桑树内生拮抗细菌Burkholderia cepacia Lu10-1的分离鉴定及其内生定殖

[目的]对从健康桑树叶片中分离到的一株内生拮抗细菌Lu10-1进行鉴定,并探讨该菌株在桑树体内的定殖.[方法]通过形态观察、生理生化指标测定及16S rRNA基因序列同源性分析,结合recA基因特异引物PCR检测法对菌株Lu10-1进行分类学鉴定;以抗利福平(Rif)和氨苄青霉素(Amp)双抗药性为标记,采用浸种、浸根、涂叶和针刺等方法接种,测定Lu10-1菌株在桑树体内的定殖.[结果]结果表明,菌株Lu10-1属于伯克霍尔德氏菌属(Burkholderia),与亲缘关系较近菌株B.cepacia(X80284)的同源性达98%,该菌株的16S rDNA序列已在GenBank中注册,登录号为EF546394;Lu10-1菌株浸种接种后,菌株在桑苗组织中的数量总体上呈现下降趋势,到第20天后菌量趋于稳定;细菌浸根接种后,菌株在茎叶部定殖的菌量均呈现出"先增后降"的趋势.[结论]内生拮抗细菌Lu10-1归属于洋葱伯克霍尔德氏菌基因型Ⅰ(Burkholderia cepacia genomovar Ⅰ);该菌株可在桑树体内长期定殖并传导,且在定殖过程中菌株的拮抗性能未改变;为将该菌株导入桑树体内进行病害的生物防治提供了理论依据.     

基因标记枯草芽孢杆菌BS-68A在黄瓜上定殖

目的:野生型枯草芽孢杆菌Bacillus subtilisBS-68能有效地防治由Pythium spp.和Fusarium oxporum引起的黄瓜立枯病和枯萎病。为了探究该菌株的生防机制,利用该菌株的黄绿荧光蛋白基因和氯霉素抗性基因标记菌株BS-68A研究其在黄瓜植株各个部位的定殖能力、种群动态和在根围的分布。方法和结果:用基因标记菌株发酵液分别对黄瓜种子进行浸种和浇穴处理,播种后30d,该菌能在黄瓜根部和茎基部定殖,不能在茎部和叶部定殖。浸种处理,该菌在茎基部的种群数量为3.1×104cfu/株,大于根部的种群数量4.1×102cfu/株;浇穴处理,该菌在茎基部的种群数量8.0×103cfu/株,低于根部的种群数量2.5×104cfu/株。     

内生细菌01-144在番茄根茎内定殖的初步研究

对内生细菌 0 1 1 4 4进行了抗药性标记 ,利用标记菌株研究了其在番茄根茎内的定殖情况。浸种与灌根处理均可使 0 1 1 4 4在番茄根茎内定殖 ,且在根内的定殖能力大于茎内 ;灌根处理还表明 ,其在茎下部的定殖能力大于茎上部 ;0 1 1 4 4定殖数量动态在根茎内均有一个“由增到减”的趋势 ,但其在根内的数量变化明显较茎内平缓。     

两株PGPR菌株的花生定殖及对根际细菌群落结构的影响北大核心CSCD

为探讨2株根际促生菌耐酪氨酸束村氏菌P9和吡咯伯克霍尔德氏菌P10对花生的促生机制。利用GFP及利福平对2个菌株进行标记、结合扫描电镜观察,追踪了2株PGPR菌株在花生组织中的定殖动态;并通过16S rRNA全长测序对菌株接种花生根际土壤的细菌多样性进行分析。结果表明,利福平标记的P9和P10菌株具有良好的遗传稳定性,其生长和促生特性与原始菌株基本一致。GFP标记菌株可在花生的根尖及其分生区定殖;利福平标记菌株可稳定定殖在土壤及花生的根、茎部,且接菌30 d后定殖数仍保持在10CFU/g数量级。与未接菌植株根际土壤相比,P9、P10及混合菌株接种组的细菌群落相似性更高;接菌组的Flavihumibacter、unidentified_Rhizobiaceae的相对丰度显著增加,芽孢杆菌属、链霉菌属等的丰度较CK有不同程度增加,溶杆菌属、无色杆菌属及假黄单胞菌属等的丰度降低。2株PGPR菌株均可通过直接定殖在植株组织中、间接影响土壤细菌群落结构而发挥对花生的促生作用,混合菌株接种效果更优。研究结果明析了2株促生菌的促生机制,并为菌株的应用提供了科学依据。     

两株香蕉枯萎病拮抗菌在香蕉体内的定殖

本研究将2株香蕉枯萎病拮抗细菌0202和1112分别涂布于含有不同浓度的利福平培养基上,筛选出抗药性标记菌株0202-r和1112-r.将香蕉枯萎病菌Foc4接种于香蕉植株根部,3 d后再将抗药性标记菌株接种在同一部位或位点,于不同时间测定拮抗细菌在根际、根内、球茎和假茎的定殖情况.研究结果显示,将拮抗细菌接种香蕉根部1～3 d后,在植株根际土壤内存在大量拮抗细菌,而根内、球茎和假茎内存在少量拮抗细菌;接种7～14 d后,在植株根内、球茎和假茎内定殖的拮抗细菌增多,并达到最大量;在接种14～21 d后,在植株根内、球茎和假茎内定殖的拮抗细菌量急剧下降;在接种28～35 d后,在植株根内、球茎和假茎内定殖的拮抗细菌量稍有回升.     

生防菌根系定殖竞争作用对西瓜枯萎病发病机理的影响

【目的】西瓜枯萎病是由西瓜专化型尖孢镰刀菌(Fusarium oxysporum f.sp.niveum)引起的一种常见的毁灭性土传病害,对镰刀菌同属非致病性菌株与致病性菌株存在的竞争作用进行研究,有助于获得新的具有生防效果的菌株,从而拓宽西瓜枯萎病生物防治的手段。【方法】利用选择性培养基和稀释平板计数法对温室盆栽试验中西瓜根际和非根际土壤及植物组织中非致病性轮枝镰刀菌菌株(Fusarium verticillioides XA)与致病性尖孢镰刀菌(Fusarium oxysporum LD)进行计数,确定其在西瓜植株根际和组织中的定殖情况。【结果】将从田间西瓜枯萎病发病植株根部分离获得的菌株XA和LD接入健康土壤中,接种菌株XA既不会引起西瓜枯萎病发病症状,也不会影响西瓜植株生物量,但接种菌株LD导致严重发病症状。与单接种LD处理相比较,双接种(XA+LD)处理地上部鲜重和地上部干重都分别增加了151.2%和110%。XA菌株能成功定殖于西瓜根系,但在茎基部没有检测到。在接种菌株LD的处理中植物组织和土壤中致病性镰刀菌的数量达到(1.58 4.85)×104CFU/g。与单接种LD处理相比,双接种菌株XA和LD处理植物茎基部、根系、根际土壤和土体土壤致病性镰刀菌的数量分别下降63.3%、66.1%、3.3%和24.4%,根系、根际土壤和土体土壤非致病性镰刀菌的数量增加到(0.35 3.84)×104CFU/g;双接种处理对西瓜枯萎病的防效达57.8%。【结论】非致病性轮枝镰刀菌菌株XA可有效降低致病性尖孢镰刀菌LD对西瓜植株的定殖侵染能力,对西瓜枯萎病具有一定的生防效果。     

辣椒内生细菌BS-1和BS-2在植物体内的定殖及鉴定

以抗利福平为标记,用浸种、涂叶和灌根方法接种,测定菌株在植物体内的定殖。结果表明,来自辣椒体内的BS_2和BS_1菌株不仅可在辣椒体内定殖,也可在番茄、茄子、黄瓜、甜瓜、西瓜、丝瓜、小白菜等植物体内定殖,BS_2菌株还可在水稻、小麦及豇豆等植物体内定殖,BS_2菌株的内生定殖宿主范围比BS_1菌株的广;另外BS_2菌株可在辣椒和白菜体内较长期定殖。用常规方法、Biolog及16S rRNA序列比较,两菌株鉴定为枯草芽杆菌内生亚种（Bacillus subtilis subsp. endophyticus）。     

巴西固氮螺菌Yu62的EGFP标记及其在小麦体内的定殖研究

以质粒pEGFP-C1为模板,采用PCR方法特异性扩增增强型绿色荧光蛋白(EGFP)基因全长序列,将其与原核表达载体pVK-100连接,构建成重组载体pVK-EGFP.利用电转化法将重组载体导入巴西固氮螺菌Yu62中,得到EGFP标记菌株.用EGFP标记菌接种小麦'小偃107'种子,室内限菌条件下培养10 d后,用荧光显微镜观测标记菌在小麦体内的定殖规律并观察接菌植株的田间生长状况.结果显示,巴西固氮螺菌Yu62能定殖于小麦根毛区、茎组织的细胞间隙等部位,而且接菌小麦'小偃107'植株在根系发育、株高、分蘖数等方面比对照有较明显的优势.研究表明,巴西固氮螺菌Yu62能够定殖于小麦根茎内,并具有促进植物生长的作用.     

接种变栖克雷伯氏菌DX120E对不同甘蔗品种的促生效应

为探究具有高固氮酶活性的变栖克雷伯氏菌DX120E回接甘蔗后的固氮能力和促生效应,以甘蔗品种B8和GT21的无菌组培苗为材料,采用根部接种的方法,研究固氮菌DX120E在甘蔗体内的定殖数量及其对甘蔗组培苗植株生长、氮代谢关键酶活性和硝态氮含量及矿质元素吸收的影响.结果表明： 固氮菌DX120E能在甘蔗根和地上部分组织内生存和定殖;接种固氮菌DX120E可以有效促进甘蔗植株生长和对矿质营养的吸收;显著提高甘蔗植株体内的硝酸还原酶(NR)活性,同时也能在一定程度上提高植株体内谷氨酰胺合成酶(GS)活性,增加硝态氮含量.表明变栖克雷伯氏菌DX120E对甘蔗具有明显的促生效应,在生物固氮肥开发方面具有较大的应用前景.     

环链虫草Cordyceps cateniannulata对番茄生长和抗氧化酶活性的影响

【目的】评估环链虫草Cordyceps cateniannulata对植物促生和植物抗氧化酶活性的影响。【方法】本研究利用浸种法将环链虫草接种于番茄植物体,在接种后的第30天和60天,通过番茄株高、根长、地上和地下部分的干鲜重指标评价其对番茄生长的影响;在接种后第10、20、30、60和90天,通过选择性培养基分析其在番茄不同组织中的生存情况,使用形态学及DNA序列比对的方法检验所分离菌株与原有菌株的一致性。在处理后的第30天,检测番茄叶片中的过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量,观察环链虫草对番茄的抗氧化酶活性影响。【结果】环链虫草可定殖于番茄幼苗且对番茄生长有显著促进作用,菌株对植株的定殖偏好性分别为根部>茎部>叶部。酶活检测结果表明,处理组番茄叶片防御酶活性均呈显著升高的趋势,其中POD、CAT、SOD活性分别比对照增加了52.21%、75.31%和158.59%,MDA含量下降了35.15%。【结论】环链虫草可以通过浸种的方法感染并定殖番茄幼苗的根、茎、叶,促进番茄幼苗的生长并提高番茄抗氧化酶活性,具有较好的田间...     

Delivery methods for introducing endophytic bacteria into maize

The effectiveness of fivemethods for delivery of ten endophyticbacteria into maize stem and root tissues wasstudied in greenhouse conditions at EmbrapaMilho Sorgo, Sete Lagoas, MG, Brazil. Thedelivery methods included seed inoculation,soil drench, foliar spray, pruned-root dip andseed inoculation + soil drench. The bacterialendophytes were previously isolated from maizeplants, and reinoculated and recovered aftertreatments from maize, cv BR201. Each of thefive methods led to establishment and recoveryof bacterial endophytes in root tissues, butonly four isolates were recovered by the seedtreatment method. All 10 isolates wererecovered by pruned-root dip and seed treatment+ soil drench. No isolates were recovered instem tissues by the seed treatment method, andin the root by foliar spray method. However,all isolates were recovered in stem tissue bypruned-root dip method. The pruned-root dip wasthe most efficient method to deliver bacterialendophytes into maize. The isolate, BR201, wasrecovered by almost all methods in root andstem tissues. The results demonstrate thatendophytic bacteria can be recovered from maizetissues following inoculation by the differentmethods described, but the delivery depends onthe methods used and the endophytic bacterialisolate.     

Development of Delivery Systems for Introducing Endophytic Bacteria into Cotton

Experiments were designed to evaluate the effectiveness of several methods for delivering 15 endophytic bacteria into cotton stem and root tissues. The delivery methods included stab-inoculation of bacteria into stems, soaking seeds in bacterial suspensions, methyl cellulose seed coating, foliar spray, bacteria-impregnated granules applied in-furrow, vacuum infiltration and pruned-root dip. The success of delivery was gaged by recovery of the bacteria from internal plant tissues 2 weeks after the plants had been grown in a glasshouse potting mix. Following stab-inoculation into stems or radicles, 10 of the bacterial endophytes which previously exhibited biological control against fusarium wilt of cotton were successfully re-isolated from 50% of the plants inoculated; however, this method was labor-intensive, involved wounding the plant and sometimes reduced plant growth. Four of the other methods established from six to eight of the 15 strains, and, with some strains, all methods effectively established endophytic bacteria, based on re-isolation of strains from internal tissues 2 weeks after inoculation. A method was developed which allowed more convenient isolation of endophytes from a large number of plants. The results suggest that introduction of beneficial endophytic strains into cotton plants could be accomplished by practical methods chosen specifically for each strain.     

The modes of action of two benzimidazoles and thiophanate-methyl used for the control of Verticillium wilt of strawberry

Benomyl, thiabendazole and thiophanate-methyl were found to be fungistatic against Verticillium dahliae in vitro. All materials suppressed wilt symptoms on artificially inoculated strawberry plants when applied as a root dip, soil drench or a foliar application. Quantitative bioassays of the amount of toxic residues and fungal colonization in tissues of treated plants after root treatment revealed that these materials or their toxic breakdown products were readily translocated to crown and leaves: they suppressed the internal spread of the pathogen throughout the plant. After foliar application there was no marked basipetal movement of material within the plant: fungal colonization was greatly reduced in treated leaves, but the roots and crowns were colonized to a similar extent to those of untreated plants.     

Methyl jasmonate stimulates polyamines biosynthesisand resistance against leaf rust in wheat plants

Abstract

Leaf rust of wheat (Triticum aestivum L.), incited by Puccinia recondita ex Desm. f. sp. tritici Eriks, is one of the most important wheat diseases in Egypt. Methyl jasmonate (MJ) is a potential plant elicitor which induces a wide range of chemical and anatomical defense reactions in conifers and might be used to increase systemic resistance against biotic damage. In the greenhouse, different concentrations of MJ (10, 20 and 30 mM) were applied as seed soaking plus foliar spray or only as foliar spray to control leaf rust and induction of secondary compound production in leaves of wheat plants. Foliar spray was applied after 30 and 50 days of sowing. Results indicated that all concentrations and treatments reduced the severity of rust disease caused by P. recondita f. sp. tritici in wheat leaves during 45 days of inoculations. Disease incidence was decreased significantly in MJ-treated plants as seed soaking plus foliar spray with 20 and 30 mM when compared to 10 mM MJ or control plants. The study revealed that, with increasing concentrations of MJ, the secondary metabolites were greatly increased. Endogenous levels of both free and conjugated putrescine, spermidine and spermine increased in response to the elicitor. Activities of polyamine biosynthetic enzymes of ornithine decarboxylase (ODC) and polyamine oxidase (PAO) displayed up to threefold increases relative to untreated control. Moreover, significant increases in activities of plant defense-related protein, enzymes as peroxidase and chitinase as well as free and conjugated phenols contents were recorded in treated plants compared with untreated and infected plants. Furthermore, MJ treatment increased the chlorophyll-a, chlorophyll-b and carotenoids pigments contents, the higher increase was obtained with combined treatment between seeds soaking plus foliar spray at 20 and 30 mM of MJ. Under field conditions, three concentrations of MJ, i.e. 10, 20 and 30 mM as combined treatment between seeds soaking plus foliar spray or only as foliar spray were applied to study their effect against rust disease. Foliar spray was applied after 30 and 80 days of sowing. Results showed that the high reduction in disease severity was obtained with combined treatments between seeds soaking plus foliar spray with MJ at 20 and 30 mM compared with other treatments and control. At the same time, all treatments increased the growth and grain yield of wheat plants. It could be suggested that combination treatment between seeds soaking plus foliar spray with methyl jasmonate might be used commercially for controlling rust disease of wheat plants under field conditions.     

Specificity and mode of action of BTS 44584 and chlormequat chloride on wheat and soybeans

The selective action of the growth retardants chlormequat chloride and BTS 44584 on the growth of wheat and soybean was investigated. Chlormequat chloride retarded the growth of wheat whether applied as a seed soak, foliar spray or soil drench, at concentrations from 10^-2 M to 10^-4 M, although effectiveness depended on the age of the plant at application. BTS 44584 retarded the growth of wheat only when applied to the seeds. In soybeans, chlormequat chloride did not retard elongation growth when applied as a seed soak but showed some effects when applied as a foliar spray and more especially as a soil drench. Irrespective of the type of application, treatment with BTS 44584 retarded growth of soybeans. Thus, the selective action of the two compounds was not solely related to different species but also to timing and mode of application.     

Effect of seaweed concentrate on growth and development of the marigoldTagetes patula

The seaweed concentrate Kelpak, made fromEcklonia maxima, and applied as a foliar spray or a root drench at transplanting, improved both the vegetative and reproductive growth of marigolds. Of particular significance is that the overall production of seeds (fruits) was increased by as much as 50% in some instances. Very low concentrations of seaweed concentrate were not always effective, while the higher dosages decreased vegetative growth.     

Effect of uniconazole and limited water on growth,water relations,and mineral nutrition of “Lalandei” Pyracantha

Pyracantha (Pyracantha coccinea M. J. Roem. Lalandei) plants were treated with uniconazole at 0.5 mg ai container^–1 as a medium drench, 150 mg ai L^–1 as a foliar spray, or left untreated. Plants from all treatments were placed under three water regimes: drought acclimated, nonacclimated and later exposed to drought, or nonstressed. Acclimated plants were conditioned by seven 4-day stress cycles (water withheld), while nonacclimated were well watered prior to a single 4-day stress cycle at the same time as the seventh drought cycle of acclimated plants. Nonstressed plants were well watered throughout the study. Nonstressed plants had higher leaf water potentials and leaf conductances than acclimated and nonacclimated plants, and transpiration rates were higher in nonacclimated than acclimated plants. Uniconazole did not affect leaf water potential, leaf conductance, or transpiration rate. Acclimated plants had smaller leaf areas and leaf, stem, and root dry weights than nonacclimated or nonstressed plants. Plants drenched with uniconazole had the lowest stem and root dry weights. Acclimated plants also contained higher N concentrations than nonacclimated or nonstressed plants, and higher P concentrations than nonacclimated plants. Uniconazole medium drench treatments increased levels of Mn and P. Calcium concentration was increased in plants receiving either medium drench or foliar applications.     

Response of chrysanthemum to uniconazole and daminozide applied as dip to cuttings or as foliar spray

Uniconazole and daminozide were used as dip on unrooted cuttings or as foliar spray on pinched Dendranthema grandiflora Tzvelev. Dalvina to control height. Stem elongation was determined on cuttings dipped in solutions of 0, 1.25, 2.5, 5, or 10 mg/L uniconazole or cuttings were dipped and later treated with foliar sprays in concentrations of 1.25/5, 1.25/10, 2.5/10, and 5/5 mg/L uniconazole, respectively. Other plants were sprayed once or twice with uniconazole at 10 mg/L. Daminozide treatments included a pre-plant dip/foliar spray application of 1000/2000 mg/L, respectively, or two foliar sprays of 2,000 mg/L. Uniconazole dip alone retarded stem elongation linearly up to 8 weeks after propagation, 5 weeks after pinching, but was not discernible from the control treatment 8 weeks after pinching. Uniconazole at 2.5/10 and 5/5 mg/L as a dip/spray combination resulted in plants 33% shorter than the control at the end of the production. Doubling uniconazole dip or spray treatments from 5 to 10 mg/L provided no additional reduction of stem elongation. The single uniconazole spray and both daminozide treatments had no effect on final height, although daminozide treatments reduced stem dry weight compared to the control. Stem dry weight was reduced by uniconazole dip/spray combinations compared to dip treatments alone. Similarly, inflorescence and root dry weights were also reduced by the highest uniconazole concentrations. Higher concentrations of uniconazole reduced transpiration on a per leaf area basis up to 47% compared to the control at the end of production. In contrast to previous work, leaf area and leaf thickness increased with some uniconazole treatments, while time to anthesis was not affected by any of the treatments.     

Cow dung extract: a medium for the growth of pseudomonads enhancing their efficiency as biofertilizer and biocontrol agent in rice

Some pseudomands are being utilized as biofertilizers and biopesticides because of their role in plant growth promotion and plant protection against root parasites, respectively. Two strains of Pseudomonas, P. jessenii LHRE62 and P. synxantha HHRE81, recovered from wheat rhizosphere, have shown their potential in field bioinoculation tests under rice-wheat and pulse-wheat rotation systems. Normally, pseudomonads are cultivated on synthetic media-like King’s B and used for inoculation on seeds/soil drench with talcum or charcoal as carrier material. Cow dung is being used for different purposes from the ancient time and has a significant role in crop growth because of the content in humic compounds and fertilizing bioelements available in it. Here, cow dung extract was tested as a growth medium for strains LHRE62 and HHRE81, in comparison with growth in King’s B medium. The log phase was delayed by 2 h as compared to growth in King’s B medium. The bacterial growth yield, lower in plain cow dung extract as compared to King’s B medium, was improved upon addition of different carbon substrates. Growth of rice var. Pant Dhan 4 in pot cultures was increased using liquid formulation of cow dung extract and bacteria as foliar spray, compared to their respective controls. Biocontrol efficacy of the bioagents was assessed by challenging rice crop with Rhizoctonia solani, a sheath blight pathogen. The growth promotion and biocontrol efficiencies were more pronounced in the case of mixed inocula of strains LHRE62 and HHRE81.     

Fungal entomopathogens as endophytes: can they promote plant growth?

Two experimental replicates were conducted to test whether strains of Beauveria brongniartii (BIPESCO2 and 2843) and Metarhizium brunneum (BIPESCO5) can endophytically colonise Vicia faba plants and improve their growth by comparing them with an endophytic strain of B. bassiana (NATURALIS^®). The plants were inoculated through foliar spray and the effect of inoculation on plant height, leaf pair number, fresh root and shoot weights was measured at 7 and 14 days post inoculation (dpi). Endophytic colonisation of different plant parts with the tested fungal strains were confirmed 7 and 14?dpi through re-isolation of inoculated fungi onto selective media and subsequent Simple Sequence Repeat (SSR) marker-based genetic identification. All tested strains were able to endophytically colonise leaves, stems, and even roots of inoculated plants 7 and 14?dpi, but per cent colonisation varied significantly among strains and plant parts within each sampling date. Foliar inoculation of plants with the tested strains increased plant height, leaf pair number, fresh shoot and root weights; however the increase was not always consistent across sampling dates in both experimental replicates. This study provides the first evidence for the endophytic colonisation of plants with two strains of B. brongniartii, an important biocontrol agent of Melolontha melolontha and other scarab beetles in several European countries, and thus extends previous reports on the ability of entomopathogenic fungi to act as endophytes. It also presents possible explanations for the lack of consistency in the plant growth promotion obtained by the foliar inoculation of entomopathogenic fungi.