一株马铃薯干腐病生防菌的鉴定及其抑菌活性研究

镰孢菌引起的马铃薯干腐病是影响马铃薯贮存期间产量与品质的重要病害之一。寻找高效低毒的防治马铃薯干腐病的生防药剂对提高马铃薯的产量与质量具有重要意义。本研究以腐皮镰孢Fusarium solani为病原菌, 测定分离自马铃薯块茎的菌株2-1-9-CJK-2的抑菌活性。该菌株发酵液、无菌发酵液、菌悬液、胞内组分以及挥发性有机物对腐皮镰孢均具有抑菌活性, 抑制率分别为93.37%、61.92%、80.82%、44.01%和49.16%。结合形态学、生理生化特征和多基因系统发育树构建鉴定其为贝莱斯芽胞杆菌Bacillus velezensis。马铃薯切片试验和马铃薯块茎试验表明, 当菌株发酵液浓度为1×109 cfu/mL时, 对病原菌的抑制效果最好。除此之外, 菌株可以分泌蛋白酶、淀粉酶、纤维素酶, 具有溶磷能力和生物膜形成能力, 但不具有溶血性, 并且对供试的其他6种病原真菌有较强的抑菌活性。研究表明贝莱斯芽胞杆菌2-1-9-CJK-2抑菌谱广, 具有生物安全性, 对腐皮镰孢有较强的抑菌活性, 在植物病害防治中具有一定的应用潜力, 可为马铃薯干腐病生防制剂的开发奠定基础。     

马铃薯内生细菌的分离及环腐病拮抗菌的筛选鉴定

 本研究从大同、太原和内蒙古等地采集马铃薯块茎,分离到240株内生细菌,通过离体抑菌作用测定,共得到55株对环腐病菌有拮抗作用的菌株,占菌株总数的22.9%,抑菌圈半径最大的可达13 mm。按抑菌圈半径大小将拮抗菌分为强、中、弱三类。从中筛选出9个对环腐病等病菌具有较强拮抗作用的内生菌株进行了细菌学鉴定,结果表明:118为荧光假单胞生物型V (Pseudomonas fluorescens biovar V);110为短小芽孢杆菌(Bacillus pumilus);085为嗜热脂肪芽孢杆菌(Bacillus stearothermophilus);069为草生欧文氏菌(Erwinia herbicola);043为草莓黄单胞菌(Xanthomonas fragariae);A-10'、T3为芽孢杆菌属(Bacillus);H1-6为荧光假单胞菌(Pseudomonas fluorescens);116为短小杆菌属(Curtobacterium)。     

南方红豆杉内生真菌的抗菌活性

从南方红豆杉中分离到549株内生真菌,以终极腐霉Pythiumultimum、立枯丝核菌Rhizocto-niasolani为靶标菌进行抗菌活性的筛选。结果表明,对终极腐霉、立枯丝核菌的半抑制稀释倍数(ID50)在10以上的活性菌株分别为20株(占总菌株的3.6%)和15株(占总菌株的2.8%)。其中矮棒曲霉Aspergillusclavatonanicus菌株F0028对这两种病原菌的活性最高(对终极腐霉、立枯丝核菌的ID50分别为278±15.0、108±18.6)。进一步研究表明,F0028发酵液在pH值为1.0~7.0之间均有较强的抑菌活性;经高温高压处理后抑菌活性仍达到了对照的(65±3.8)%。发酵液经高效液相色谱纯化得到5种化合物,活性测定表明,5种化合物对终极腐霉、立枯丝核菌均有不同程度的抑制作用,半抑制浓度(EC50)在7.0~45μg/mL之间。     

解淀粉芽胞杆菌MH71抗菌物质理化特性及对番茄灰霉病菌的抑菌活性

解淀粉芽胞杆菌Bacillus amyloliquefaciens MH71是一株高效广谱拮抗活性菌株,明确该菌株发酵液中活性物质的理化特性和抑菌活性,为解淀粉芽胞杆菌生物农药开发应用奠定坚实的基础。本研究在对菌株MH71抑菌活性物质理化特性研究的基础上,以番茄灰霉病菌为供试致病菌,探究了抗菌活性物质对番茄灰霉病菌的抑菌作用;采用酸沉淀和硫酸铵沉淀分别获得脂肽类和蛋白类活性物质,SDS–PAGE电泳和HPLC分别对活性物质进行检测。该菌活性物质具有较强的耐热性和pH适应性,100 ℃处理60 min和121 ℃处理30 min后抑菌活性分别为原有活性的88.98%和79.60%,在pH 3～11时均有抑菌活性,且对紫外线、蛋白酶K、胃蛋白酶和胰蛋白酶不敏感。该抗菌活性物质对番茄灰霉病菌菌丝的毒力测定结果表明,不同浓度发酵液处理的番茄灰霉病菌菌丝呈现出较多局部膨大或溢缩等异常菌丝。其发酵液经酸沉淀法得到抗菌脂肽类活性物质,经硫酸铵沉淀法得到抗菌蛋白,脂肽类的抑菌活性高于抗菌蛋白类物质,SDS–PAGE电泳检测,得到约40 kD的蛋白条带,HPLC检测表明该抗菌物质中有surfactin存在。     

特基拉芽胞杆菌JN-369抗菌粗蛋白的理化性质和抑菌效果

特基拉芽胞杆菌Bacillus tequilensis JN-369是1株对稻瘟病菌Magnaporthe oryzae有较好拮抗作用的植物内生细菌,其发酵液经硫酸铵沉淀后得到抗菌粗蛋白。为明确菌株JN-369抗菌粗蛋白的抑菌机理,首先确定了该抗菌粗蛋白抑菌谱、热稳定性、酸碱稳定性、紫外稳定性以及酶稳定性,并观察研究了抗菌粗蛋白对稻瘟病菌菌丝和分生孢子的抑制作用。并采用针刺离体接种法测定了40%稻瘟灵EC、25%三环唑WP及不同浓度JN-369抗菌粗蛋白对水稻稻瘟病的拮抗效果。结果表明：菌株JN-369抗菌粗蛋白抑菌谱广,对葡萄灰霉病菌、草莓灰霉病菌和水稻稻瘟病菌等13种植物病原真菌及卵菌表现出抑制作用,而对油菜菌核病菌没有抑制作用;该粗蛋白理化性质相对稳定,具有应用于粮食作物生物防治的潜力;对稻瘟病菌的菌丝生长和分生孢子萌发有双重抑制作用,能使病原菌菌丝细胞膨大,并可抑制病菌分生孢子的萌发;离体防效试验显示,供试杀菌剂对水稻稻瘟病均有显著的拮抗效果,菌株JN-369抗菌粗蛋白在浓度为50～100 μg/mL时对稻瘟病的拮抗效果最好,该菌株有望开发为防治稻瘟病的生防菌株。     

暗黑链霉菌PY-1抑菌活性产物的结构鉴定及其对葡萄霜霉病菌的抑制作用

 从暗黑链霉菌(Streptomyces atratus)PY-1菌株发酵液中分离鉴定抑菌活性产物,评价其对葡萄霜霉病菌(Plasmopara viticola)的抑制作用,为该菌株及其产生的抑菌活性物质的应用提供依据。PY-1菌株发酵液经二氯甲烷萃取,减压浓缩获得抑菌粗提物,稳定性测定结果显示,避光条件下,15℃~65℃、pH 6~10、4℃或常温保存6个月,抑菌活性稳定。采用硅胶柱层析、Sephadex LH-2柱层析、薄层层析和HPLC等技术,对抑菌粗提物进一步分离纯化,得到2种抑菌活性组分;采用ESI-MS、¹H NMR、¹³C NMR等波谱分析技术,对活性组分进行结构鉴定,2种活性组分分别鉴定为5-乙酸环已基酰亚胺(5-acetoxycycloheximide)和环己酰亚胺(cycloheximide)。采用离体叶片法测定2种组分的抑菌活性,不同浓度(10⁴ ng·mL^-1、10² ng·mL^-1、1 ng·mL^-1)的5-乙酸环已基酰亚胺和环己酰亚胺对葡萄霜霉病菌的抑制作用分别为92.59%、86.30%、64.81%和97.04%、91.85%、84.07%。     

一株拮抗链霉菌SM3-7的鉴定及其对玉米茎基腐病的防效

为获得对玉米茎基腐病具有良好防效的拮抗菌株,从玉米根际土壤中分离以玉米茎基腐病拟轮枝镰孢菌Fusarium verticillioides为靶标菌的拮抗放线菌菌株。采用平板对峙法测定不同菌株的抑菌活性,用抑菌圈法筛选效果良好的拮抗菌株并进行种类鉴定、抑菌谱和防效测定。结果表明,从土壤中分离并筛选获得1株对拟轮枝镰孢菌具有明显抑制作用的菌株SM3-7。经形态学、生理生化特征和16S rDNA系统发育分析,将菌株SM3-7鉴定为核粒链霉菌Streptomyces sclerogranulatus。该菌株抑菌谱广,对玉米上的8种植物病原菌有良好的抑制作用,对拟轮枝镰孢菌抑菌圈直径可达（28.03±0.45mm）。盆栽防效结果表明,菌株SM3-7发酵液对抗病品种“良玉99”和感病品种“全玉1233”的玉米茎基腐病14d防效分别达77.28%和76.12%,21d防效达72.20%和71.89%。核粒链霉菌菌株SM3-7发酵液可有效防治玉米茎基腐病,为利用微生物防治玉米茎基腐病拟轮枝镰孢菌提供参考。     

枯草芽孢杆菌B9601-Y2抑菌蛋白活性及产生条件的研究

 枯草芽孢杆菌B9601-Y₂抑菌蛋白可以使棉花红腐病原菌菌丝畸形,呈念珠状,胞壁穿孔,不规则消解,内含物外泄,失活.B9601-Y₂菌株产抑菌蛋白的最适培养条件为:pH 7.0,37℃培养48 h.菌株的马铃薯蔗糖培养液经(NH₄)₂SO₄至80%饱和度盐析能得到0.1 mg/mL以上的粗蛋白,粗蛋白的产量与培养过程中供氧条件、产菌量呈正相关.粗蛋白能耐80℃高温和pH 9.0的碱性,在4℃下,抑菌活性60 d内基本不变.     

宁夏干旱荒漠区苦豆子内生颉颃细菌的筛选及其抑菌物质研究初报

通过测定菌体及胞外分泌物抑茵活性,从354株苦豆子内生细菌中筛选出对黄瓜枯萎病、番茄枯萎病和茄子枯萎病病菌有较强颉颃活性的菌株.对抗菌活性显著的KDB342菌株的胞外抑菌物质进行了初步研究.结果表明,KDB342株胞外分泌物抑菌活性、最高稀释倍数为100倍;其抑菌物质具有一定的热稳定性,100℃处理10 min仍有部分抑菌活性,120 ℃则完全丧失活性;其抗茵活性物质为蛋白类物质,可通过(NH4)2SO4盐析和浓缩,75%硫酸铵提取的粗蛋白抗菌活性最强.     

假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究

本研究采用报告菌平板法及β-半乳糖苷酶活性测定并筛选到10株具有较强降解酰基高丝氨酸内酯（AHLs）能力的细菌,其中菌株WX14降解AHLs能力较强,且菌体和菌体外泌物均具有降解活性。利用PCR法从菌株WX14中克隆到hacA和hacB两个群体感应淬灭酶基因,基因全长分别为2286和2370 bp,氨基酸序列比对分析结果表明,HacA属于阿库来菌素A酰化酶蛋白家族,HacB属于青霉素G酰化酶蛋白家族。这两个基因编码产物均为N末端亲核（N-terminal nucleophile,Ntn）水解酶家族成员。将hacA和hacB导入到软腐果胶杆菌Pectobacterium carotovorum后,可减弱该细菌在马铃薯块和白菜上的致病性,因此这2种AHLs降解酶对依赖于群体感应的软腐病有一定防病作用。经16S rDNA序列同源性分析鉴定,3株为假节杆菌Pseudarthrobacter spp.、1株节杆菌Paenarthrobacter sp.、4株假单胞菌Pseudomonas和2株芽胞杆菌Bacillus spp.。     

Host Specificity of Phytophthora infestans on Tomato and Potato in Ecuador

ABSTRACT Sixty Ecuadorian isolates of Phytophthora infestans from potato and 60 isolates from tomato were compared for dilocus allozyme genotype, mitochondrial DNA haplotype, mating type, and specific virulence on 11 potato R-gene differential plants and four tomato cultivars, two of which contained different Ph genes. Restriction fragment length polymorphism (RFLP) fingerprints of subsamples of isolates from each host were compared by using RG57 as the probe. All potato isolates had the allozyme genotype, haplotype, and mating type of the clonal lineage EC-1, which had been previously described in Ecuador. With the same markers, only one isolate from tomato was classified as EC-1; all others belonged to the globally distributed US-1 clonal lineage. RFLP fingerprints of isolate subsets corroborated this clonal lineage classification. Specific virulence on potato differentials was broadest among potato isolates, while specific virulence on tomato cultivars was broadest among tomato isolates. Some tomato isolates infected all tomato differentials but no potato differentials, indicating that specific virulence for the two hosts is probably controlled by different avirulence genes in P. infestans. In two separate experiments, the diameters of lesions caused by nine isolates from potato and 10 from tomato were compared on three tomato and three potato cultivars. All isolates produced larger lesions on the host from which they were isolated. No isolates were found that were highly aggressive on both tomato and potato. We conclude that there are two different populations of P. infestans in Ecuador and that they are separated by host.     

Genetic diversity and host differentiation among isolates of Phytophthora infestans from cultivated potato and wild solanaceous hosts in Peru

To test the hypothesis that isolates of Phytophthora infestans attacking wild Solanaceae exhibit specialization for particular host species, 115 isolates of P. infestans were collected from cultivated potatoes, nontuber-bearing Solanum spp. of the Basarthrum section and wild tomatoes from five departments in the northern and central highlands of Peru, and characterized using several neutral markers. All isolates belonged to one of four clonal lineages described previously in Peru: EC-1, US-1, PE-3 and PE-7. There was a strong association of three lineages with host species: PE-3 was only isolated from cultivated potato, while PE-7 and US-1 were only isolated from nontuber-bearing Solanum spp. ( Basarthrum section and wild tomatoes). EC-1 was isolated from all host groups sampled. A subset ( n  = 74) of the isolates was evaluated for metalaxyl resistance. High levels of resistance were found almost exclusively in EC-1 and PE-3, while US-1 and PE-7 isolates were generally sensitive. In a detached-leaf assay for lesion diameter using five EC-1 isolates from S. caripense and seven EC-1 isolates from cultivated potato, there was a significant interaction between isolate origin and inoculated host, caused by higher aggressiveness of EC-1 from cultivated potato on its host of origin. In a comparison of EC-1 (seven isolates from cultivated potato) and US-1 (three isolates from S. caripense ), each pathogen lineage was more aggressive on its original host species, causing a highly significant interaction between isolate origin and inoculated host. Wild tomatoes and nontuber-bearing Solanum spp. harbour several pathogen lineages in Peru and could serve as reservoirs of inoculum that might contribute to epidemics on potato or tomato. Potential risks associated with the use of wild Solanum hosts as sources of resistance to P. infestans are discussed .     

玉米马铃薯间作群体的蒸腾量和蒸腾效率研究

以玉米(Zea mays L.)和马铃薯(Solanum tuberosum L.)间作为研究对象,结合大田和盆栽试验,通过观测土壤蒸发、土壤含水量和作物蒸腾量,分析作物的水分竞争能力和间作的产量优势,研究间作对作物水分利用的影响特征与机理。大田试验结果表明:1玉米与马铃薯在同一时期的蒸腾量差异导致了两作物种植区域之间的土壤含水量差异;2间作的蒸腾量4 424.07 t·hm-2显著大于单作玉米和单作马铃薯蒸腾量的加权平均值3612.27 t·hm-2(P0.01);3间作的土地当量比LER(land equivalent ratio)1(P0.01),说明间作表现了增产优势。盆栽试验结果表明:1玉米和马铃薯在同一时期的蒸腾量存在差异,这意味着两作物种植区域的土壤含水量也存在差异(盆栽试验已基本消除土壤蒸发,土壤含水量主要受作物蒸腾影响);2间作的蒸腾量51.79 kg·盆-1显著大于单作玉米和单作马铃薯蒸腾量的加权平均值48.36 kg·盆-1(P=0.011);3作物的种间相对竞争力RC(Relative competitive abilty)1(P0.001),说明玉米在种间水分竞争中占据优势地位;4玉米的蒸腾效率5.38 g·kg-1显著大于马铃薯的3.68 g·kg-1(P0.001);5间作的蒸腾效率4.82 g·kg-1显著大于单作玉米和单作马铃薯蒸腾效率的加权平均值4.53 g·kg-1(P0.001);6间作的土地当量比LER1(P=0.001),说明间作表现了增产优势。以上结果说明:玉米和马铃薯在同一时期的蒸腾量差异可以导致它们种植区域之间的土壤含水量差异,进而使得在间作中,一种作物可以利用另一种作物种植区域的较多土壤水分,所以间作可以提高作物群体蒸腾量;玉米既具有较高的蒸腾效率,又在种间水分竞争中占据优势地位,因此玉米马铃薯间作可以提高作物群体蒸腾效率;蒸腾量和蒸腾效率的协同提高是该间作表现增产优势的重要原因。     

马铃薯种苗复合感染病毒多重RT-PCR同步快速检测

 基于马铃薯病毒ssRNA的快速制备方法,根据马铃薯病毒CP基因序列设计PVX、PVS、PVY和PLRV特异性引物对、P1基因序列设计PVA特异性引物对,建立了多重RT PCR快速检测体系,可以同步检出复合侵染马铃薯种苗主要病毒,灵敏度比传统的ELISA至少高100倍。结合生物活性稳定剂研制的固相化检测试剂盒,已用于四川和重庆等15个县市田间与苗圃248个马铃薯显症或无症样品的实际检测,表明四川和重庆地区2~3种马铃薯病毒往往复合侵染(PVX、PVA和PVS三种病毒复合侵染或PLRV和PVY二种病毒复合侵染)。     

SSR assessment of Phytophthora infestans populations on tomato and potato in British gardens demonstrates high diversity but no evidence for host specialization

Phytophthora infestans populations can differ in composition as a result of host specialization on tomato and potato hosts. In Great Britain many amateur gardeners grow outdoor tomatoes but there is little or no commercial tomato production outdoors. This study analysed isolates of P. infestans from British gardens with 12 multiplexed simple sequence repeat markers that are used to monitor the disease on commercial potato crops. Samples of P. infestans from tomato hosts were collected in 3 years and from potato in 1 year from across Great Britain. Seven previously unreported clonal lineages were detected in garden populations and higher frequencies of unique clonal lineages (28–40%) were present compared with populations from British commercial potato crops reported elsewhere. Garden populations had a lower proportion (11–48% less) of the most common lineages (13_A2 and 6_A1) that together made up at least 86% of the commercial potato populations during the sampling period. Host species accounted for only 2·0% of molecular variance detected between garden potato‐ and tomato‐hosted samples. No significant difference in clonal lineage composition was found between host species in Great Britain and this could be due to the whole P. infestans population overwintering on potato. British garden populations on both hosts were much more diverse than those on commercial potato crops; this finding may be influenced by less frequent fungicide use by gardeners and a higher diversity of unsprayed susceptible potato cultivars, enabling metalaxyl‐sensitive and less aggressive genotypes to survive in gardens.     

宁夏马铃薯晚疫病菌交配型和生理小种研究

为明确宁夏回族自治区马铃薯主产区晚疫病菌的交配型种类以及生理小种的类型、组成和分布,为宁夏有针对性地选育马铃薯抗晚疫病品种提供科学依据。利用A1、A2交配型标准菌株和含有11个主效抗性基因的鉴别寄主,对2018年-2019年从原州区、泾源县、彭阳县、隆德县、西吉县、盐池县、海原县采集得到的130个马铃薯晚疫病菌菌株进行交配型和生理小种鉴定。结果表明,宁夏马铃薯晚疫病菌群体的交配型和生理小种存在多样性,采集地交配型有A1、A2、SF(自育型)3类,分别占被测菌株的30%、63.8%、6.2%;生理小种有8种类型,其中生理小种1.2.3.4.5.6.7.8.9.10.11发生频率最高,占被测菌株总数的44.62%,是宁夏马铃薯主栽区的晚疫病菌优势小种,在各测试地均有分布;次优势小种为3.4.10和1.2.3.5.6.7.8.11,发生频率分别为12.31%和10.77%。宁夏不同种植区域马铃薯晚疫病菌交配型组成复杂,采集地组成差异较大,但西吉县、原州区交配型年度间的变化基本趋于一致。宁夏马铃薯晚疫病菌群体组成日趋复杂,生产中要合理布局已有抗病品种,挖掘培育抗病水平高的新品种,综合防控马铃薯...     

Oospores in cultures of Phytophthora infestans resulting from selfing induced by the presence of P. drechsleri isolated from blighted potato foliage

A Phytophthora species isolated from blighted potato foliage was identified as P. drechsleri , mating type A2. The isolate was found to be weakly pathogenic on a range of hosts. Pairings in vitro of P. drechsleri with British isolates of P. infestans mating type A1 when separated by a polycarbonate membrane resulted in selfed oospores of both species. The possibility that A1 P. infestans is induced to self in the field and the genetic consequences of such selfing are discussed.     

Can weed hosts increase aggressiveness of Phytophthora infestans on potato?

Potato late blight, caused by Phytophthora infestans, is a major disease in potato production throughout the world. In southern Sweden, hairy nightshade (Solanum physalifolium), an alternative non-crop host to the pathogen, is an increasing weed problem. Single-lesion leaves infected by P. infestans were collected from potato and hairy nightshade to determine phenotypic and genotypic population differentiation of P. infestans between the two hosts. Genotypic variation was estimated using microsatellites as markers. The results showed no genotypic differentiation in the samples between the two hosts. Aggressiveness tests were performed using the sampled isolates to cross-inoculate potato and hairy nightshade. The proportion of infected leaves, latency period, lesion growth rate, and sporulation capacity were measured. For isolates from hairy nightshade, the odds of infection were higher on both hosts combined. When tested on potato leaves, isolates from hairy nightshade showed a significantly shorter latency period and higher sporulation capacity compared with isolates from potato. This indicates that an alternative host can filter populations of P. infestans toward a higher aggressiveness, which could lead to increasing problems in controlling potato late blight.     

Phenotypic variation within a clonal lineage of Phytophthora infestans infecting both tomato and potato in Nicaragua

Late blight caused by Phytophthora infestans (Mont.) de Bary is a constraint to both potato and tomato crops in Nicaragua. The hypothesis that the Nicaraguan population of P. infestans is genotypically and phenotypically diverse and potentially subdivided based on host association was tested. A collection of isolates was analyzed using genotypic markers (microsatellites and mitochondrial DNA haplotype) and phenotypic markers (mating type, virulence, and fungicide sensitivity). The genotypic analysis revealed no polymorphism in 121 of 132 isolates of P. infestans tested. Only the Ia haplotype and the A2 mating type were detected. Most of the tested isolates were resistant to metalaxyl. The virulence testing showed variation among isolates of P. infestans. No evidence was found of population differentiation among potato and tomato isolates of P. infestans based on the genotypic and phenotypic analysis. We conclude that the Nicaraguan population of P. infestans consists of a single clonal lineage (NI-1) which belongs to the A2 mating type and the Ia mitochondrial DNA haplotype. Moreover, based on the markers used, this population of P. infestans does not resemble the population in countries from which potato seed is imported to Nicaragua or the population in neighboring countries. The data presented here indicate that the NI-1 clonal lineage is the primary pathogen on both potato and tomato, and its success on both host species is unique in a South American context.     

山西省窖藏薯类块茎中2种短体线虫的种类鉴定

本文采用传统形态学与分子生物学相结合的方法,对采自山西省窖藏马铃薯和薯蓣块茎中的短体线虫进行了种类鉴定.结果 表明,从马铃薯块茎中分离出的短体线虫形态学特征与斯克里布纳短体线虫Pratylenchus scrib-neri一致,其SSU序列与P.scribneri美国群体相似性达99.8％.从薯蓣块茎中分离出的短体线虫...