基的三角瓶中,在28℃、200 r/min条件下培养36 h。菌丝经8层无菌纱布过滤,以0.6 mol·L^-1 NaCl冲洗至显微镜下观察无孢子。称取0.5 g菌丝放入已灭菌的50 mL离心管中,加入10 mL酶解液（蜗牛酶﹕纤维素酶=1﹕1）37℃酶解2 h。以布氏漏斗和6层擦镜纸过滤,并以0.6 mol·L^-1 NaCl冲洗,离心（4℃、5 000 r/min、10 min）去除酶解液,0.6 mol·L^-1 NaCl洗涤2次后悬浮原生质体于5 mL的0.6 mol·L^-1 NaCl中。在显微镜下以血球计数板计数,调整原生质体浓度至 1.0×10⁶个/mL,4℃冰箱保存待用^[23]。

1.2.2 原生质体紫外诱变 将浓度为1.0×10⁶个/mL的原生质体在距离紫外灯20 cm处分别照射0、0.5、1.0、1.5、2.0、2.5、3.0和3.5 min,分别取50 μL涂布于原生质体再生培养基,28℃避光培养至长出单菌落。计算诱变致死率,致死率= [1-（诱变后原生质体再生菌落数/未诱变原生质体再生菌落数）]×100%。选取合适的剂量进行诱变处理,并在红光下涂布于原生质体再生培养基,28℃黑暗培养,挑取单菌落纯化待用。

1.2.3 对扣试验 将ACCC 33109、ACCC 37438和突变株分别在PDA上活化,7 d后以打孔器（Φ=6 mm）分别在菌落边缘打取菌饼并接种到PDA中央,二者对扣,中间以无菌玻璃纸隔开,用两层封口膜密封,28℃恒温培养。对扣无ACCC 33109野生株和突变株菌饼的ACCC 37438为对照,每个处理设置3个重复,连续5 d测量菌落半径,并计算抑菌率。抑菌率=（对照菌落直径-处理菌落直径）/对照菌落直径×100%。

1.2.4 ACCC 33109和MU153对黄瓜促生防病效果 以打孔器（Φ=6 mm）获得活化好的ACCC 33109、MU153和ACCC 37438菌饼,接种到PDA培养基,7 d后分别用无菌水洗下孢子,用6层纱布和2层擦镜纸过滤至显微镜下观察无菌丝,调整孢子浓度为1.0×10 ⁷ conidia/mL备用。盆栽试验共设置2个对照和4个处理（表1）,每个处理10个重复。移栽黄瓜幼苗后观察记录黄瓜发病死亡情况,6周后统计黄瓜枯萎病发病级数,并计算病情指数、发病率和相对防治效果。黄瓜枯萎病发病级数：0级：黄瓜不发病;1级：叶片轻微黄化萎蔫;2级：叶片轻度黄化萎蔫,植株矮化;3级：叶片重度黄化萎蔫,植株明显矮化;4级：植株死亡。病情指数=100×∑（病情级值×该级病情株数）/（病情最高级值×总株数）;发病率=发病植株数/调查总株数×100%;相对防治效果=（对照区病情指数-处理区病情指数）/对照区病情指数×100%^[24]。

Table 1
表1
表1盆栽试验设计
Table 1Design of the pot experiment

	处理Treatment
无菌水对照CK1	等量无菌水 Sterile ddH2O
病原菌对照CK2	ACCC 37438孢子悬浮液 ACCC 37438 spore suspension (1.0×10 7 conidia/g soil)
处理1 Treatment 1	ACCC 33109孢子悬浮液ACCC 33109 spore suspension (1.0×10 7 conidia/g soil)
处理2 Treatment 2	ACCC 37438和ACCC 33109孢子混合液 ACCC 37438 and ACCC 33109 spore suspensions (each at 1.0×10 7 conidia/g soil)
处理3 Treatment 3	MU153孢子悬浮液MU153 spore suspension (1.0×10 7 conidia/g soil)
处理4 Treatment 4	ACCC 37438和MU153孢子混合液ACCC 37438 and MU153 spore suspensions (each at 1.0×10 7 conidia/g soil)

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1.2.5 菌落形态和分生孢子梗结构比较 将ACCC 33109和突变株MU153、MU792在PDA上活化7 d,以打孔器（Φ=6 mm）获得菌饼,分别接种到CMA、PDA和SNA培养基中央,28℃黑暗培养,每天在体式显微镜下观察记录各菌株生长和形态变化。

采用插片法,将ACCC 33109和突变株MU153、MU792的菌饼分别接种到PDA培养基中央,同时将无菌盖玻片斜插到PDA培养基中,28℃黑暗培养3 d,光学显微镜下观察菌丝、分生孢子和分生孢子梗的形态特征。

1.2.6 营养利用比较 Omnilog表型分析：将ACCC 33109和突变株MU153、MU792在PDA上活化7 d,用无菌棉签沾取孢子并转入FF-IF接种液,调节菌悬液浊度至75% T,然后将100 μL接种液加入到FF板微孔中,在Omnilog培养箱26℃条件下培养7 d。利用Data File Converter、OL_FM_12和OL_PR_12软件以Area为参数进行数据分析,Area值越大表明菌株对该底物利用率越高。

优化培养基的抑菌效率：依据Omnilog表型分析结果,分别将合成培养基中的碳源果糖换为α-D-半乳糖、糊精、麦芽糖、α-D-葡萄糖和吐温-80进行ACCC 33109和突变株MU153、MU792与ACCC 37438的对扣试验。每个处理设置3个重复,连续5 d计算抑菌率。

1.3 数据处理与分析

采用Microsoft Excel 2003和SPSS v. 24软件进行数据整理、统计分析与图表制作。

2 结果

2.1 紫外诱变时间及诱变菌株获得

当紫外诱变微生物的致死率为75%—80%时,正突变率较高^[25]。非洲哈茨木霉ACCC 33109的原生质体经紫外灯（25 W）照射1.0—1.5 min后致死率上升最快。紫外诱变2.0 min时的致死率为76.63%,是紫外诱变的最佳时间（图1）。经多批次原生质体紫外诱变,优先挑取生长较快、菌落直径较大且菌丝较密的菌落,涂布于原生质体再生培养基,最终获得828个突变株,分别记为MU1—MU828。

图1

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图1紫外诱变时间对非洲哈茨木霉ACCC 33109原生质体致死率的影响

Fig. 1Effects of UV mutagenesis duration on the protoplast lethality rate of T. afroharzianum ACCC 33109



基于平板对扣试验,非洲哈茨木霉ACCC 33109及其突变株均能不同程度地抑制尖镰孢ACCC 37438的菌丝生长,且抑制效果随时间的延长而增强,表现为病原菌菌丝生长缓慢,色素减少,菌落边缘菌丝稀疏且不规则生长（图2）。突变株均能产生具有抑菌作用的挥发性有机物,抑制效果差异较大。其中抑菌率＜10%的有146株,10%—20%有270株,20%—30%有315株,30%—40%有70株,40%—50%有25株,＞50%有2株,30个突变株的抑菌率高于野生株ACCC 33109（37.18%）。其中,突变株MU153和MU792经过多次传代培养性状稳定,抑菌率分别为53.86%和15.83%,且菌落形态与野生株有明显差异。

图2

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图2非洲哈茨木霉挥发性有机物对尖镰孢的抑菌活性（28℃, 5 d）

Fig. 2Inhibitory activities of the VOCs produced by T. afroharzianum against F. oxysporum (28℃, 5 d)

2.2 ACCC 33109和MU153对黄瓜促生和防病效果

盆栽试验中,CK2（病原菌对照）、处理2和处理4均在第2周开始发病,表现为黄瓜根部开始变为褐色等,CK1（清水对照）、处理1和处理3生长正常。第3周处理2和处理4发病情况减轻,CK2发病情况变重,表现为植株矮小、根部和茎基部褐色加深和叶片开始黄化等,CK1、处理1和处理3生长正常。第4周CK2黄瓜开始出现整株枯萎死亡现象,CK1、处理1、处理2、处理3和处理4生长正常。

图3

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图3非洲哈茨木霉防治黄瓜枯萎病盆栽试验

Fig. 3A pot experiment of T. afroharzianum against cucumber fusarium wilt



6周后,处理2和处理4的发病率较CK2分别下降了55.56%和66.67%,病情指数分别下降了38.89和47.23。突变株MU153对黄瓜枯萎病的相对防治效果为89.69%,相对野生株ACCC 33109提高了15.88% （表2）。ACCC 33109和MU153不仅可以防治黄瓜枯萎病,而且可以促进黄瓜生长,表现为植株株高增大、叶片增多和须根发达等（图3）。

Table 2
表2
表2黄瓜枯萎病发病率、病情指数和相对防治效果
Table 2Incidence rate, disease index and relative control effect of cucumber fusarium wilt

处理 Treatment	发病率 Incidence rate (%)	病情指数 Disease index	相对防治效果 Relative control effect (%)
无菌水对照CK1	0	0	-
病原菌对照CK2	88.89±19.24	52.78±4.81	-
处理1 Treatment 1	0	0	-
处理2 Treatment 2	33.33±0	13.89±4.82	73.81±8.59
处理3 Treatment 3	0	0	-
处理4 Treatment 4	22.22±19.24	5.55±4.81	89.69±9.01

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2.3 野生菌株和突变菌株形态特征比较

非洲哈茨木霉ACCC 33109和突变株MU153、MU792在CMA、PDA和SNA培养基28℃培养3 d后除SNA上生长的MU792外均长满平板,但菌落形态有较大差异（图4）。在CMA培养基上,ACCC 33109菌落白色,产生不明显轮纹,菌落圆形,气生菌丝丰富,为绒毛状,不产生色素;MU153菌落初为白色,第3天变为浅绿色,菌落圆形,气生菌丝丰富,菌落背面颜色由无色变为浅黄色;MU792菌落始终为白色,呈放射状,形成明显的同心轮纹,气生菌丝较少,不产生色素。在PDA培养基上,ACCC 33109菌落白色、圆形,气生菌丝丰富,为絮状,不产生色素;MU153菌落初为白色,后变为绿色,菌落圆形,气生菌丝丰富,菌落背面颜色由无色变为浅绿色;MU792菌落始终为白色,气生菌丝丰富,为絮状,不产生色素。在SNA培养基上,ACCC 33109菌落由白色变为浅绿色,呈放射状,菌落边缘不规则,气生菌丝稀疏,不产生色素;MU153菌落由白色变为浅绿色,呈放射状,气生菌丝稀疏,呈绒毛状,菌落背面颜色由白色变为浅绿色;MU792生长较慢,培养3 d后菌落半径3.8—4.0 cm,菌落始终为白色,气生菌丝丰富,呈绒毛状,不产生色素。

图4

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图4非洲哈茨木霉ACCC 33109、MU153和MU792在不同培养基上的菌落形态

Fig. 4Colony morphology of T. afroharzianum ACCC 33109, MU153 and MU792 on CMA, PDA, and SNA plates



非洲哈茨木霉ACCC 33109、MU153和MU792在PDA平板28℃培养3 d后均产生分生孢子梗和分生孢子（图5）。原生质体紫外诱变提高了MU153的瓶梗长度、瓶梗长/宽、支持细胞宽度、支持细胞长度、孢子长、宽和孢子长/宽,降低了MU153分生孢子梗最宽处和基部宽;提高了MU792的瓶梗长/宽、支持细胞长度、孢子长和宽,降低了MU792分生孢子梗最宽处、基部宽、支持细胞宽度和孢子长/宽（表3）。

图5

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图5非洲哈茨木霉ACCC 33109、MU153和MU792的分生孢子梗结构

Fig. 5Conidiogenous structures of T. afroharzianum ACCC 33109, MU153 and MU792



Table 3
表3
表3非洲哈茨木霉ACCC 33109、MU153和MU792的分生孢子梗相关数据
Table 3Conidiogenous data of T. afroharzianum ACCC 33109, MU153 and MU792

参数Parameter	ACCC 33109	MU153	MU792
瓶梗长度Phialide length (μm)	9.44±1.60	10.61±2.15	8.95±2.03
瓶梗最宽处Phialide maximum width (μm)	3.00±0.33	2.71±0.39	2.55±0.28
瓶梗长/宽Phialide length/width ratio	3.16±0.65	3.99±1.01	3.58±0.99
瓶梗基部宽度Phialide base width (μm)	2.19±0.33	2.03±0.34	1.79±0.28
支持细胞宽度Supporting cell width (μm)	2.68±0.45	2.72±0.42	2.56±0.37
支持细胞长度Supporting cell length (μm)	10.68±3.49	12.73±3.46	11.43±3.93
孢子长度Conidium length (μm)	2.93±0.44	3.09±0.40	3.04±0.40
孢子宽度Conidium width (μm)	2.36±0.22	2.44±0.19	2.58±0.34
孢子长/宽Conidium length/width ratio	1.24±0.17	1.27±0.15	1.19±0.16

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2.4 野生菌株和突变菌株对碳源代谢表型分析

图6结果表明ACCC 33109、MU153和MU792均可代谢FF板中所有物质,利用率有显著差别,MU153对FF板中46种物质的代谢能力高于ACCC 33109,达到显著水平的有2种,分别为甘油和麦芽糖。MU792对FF板中69种物质的代谢能力低于ACCC 33109,达到显著水平的物质有8种,分别为β-环糊精、D-半乳糖醛酸、D-蜜二糖、水苏糖、木糖醇、D-糖质酸、琥珀酰胺酸和琥珀酸,达到极显著水平的物质有4种,分别为D-棉子糖、γ-羟基丁酸、对羟基苯乙酸和葵二酸。ACCC 33109利用率低于MU153且高于MU792的物质有29种,达到显著水平的物质有2种,分别为α-D-半乳糖和吐温-80,达到极显著水平的物质有1种,为D-葡萄糖醛酸（表4）。

图6

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图6非洲哈茨木霉碳源代谢热图（26℃, 7 d）

1：水Water;2：吐温-80 Tween-80;3：N-乙酰-D-半乳糖胺N-Acetyl-D-galactosamine;4：N-乙酰-D-葡萄糖胺N-Acetyl-D-glucosamine;5：N-乙酰-D-甘露糖胺N-Acetyl-D-mannosamine;6：核糖醇Ribitol;7：扁桃苷Amygdalin;8：D-阿拉伯糖D-Arabinose;9：L-阿拉伯糖L-Arabinose;10：D-阿拉伯醇 D-Arabinol;11：熊果苷Arbutin;12：纤维二糖Cellobiose;13：α-环糊精α-Cyclodextrins;14：β-环糊精β-Cyclodextrin;15：糊精Dextrin;16：1-赤藓糖醇1-Erythritol;17：D-果糖D-Fructose;18：L-岩藻糖L-Trehalose;19：α-D-半乳糖α-D-Galactose;20：D-半乳糖醛酸D-Galacturonic acid;21：龙胆二糖Gentiobiose;22：D-葡萄糖酸D-Gluconic acid;23：D-葡萄糖胺D-Glucosamine;24：α-D-葡萄糖α-D-Glucose;25：葡萄糖-1磷酸Glucose-1- phosphate;26：葡糖苷酸Glucosiduronide;27：D-葡萄糖醛酸D-Glucuronic acid;28：甘油Glycerol;29：糖原Glucogen;30：肌醇Inositol;31：D-2-酮-D-葡萄糖酸D-2-Ketone-D-gluconic acid;32：α-D-乳糖α-D-Galactose;33：乳果糖Lactulose;34：麦芽糖醇Maltitol;35：麦芽糖Maltose;36：麦芽三糖Maltotriose;37：D-甘露醇D-Mannitol;38：D-甘露糖D-Mannose;39：D-松三糖D-Melezitose;40：D-蜜二糖D-Melibiose;41：α-甲基-D-半乳糖苷α-Methyl-D-galactoside;42：β-甲基-D-半乳糖苷β-Methyl-D-galactoside;43：α-甲基-D-葡萄糖苷α-Methyl-D-glucoside;44：β-甲基-D-葡萄糖苷β-Methyl-D-glucoside;45：异麦芽酮糖Isomaltulose;46：D-阿洛酮糖D-Psicose;47：D-棉子糖D-Raffinose;48：L-鼠李糖L-Rhamnose;49：D-核糖D-Ribose;50：水杨苷Salicin;51：景天庚酮糖Sedoheptulose;52：D-山梨醇D-Sorbitol;53：L-山梨糖L-Sorbose;54：水苏糖Tachyose;55：蔗糖Sucrose;56：D-塔格糖D-Tagatose;57：D-海藻糖D-Trehalose;58：松二糖Turanose;59：木糖醇Xylitol;60：D-木糖D-Xylose;61：γ-氨基丁酸γ-Aminobutyric acid;62：溴代丁二酸Bromosuccinic acid;63：富马酸Fumaric acid;64：β-羟丁酸β-Hydroxybutyric acid;65：γ-羟丁酸 γ-Hydroxybutyric acid;66：对羟基苯乙酸4-Hydroxyphenylacetic acid;67：α-酮戊二酸α-Ketoglutaric acid;68：D-乳酸甲酯D-Methyl lactate;69：L-乳酸L-Lactic acid;70：D-苹果酸D-Malic acid;71：L-苹果酸L-Malic acid;72：奎尼酸Quinic acid;73：D-糖质酸D-Saccharic acid;74：葵二酸 Sebacic acid;75：琥珀酰胺酸Succinamic acid;76：琥珀酸Succinic acid;77：琥珀酸单甲酯Succinic acid monomethyl ester;78：N-酰-谷氨酸 N-Acetyl-glutamic acid;79：丙酰胺Propanamide;80：L-丙氨酸L-Alanine;81：L-丙酰胺-谷氨酸L-Alanyl-glycine;82：L-天冬氨酰L-Aspartyl;83：L-天冬氨酸L-Aspartic acid;84：L-谷氨酸L-Glutamic acid;85：谷氨酰-L-谷氨酸Glutamyl-L-glutamic acid;86：L-鸟氨酸L-Ornithine;87：L-苯基丙氨酸L-Phenylalanine;88：L-脯氨酸L-Proline;89：L-焦谷氨酸L-Pyroglutamic acid;90：L-丝氨酸L-Serine;91：L-苏氨酸L-Threonine;92：二胺乙醇Diethanolamine;93：腐胺Putrescine;94：腺苷Adenosine;95：鸟苷Guanosine;96：5-磷酸腺苷Adenosine 5-phosphate
Fig. 6Heatmap of carbon utilization of T. afroharzianum (26℃, 7 d)



Table 4
表4
表4非洲哈茨木霉ACCC 33109代谢能力高于MU792低于MU153的物质
Table 4Substances utilized by T. afroharzianum ACCC 33109 at higher rate than MU792 but lower than MU153

编号Number	底物 Substrate	面积Area
		ACCC 33109	MU153	MU792
1	麦芽三糖Maltotriose	59165	62095	57507
2	甘油Glycerol	60735	61987	58852
3	β-甲基-D-葡萄糖苷β-Methyl-D-glucoside	58513	58843	57838
4	1-赤藓糖醇1-Erythritol	57976	58760	53527
5	D-核糖D-Ribose	56959	58054	56236
6	D-海藻糖D-Trehalose	56009	57807	50790
7	N-乙酰-D-葡萄糖胺N-Acetyl-D-glucosamine	56061	57514	46953
8	α-D-乳糖α-D-Lactose	56331	57189	55604
9	糊精Dextrin	54890	57178	50037
10	α-D-半乳糖α-D-Galactose	53950	56233*	51843
11	肌醇Inositol	54608	55987	51558
12	D-果糖D-Fructose	54745	55594	50364
13	α-D-葡萄糖α-D-Glucose	52286	55493	50449
14	麦芽糖Maltose	50178	54487	48708
15	N-乙酰-谷氨酸N-Acetyl-glutamic acid	53022	53290	51819
16	L-鼠李糖L-Rhamnose	51683	52537	49096
17	D-葡萄糖胺D-Glucosamine	51967	52333	49382
18	腺苷Adenosine	51170	51702	50163
19	吐温-80 Tween-80	48707	51471*	41716
20	扁桃苷Amygdalin	49287	50321	44435
21	鸟苷Guanosine	47915	49703	41058
22	腐胺Putrescine	49574	49667	45904
23	D-葡萄糖醛酸D-Gluconic acid	46310	48453**	44423
24	D-苹果酸D-Malic acid	47934	48074	46185
25	L-阿拉伯糖L-Arabinose	46014	47723	40849
26	核糖醇Ribitol	45415	46865	41124
27	N-乙酰-D-甘露糖胺N-Acetyl-D-mannosamine	42902	44170	39655
28	二胺乙醇Diethanolamine	38883	43295	35369
29	葡糖苷酸Glucosiduronide	38550	39477	31867

*：P≤0.05;**：P≤0.01;Area指荧光动力曲线下的面积Area is that under the kinetic curve of dye formation

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2.5 非洲哈茨木霉代谢底物种类对其抑菌活性影响

MU153基于α-D-半乳糖、糊精、麦芽糖、α-D-葡萄糖和吐温-80 5种底物产生的挥发性有机物对尖镰孢的抑菌率均明显高于野生株,MU792产生挥发性有机物对尖镰孢的抑菌率均低于野生株。以α-D-葡萄糖为底物时,ACCC 33109、MU153和MU792产生的挥发性有机物对尖镰孢的抑菌率均高于其他4种碳源,并且MU153抑菌率达到了最高为56.17%（图7）。

图7

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图7不同碳源条件下非洲哈茨木霉ACCC 33109、MU153和MU792抑菌作用（28℃, 5 d）

柱上不同字母表示在5%水平下差异显著
Fig. 7Effects of different carbon sources on the inhibitory activities of the VOCs produced by T. afroharzianum ACCC 33109, MU153 and MU792 (28℃, 5 d) Different letters on the bars indicate significant differences at the 5% level

3 讨论

木霉抑菌性挥发性有机物种类鉴别和产生机制是近年来的研究热点和难点。CHEN等^[26]研究发现,盖姆斯木霉（Trichoderma gamsii）YIM PH30019可产生二甲基二硫醚、二苯并吡喃、甲硫醇和酮类等挥发性物质抑制三七根腐病;张雯雯等^[27]研究表明,11株木霉菌产生的挥发性有机物均可抑制尖镰孢的生长,经GC-MS检测共发现257种挥发性有机物;LI等^[28]研究发现,哈茨木霉和深绿木霉（Trichoderma atroviride）产生的28种挥发性有机物可抑制尖镰孢的生长,对扣试验表明尖镰孢还可诱导木霉挥发性有机物的产生。

本研究中,分别以α-D-半乳糖、糊精、麦芽糖、α-D-葡萄糖和吐温-80为碳源时,非洲哈茨木霉突变株MU153抑菌率均高于野生株ACCC 33109,MU792抑菌率均低于ACCC 33109,其抑菌活性与碳源利用效率呈正相关。以α-D-葡萄糖为底物时,ACCC 33109、MU153和MU792抑菌率最高,分别为48.08%、56.17%和40.94%。分析原因可能是葡萄糖作为单糖最易被菌株吸收利用,从而菌株生长变快,菌丝量增多,产生大量的抑菌挥发性物质,这与文献报道一致。如詹艺舒等^[29]研究发现,甲基营养型芽孢杆菌（Bacillus methylotrophicus）以α-D-葡萄糖为碳源时,菌体量高达8.35 g·mL^-1,对康氏木霉（Trichoderma koningii）抑菌率最高,为58.56%,显著高于乳糖、蔗糖和可溶性淀粉等碳源;蔡邵宁^[30]研究表明,蛹虫草（Cordyceps militaris）以α-D-葡萄糖为单一碳源培养16 d时,菌丝干重超过7 g·L^-1,对大肠杆菌（Escherichia coli）、金黄色葡萄球菌（Staphylococcus aureus）和枯草芽孢杆菌（Bacillus subtilis）的抑制效果最好,抑菌率高于蔗糖和淀粉;吴惠贞等^[31]研究表明,罗伊氏乳杆菌（Lactobacillus reuteri）分别以葡萄糖、蔗糖和麦芽糖为碳源时,活菌数均在10⁹ CFU/mL以上,对大肠杆菌的抑菌圈均在7.65 mm以上,抑菌率高于甘油和果糖等碳源。紫外诱变不仅改变了菌株对碳源的利用率,可能还改变了菌株对碳源的利用方式,使诱变菌株产生了不同的抑菌挥发性物质。MUKHERJEE等^[32]研究表明,绿色木霉的诱变突变株G2以葡萄糖为碳源时,其代谢产物是野生株的2—3倍,包含绿胶霉素类物质和一些尚未鉴定的物质。

4 结论

通过原生质体紫外诱变和对扣法获得一株高产抑菌挥发性有机物的非洲哈茨木霉突变株MU153。与野生株ACCC 33109相比,该菌株抑菌率提高了16.68%,且菌落形态、菌落颜色以及分生孢子梗均有显著变化;以最适底物α-D-葡萄糖为碳源时,MU153抑菌率高达56.17%;盆栽试验中MU153具有抑菌、促生的双重效果,是一株具有重要应用潜力的生防菌。

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[1] SCHALCHLI H, TORTELLA G R, RUBILAR O, PARRA L, HORMAZABAL E, QUIROZ A . Fungal volatiles: An environmentally friendly tool to control pathogenic microorganisms in plants
Critical Reviews in Biotechnology, 2014,36(1):144-152. DOI: 10.3109/ 07388551.2014.946466.
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Fungi are an extraordinary and immensely diverse group of microorganisms that colonize many habitats even competing with other microorganisms. Fungi have received recognition for interesting metabolic activities that have an enormous variety of biotechnological applications. Previously, volatile organic compounds produced by fungi (FVOCs) have been demonstrated to have a great capacity for use as antagonist products against plant pathogens. However, in recent years, FVOCs have been received attention as potential alternatives to the use of traditional pesticides and, therefore, as important eco-friendly biotechnological tools to control plant pathogens. Therefore, highlighting the current state of knowledge of these fascinating FVOCs, the actual detection techniques and the bioactivity against plant pathogens is essential to the discovery of new products that can be used as biopesticides.

[2] SCHALCHLI H, HORMAZABAL E, BECERRA J, BIRKETT M, ALVEAR M, VIDAL J, QUIROZ A . Antifungal activity of volatile metabolites emitted by mycelial cultures of saprophytic fungi
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[3] 李梅云, 李天飞, 王革, 刘开启 . 木霉对烟草黑胫病菌的拮抗机制
植物保护学报, 2002,29(4):309-312. DOI: 10.13802/j.cnki.zwbhxb.2002.04.005.
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采用平板对峙法、水解酶平板活性测定、圆孔滤液法与游动孢子萌发检测、挥发性抑菌物检测等方法探讨木霉生防菌株对烟草疫霉Phytophthora nicotianae Brada de Hann的拮抗作用机制。结果表明,TR13、TR14、TR17对烟草疫霉有竞争、重寄生和抗生作用,但不同木霉菌株对烟草疫霉的拮抗作用有所不同,TR13、TR14的竞争作用较强;水解酶平板活性测定表明,3个木霉菌株均产生β-1,3-葡聚糖酶、纤维素酶,两种酶均参与烟草疫霉细胞壁的消解,以TR13与TR14酶的活性较高;3个菌株均产生非挥发性抗生素抑制烟草疫霉菌孢子萌发,但对疫霉菌菌丝的生长影响不大;TR13、TR14几乎不产生挥发性抗生素,TR17则产生挥发性抗生素,明显抑制疫霉菌生长。
LI M Y, LI T F, WANG G, LIU K Q . Antagonistic mechanism of Trichoderma spp. against Phytophthora nicotianae
Acta Phytophylacica Sinica, 2002,29(4):309-312. DOI: 10.13802/j.cnki.zwbhxb.2002.04.005. (in Chinese)
URL [本文引用: 1]
采用平板对峙法、水解酶平板活性测定、圆孔滤液法与游动孢子萌发检测、挥发性抑菌物检测等方法探讨木霉生防菌株对烟草疫霉Phytophthora nicotianae Brada de Hann的拮抗作用机制。结果表明,TR13、TR14、TR17对烟草疫霉有竞争、重寄生和抗生作用,但不同木霉菌株对烟草疫霉的拮抗作用有所不同,TR13、TR14的竞争作用较强;水解酶平板活性测定表明,3个木霉菌株均产生β-1,3-葡聚糖酶、纤维素酶,两种酶均参与烟草疫霉细胞壁的消解,以TR13与TR14酶的活性较高;3个菌株均产生非挥发性抗生素抑制烟草疫霉菌孢子萌发,但对疫霉菌菌丝的生长影响不大;TR13、TR14几乎不产生挥发性抗生素,TR17则产生挥发性抗生素,明显抑制疫霉菌生长。

[4] SAWANT I S, WADKAR P N, GHULE S B, SALUNKHE V P, CHAVAN V, SAWANT S D . Induction of systemic resistance in grapevines against powdery mildew by Trichoderma asperelloides strains
Australasian Plant Pathology, 2020,49:107-117. DOI: 10.1007/s13313-020-00679-8.
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[5] PRABHAKARAN N, PRAMEELADEVI T, SATHIYABAMA M, KAMIL D . Screening of different Trichoderma species against agriculturally important foliar plant pathogens
Journal of Environmental Biology, 2015,36(1):191-198.
URLPMID:26536792 [本文引用: 1]
Different isolates of Trichoderma were isolated from soil samples which were collected from different part of India. These isolates were grouped into four Trichoderma species viz., Trichoderma asperellum (Ta), T. harzianum (Th), T. pseudokoningii (Tp) and T. longibrachiatum (Tl) based on their morphological characters. Identification of the above isolates was also confirmed through ITS region analysis. These Trichoderma isolates were tested for in vitro biological control of Alternaria solani, Bipolaris oryzae, Pyricularia oryzae and Sclerotinia scierotiorum which cause serious diseases like early blight (target spot) of tomato and potato, brown leaf spot disease in rice, rice blast disease, and white mold disease in different plants. Under in vitro conditions, all the four species of Trichoderma (10 isolates) proved 100% potential inhibition against rice blast pathogen Pyracularia oryzae. T. harzianum (Th-01) and T. asperellum (Ta-10) were effective with 86.6% and 97.7%, growth inhibition of B. oryzae, respectively. Among others, T. pseudokoningii (Tp-08) and T. Iongibrachiatum (Tl-09) species were particularly efficient in inhibiting growth of S. sclerotiorum by 97.8% and 93.3%. T. Iongibrachiatum (TI-06 and TI-07) inhibited maximum mycelial growth of A. solani by 87.6% and 84.75. However, all the T. harzianum isolates showed significantly higher inhibition against S. sclerotiorum (CD value 9.430), causing white mold disease. This study led to the selection of potential Trichoderma isolates against rice blast, early blight, brown leaf spot in rice and white mold disease in different crops.

[6] 邹佳迅, 范晓旭, 宋福强 . 木霉 ( Trichoderma spp.) 对植物土传病害生防机制的研究进展
大豆科学, 2017,36(6):146-153. DOI: 10.11861/j.issn.1000-9841.2017.06.0970.
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ZOU J X, FAN X X, SONG F Q . Biocontrol mechanism of Trichoderma spp. against soilborn plant disease
Soybean Sciences, 2017,36(6):146-153. DOI: 10.11861/j.issn.1000-9841.2017.06.0970. (in Chinese)
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[7] LI N, ALFIKY A, VAUGHAN M M, KANG S . Stop and smell the fungi: Fungal volatile metabolites are overlooked signals involved in fungal interaction with plants
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[8] KHALIL S, ALSANIUS B W . Utilisation of carbon sources by Pythium. Phytophthora an. Fusarium species as determined by Biolog? microplate assay
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[9] 汪汉成, 王茂胜, 黄艳飞, 王进, 商胜华, 张长青 . 烟草青枯病拮抗菌株X-60的分离鉴定及其表型组学分析
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WANG H C, WANG M S, HUANG Y F, WANG J, SHANG S H, ZHANG C Q . Isolation, identification and phenotype microarrays analysis of an antagonistic bacterial strain X-60 against tobacco bacterial wilt
Acta Phytopathologica Sinica, 2016,46(3):409-419. DOI: 10.13926/j.cnki.apps.2016.03.015. (in Chinese)
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[10] YONG F M, LIM G . Effect of carbon source on aroma production by Trichoderma viride
MIRCEN Journal of Applied Microbiology and Biotechnology, 1986,2(4):483-487. DOI: 10.1007/bf00933371.
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[11] EFFMERT U, KALDERáS J, WARNKE R, PIECHULLA B. Volatile mediated interactions between bacteria and fungi in the soil
Journal of Chemical Ecology, 2012,38(6):665-703. DOI: 10.1007/s10886- 012-0135-5.
DOI:10.1007/s10886-012-0135-5URL [本文引用: 1]
Soil is one of the major habitats of bacteria and fungi. In this arena their interactions are part of a communication network that keeps microhabitats in balance. Prominent mediator molecules of these inter- and intraorganismic relationships are inorganic and organic microbial volatile compounds (mVOCs). In this review the state of the art regarding the wealth of mVOC emission is presented. To date, ca. 300 bacteria and fungi were described as VOC producers and approximately 800 mVOCs were compiled in DOVE-MO (database of volatiles emitted by microorganisms). Furthermore, this paper summarizes morphological and phenotypical alterations and reactions that occur in the organisms due to the presence of mVOCs. These effects might provide clues for elucidating the biological and ecological significance of mVOC emissions and will help to unravel the entirety of belowgroundaEuroe volatile-wired' interactions.

[12] LI M F, LI G H, ZHANG K Q . Non-volatile metabolites from Trichoderma spp
Metabolites, 2019,9(3):E58. DOI: 10.3390/ metabo9030058.
DOI:10.3390/metabo9030058URLPMID:30909487 [本文引用: 1]
The genus Trichoderma is comprised of many common fungi species that are distributed worldwide across many ecosystems. Trichoderma species are well-known producers of secondary metabolites with a variety of biological activities. Their potential use as biocontrol agents has been known for many years. Several reviews about metabolites from Trichoderma have been published. These reviews are based on their structural type, biological activity, or fungal origin. In this review, we summarize the secondary metabolites per Trichoderma species and elaborate on approximately 390 non-volatile compounds from 20 known species and various unidentified species.

[13] GHISALBERTI E L, SIVASITHAMPARAM K . Antifungal antibiotics produced by Trichoderma spp
Soil Biology and Biochemistry, 1991,23(11):1011-1020. DOI: 10.1016/0038-07179(91)90036-J.
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[14] NIELSEN K F, GR?FENHAN T, ZAFARI D, THRANE U. Trichothecene production by Trichoderma brevicompactum
Journal of Agricultural and Food Chemistry, 2005,53(21):8190-8196. DOI: 10.1021/jf051279b.
DOI:10.1021/jf051279bURLPMID:16218663 [本文引用: 1]
Trichoderma brevicompactum, T. viride, T. harzianum, T. atroviride, T. longibrachiatum, T. erinaceum, T. citrinoviride, and Hypocrea lutea were screened for production of trichothecenes after growth on one or several solid and liquid media. Trichothecenes were detected by liquid chromatography combined with online UV/vis spectroscopy and electrospray high-resolution mass spectrometry. T. brevicompactum produced trichodermin and/or harzianum A on all media investigated, with liquid media yielding the largest amounts. Detection of octa-2Z,4E,6E-trienedioic acid in the harzianum-A-producing strains indicated that harzianum A was synthesized directly by esterification of trichodermol with octa-2Z,4E,6E-trienedioic acid. Both the T. viride strain from which trichodermin was originally isolated and the T. harzianum strain from which harzianum A was originally isolated were shown to belong to T. brevicompactum based on four independent criteria: metabolite profiles, micromorphology, macromorphology on yeast extract sucrose agar and potato dextrose agar, and DNA sequences of the ITS1/ITS2 regions of the nuclear ribosomal DNA.

[15] RUIZ N, WIELGOSZ-COLLIN G, POIRIER L, GROVEL O, PETIT K E, MOHAMED-BENKADA M, PONT T R, BISSETT J, VéRITé P, BARNATHAN G, POUCHUSA Y F. New trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma longibrachiatum
Peptides, 2007,28(7):1351-1358. DOI: 10.1016/j.peptides.2007.05.012.
DOI:10.1016/j.peptides.2007.05.012URL [本文引用: 1]

Abstract

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MSⁿ, CID-MSⁿ and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.

[16] PAZ Z, GERSON U, SZTEJNBERG A . Assaying three new fungi against citrus mites in the laboratory, and a field trial
BioControl, 2007,52(6):855-862. DOI: 10.1007/s10526-006-9060-2.
DOI:10.1007/s10526-006-9060-2URL [本文引用: 1]
The Basidiomycotine fungi Meira geulakonigii, Meira argovae and Acaromyces ingoldii were assayed in the laboratory against five species of herbivorous mites: Phyllocoptruta oleivora (Eriophyidae), Panonychus citri, Eutetranychus orientalis, Tetranychus urticae and Tetranychus cinnabarinus (all four Tetranychidae). All fungi caused significantly high mortality rates (as compared to controls) after 14days, some after 1week. Phyllocoptruta oleivora was the most susceptible, showing >80% mortality even after 1week. In a field trial, grapefruits sprayed either once a month or once a season with M. geulakonigii had significantly fewer P. oleivora and less damage than unsprayed fruit. These results suggest that M. geulakonigii may protect grapefruits against the injurious P. oleivora.

[17] HARMAN G E, HOWELL C R, VITERBO A, CHET I, LORITO M . Trichoderma species-opportunistic, avirulent plant symbionts
Nature Reviews. Microbiology, 2004,2(1):43-56. DOI: 10.1038/nrmicro797.
DOI:10.1038/nrmicro797URLPMID:15035008 [本文引用: 1]
Trichoderma spp. are free-living fungi that are common in soil and root ecosystems. Recent discoveries show that they are opportunistic, avirulent plant symbionts, as well as being parasites of other fungi. At least some strains establish robust and long-lasting colonizations of root surfaces and penetrate into the epidermis and a few cells below this level. They produce or release a variety of compounds that induce localized or systemic resistance responses, and this explains their lack of pathogenicity to plants. These root-microorganism associations cause substantial changes to the plant proteome and metabolism. Plants are protected from numerous classes of plant pathogen by responses that are similar to systemic acquired resistance and rhizobacteria-induced systemic resistance. Root colonization by Trichoderma spp. also frequently enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients.

[18] LORITO M, WOO S L, HARMAN G E, MONTE E . Translational research on Trichoderma: From omics to the field
Annual Review of Phytopathology, 2010,48:395-417. DOI: 10.1146/annurev-phyto-073009-114314.
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Structural and functional genomics investigations are making an important impact on the current understanding and application of microbial agents used for plant disease control. Here, we review the case of Trichoderma spp., the most widely applied biocontrol fungi, which have been extensively studied using a variety of research approaches, including genomics, transcriptomics, proteomics, metabolomics, etc. Known for almost a century for their beneficial effects on plants and the soil, these fungi are the subject of investigations that represent a successful case of translational research, in which 'omics-generated novel understanding is directly translated in to new or improved crop treatments and management methods. We present an overview of the latest discoveries on the Trichoderma expressome and metabolome, of the complex and diverse biotic interactions established in nature by these microbes, and of their proven or potential importance to agriculture and industry.

[19] RAUT I, BADEA-DONI M, CALIN M, OANCEA F, VASILESCU G, SESAN T E, JECU L . Effect of volatile and non-volatile metabolites from Trichoderma spp. against important phytopathogens
Revista de Chimie-Bucharest-Original Edition, 2014,65(11):1285-1288.
[本文引用: 1]

[20] BELéN RUBIO M, PARDAL A J, CARDOZA R E, GUTIéRREZ S, MONTE E, HERMOSA R . Involvement of the transcriptional coactivator ThMBF1 in the biocontrol activity of Trichoderma harzianum
Frontiers in Microbiology, 2017,8:2273. DOI: 10.3389/ fmicb.2017.02273.
DOI:10.3389/fmicb.2017.02273URLPMID:29201024 [本文引用: 1]
Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1) homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea (BC) in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC) with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1). Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the necrotic leaf lesions caused by BC while plants treated with the Thmbf1-overexpressing transformants exhibited an increased susceptibility to this pathogen. The percentages of Fusarium wilt disease incidence and values of aboveground dry weight showed that T34 did not have biocontrol activity against FO, at least in the 'Moneymaker' tomato variety, and that Thmbf1 overexpression increased the incidence of this disease. Our results show that the Thmbf1 overexpression in T34 negatively affects its biocontrol mechanisms.

[21] PACHAURI S, CHATTERJEE S, KUMAR V, MUKHERJEE P K . A dedicated glyceraldehyde-3-phosphate dehydrogenase is involved in the biosynthesis of volatile sesquiterpenes in Trichoderma virens— evidence for the role of a fungal GAPDH in secondary metabolism
Current Genetics, 2018,65(1):243-252. DOI: 10.1007/s00294-018- 0868-y.
DOI:10.1007/s00294-018-0868-yURLPMID:30046843 [本文引用: 1]
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the sixth step of glycolysis, and is also known to perform other (moonlighting) activities in animal cells. We have earlier identified an additional GAPDH gene in Trichoderma virens genome. This gene is consistently associated with the vir cluster responsible for biosynthesis of a range of volatile sesquiterpenes in Trichoderma virens. This gene is also associated with an orthologous gene cluster in Aspergillus spp. Both glycolytic GAPDH and the vir cluster-associated GAPDH show more than 80% similarity with essentially conserved NAD(+) cofactor- and substrate-binding sites. However, a conserved indel is consistently present only in GAPDH associated with the vir cluster, both in T. virens and Aspergillus spp. Using gene knockout, we demonstrate here that the vir cluster-associated GAPDH is involved in biosynthesis of volatile sesquiterpenes in T. virens. We thus, for the first time, elucidate the non-glycolytic role of a GAPDH in a fungal system, and also prove for the first time that a GAPDH, a primary metabolism protein, is involved in secondary metabolism.

[22] QIN W T, ZHUANG W Y . Seven new species of Trichoderma(Hypocreales) in the Harzianum and Strictipile clades
Phytotaxa, 2017,305(3):121-139. DOI: 10.11646/phytotaxa.305.3.1.
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[23] 吕天晓, 徐凤花, 李世贵, 顾金刚, 姜瑞波, 牛永春 . 生防木霉菌原生质体的制备及再生研究
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Lü T X, XU F H, LI S G, GU J G, JIANG R B, NIU Y C . The research on protoplast preparation and regeneration of biocontrolTrichoderma spp. strain
Biotechnology Bulletin, 2009(4):130-134. DOI: CNKI:SUN:SWJT.0.2009-04-032. (in Chinese)
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[24] 李书强, 李林会, 沈江浩, 景焕, 张明珠, 杜晓端, 芦国嫣 . 生防菌对黄瓜枯萎病防效及其对黄瓜诱导抗性测定
河北科师范学院学报, 2017,31(1):53-58. DOI: 10.3969/J.ISSN.1672-7983.2017.01.011.
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LI S Q, LI L H, SHEN J H, JING H, ZHANG M Z, DU X D, LU G Y . Efficacy of biocontrol agents to cucumber fusarium wilt and their induced resistance
Journal of Hebei Normal University of Science and Technology, 2017,31(1):53-58. DOI: 10.3969/J.ISSN.1672-7983.2017.01.011. (in Chinese)
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[25] 袁成凌, 姚建铭, 王纪, 余增亮 . 低能离子注入在花生四烯酸(aa)高产菌株选育中的研究
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Journal of Radiation Research and Radiation Processing, 2003,21(4):237-242. DOI: 10.3969/j.issn.1000-3436.2003.04.004. (in Chinese)
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[26] CHEN J L, SUN S Z, MIAO C P, WU K, CHEN Y W, XU L H, GUAN H L, ZHAO L X . Endophytic Trichoderma gamsii YIM PH30019: A promising biocontrol agent with hyperosmolar, mycoparasitism, and antagonistic activities of induced volatile organic compounds on root-rot pathogenic fungi o. Panax notoginseng
Journal of Ginseng Research, 2016,40(4):315-324. DOI: 10.1016/j.jgr.2015.09.006.
DOI:10.1016/j.jgr.2015.09.006URLPMID:27746683 [本文引用: 1]
BACKGROUND: Biocontrol agents are regarded as promising and environmental friendly approaches as agrochemicals for phytodiseases that cause serious environmental and health problems. Trichoderma species have been widely used in suppression of soil-borne pathogens. In this study, an endophytic fungus, Trichoderma gamsii YIM PH30019, from healthy Panax notoginseng root was investigated for its biocontrol potential. METHODS: In vitro detached healthy roots, and pot and field experiments were used to investigate the pathogenicity and biocontrol efficacy of T. gamsii YIM PH30019 to the host plant. The antagonistic mechanisms against test phytopathogens were analyzed using dual culture, scanning electron microscopy, and volatile organic compounds (VOCs). Tolerance to chemical fertilizers was also tested in a series of concentrations. RESULTS: The results indicated that T. gamsii YIM PH30019 was nonpathogenic to the host, presented appreciable biocontrol efficacy, and could tolerate chemical fertilizer concentrations of up to 20%. T. gamsii YIM PH30019 displayed antagonistic activities against the pathogenic fungi of P. notoginseng via production of VOCs. On the basis of gas chromatography-mass spectrometry, VOCs were identified as dimethyl disulfide, dibenzofuran, methanethiol, ketones, etc., which are effective ingredients for antagonistic activity. T. gamsii YIM PH30019 was able to improve the seedlings' emergence and protect P. notoginseng plants from soil-borne disease in the continuous cropping field tests. CONCLUSION: The results suggest that the endophytic fungus T. gamsii YIM PH30019 may have a good potential as a biological control agent against notoginseng phytodiseases and can provide a clue to further illuminate the interactions between Trichoderma and phytopathogens.

[27] 张雯雯, 国振宇, 赵晓迪, 龚明波, 李世贵, 顾金刚, 杨礼富 . 木霉菌株挥发性物质拮抗尖孢镰刀菌的效果及其鉴定
热带作物学报, 2017,38(4):704-715. DOI: 10.3969/j.issn.1000-2561.2017.04.019.
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ZHANG W W, GUO Z Y, ZHAO X D, GONG M B, LI S G, GU J G, YANG L F . Effect and identification of volatile compounds from Trichoderma against Fusarium oxysporum
Chinese Journal of Tropical Crops, 2017,38(4):704-715. DOI: 10.3969/j.issn.1000-2561.2017.04.019. (in Chinese)
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[28] LI N X, ALFIKY A, WANG W Z, ISLAM M, NOUROLLAHI K, LIU X, KANG S . Volatile compound-mediated recognition and inhibition between Trichoderma biocontrol agents an. Fusarium oxysporum
Frontiers in Microbiology, 2018,9:2614. DOI: 10.3389/fmicb.2018. 02614.
DOI:10.3389/fmicb.2018.02614URLPMID:30455673 [本文引用: 1]
Certain Trichoderma strains protect plants from diverse pathogens using multiple mechanisms. We report a novel mechanism that may potentially play an important role in Trichoderma-based biocontrol. Trichoderma virens and T. viride significantly increased the amount/activity of secreted antifungal metabolites in response to volatile compounds (VCs) produced by 13 strains of Fusarium oxysporum, a soilborne fungus that infects diverse plants. This response suggests that both Trichoderma spp. recognize the presence of F. oxysporum by sensing pathogen VCs and prepare for attacking pathogens. However, T. asperellum did not respond to any, while T. harzianum responded to VCs from only a few strains. Gene expression analysis via qPCR showed up-regulation of several biocontrol-associated genes in T. virens in response to F. oxysporum VCs. Analysis of VCs from seven F. oxysporum strains tentatively identified a total of 28 compounds, including six that were produced by all of them. All four Trichoderma species produced VCs that inhibited F. oxysporum growth. Analysis of VCs produced by T. virens and T. harzianum revealed the production of compounds that had been reported to display antifungal activity. F. oxysporum also recognizes Trichoderma spp. by sensing their VCs and releases VCs that inhibit Trichoderma, suggesting that both types of VC-mediated interaction are common among fungi.

[29] 詹艺舒, 李婕, 褚秀丹, 蔡志英, 纪鹏伟, 陈炳智, 江玉姬 . 一株真菌拮抗细菌Z21的筛选与鉴定及其发酵条件优化
微生物学通报, 2020,47(5):1503-1514. DOI: 10.13344/j.microbiol.china.190622.
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ZHAN Y S, LI J, CHU X D, CAI Z Y, JI P W, CHEN B Z, JIANG Y J. Screen, identification and fermentation optimization of an antifungal bacterium Z21
Microbiology China, 2020, 47(5): 1503-1514. 10.13344/j.microbiol.china.190622. (in Chinese)
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[30] 蔡昭宁 . 不同碳源对蛹虫草液体发酵代谢组的影响及发酵液抑菌能力探究
[D]. 重庆: 西南大学, 2016.
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CAI Z N . Effects of different carbon sources on metabolome of Cordyceps militaris fermentation and preliminary study on the antibacterial ability of the zymotic fluid
[D]. Chongqing: Southwest University, 2016. (in Chinese)
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[31] 吴惠贞, 夏枫耿, 陈中, 黄魁英, 林伟峰 . 碳源与罗伊氏乳杆菌LYS-1发酵上清液抑菌效果的关系
现代食品科技, 2020,36(4):125-131. DOI: 10.13982 /j.mfst.1673-9078.2020.4.016.
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WU H Z, XIA F G, CHEN Z, HUANG K Y, LIN W F . The relation between carbon source and the antimicrobial effect of Lactobacillus reuteri fermentation supernatant
Modern Food Science and Technology, 2020,36(4):125-131. DOI: 10.13982/j.mfst.1673-9078.2020.4.016. (in Chinese)
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[32] MUKHERJEE P K, MEHETRE S T, SHERKHANE P D, MUTHUKATHAN G, GHOSH A, KOTASTHANE A S, KHARE N, RATHOD P, SHARMA K K, NATH R, TEWARI A K, BHATTACHARYYA S, ARYA M, PATHAK D, WASNIKAR A R, TIWARI R K S, SAXENA D R,. A novel seed-dressing formulation based on an improved mutant strain of Trichoderma virens, and its field evaluation
Frontiers in Microbiology, 2019,10:1910. DOI: 10.3389/fmicb.2019.01910.
DOI:10.3389/fmicb.2019.01910URLPMID:31543866 [本文引用: 1]
Using gamma-ray-induced mutagenesis, we have developed a mutant (named G2) of Trichoderma virens that produced two- to three-fold excesses of secondary metabolites, including viridin, viridiol, and some yet-to-be identified compounds. Consequently, this mutant had improved antibiosis against the oomycete test pathogen Pythium aphanidermatum. A transcriptome analysis of the mutant vis-a-vis the wild-type strain showed upregulation of several secondary-metabolism-related genes. In addition, many genes predicted to be involved in mycoparasitism and plant interactions were also upregulated. We used tamarind seeds as a mass multiplication medium in solid-state fermentation and, using talcum powder as a carrier, developed a novel seed dressing formulation. A comparative evaluation of the wild type and the mutant in greenhouse under high disease pressure (using the test pathogen Sclerotium rolfsii) revealed superiority of the mutant over wild type in protecting chickpea (Cicer arietinum) seeds and seedlings from infection. We then undertook extensive field evaluation (replicated micro-plot trials, on-farm demonstration trials, and large-scale trials in farmers' fields) of our mutant-based formulation (named TrichoBARC) for management of collar rot (S. rolfsii) in chickpea and lentil (Lens culinaris) over multiple locations in India. In certain experiments, other available formulations were included for comparison. This formulation consistently, over multiple locations and years, improved seed germination, reduced seedling mortality, and improved plant growth and yield. We also noticed growth promotion, improved pod bearing, and early flowering (7-10 days) in TrichoBARC-treated chickpea and lentil plants under field conditions. In toxicological studies in animal models, this formulation exhibited no toxicity to mammals, birds, or fish.