番茄内生细菌种群动态分析及拮抗菌株的筛选

摘要：采用研磨组织稀释分离法对3个种植地区的番茄内生细菌的种群分布、数量变化规律进行了初步探讨。共分离、纯化得到内生细菌374株,经形态及理化特征鉴定,分属10个不同的属,13个种。其中假单孢菌属Pseudomonas sp.,黄单孢菌属Xanthomonas sp.;肠杆菌属Entenobacter sp.和芽孢杆菌属Bacillus sp.为优势种群。内生细菌在番茄植株种群数量从0.21x104 到13.82x104 cfu.g -1鲜重之间,但不同品种、组织、生育期及种植地,内生细菌的种群和数量各不相同,各组织中内生细菌的种群及数量根中最多,其次是茎和叶,成株期内生细菌无论种类和数量都明显高于苗期。采用平板对峙法从分离的内生细菌中筛选出对番茄灰霉病菌具有体外拮抗活性的菌株32个,占菌株总数的8.56%; 25个菌株对叶霉病菌有抑制作用,占菌株总数的6.68%;对灰霉病菌和叶霉病菌都具有拮抗作用的菌株20个,占菌株总数的5.35%。对灰霉病菌有强拮抗作用的菌株12个,对叶霉病菌有强拮抗作用的菌株仅1个,其中芽孢杆菌是主要的类群。     

绿色荧光蛋白标记的枯草芽胞杆菌R31在西芹根际定殖研究

为了更直观地了解植物内生枯草芽胞杆菌R31在西芹根际定殖情况,评估其定殖能力,为西芹黄萎病生物防治提供依据。通过原生质体转化法将携带绿色荧光蛋白基因的质粒pHT315-gfp导入枯草芽胞杆菌R31细胞中,对菌株进行gfp标记,然后利用gfp标记菌株灌根处理西芹苗,研究其在西芹根际的定殖形式和能力。结果获得了携带pHT315-gfp质粒的R31-gfp菌株,该质粒可以在R31细胞中稳定表达,标记菌R31-gfp在蓝光激发下可以稳定发出强的绿色荧光。利用标记菌接种西芹幼苗后,可以在根系表皮检测到绿色荧光膜。对其定殖量的检测发现,接种3天后标记菌在根表和根际土中稳定定殖,定殖量分别达到1.4×10⁴ cfu/g和7.6×10⁴ cfu/g,并长期以该水平稳定定殖,到第65天仍可在根表和根际土检测到9.1×10⁴ cfu/g和3.17×10⁵ cfu/g的R31-gfp。由此得出,R31可以在西芹根表形成菌膜,并在根际长期稳定定殖。     

黄瓜白粉病的生防细菌筛选及鉴定

为开发黄瓜白粉病的生防资源,与三七根腐病菌、禾谷镰刀菌和柑橘炭疽病菌进行平板对峙培养,从25株黄瓜根际土细菌和91株烟草内生细菌中筛选出具拮抗功能的菌株;以盆栽试验从拮抗功能菌株中筛选能够防治黄瓜白粉病的生防菌株;用PCR扩增生防菌株的gyrB基因,结合菌株的菌落形态和部分生理生化特征,确定生防菌株分类地位。结果表明,在116株对峙培养细菌中,18株对三七根腐病菌、禾谷镰刀菌和柑橘炭疽病菌有较好拮抗效果。在盆栽试验中,烟草内生菌YN-201490和黄瓜根际菌株WL-1对黄瓜白粉病防效较好,其中YN-201490的1.81×10⁸ cfu/mL和WL-1的1.6×10⁸ cfu/mL稀释液连续施用3次后的防效分别达64.86%和61.26%。最终YN-201490被鉴定为枯草芽孢杆菌,WL-1被鉴定为解淀粉芽孢杆菌。     

烟草内生细菌YN2014042对烟草、玉米的定殖和促生作用研究

为了开发利用烟草内生解淀粉芽孢杆菌YN2014042(YN42),对其在植物上的定殖和促生作用进行了研究。用自然转化法将携带绿色荧光蛋白基因的质粒pHAPII成功导入YN42细胞中,获得质粒稳定遗传和对烟草黑胫病菌、玉米穗腐病禾谷镰刀菌等13种病原真菌的拮抗能力与野生型YN42相同的GFP标记菌株YN42-GFP。用1.00×10⁷ CFU/g的YN42-GFP分别浇灌烟草和玉米幼苗,在第50天时烟草根、茎和叶组织中仍回收到1.89×10⁶ CFU/g、1.07×10⁴ CFU/g和0.93×10³ CFU/g的菌落;到第25天时玉米叶片中可回收到6.67×10³ CFU/g的菌落;说明YN42在来源寄主和非来源寄主植物中都能稳定地定殖。将YN42添加到烟草育苗基质中制成微生物功能基质,与普通基质对比,1.00×10⁷ CFU/g的菌株基质上的45天烟苗型高、地上部鲜重分别增长450.58%、756.52%;对玉米灌根30天处理,YN42处理的玉米苗型高和地上部鲜重分别增加19.37%和61.42%。研究结果表明YN42对多种病原菌有抑制作用,能在烟草和玉米上稳定定殖,对烟草和玉米的促生作用显著,在农业上具有良好的应用前景。     

一株巨大芽孢杆菌发酵培养基的优化及解磷效果研究

对巨大芽孢杆菌进行发酵培养基优化及解磷效果研究,旨在为巨大芽孢杆菌的深入研究和生产应用奠定理论基础。以巨大芽孢杆菌为试材,用正交实验优化其发酵培养基条件,用钼锑抗比色法测定其解磷效果。确定最佳培养基及条件为：麸皮1%,豆粕粉0.5%,氯化钠1%,MnSO4 0.05%,pH 7.0,温度30℃,接种量8%,转速200 r/min,发酵有效活菌数达到29×108 cfu/mL;50 L发酵罐26 h基本达到终点,有效活菌数达到48×108 cfu/mL,芽孢率≥95%;其分别在卵磷脂和磷酸钙液体培养基中培养5天后,上清液中有效磷含量为1.67 mg/L和83 mg/L,分别是对照组的26倍和17倍;田间试验处理2周后,土壤有效磷含量为165.3 mg/kg。通过本研究,其有效活菌数比原配方提高7倍,能够有效降解有机磷和无机磷,可以明显提高土壤有效磷含量。     

解淀粉芽孢杆菌悬浮剂研制成功

<正>云南农业大学科研人员近期通过对润湿分散剂、黏度调节剂、UV保护剂、防腐剂、抗冻剂和消泡剂的筛选,确定了5.0×109cfu/mL解淀粉芽孢杆菌B9601-Y2悬浮剂的最佳配方为:Y2菌粉:5.00%,农乳34:2.50%,D-425:2.00%,OP-10:1.50%,黄原胶:0.15%,尿素:0.15%,对羟基苯甲酸     

解淀粉芽孢杆菌菌株P-r21抑菌广谱性及对番茄灰霉病的防治

为明确内生解淀粉芽孢杆菌(Bacillus amyloliquefaciens)菌株P-r21的抑菌广谱性及对番茄灰霉病的防治效果,本研究优化了菌株P-r21发酵条件,利用平板对峙法测定了该菌株对6种病原菌的抑菌活性及对番茄灰霉病菌(Botrytis cinerea)孢子萌发的抑制作用,并通过离体接种试验研究了对番茄灰霉病的防效。结果表明：菌株P-r21最佳发酵条件为在PYJ中培养初始pH 6.0,培养时间为12 h。菌株P-r21对供试的6种植物病原菌(Botrytis cinerea、Fusarium proliferatum、Rhizoctonia cerealis、Gaeumannomyces graminis、Fusarium graminearum、Colletotrichum karstii)的生长均表现出抑制作用;对C. karstii和B.cinerea抑制作用最强,抑菌率达到88.0%和84.5%。发酵滤液对B. cinerea孢子萌发抑制率达80.6%,对叶片和果实灰霉病的防效达到100%和80.0%;发酵液10倍稀释后防效也可达到67.1%和60.7%。本研究明...     

抗香蕉枯萎病甲基营养型芽孢杆菌的鉴定及定殖

为系统评价分离自天然饼肥发酵液4-L-16菌株的抗真菌活性及定殖规律。以尖饱镰刀菌Race 4(Fusarium oxysporum f.sp.cubense Race 4)等9种病原菌为靶标菌株,采用平板对峙、含药介质法检测菌株抗真菌活性,并通过革兰氏染色、形态观察、生理生化特征和16S rDNA序列分析对其进行鉴定。结果表明:4-L-16菌株对9种植物病原菌具有广谱抗性,菌丝生长抑制率可达28.05%~76.07%,孢子萌发抑制率达24.44%~70.00%,其中对香蕉枯萎病菌的菌丝生长和孢子萌发抑制率分别为76.07%和70%,经鉴定该菌株为甲基营养型芽孢杆菌Bacillus methylotrophicus。经抗生素标记,获得抗600μg/mL利福平的标记菌株,盆栽接种试验表明标记菌在香蕉根际土壤和植株体内具有良好定殖能力,接种第10天土壤中菌量达到最大值为6.67×10~6CFU/g,随时间延长,定殖数量逐渐降低,25天后数量趋于稳定,为2.05×10~6CFU/g。香蕉植株内定殖以根系最多,为4.38×10~3CFU/g。甲基营养型芽孢杆菌4-L-16菌株广谱抗真菌活性、良好的定殖能力,已成为香蕉枯萎病生物防治新的菌种资源。     

生物农药武夷菌素对保护地番茄灰霉病的防治效果

为明确生物农药武夷菌素对番茄灰霉病的防治作用,通过室内毒力测定、温室盆栽和田间试验研究了武夷菌素对灰霉病菌的抑制作用和对灰霉病的防治效果。毒力测定结果表明武夷菌素能明显抑制灰霉病菌菌丝的生长,EC50为10.0802 μg/mL。温室试验表明,武夷菌素对番茄灰霉病有保护和治疗作用,而且保护作用优于治疗作用;武夷菌素对灰霉病有较长的持效期,400倍液处理番茄8天后再接种的防效仍然达61.32%。田间试验表明,对保护地番茄间隔7天、连续施药3次,第3次施药15天后2%武夷菌素100倍液的防效达83.93%,显著高于35%腐霉利悬浮剂600倍液的防效。此外对番茄果实品质的测定结果表明,使用2%武夷菌素后番茄除有机酸的含量无明显变化外,可溶性固形物、维生素C和可溶性糖等含量都有一定程度的升高。试验表明武夷菌素对番茄灰霉病有很好的防治作用,同时可以提高番茄果实品质,为番茄生产上合理应用生物农药提供了依据。     

拮抗BS-208菌株对番茄灰霉病诱导抗性的初步研究

本文对拮抗BS-208菌株防治番茄灰霉病的作用机制进行了初步研究。通过在番茄苗上接种番茄灰霉病菌和拮抗枯草芽孢杆菌BS-208,测定PAL、SOD、POD和PPO的活性变化及MDA含量。结果表明,接种BS-208菌株发酵液处理48h后PAL、POD和PPO活性均升高,其中PAL和POD活性在处理后48h达到最大值,均为0．72,PPO活性在处理后96h达到最大值,为4．5,这些酶活性的升高增强了番茄抵御病原菌侵入的能力;而MDA含量与对照相比无明显变化,表明接种BS-208菌株后对番茄植株没有伤害。     

天然酚类化合物与两种化学杀菌剂的联合抑菌活性研究

为探索天然酚类化合物百里香酚、和厚朴酚与化学合成杀菌剂百菌清、啶酰菌胺混配的增效作用,筛选出能够防治灰霉病的混剂,本研究将百里香酚、和厚朴酚分别与百菌清、啶酰菌胺以不同比例混配,采用菌丝生长速率法测定其对灰葡萄孢菌Botrytis cinerea的抑菌活性,并将Bliss法、Mansour法和孙云沛法结合,以优化筛选方案,评价混剂的联合毒力。实验结果表明:将Bliss法、Mansour法和孙云沛法结合使用能够有效降低筛选工作量,且准确率较高;百里香酚和百菌清以质量比5:1、2:3、1:5和1:10比例混配,和厚朴酚和百菌清以质量比10:1、5:1、3:2、2:3和1:10比例混配均表现出协同增效作用;和厚朴酚和百菌清以10:1比例混配时协同增效作用最明显,其CTC值为309.15,以1:10比例混配时抑菌活性最高,其对B. cinerea的EC₅₀为0.29 mg/L,高于杀菌剂百菌清。因此,将定性分析方法与定量分析方法结合使用是筛选杀菌剂混剂配方的合理方案,天然酚类化合物百里香酚、和厚朴酚与百菌清以合理比例混配所得混剂具有防治灰霉病的潜力。     

Metschnikowia pulcherrima strain MACH1 outcompetes Botrytis cinerea, Alternaria alternata and Penicillium expansum in apples through iron depletion

A new strain of Metschnikowia pulcherrima (MACH1) was studied for its efficacy as a biocontrol agent against Botrytis cinerea, Penicillium expansum and Alternaria alternata on apples stored for 8 months at 1 °C. The results of two semi-commercial trials demonstrated the efficacy of the biocontrol strain MACH1. In order to understand the mechanism of action involved, the yeast strain was investigated for its competitive ability for iron against postharvest pathogens of apple. M. pulcherrima strain MACH1 was cultivated on PDA with different concentrations of iron (supplemented as FeCl₃) against A. alternata and B. cinerea. The yeast strain MACH1 produced a wider pigmented inhibition zone against both pathogens in low iron amendments while less inhibition was measured with increased iron concentrations. At the coloured inhibition zone, B. cinerea and A. alternata conidia did not germinate and mycelial degeneration was observed. In addition, a high reduction in infection by both pathogens was recorded in apples treated with M. pulcherrima strain MACH1 supplemented with low iron amendments compared to higher iron concentrations. The same experiments were carried out in vivo and in vitro against P. expansum. M. pulcherrima strain MACH1 amended with low iron concentration (5 μg mL⁻¹ FeCl₃), showing modest lesion diameter reduction and no effect on P. expansum under increased iron and without iron amendments. This study illustrated that iron depletion by the yeast strain MACH1 under low iron conditions could reduce the growth of some postharvest pathogens in vitro and in vivo. Although, iron depletion seems to be a primary mode of action against the postharvest pathogens studied, other mechanisms of action cannot be excluded in the biocontrol employed by M. pulcherrima strain MACH1.     

Control of postharvest rots of sweet cherries and table grapes with endophytic isolates of Aureobasidium pullulans

Fifty-one endophytic isolates of Aureobasidium pullulans were obtained from the flesh of sweet cherries and extensively screened to evaluate their biocontrol activity against postharvest rots of sweet cherries and table grapes. Preliminary analysis of all isolates by randomly amplified polymorphic DNA (RAPD) with three different primers showed the presence of a high genetic variability and enabled isolates not showing any genetic difference to be discarded. Thirty-five isolates with different RAPD electrophoresis patterns had a wide range of biocontrol activity against Botrytis cinerea and Monilinia laxa on single-wounded berries of sweet cherries and table grapes with a reduction of decay from 10 to 100%. Two isolates (533 and 547) significantly reduced B. cinerea on table grape berries also when applied 6, 12, and 24 h after the pathogen inoculation. In a 2-year period of investigation (1998–1999), a reduction of total rots ranging from 32 to 80% (sweet cherries) and from 59 to 64% (table grape) was achieved with isolates 533 and 547 applied after harvest. Preharvest applications of isolate 547 significantly reduced postharvest rots of sweet cherries and table grapes by 47 and 38%, respectively. On the whole, isolates 533 and 547 were more effective than A. pullulans L47, a biocontrol agent of postharvest diseases with a known activity. Population studies demonstrated that isolate 547 was able to survive under field conditions, to increase its population during cold storage, and to penetrate the flesh of sweet cherries when applied during flowering.     

Physio-pathological responses of cabbage stored under controlled atmospheres

The response of cabbage to controlled atmospheres (CA) of 1–3% O₂ and 1–10% CO₂; 3% O₂ and 5% CO₂ was studied in relation to the incidence of physiological and pathological disorders. Fungal infections, pepper spot, low oxygen injury, weight loss, colour, odour, flavour and ethanol were evaluated. The findings show that CA did not reduce Botrytis cinerea rot in comparison to low temperature storage in air. PVC film and CA, on the other hand, reduced pepper spot by over 50% with respect to the air control. In particular, pepper spot was eliminated by high CO₂ (10%) levels. Low O₂ (1% O₂ and 1% CO₂) atmospheres caused 33 and 50% injury respectively after 89 and 109 days of storage. CA and PVC film reduced weight loss to 1%, as compared with 11% in the heads in air, which had shrivelled. The combination of 3% O₂/5% CO₂ and PVC film delayed yellowing with respect to air control. Concentrations of 1–3% O₂ and 10% CO₂ resulted in off odours and flavours after 74 days of storage. This result was confirmed by a considerable increase in ethanol concentrations. The most effective concentration tested was 3%O₂/5%CO₂ although, in general, infection by B. cinerea limited the storage life.     

江苏省域草莓灰霉病菌种群多药剂抗性检测

本研究旨在阐明江苏省草莓主产区草莓灰霉病菌种群对常用杀菌剂的敏感性。采用区分剂量法测定了2019和2020年采集的236株草莓灰霉病菌(Botrytis cinerea)对13种杀菌剂的抗性,并对菌株抗性频率和多药剂抗性进行了分析。结果表明：所有灰霉菌株对咯菌腈(fludioxonil,Flu)表现敏感,而对多菌灵(carbendazim,Car)、吡唑醚菌酯(pyraclostrobin,Pyra)、扑海因(iprodione,Ipr)、腐霉利(procymidone,Pro)、嘧霉胺(pyrimethanil,Pyr)产生了较高的抗药性,其抗性频率分别为58.05%、55.01%、51.44%、45.80%、33.94%;灰霉病菌种群在地区间对不同药剂的抗药性水平不同,溧水、昆山、姜堰、铜山田间以抗性菌株为优势种群,无锡和常州新北地区以敏感菌株为主;本研究在区分计量浓度下,根据各药剂抑菌率分布状况,将供试药剂分为敏感药剂、敏感性下降药剂和抗性药剂3种类型;本研究共检测到9种抗药性类型：Pyra^R(7.32%)、Car^R(6.50%)、Pyr^R(0.81%)、Car^R Pyr^R(3.25%)、Car^RPyra^R(14.63%)、Car^RBos^R(1.63%)、Car^RPyra^RPyr^R(23.58%)、Car^RBos^RPyra^R(7.31%)、Car^RBos^RPyra^RPyr^R(34.15%)。江苏省草莓灰霉病菌种群对多菌灵和吡唑醚菌酯基本丧失了敏感性,对扑海因、腐霉利、啶酰菌胺、戊唑醇等敏感性下降,多药剂抗药性种群以Car^RPyra^RPyr^R和Car^RBos^RPyra^R Pyr^R为主,抗性治理措施中推荐使用不同类型药剂交替防治灰霉病。     

Effect of high-pressure hot water washing treatment on fruit quality, insects, and disease in apples and pears: Part II. Effect on postharvest decay of d’Anjou pear fruit

A hot water pressure process (HWP) was evaluated for its effect on conidia of Penicillium expansum and on development of blue mold, gray mold, and mucor rot of d’Anjou pear fruit. Spores were removed from the water system through dilution and also as a result of hot water in the system that was lethal to the spores. When the system was heated, viable spores were not detected after 2–4 h of operation. Reductions in decay in the HWP system were 36, 29, and 13% for Botrytis cinerea, Mucor piriformis, and P. expansum, respectively. The response of P. expansum appeared related to the length of time fruit was in cold storage. Heat injury was observed on fruit treated with 40 and 50 °C water but not on fruit at 30 °C nozzle temperature. The HWP system described in this study should be considered as a component of an integrated decay control strategy.     

Integration of biocontrol yeast and thiabendazole protects stored apples from fungicide sensitive and resistant isolates of Botrytis cinerea

Three selected biocontrol yeasts, known for their elevated antagonistic activity against several fungal pathogens, were tested in vitro for compatibility with common fungicides. The antagonists were resistant to several fungicides, but they were inhibited by triazoles and dithiocarbamates. The yeast isolate LS28 (Cryptococcus laurentii), which tolerated in vitro high rates of benzimidazoles, was tested, alone or in combination with a low dose (10% of the full label rate) of thiabendazole, against grey mould on stored apples. Wounded fruit were inoculated with thiabendazole resistant and sensitive isolates of Botrytis cinerea, applied separately or as a mixture. A more effective and prolonged control of fungal decay was always exerted by the biocontrol yeast applied together with thiabendazole at a low dose. This treatment provided synergistic effects and was markedly better than treatments applied separately, whereas the fungicide applied alone at the highest label dose was ineffective in the presence of the isolate of B. cinerea resistant to thiabendazole. The biocontrol yeast applied alone provided significant protection of apples stored for up to 12 days at 20 °C (from 39 to 81% less decay than untreated control), whereas the fungicide alone at low or high dose was always ineffective in the presence of the B. cinerea isolate resistant to benzimidazoles. The integrated treatment was highly effective and durable showing high reduction of decay (from 66 to 92%) even after 18 days of storage. This study suggests that the integration of a biocontrol agent with a low rate of fungicide may be a useful alternative strategy to manage both sensitive and resistant isolates of fungal pathogens efficiently and to reduce risks involved with extensive use of synthetic chemicals.     

HPLC法测定发芽糙米中γ-氨基丁酸中不同衍生方式的比较研究

针对HPLC法测定发芽糙米中γ-氨基丁酸(GABA)所用衍生剂缺乏系统的比较分析,研究了3种常见衍生剂及其色谱方法比较。以邻苯二甲醛(OPA)、丹磺酰氯(Dansyl-Cl)、6-氨基喹啉基-N-羟基琥珀酰亚氨基氨基甲酸酯(AQC)3种物质作为柱前衍生剂,色谱柱为intertsil ODS-C18柱（前两者使用）、Waters AccQ Tag氨基酸分析柱,均为梯度洗脱,紫外吸收波长分别为332 nm、386 nm、250 nm,线性方程分别为Y=9.0×10⁶X+2062.5(R²=0.9989)、Y=7.0×10⁶X+11337(R²=0.9982)、Y=7.1×10⁶X+10025(R²=0.9999),检出限分别为0.005 mg/mL、0.01 mg/mL、0.001 mg/mL,同一样品中测得GABA峰面积的RSD分别为1.16%、1.98%、0.94%,加标回收率分别为98.5%~106.9%、95.8%~110.6%、99.4%~103.4%,同一样品中测得GABA含量分别为11.24、10.67、13.47 mg/100 g干重。从上述结果得知,HPLC法测定GABA的3种衍生剂中,测定结果最准确的是AQC,其次是OPA和Dansyl-Cl。AQC作为一种新型衍生剂,在γ-氨基丁酸乃至氨基酸分析中前景广阔。