紫外-氯化锂复合诱变哈茨木霉产生多菌灵抗药性菌株的研究

多菌灵是植物病害防治中常用的化学杀菌剂,野生型哈茨木霉对多菌灵比较敏感。为了能够更好地将哈茨木霉菌应用于实践,本试验采用紫外线-氯化锂复合诱变的方法,实现哈茨木霉菌株对多菌灵的抗性改良。试验共获得315株正向突变菌株,其中hcb-35菌株抗性能力较强。利用毒力测定方法检测了多菌灵对hcb-35有效抑菌中浓度,及hcb-35对多菌灵的抗药遗传稳定性;并利用对峙试验及显微观察检测其抑菌能力。结果显示,与哈茨木霉出发菌株hc相比,多菌灵对哈茨木霉突变株hcb-35菌株的有效抑菌中浓度提升285%;连续转接12代后,hcb-35菌株抗药性相对稳定且抑菌效果较出发菌株无明显差异,表明应用紫外-氯化锂复合诱变哈茨木霉可以获得遗传稳定的耐多菌灵突变株。     

哈茨木霉菌对水稻幼苗根际土壤微生物和酶活性的影响

采用钵盘育苗试验,研究了哈茨木霉菌在调节水稻苗床土壤微生物群落及土壤酶活性中的作用。研究结果表明：哈茨木霉菌能有效调节水稻幼苗根际土壤微生物群落组成;哈茨木霉菌对细菌、真菌和放线菌数量的影响程度明显不同,播种28天后,哈茨木霉菌接种处理的水稻幼苗根际土壤细菌和放线菌数量较对照分别增加50.70%和48.56%,而真菌数量较对照减少16.15%,并且哈茨木霉菌数量较刚播种时增加了138.46%;哈茨木霉菌也能显著提高土壤脲酶、磷酸酶和蔗糖酶的活性,分别较对照提高8.55%、18.31%和49.61%。研究表明,哈茨木霉菌具有改善土壤微生态环境的作用。     

哈茨木霉T-H-30菌株对黄瓜白粉病的防治效果及其促生作用

用哈茨木霉菌株T-H-30在黄瓜上进行促生作用及生物防治效果研究。结果表明,菌株T-H-30对黄瓜有明显的促生作用,能增加黄瓜的叶片数及藤蔓的长度,从而有效提高黄瓜的产量;菌株T-H-30对黄瓜白粉病具有良好的生物防治效果,可有效控制黄瓜白粉病在大田的发展,相对防效达69.9%～78.8%,且对供试植株安全。     

两株哈茨木霉菌株防治水稻纹枯病及促进水稻生长的潜力研究

 由茄丝核菌(Rhizoctonia solani)引起的纹枯病是水稻生产中的重要真菌病害,严重影响水稻产量及品质。本研究评估了两株哈茨木霉菌株3S1-13和4S2-46对纹枯病生防潜力及其对水稻种子萌发和幼苗生长的促生效果。研究表明两株哈茨木霉菌株在马铃薯葡萄糖琼脂培养基(PDA)上不产生分生孢子,但在含有1%酵母粉的PDA上能恢复产分生孢子的能力。菌株3S1-13和4S2-46发酵液对茄丝核菌的菌丝生长及菌核形成均有较强抑制作用,具有防治水稻纹枯病潜力,且发酵液防效显著高于孢子液的防效。菌株3S1-13和4S2-46发酵液处理后的水稻种子发芽率和幼苗的根系、鲜重均显著高于对照,显示出有利于促进种子发芽与生长的作用。研究结果表明两株哈茨木霉菌株发酵液不仅可以用于水稻纹枯病的生物防治,而且可以促进水稻种子发芽及生长,具有较好的应用潜力。     

哈茨木霉SH2303防治玉米小斑病的初步研究

哈茨木霉SH2303是本实验室分离获得的1株具有生防效果的菌株,该菌株能够较好的防治玉米小斑病。通过离体叶片试验确定哈茨木霉SH2303诱导玉米抗小斑病的持效期达15 d。盆栽及大田试验表明,防治玉米小斑病防效分别达到78.1%和56.3%。盆栽试验表明,哈茨木霉 SH2303处理的叶片在挑战接种小斑病菌后第36 h,玉米体内防御反应酶系PAL和SOD活性达到峰值。同时,防御基因Pal和Sod的表达水平也明显上升。综合分析表明,哈茨木霉SH2303的诱导抗性作用是防治玉米小斑病的重要机制之一。     

枸骨内生真菌抗菌代谢产物的鉴定及活性研究

对一株枸骨内生真菌哈茨木霉Trichoderma harzianum抗菌活性成分的分离、鉴定及其抗菌生物活性进行了研究。该真菌发酵液经乙酸乙酯萃取、正相硅胶和ODS-反相色谱分离纯化得到一单端孢霉烯化合物,经红外、质谱和核磁分析,鉴定为:4β-乙酰基-12,13-环氧-9-单端孢霉烯(trichodermin),这是首次从哈茨木霉菌代谢产物中分离出该化合物。Trichodermin对蕃茄早疫病和黄瓜立枯病病原菌的离体抑制活性EC50值分别为3.35和3.59 mg/L;活体测定结果表明,在 100 mg/L的剂量下对两种病的保护效果分别为97.8% 和98.1%,治疗效果为96.7%和97.3%。     

木霉分生孢子和厚垣孢子对黄瓜叶片抗氧化系统及枯萎病防效的影响

采用前期筛选出的对黄瓜枯萎病菌有较好拮抗作用的3株木霉菌,即哈茨木霉菌(Trichoderma harzianum)809、拟康氏木霉菌(Trichoderma pseudokoningii)886和棘孢木霉菌(Trichoderma asperellum)525,利用盆栽试验,测定了木霉菌分生孢子和厚垣孢子对黄瓜幼苗抗氧化能力及对枯萎病防效的影响。结果显示:3株木霉菌分生孢子和厚垣孢子对黄瓜枯萎病的盆栽防效均在66.81%以上,且以拟康氏木霉菌886厚垣孢子防效最高,达到81.46%;当黄瓜幼苗长至三叶一心时,与CK(即只接种病原菌)相比,经哈茨木霉菌809、拟康氏木霉菌886、棘孢木霉菌525分生孢子以及厚垣孢子处理后,黄瓜幼苗叶片相对电导率和丙二醛(MDA)含量均呈下降趋势,而保护性酶包括过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、超氧化物歧化酶(SOD)活性则呈上升趋势,其中以拟康氏木霉菌886厚垣孢子的变化幅度最显著;拟康氏木霉菌886厚垣孢子处理的黄瓜幼苗叶片相对电导率、MDA含量分别比CK下降了47.74%、41.40%;而叶片中的POD、CAT、APX、SOD活性则分别比CK增加了318.11%、155.36%、157.09%和300.34%。研究表明3株木霉菌分生孢子和厚垣孢子均能通过改善黄瓜幼苗叶片抗氧化能力,增加保护酶活性,提高了对黄瓜枯萎病的防治效果。     

木霉对土传病原真菌的拮抗作用

 分别在体外及温室测定了筛选菌株哈茨木霉Trichoderma harzianum(T₈₂)和Tricho-derma sp.(NF9)对土传病原真菌的拮抗作用。体外测定表明,木霉菌株T₈₂和NF₉对白绢病菌Sclerotium rolfsii,立枯丝核菌Rhizoctonia solani,瓜果腐霉Pythium aphanidermatum刺腐霉P.spinosum和尖镰孢Fusarium oxysporum在对崎培养中的拮抗系数分别为2或2~3和2。温室测定表明,用0、6%(W/W)T₈₂麸皮培养物(10⁷cfu/g)处理土壤。在人工接种白绢病菌,立枯丝核菌及瓜果腐霉20天后,黄瓜发病率分别比未用木霉处理的对照减少46、5%,28.4%和81。2%;用T₈₂和NF₉木霉孢子悬浮液(10⁸cfu/ml)处理黄瓜种子,人工接种白绢病菌11天后,黄瓜成苗率分别比未用木霉处理的对照增加14%的20%。分别在光学显微镜和扫描电镜下观察到木霉T₈₂对白绢病菌菌丝和菌核的重寄生以及木霉T₈₂和NF₉对立枯丝核菌菌丝的缠绕。穿入及寄生。作者认为重寄生可能是试验木霉菌株T₈₂和NF₉对白绢病菌和立枯丝核菌的主要拮抗机制。     

一株具有诱导抗性木霉菌株的筛选及其对黄瓜灰霉病诱导抗性的初步研究

从植物根部土壤中分离到一株能够诱导黄瓜产生系统抗性的木霉Tr-92,通过形态观察和18SrDNA序列分析对其进行了分类学鉴定,并进行了诱导黄瓜产生系统抗性能力的初步研究.结果表明,菌株Tr-92的菌落形态和显微形态与木霉一致,18SrDNA序列与哈茨木霉的相似性为99％,初步鉴定为哈茨木霉.利用该菌株孢子悬浮液对5叶期黄瓜苗进行根部诱导接种,24 h后灰霉病菌挑战接种,对灰霉病的防效达56％,叶片中抗性相关酶过氧化物酶、多酚氧化酶、β-1,3葡聚糖酶、苯丙氨酸解氨酶、几丁质酶的酶活性显著上升.表明该菌在黄瓜根部灌施可以诱导叶部系统抗性相关酶的活性升高,可为植物病害的生物防治提供适宜的菌种资源.     

木霉分生孢子和厚垣孢子对黄瓜幼苗生理特性及枯萎病防效的影响

采用前期筛选出的对黄瓜枯萎病菌有较好拮抗作用的3株木霉菌,即哈茨木霉Trichoderma harzianum 809、拟康氏木霉Trichoderma pseudokoningii 886和棘孢木霉Trichoderma asperellum 525,利用盆栽试验,测定了木霉菌孢子不同类型施用对黄瓜幼苗生长、生理特性及枯萎病防效的影响。结果表明:哈茨木霉809、拟康氏木霉886、棘孢木霉525分生孢子和厚垣孢子对黄瓜枯萎病的盆栽防效均在66.81%以上,且以拟康氏木霉886厚垣孢子防效最高,达到81.46%。当黄瓜幼苗长至三叶一心时,3株木霉菌分生孢子和厚垣孢子处理的黄瓜幼苗株高、茎粗、根体积、叶面积、全株鲜质量均比CK显著上升,以厚垣孢子886的促进效果最显著,其株高、茎粗、根体积、叶面积、全株鲜质量分别较CK增加了85.33%、108.43%、235.29%、144.38%、272.32%。同时,3株木霉菌分生孢子和厚垣孢子处理的黄瓜幼苗叶绿素含量、根系活力、叶片可溶性糖含量、叶片可溶性蛋白含量也均比CK显著上升,厚垣孢子886的促进效果仍最显著,其叶绿素含量、根系活力、叶片可溶性糖含量、叶片可溶性蛋白含量分别较CK增加了97.47%、86.05%、172.49%、201.91%。     

冬青卫矛内生放线菌YDG17菌株发酵液抑菌活性研究

从冬青卫矛植株中筛选得到一株内生链霉菌YDG17,其发酵液对多种植物病原真菌有较强的抑制作用。室内生物测定结果表明,YDG17菌株发酵液对供试的11种植物病原真菌菌丝生长均有一定的抑制作用,其中对小麦赤霉病菌Fusarium graminearum、番茄灰霉病菌Fulvia fulvum和番茄早疫病菌Alternaria solani的抑制作用最强,EC50值分别为259.98、336.13和100.72 mg/L;对供试的3种植物病原真菌孢子萌发也均有一定的抑制作用,其中对番茄灰霉病菌孢子萌发的抑制作用最强,EC50值为87.84 mg/L。离体子叶法测定结果表明,发酵液原液对番茄灰霉病的保护效果为97.62%,治疗效果为79.63%。盆栽试验结果表明,发酵液原液对番茄灰霉病的保护效果为71.34%,治疗效果为64.23%。捷克八溶剂系统纸色谱测定结果表明,该发酵液的主要活性物质为碱性水溶性抗生素。选用弱酸性离子交换树脂吸附法对YDG17发酵液活性成分进行了初步分离,并对其活性馏分进行ESI-MS/MS分析,表明其活性物质主要为链丝菌素类化合物,该类化合物为首次从植物内生菌中分离得到。     

哈茨木霉对黄瓜尖孢镰刀菌的抑制作用和抗性相关基因表达

采用室内筛选与田间防效相结合的方法,对哈茨木霉抑制黄瓜尖孢镰刀菌的拮抗机制进行了研究。对峙培养结果显示,木霉菌和病原菌间均形成了较明显的抑菌圈,对病原菌的抑菌率达66.7%~85.8%,其中菌株TG、TM对枯萎病菌抑制作用较强。木霉菌可有效提高根系对病原菌的抗性,黄瓜植株接种枯萎病菌后,根系细胞大量死亡,而先接种木霉菌再接种病原菌后则减小了对根系的伤害。田间防效试验结果表明,TG和TM孢子悬浮液浓度在108个/mL时防效最好,分别为54.9%和49.4%。接种木霉菌植株根系抗性基因的表达量均高于对照植株,呈双峰趋势。在第6天时抗性基因表达量最高,WRKY6、MYB、PR-1、PAL、GST和GLU的表达量分别为对照的5.15、5.22、6.07、6.00、3.16、和16.15倍。表明木霉菌通过激活与胁迫相关的基因表达提高了对病原菌的抗性。     

Changes induced by Trichoderma harzianum in suppressive compost controlling Fusarium wilt

The addition of species of Trichoderma to compost is a widespread technique used to control different plant diseases. The biological control activity of these species is mainly attributable to a combination of several mechanisms of action, which may affect the microbiota involved in the suppressiveness of compost. This study was therefore performed to determine the effect of inoculation of Trichoderma harzianum (T. harzianum) on compost, focusing on bacterial community structure (16S rRNA) and chitinase gene diversity. In addition, the ability of vineyard pruning waste compost, amended (GCTh) or not (GC) with T. harzianum, to suppress Fusarium wilt was evaluated. The addition of T. harzianum resulted in a high relative abundance of certain chitinolytic bacteria as well as in remarkable protection against Fusarium oxysporum comparable to that induced by compost GC. Moreover, variations in the abiotic characteristics of the media, such as pH, C, N and iron levels, were observed. Despite the lower diversity of chitinolytic bacteria found in GCTh, the high relative abundance of Streptomyces spp. may be involved in the suppressiveness of this growing media. The higher degree of compost suppressiveness achieved after the addition of T. harzianum may be due not only to its biocontrol ability, but also to changes promoted in both abiotic and biotic characteristics of the growing media.     

土传黄瓜立枯病高效拮抗菌的筛选鉴定及其生物效应

采用平板对峙法从黄瓜根际土壤中分离出的400余株细菌菌株中筛选出16株对立枯丝核菌Rhizoctonia solani具有拮抗效果的菌株,抑菌带直径在0.81～1.93 cm之间。并从中选出3株抑菌带直径在1.6cm以上的菌株N33、N35和N43,结合形态、生理生化特性及16S rDNA序列比对分析,鉴定N33菌株为假单胞菌属Pseudomonas sp.,N35和N43菌株为芽胞杆菌属Bacillus sp.。通过在黄瓜育苗基质中添加选育的高效拮抗菌株,观测其对黄瓜苗生长的促进作用以及对立枯病的防治作用,3株菌株均具有促进黄瓜苗期生长和防治苗期立枯病的作用,其中N43菌株促生及防病效果均最显著,地上部鲜重比对照处理增加62.16%,防治效果达62%。     

Selective accumulation of Trichoderma species in soils suppressive to radish damping-off disease after repeated inoculations with Rhizoctonia solani, binucleate Rhizoctonia and Sclerotium rolfsii

Artificial suppression of radish damping-off disease was induced by repeated soil inoculations with Rhizoctonia solani, binucleate Rhizoctonia (BNR) and Sclerotium rolfsii in pot systems. Soils repeatedly inoculated with R. solani and BNR showed suppressive to disease caused by R. solani and S. rolfsii, while soils repeatedly inoculated with S. rolfsii were suppressive to disease caused by S. rolfsii but not by R. solani. Species of Trichoderma were consistently isolated from soils repeatedly inoculated with R. solani, BNR and S. rolfsii. These Trichoderma spp. accumulated selectively in relation to the fungal species that was repeatedly added to the soils. The ratios of the frequencies of T. viride, T. harzianum and T. hamatum were 5:2:2 and 8:5:2 in soils repeatedly inoculated with R. solani and BNR, respectively. In S. rolfsii- inoculated soils, T. koningii was predominantly isolated. T. viride, T. harzianum and T. hamatum isolates obtained from either R. solani or BNR after repeated additions to the soils suppressed radish damping-off disease caused after challenge inoculations with R. solani or S. rolfsii. Among the Trichoderma species, T. viride consistently yielded high levels of suppression. However, isolates of T. koningii obtained from S. rolfsii-infested soils suppressed disease caused by S. rolfsii but failed to suppress disease caused by R. solani. Generally, the species of Trichoderma accumulated in a selective pattern that was closely related to the species of fungal pathogen used to induce the suppressive soil.     

嘧菌酯对番茄早疫病菌的抑制作用

采用菌丝生长速率法和孢子萌发法分别测定了嘧菌酯对番茄早疫病菌Alternaria solani菌丝生长的抑制作用及其对分生孢子萌发的影响,同时测定了其对菌丝呼吸速率的影响。结果表明,嘧菌酯抑制番茄早疫病菌分生孢子萌发的作用强于对菌丝生长的抑制作用。抑制菌丝生长的剂量-反应曲线与抑制菌丝呼吸的剂量-反应曲线趋势相似,但菌丝在经嘧菌酯处理5~7 h后呼吸速率恢复到对照水平,相比之下,嘧菌酯却能一直抑制菌丝的生长。因此,采用孢子萌发法测定番茄早疫病菌对嘧菌酯的敏感性比用菌丝生长速率法测定更为适宜。水杨肟酸与嘧菌酯在抑制番茄早疫病菌菌丝生长和分生孢子萌发方面有协同作用,对菌丝生长的平均协同系数为7.24。随处理时间延长,药剂对菌丝耗氧的抑制作用下降,但仍抑制菌丝生长。研究表明:呼吸作用对药剂的敏感性随着处理的延长而下降的机理不是因为旁路氧化途径增强, 也不是因为基质中药剂效力的下降,而是存在其它机制。     

High activities and mRNA expression of pyrophosphate-fructose-6-phosphate-phosphotransferase and 6-phosphofructokinase are induced as a response to Rhizoctonia solani infection in rice leaf sheaths

Rice sheath blight disease caused by Rhizoctonia solani Kuhn results in significant yield and quality losses in rice growing areas worldwide. The glycolytic pathway is important in the resistance response to R. solani infection in rice. This study examined one of the regulatory steps in this pathway catalyzed by pyrophosphate-fructose-6-phosphate-phosphotransferase (PFP) and 6-phosphofructokinase (PFK). PFP and PFK activity in R. solani-infected rice plants increased. The mRNA expression of PFP/PFK isozymes showed that PFK 1, 2, 4 and 5 in the resistant line at 1 dpi were high as compared to the gradual increase observed in the expression of all PFP isozymes. Also PFK 1, PFK 3, PFK 4, PFK 5, PFP 2 and PFP 5 were adaptive to sheath blight disease infection and linked to defence response while, the expressions of PFK 2, PFP 1, PFP 3 and PFP 4 although adaptive, were not specific to R. solani infection. These observations provide evidence that (a) both PFP and PFK have isozymes that play an adaptive role after R. solani infection but while those of PFK are expressed at higher levels within a short time after infection those of PFP are expressed gradually, (b) the adaptive activation of PFP in R. solani-infected rice plants is correlated with the paired expression of its α- and β-subunits as shown by PFP 2 and PFP 5, and (c) the expression of some α-subunits is not specific to R. solani infection as shown by PFP 1, PFP 3 and PFP 4.     

Integrated control of soilborne and bulbborne pathogens in Iris

Coating iris bulbs with a preparation ofTrichoderma harzianum was highly effective under greenhouse conditions in reducing incidence of diseases caused byRhizoctonia solani andSclerotium rolfsii. In field experiments with irises for bulb production, the incidence ofR. solani in plants and bulbs was effectively reduced (up to 93%), and the yield increased (by 35–41%), by applyingT. harzianum either as a bulb coating or broadcast application (biological treatment), treating soil with pentachloronitrobenzene (PCNB; quintozene) (chemical treatment), or solarizing the soil by mulching it with transparent polyethylene sheets (physical treatment) prior to planting. Combined treatments,i.e., chemical-biological or physicalbiological, were the most effective.T. harzianum bulb treatment and broadcast application in field plots increasedTrichoderma population density in the soil by 4–27 times.     

Effect of chitin on biological control activity of Bacillus spp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants

Two bacterial isolates and one strain of Trichoderma harzianum were tested alone and in combination with chitin for efficacy in control of root rot disease caused by Phytophthora capsici and Rhizoctonia solani in pepper plants under greenhouse conditions. These bacteria (Bacillus subtilis HS93 and B. licheniformis LS674) were isolated from repeatedly washed roots of pepper plants. In in vitro assays, HS93, LS674 and T. harzianum were antagonistic against P. capsici and R. solani and produced high levels of chitinase. Seed treatment and root drenching with bacterial suspensions of HS93 with 0.5% chitin was more effective against Phytophthora and Rhizoctonia root rot than addition of the organisms without chitin. LS674 and T. harzianum reduced Rhizoctonia but not Phytophthora root rot. In two greenhouse tests, seed treatment and root drenching with HS93 amended with chitin enhanced its biocontrol activity against P. capsici but not on R. solani. The effects of LS674 and T. harzianum against R. solani were significantly enhanced when they were used as suspensions with 0.5% chitin for root drenching, but this had no effect on P. capsici. In both greenhouse experiments, the use of 0.5% chitin alone for root drenching reduced Rhizoctonia root rot. Reduction of root rot disease was accompanied by increased yield. These results show that the antagonistic activity of HS93, LS674 and T. harzianum may be stimulated by chitin resulting in significant improvements in their effectiveness against pathogens.     

Inhibition of plant pathogens in vitro and in vivo with essential oil and organic extracts of Cestrum nocturnum L.

The efficacy of the essential oil and various organic extracts from flowers of Cestrum nocturnum L. was evaluated for controlling the growth of some important phytopathogenic fungi. The oil (1000 ppm) and the organic extracts (1500 μg/disc) revealed antifungal effects against Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum in the growth inhibition range of 59.2-80.6% and 46.6-78.9%, respectively, and their MIC values were ranged from 62.5 to 500 and 125 to 1000 μg/mL. The essential oil had a remarkable effect on spore germination of all the plant pathogens with concentration and time-dependent kinetic inhibition of P. capsici. Further, the oil displayed remarkable in vivo antifungal effect up to 82.4-100% disease suppression efficacy on greenhouse-grown pepper plants. The results obtained from this study may contribute to the development of new antifungal agents to protect the crops from fungal diseases.