Effects of phosphate solubilization and phytohormone production of Trichoderma asperellum Q1 on promoting cucumber growth under salt stress

Salinity is one of the major abiotic stresses limiting crop growth and yield.This study investigated the underlying mechanisms of Trichoderma asperellum Q1 in promoting cucumber growth under salt stress, including the abilities of the strain to solubilize phosphate and to produce phytohormone.The results showed that T.asperellum Q1 could solubilize inorganic or organic phosphate and the activities of phosphatases and phytase could be detected in the culture supernatant.In hydroponic experiments, the growth of cucumber seedlings was increased in the hydroponic system treated by culture filtrate of strain Q1 with tricalcium phosphate or calcium phytate under salt stress.This strain also exhibited the ability to produce indole acetic acid（IAA）, gibberellic acid（GA） and abscisic acid（ABA） in liquid medium without any inducers.The levels of those three phytohormones in cucumber seedling leaves also increased after inoculated with this strain, along with increased root growth and root activities of the plant.These results demonstrated the mechanisms of T.asperellum Q1 in alleviating the suppression effect of salt stress involving the change of phytohormone levels in cucumber plant and its ability of phosphate solubilization.

Soil application of Trichoderma asperellum GDFS1009 granules promotes growth and resistance to Fusarium graminearum in maize

Of diseases affecting maize(Zea mays), Fusarium graminearum is one of the most common pathogenic fungi that cause stalk rot. In the present study, the Trichoderma asperellum GDFS1009 strain was shown to be an effective biocontrol agent against stalk rot. In a confrontation culture test, Trichoderma strain displayed an approximately 60% inhibition rate on the mycelial growth of F. graminearum. In pot trials, the application of 2 g/pot of T. asperellum GDFS1009 granules had the best control effect on stalk rot at the seedling stage(up to 53.7%), while the average plant height and fresh weight were also significantly improved. Additionally when fertilizer was added at 8 g/pot, the application of 3 g/pot of Trichoderma granules had the best control effect on maize stalk rot(40.95%). In field trials, when inoculating F. graminearum alone, the disease index for inoculating was 62.45, but only 31.43 after treatment with T. asperellum GDFS1009 granules, suggesting a control efficiency of 49.67%. Furthermore, in a naturally F. graminearum-infected field, Trichoderma granules, when applied for 3 consecutive years, showed significant control of stalk rot and increased yields.

Effect of acid phosphatase produced by Trichoderma asperellum Q1 on growth of Arabidopsis under salt stress

Salt stress is a major environmental factor that inhibits crop growth.Trichoderma spp.are the most efficient biocontrol fungi and some of the strains can stimulate plant growth.Phosphate solubilization is known as one of the main mechanisms in promoting plant growth,but the underlying mechanisms of phosphate solubilization in the salinity still need to be explored.The Trichoderma asperellum Q1 isolated and identified in our lab is a beneficial rhizosphere biocontrol fungus with a high phosphate solubilization activity.It could produce acid and alkaline phosphatases when using insoluble organic phosphorus as the sole phosphorus source,the salt stress increased the phosphorus-solubilization ability of the strain and the activities of the two enzymes.Furthermore,an acid phosphatase was purified from the fermentation broth by ammonium sulphate precipitation,ion-exchange,and gel filtration chromatography.Its molecular weight was 55 k Da as determined by SDS-PAGE.The purified acid phosphatase was used to investigate growth performance of Arabidopsis thaliana by plate assay and the result showed that it contributed to Arabidopsis growth by transforming organic phosphate into a soluble inorganic form under salt stress.To our knowledge,this is the first report on acid phosphatase purification from T.asperellum and its function in regulation of plant growth under salt stress.

Biocontrol potential of Trichoderma asperellum mutants T39 and T45 and their growth promotion of poplar seedlings

This study investigates the biocontrol potential of Trichoderma asperellum mutants against Rhizoctonia solani, Alternaria alternata, and Fusarium oxysporum and growth promotion of Populus davidiana 9 P. alba var.pyramidalis(Pd Pap poplar) seedlings. A T-DNA insertion mutant library of T. asperellum was constructed using Agrobacterium tumefaciens-mediated transformation.Sixty-five positive transformants(T1–T65) were obtained.Growth rates of the mutants T39 and T45 were the same,39.68% faster than the WT. In toxin tolerance tests, only T39 had greater tolerance to A. alternata fermentation broth than the WT, but mutant T45 had the same tolerance as the WT to all fermentation broths. Furthermore, T39 and T45 had a greater antagonistic ability than the WT strain against R. solani and A. alternata. The inhibition rate of the mutants T39 and T45 against A. alternata are 73.92% and80.76%, respectively, and 63.51% and 63.74%, respectively. Furthermore, the three strains increased the activities of superoxide dismutases, peroxidase, catalase(CAT)and phenylalanine ammonia lyase(PAL) in Pd Pap seedling leaves. CAT and PAL activity in the Pd Pap seedling leaveswas 11.25 and 5.50 times higher, respectively, in the presence of T39 than in the control group and 12 and 6.35 times higher, respectively, in the presence of T45 than in the control group. All three strains promoted seedling growth and the root and stem development, especially mutant T45. Mutants T39 and T45 reduced the incidence of pathogenic fungi in poplar and stimulated poplar seedling growth.

Inoculation with chlamydospores of Trichoderma asperellum SM-12F1 accelerated arsenic volatilization and influenced arsenic availability in soils

Fungi capable of arsenic（As） accumulation and volatilization are hoped to tackle As-contaminated environment in the future. However, little data is available regarding their performances in field soils. In this study, the chlamydospores of Trichoderma asperellum SM-12F1 capable of As resistance, accumulation, and volatilization were inoculated into As-contaminated Chenzhou（CZ） and Shimen（SM） soils, and subsequently As volatilization and availability were assessed. The results indicated that T. asperellum SM-12F1 could reproduce well in As-contaminated soils. After cultivated for 42 days, the colony forming units（cfu） of T. asperellum SM-12F1 in CZ and SM soils reached 10^10–10^11 cfu g^–1 fresh soil when inoculated at a rate of 5.0%. Inoculation with chlamydospores of T. asperellum SM-12F1 could significantly accelerate As volatilization from soils. The contents of volatilized As from CZ and SM soils after being inoculated with chlamydospores at a rate of 5.0% for 42 days were 2.0 and 0.6 μg kg^–1, respectively, which were about 27.5 and 2.5 times higher than their corresponding controls of no inoculation（CZ, 0.1 μg kg^–1; SM, 0.3 μg kg^–1）. Furthermore, the available As content in SM soils was decreased by 23.7%, and that in CZ soils increased by 3.3% compared with their corresponding controls. Further studies showed that soil p H values significantly decreased as a function of cultivation time or the inoculation level of chlamydospores. The p H values in CZ and SM soils after being inoculated with 5.0% of chlamydospores for 42 days were 6.04 and 6.02, respectively, which were lowered by 0.34 and 1.21 compared with their corresponding controls（CZ, 6.38; SM, 7.23）. The changes in soil p H and As-binding fractions after inoculation might be responsible for the changes in As availability. These observations could shed light on the future remediation of As-contaminated soils using fungi.

Screening,Identification,Metal-tolerance Cadmium Biosorption and Application of a Cadmium-Resistant Trichoderma asperellum

This study was designed to screen and identify cadmium-resistant fungi and characterize its cadmium adsorption.A cadmium-resistant strain(HD228)was isolated from cadmium-polluted paddy soil by using the Cd^2+concentration gradient pressure domestication method.According to the morphological characteristics and the analysis of internal transcribed spacers(ITS)region gene sequence phylogenesis,the strain was identified as Trichoderma asperellum.This strain was resistant to Cd at 22 mmol/L with a Cd adsorption rate of up to 79.88%,and it was also resistant to other heavy metals such as Pb,Zn and Cu.The colony diameter decreased as the heavy metal concentration increased,and the colony was out-of-shape when the Cd^2+concentration was 20 mmol/L.The strain HD228 was found to grow well at pH 4 to 8 and a temperature between 20 and 35℃.The optimal growth conditions were established to be pH 5 and temperature of 30℃.Fermented liquid of the strain is neither disease-causing nor inhibitory to rice seedling emergence,and indeed it improves rice seedling and root growth and enhances rice’s detoxification ability under Cd stress.Thus,the Cd-resistant fungus HD228 has the potential for the treatment of Cd-polluted rice paddies.

棘孢木霉(Trichoderma asperellum)产聚半乳糖醛酸酶的特性

聚半乳糖醛酸酶是水解D-半乳糖醛酸α-1,4-糖苷键的酶,在食品工业特别是果蔬加工中具有重要意义。该文研究棘孢木霉(14636)所产聚半乳糖醛酸酶的酶学特性,采用硫酸铵沉淀法、透析袋透析,对粗酶液进行纯化,并以聚合烯酰胺凝胶电泳(sodium dodecyl sulfate polyamide gel electrophoresis,SDS-PAGE)确定其分子质量大小。实验结果表明,该酶的活性区域在29.29-50.46 k Da,聚半乳糖醛酸酶的最适反应pH为4.0,在pH 3.0-5.0稳定性较好,最适温度为40℃,具有一定的热稳定性;在乙酸乙酸钠缓冲溶液中活性较高。催化性能表明,果胶是聚半乳糖醛酸酶的最佳底物,Km值为0.74 mg/m L,Vmax为3 100μg/min;Mn^2+、Mg^2+、Cu^2+、Ca^2+和Tritonx-100对酶有激活作用,Ca^2+和Tritonx-100激活作用较强,Co^2+、Zn^2+、Ba^2+、Li^+、Fe^2+、SDS和Tween-80对该酶有不同的抑制作用;贮藏特性研究表明,该酶即使在30℃下30 d仍保留70%以上酶活性。研究结果为果蔬清洁加工领域新型酶制剂的开发提供理论依据。

Preparation on the basis of Trichoderma asperellum in the system of biological protection of wheat from Fusarium ear scab

During the last century, as the area of wheat grown under advanced grain husbandry has increased worldwide, so too has the importance of Fusarium ear scab (FES) (synonym, Fusarium head blight) caused by several species of the fungus Fusarium. Yield losses due to FES can total 20%-40% and more depending on climatic conditions. During the last twenty years epidemics of FES in cereals have become chronic all over the world, including the United States and Russia. The most destructive of these were observed in 1982, 1986, 1990-1996 in USA and in the south of Russia in 1982, 1984, 1988, 1992. The harmful effect of FES is manifested not only in reduced grain yields, but also in the contamination of grains and grain products with mycotoxins, such as deoxynivalenol (DON) and its derivatives (3-alfa acetyl-DON, 15-alfa acetyl-DON), T-2 toxin and zearalenone. Standard means to control FES (cultural control methodologies, chemical pesticides, and FES resistant varieties) have little effect or are not practical and rarely reduce the accumulation of mycotoxins in grain. We have developed a new technique to reduce FES using biological preparations. The technique utilizes wheat seed pretreatment with a biofungicide “Mycol” in combination with spraying wheat plants during flowering with a yeast preparation. Technology for production of Mycol on the basis of Trichoderma asperellum strain GJS 03-35 (systematics by Samuels) has been developed. This strain shows hyperparasitic activity against a wide spectrum of plant pathogens, including Fusarium graminearum, a causative agent of FES in wheat. Experiments conducted in the United States demonstrated that spraying wheat plants during flowering with the patented yeast Cryptococcus nodaensis OH 182.9 (NRRL Y-30216) reliably reduces FES development. Tests of the Mycol preparation and the yeast OH 182.9 (EOD) have been performed on the spring wheat “Ivolga” in greenhouse conditions (the Moscow region) and on the winter wheat “Kupava” in field trials in the North Caucasian region. An isolate of F. graminearum was used to insure adequate levels of disease development in greenhouse and field experiments. FES disease severity and incidence, as well as mycotoxin accumulation in wheat grains was studied for single or combination treatments with the biological preparations. Mycol (in concentrations 0.1, 0.5, 1.0, 2.0 kg/t of seeds) was used for wheat seed pretreatment. The yeast preparation EOD (2.0×107 cfu/mL) was applied by spraying wheat plants during flowering. Chemical pesticides (Raxyl, TMTD) and a biological preparation Agat-25K were used as alternative control seed treatments. In greenhouse experiments, inoculations of heads with either biological preparation 4 h prior to inoculation with conidia of F. graminearum significantly reduced FES severity. Application with Mycol reduced DON in wheat grains by 6 to 11 fold. EOD alone or, to a lesser extent, in combination was also highly effective in reducing DON content. For treatments consisting of Mycol and EOD, 1000 grain weights were equivalent or higher than for control plants (both infected, and not infected). Wheat seeds obtained from the plants protected by these biological preparations germinated rapidly and possessed high germination rates compared to the FES control. In field trials, Mycol treatments clearly reduced FES symptoms, apparently providing an immunizing effect against FES. Mycol reduced FES severity and enhanced yield of the wheat varieties used. The effect of Mycol used at a minimum test-dose (0.1 kg/t) was not so pronounced. The greatest reduction of FES development was observed at a dose of Mycol of 1.0 kg per 1 t of seeds used in combination with EOD spraying. Experimental results support the contention that the offered technology has good prospects in controlling wheat Fusarium ear scab.

棘孢木霉Trichoderma asperellum在土壤中定殖量的荧光定量PCR检测

为快速准确检测棘孢木霉Trichoderma asperellum在土壤中的定殖量,构建了荧光定量PCR检测体系,并运用该体系对防治黄瓜枯萎病后土壤中的棘孢木霉数量进行了检测。结果表明:所建立的荧光定量PCR检测体系对棘孢木霉DNA特异性强,R^2为0.998,检测限点为15 fg/μL;在灭菌土壤中检测到的棘孢木霉DNA的拷贝数大于1 866 fg/μL时,对黄瓜枯萎病的防治效果高于64.29%;在黄瓜整个生育期内,检测到土壤中的棘孢木霉定殖量呈现先降后升的变化趋势,第7天时,棘孢木霉DNA拷贝数为320 ng/μL,56 d后棘孢木霉DNA拷贝数迅速上升,最高可达5.16×10~4ng/μL,且对黄瓜枯萎病的防治效果为64.29%~76.81%。研究表明,荧光定量PCR方法检测土壤中棘孢木霉数量具有快速、灵敏度高、可靠性强等优点,可用于检测生防菌棘孢木霉施用后的定殖量和生防效果。

Structural Elucidation and Total Synthesis of Trichodermotin A,A Natural α-Glucosidase Inhibitor from Trichoderma asperellum

A pair of alkaloid enantiomers possessing a novel 1-oxaspiro[4.4]non-3-ene-2,7-dione skeleton,trichodermotin A(1),was obtained from the fungus Trichoderma asperellum.Spectroscopic data,X-ray diffraction,and ECD calculations were used to establish its structure and absolute configuration.(−)-1 showed significantα-glucosidase inhibitory activity(IC_(50)=10.1μmol/L vs.60.1μmol/L of positive control).A plausible biosynthetic pathway originating from L-β-phenylalanine was proposed,and a facile total synthesis was further accomplished.The key reaction of our synthetic strategy was a domino aza-Michael/lactonization in one pot,leading to the pivotal 4-amino-oxaspiro[4.4]octane scaffold.

河口底泥来源真菌Trichoderma asperellum对壬基酚的降解路径

从壬基酚(Nonylphenol,NP)污染严重的李村河口底泥中分离纯化出可实验室培养的真菌,以高浓度NP为环境选择压力筛选出了一株目标菌株,18S r DNA确定其归属为棘孢木霉(Trichoderma asperellum)。实验室内研究了该菌株对NP的生物降解过程,LC/MS分析其代谢产物,据此提出了NP可能的生物降解路径。菌株的生长曲线表明NP能促进其生长,该菌株3 d对5 mg/L NP的降解率为71.4%,7 d的降解率达到87.2%,14 d则达到了92.2%。LC/MS分析确定了NP四种代谢产物,分别是2-甲基-1-苯基丁醇、3,5-二羟基苯甲酸、苯二酚和苯甲醚(或苄醇)。提出了两种NP可能的生物降解路径,Ⅰ是最终转化成苯二酚,Ⅱ是最终转化成苯甲醚或苄醇。

棘孢木霉与30%霜霉·嘧菌酯协同防治烟草镰刀菌根腐病

为筛选防治烟草镰刀菌根腐病高效、安全的复配剂,采用菌丝生长速率法测定生防棘孢木霉Tr-0111、化学杀菌剂30%霜霉·嘧菌酯对尖孢镰刀菌(Fusarium oxysporum)的毒力及两者的相容性,同时测定两者混配对尖孢镰刀菌的毒力系数,并通过盆栽试验评价其对烟草镰刀菌根腐病的防治效果。结果表明,30%霜霉·嘧菌酯和棘孢木霉Tr-0111对尖孢镰刀菌均具有较强的毒力,其EC50值分别为0.0643 mL/L、2.36×10^(2)cfu/mL,且两者相容性好。除V30%霜霉·嘧菌酯∶V_(Tr-0111)=4∶6时无增效作用,其他8个混配比例均具有增效作用,其中V_(30%霜霉·嘧菌酯)∶V_(Tr-0111)=7∶3时增效比率最高,为1.16,抑菌率为68.37%。盆栽试验结果表明,V_(30%霜霉·嘧菌酯)∶V_(Tr-0111)=5∶5时,对烟草镰刀菌根腐病的防效最好,为78.18%,其次为7∶3和1∶9,防效分别为77.27%和72.73%,均显著高于单一使用30%霜霉·嘧菌酯和棘孢木霉Tr-0111。因此,可以将0.0643 mL/L 30%霜霉·嘧菌酯和2.36×10^(2)cfu/mL棘孢木霉Tr-0111以5∶5比例混匀复配应用于烟田防治烟草镰刀菌根腐病,减少化学农药使用。

棘孢木霉PT-29与枯草芽孢杆菌S-16共培养对马铃薯枯萎病的防控作用

马铃薯枯萎病是一种土传真菌病害,可对马铃薯整个生育期造成严重危害,在全国主要马铃薯产区都有不同程度的发生。为减少化学药剂防治马铃薯枯萎病带来的弊端,提高生防菌剂效果,研究了棘孢木霉(Trichoderma asperellum)PT-29与枯草芽孢杆菌(Bacillus subtilis)S-16对马铃薯枯萎病主要致病菌尖孢镰刀菌(Fusarium oxysporum)的抑菌效果,并通过盆栽试验测定了2株菌共培养发酵液对马铃薯枯萎病的防治效果及对马铃薯相关防御酶活性的影响。结果表明,2株菌单培养和3种比例共培养发酵液对尖孢镰刀菌均有抑制效果且以PT-29与S-16共培养发酵液组合B1T1(比例为1∶1)抑制效果最佳;单培养S-16的发酵液(B)处理对马铃薯枯萎病的防效为60.09%,单培养PT-29的发酵液(T)处理对马铃薯枯萎病的防效为54.85%,B1T1处理对马铃薯枯萎病的防效达到73.44%。此外,B1T1+FO(接种尖孢镰刀菌)处理的马铃薯叶片过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)活性明显高于FO处理,峰值相比于FO处理分别提高56.44%、766.89%、111.84%、179.34%。综上所述,PT-29与S-16共培养发酵液对马铃薯枯萎病的防治效果高于单培养发酵液,为复合微生物防治马铃薯枯萎病提供了理论支持。

棘孢木霉降解纤维素能力评价及其对玉米秸秆的降解效果

秸秆在自然条件下降解速度慢,容易造成环境污染及资源浪费。为了提高玉米秸秆的还田率和土壤肥力,本研究利用刚果红染色法、滤纸崩解实验等多种方法对实验室分离鉴定保藏的菌株棘孢木霉(Trichoderma asperel-lum)ZZY的降解纤维素能力进行综合评价。结果表明:棘孢木霉中的羧甲基纤维素酶(CMCase)、β-葡萄糖苷酶(β-Gase)和滤纸酶(FPA)的酶活力分别为(22.67±1.95)U/mL、(14.96±1.11)U/mL和(14.56±0.79)U/mL。随后利用盆栽实验将其孢子粉制备的可湿性粉剂施用在玉米秸秆上,与对照组相比,在15~120 d内显著加快了玉米秸秆的降解速度,菌剂组在120 d时达到了最好的降解效果,玉米秸秆的干质量、半纤维素、纤维素、木质素的降解率分别达到(82.87±5.12)%、(81.96±5.02)%、(86.75±4.72)%、(65.11±6.21)%;土壤中的营养成分也显著提高,有机质、全氮、有效磷、速效钾的含量分别达到(30.83±0.22)g/kg、(3.03±0.12)g/kg、(21.82±0.13)mg/kg、(410.30±0.84)mg/kg。这说明棘孢木霉ZZY具有良好的降解纤维素的能力,其制备的可湿性粉剂能有效降解玉米秸秆,提高秸秆还田率,提升土壤肥力。

棘孢木霉TspyrG基因选择标记转化体系的建立

旨在从棘孢木霉Ts93中克隆获得尿嘧啶合成关键酶乳清酸核苷-5′-磷酸脱羧酶基因(TspyrG),该基因无内含子,开放式阅读框大小为1140 bp,编码1个379氨基酸的蛋白。通过农杆菌介导技术,获得棘孢木霉Ts93 TspyrG基因缺失突变株ΔTspyrG,突变株在不含尿嘧啶的培养基上无法生长。同时,以PCAMBIA1300质粒为骨架,构建转化载体1300pyrG。以ΔTspyrG突变株为受体菌,成功构建以尿嘧啶营养缺陷为选择标记的木霉菌农杆菌转化系统。对绿色荧光蛋白(eGFP)的转化试验表明,该系统转化效率与抗生素选择标记转化系统无明显差异。

耐热嗜酸β-甘露聚糖酶TaMan5A在毕赤酵母中高效表达及酶学性质研究

甘露聚糖酶是一类将甘露聚糖降解为短链甘露寡糖及甘露糖的水解酶。为了开发耐酸耐高温的工程酶,从棘孢木霉(Trichodema asperellum)ND-1中克隆甘露聚糖酶基因TaMan5A并在毕赤酵母(Komagataella phaffii)X-33中成功表达。结果显示,该酶的蛋白质分子质量约为67 kDa。最适pH值和温度分别为4.0和65℃,在pH 2.0~6.0具有较强的稳定性,孵育1 h后仍具有80%的活性,且在20~65℃时,残留酶活力均能达到90%以上。1 mmol/L Co^(2+)、脲和5 mmol/L SDS、Zn^(2+)、Fe^(2+)、Mn^(2+)、Ni^(2+)对TaMan5A的活力都有不同程度的激活作用。由于玉米秸秆富含纤维素与半纤维素,因此进一步确定了TaMan5A水解秸秆的最佳组合条件(玉米秸秆用量0.5 g,TaMan5A用量0.5 g,pH 4.0,水解2 d),并得到玉米秸秆用量和TaMan5A用量对玉米秸秆降解效率影响最大。该研究首次从棘孢木霉ND-1中克隆出TaMan5A基因,实验结果表明,TaMan5A是一种嗜酸、耐高温、热稳定性强的酶,具有较大的应用前景,值得进一步研究。

高产纤维素酶菌株的筛选、固态发酵条件优化及其酶学性质研究

该研究从实验室保藏的12株真菌菌株中筛选高产纤维素酶的菌株,并通过形态学观察及分子生物学技术对其进行菌种鉴定。以纤维素酶活力为评价指标,通过单因素和正交试验优化筛选菌株固态发酵产酶条件,并将其与已报道高产纤维素酶菌株的纤维素酶活力进行比较,最后对其粗纤维素酶的酶学性质进行初步研究。结果表明,筛选鉴定得到一株高产纤维素酶的棘孢木霉(Trichoderma asperellum)BW-6,其滤纸酶(FPase)和羧甲基纤维素酶(CMCase)活力分别达到1.99 U/g和7.02 U/g,其最佳固态发酵产酶条件为:以麦麸+甘蔗渣+稻秆(2.0 g+1.5 g+1.5 g)为固态发酵基质,基础盐溶液培养基中以0.5%蛋白胨为氮源,发酵液添加量6 mL,26℃静置培养5 d。在此条件下,滤纸酶活力和羧甲基纤维素酶活力分别达到2.81 U/g和10.15 U/g,分别是优化前的1.41倍和1.46倍,且极显著高于同等条件下草酸青霉(Penicillium oxalicum)EU2101和EU2106的纤维素酶活力(P<0.01),表明菌株BW-6具有工业化应用潜能。此外,菌株BW-6所产纤维素酶的最适反应温度和pH分别为50℃和4.0,在45℃和pH 5.5~6.0条件下稳定。

脂肪酸类天然产物2E,4E-癸二烯酸对烟草根际微生物群落的影响

【目的】脂肪酸类化合物2E,4E-癸二烯酸(2E,4E-decadienoic acid,DDA)为枯草芽孢杆菌(Bacillussubtilis)Tpb55与棘孢木霉(Trichodermaasperellum)HG1共培养发酵产物,对疫霉具有特异性抑制作用。论文旨在评价DDA防治烟草疫霉(Phytophthoranicotianae)引起的烟草黑胫病的效果及其对根际土壤微生物群落的影响,为该类天然产物在植物疫霉病害防治上的应用提供依据。【方法】通过温室盆栽试验测定DDA对烟草黑胫病的防治效果,采用实时荧光定量PCR技术检测处理后根际土壤中烟草疫霉DNA拷贝数,利用Illumina Hiseq高通量测序分析烟草根际土壤细菌、真菌和卵菌群落结构,基于冗余分析和Mantel检验研究土壤养分因子与微生物群落结构的相关性。【结果】与对照(0.5%DMSO)处理相比,1.25 g·L-1的DDA溶液灌根处理组烟草黑胫病病情指数和烟草根际烟草疫霉DNA拷贝数分别降低77.06%和84.35%,土壤pH由6.72增至6.82,电导率增加102.11%,有机质含量则降低13.05%。DDA灌根处理对烟草根际土壤真菌和细菌多样性和丰富度无显著影响,但卵菌的OTU水平、香农指数、Chao1指数和ACE指数显著增加,辛普森指数显著降低。主坐标分析结果显示,DDA处理显著影响烟草根际卵菌群落结构(r=0.667,P=0.028),对真菌(r=0.259,P=0.305)和细菌(r=0.593,P=0.098)则无显著影响。DDA处理组烟草根际土壤Phycicoccus、地杆菌属等革兰氏阳性细菌菌属的相对丰度显著增加,德沃斯氏菌属、鲍特氏菌属、假黄单胞菌属等革兰氏阴性细菌菌属丰度显著降低,曲霉属、Albifimbria、Arcopilus、树粉孢属等植物有益真菌菌属得到显著富集,疫霉属相对丰度显著下降、腐霉属丰度显著上升。Mantel检验结果表明,施用DDA引起的土壤理化性质变化对根际微生物群落无显著影响。【结论】2E,4E-癸二烯酸(DDA)作为一种新型天然脂肪酸化合物,能够有效防治烟草黑胫病、降低烟草根际黑胫病菌数量,并特异性调控烟草根际卵菌群落结构,具有防治作物卵菌病害的应用潜力。

盆栽蓝莓根际接种内生真菌的效应

自然条件下,内生真菌能促进蓝莓生长。从正常生长蓝莓根系分离、筛选获得两株内生真菌棘孢木霉(Trichoderma asperellum)和卷枝毛霉(Mucor circinelloides),将其接种至盆栽蓝莓根际灭菌基质中,分析其对蓝莓植物形态指标、光合能力、根系生长和生物量的影响。结果发现,与不接菌(CK)相比,接种棘孢木霉和卷枝毛霉的叶片SPAD值、叶片净光合速率、根长、体积、根表面积、根系活力、株高、分枝数、地径、叶片数和生物量均显著增加。人工接种棘孢木霉和卷枝毛霉能够显著促进蓝莓生长发育,具有良好应用前景。

棘孢木霉菌株PZ6对香蕉促生效应及枯萎病室内防效的影响

【目的】研究棘孢木霉菌株PZ6对香蕉植株促生效应、抗氧化酶活性及枯萎病菌室内防效的影响,为香蕉枯萎病的防控及PZ6菌株的合理开发利用提供理论依据。【方法】以清水为对照(CK),分别设PZ6孢子液与枯萎病菌菌液(FOC4)5个不同组合处理:(1)PZ6;(2)PZ6+FOC4;(3)PZ6(3 d)+FOC4;(4)FOC4(3 d)+PZ6;(5)FOC4,采用盆栽伤根淋灌法,于6~7叶期对香蕉苗根际进行接种处理;不同时期调查不同处理香蕉苗植株性状,测定香蕉苗根系活力及叶片超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性;通过解剖球茎考察病情指数,评价PZ6对枯萎病的室内防效。【结果】与其他处理相比,单施PZ6菌液处理可极显著提高香蕉苗新增株高和根系活力(P<0.01,下同),两者分别为6.33 cm和487.43μg TTF/g·h;与FOC4处理相比,配施PZ6菌液的FOC4菌液处理也可极显著提高香蕉苗根系活力。接种FOC4菌液50 d后,不同处理香蕉苗球茎枯萎病发病指数和防治效果均以PZ6(3 d)+FOC4处理表现最佳,其病情指数为37.50,防治效果为48.28%,其次为PZ6+FOC4处理,而FOC4(3 d)+PZ6处理表现较差。处理45 d后,除PZ6处理与CK间的叶片POD活性无显著差异(P<0.05)外,不同处理的香蕉苗叶片SOD、POD和CAT活性均与CK间呈极显著差异;除CK外,不同处理间不同酶活性呈一定的变化规律。叶片SOD活性与根系活力表现相似,均以PZ6处理最高(423.71 U/g FW·h),其次为PZ6(3 d)+FOC4、PZ6+FOC4、FOC4(3 d)+PZ6和FOC4处理;叶片POD活性表现相反,以FOC4处理最高(355.07 U/g·min),比对照极显著增加82.33 U/g·min,以处理PZ6最低(273.84 U/g·min)。【结论】棘孢木霉PZ6菌株可在一定程度上提高香蕉苗对枯萎病菌的防御能力,提前施用PZ6菌株可有效阻止病原菌FOC4侵入香蕉苗,延缓植株发病。