当归根腐病主要病原菌生物学特性及室内毒力测定

为探究当归(Angelica sinensis)根腐病的病原菌种类、生物学特性并筛选其防治药剂。采用常规组织法分离培养病原菌,利用柯赫氏法则验证其致病性,通过形态学和分子生物学相结合的方法进行病原菌的鉴定、生物学特性分析;采用菌丝生长速率法对病原菌进行室内毒力测定。结果表明,当归根腐病菌菌落毛毡状,在三种培养基上大型分生孢子呈镰刀形,大小为(27.5～47.5)μm×(3.8～7.5)μm(n=50),隔膜数1～5;小型分生孢子在PDA和SNA培养基上呈椭圆形、肾型、纺锤形,在CLA培养基上肾型,(3.8～15.0)μm×(2.5～7.5)μm(n=50),隔膜数0～3;厚垣孢子在CLA培养基上呈串生,PDA培养基上呈不规则形,SNA培养基上厚垣孢子着生方式多样,串生、圆形、不规则形,大小为(2.5～3.8)μm×(2.5～5.0)μm(n=50)。EF-1α、RPB1和RPB2联合序列系统发育树表明,该菌为锐顶镰刀菌(Fusarium acuminatum),其最适生长温度为25℃,最适生长初始pH为9,光照对其生长无显著影响。室内毒力测定显示10%苯醚甲环唑(WG)对其抑制效果最好...     

四川盆地核桃黑斑病病原菌的分离、鉴定与核桃抗病性评价

为鉴定引起四川盆地地区核桃黑斑病的病原菌,采用组织分离法对病原菌进行分离,利用柯赫氏法则验证其致病性,依据菌株形态学和基于16S rDNA基因序列分析对病原菌进行鉴定;同时,利用分离的菌株对18个栽培品种(无性系)进行抗病性评价。结果表明,分离菌株的菌落形态与黄单胞杆菌属(Xanthomonas)相似,其16S rDNA序列与树生黄单胞杆菌(X. arboricola)的(登录号为KP340804.1)同源性高达99%,因此,将引起四川盆地地区核桃黑斑病的病原菌鉴定为树生黄单胞杆菌。18个核桃栽培品种(无性系)的田间侵染发病率和病情指数分别为35.07%～78.57%和17.71%～51.96%,变异系数分别为17.62%和28.78%,并以此为基础评价出5个高抗病品种(无性系)。这为核桃黑斑病致病机理研究和抗病新品种的选育奠定基础。     

河北省冬枣黑斑病病原菌的分离鉴定及生物防治初探

冬枣黑斑病是冬枣重要病害之一,目前多以化学农药进行防治,给自然环境和人类健康带来了严重的威胁。河北省是冬枣种植大省,进行冬枣黑斑病病原菌的分离鉴定对冬枣黑斑病的有效防治具有重要意义。2014年8月至10月从河北省沧州、衡水、邯郸、邢台等地采集冬枣黑斑病病果,采用PDA培养基进行病原菌分离,从病样中共分离出2株分离频率较高的真菌,经过回接和再分离实验筛选出河北省冬枣黑斑病的致病菌株,经形态学和ITS序列分析初步确定该菌为细交链格孢(Alternaria alternata)。以枯草芽胞杆菌J18进行冬枣采后黑斑病的防治,浓度为1×108cfu/m L的菌液对病害的防效为80.67%。     

莴笋炭疽病病原鉴定

【背景】2018年7-8月,甘肃省兰州市永登县武胜驿镇种植的莴笋大面积发生炭疽病,约40%的地块发病,绝收面积达10%。【目的】明确莴笋炭疽病的病原。【方法】采用组织分离法进行病原菌分离;通过Koch’s法则明确分出病菌的致病性;采用形态学和分子生物学方法对病原菌进行种类鉴定。【结果】分离得到形态特征一致的真菌菌株3株。在PDA平板上20°C培养7 d,病菌分生孢子无色,单隔,梭形,(10.44-19.40)μm×(2.61-4.48)μm。代表性菌株Lett-11接种莴笋离体叶片可引起与自然发病相似的症状。BLASTn分析结果显示,菌株Lett-11 (GenBank登录号MK252097)的r DNA-ITS序列与莴苣盘二孢菌Marssonina panattoniana strain CBS 163.25 (GenBank登录号MH854831.1)的序列相似性达99%。【结论】引起莴笋炭疽病的病原菌被鉴定为莴苣盘二孢菌Mar. panattoniana [Synonymy:Microdochium panattonianum],这是莴苣盘二孢菌引起莴笋炭疽病在甘肃的首次报道。     

冠耳霉的分离和鉴定

本文报道了在福建南平发现的感染毛蠓(PsycAoda sp.)成虫的冠耳霉(Conidiobolutcoronatus)有关分离、培养和鉴定等工作。在虫尸上,分生孢子卵圆形至梨形,大小13.6—30.6×13.6—20.4μm,平均23.6×16.8μm。未见囊状体、假根及休眠孢子。易于人工培养。在培养基上初生分生孢子球形,直径26.4—49.6μm,平均37.8μm。初生分生孢子容易萌发,形成次生分生孢子、以及三生分生孢子、四生分生孢子。许多小型分生孢子外生于初生分生孢子上,卵形,平均大小16.5×13.2μm。分生孢子还可以转化成长柔毛分生孢子。     

昆明美人蕉黑斑病菌——链格孢Alternaria alternata

美人蕉是重要的园林绿化植物和生物资源。为了明确美人蕉黑斑病的病原菌种类,并为病害防治提供理论指导,调查了昆明市美人蕉黑斑病的发生情况,采用组织分离法对病原物进行了分离和纯化,并进行了致病性测定。通过形态学和rDNA ITS序列分析,将昆明美人蕉黑斑病的病原菌鉴定为链格孢Alternaria alternata。     

白及锈病病原菌的鉴定及抗锈病资源筛选

为鉴定引起广西种植区白及锈病的病原菌种类且筛选抗锈病的白及资源,该研究对白及锈病病原菌进行分离,并采用形态学和分子生物学的方法对病原菌进行鉴定,同时通过人工接种病原菌法对23份白及进行锈病抗性评价以及筛选抗锈病的白及资源。结果表明:(1)从白及感病叶片中分离的锈病病原菌X2夏孢子呈金黄色,卵圆形或椭圆形,大小为(21.43～30.95)μm ×(13.10～19.05)μm; 冬孢子呈橘红色、红褐色,倒卵形或棍棒状,大小为(17.25～30)μm ×(5.5～6.65)μm。(2)把菌株X2全长689 bp的ITS序列(OQ826009)与GenBank 已登陆的序列进行相似性分析发现,菌株X2与Coleosporium sp.(KY783686.1)匹配度最高,序列一致性为95.86%,但系统发育树表明,X2与Coleosporium bletiae(MN108161.1,OP363680.1)聚为一类群; 结合形态学和分子生物学的方法,鉴定菌株X2为Coleosporium bletiae。(3)人工接种菌株X2 14 d后,23份白及的病情指数范围在0～70.7之间,并根据病情指数将 23 份白及划分为 6 个抗性等级,即表现为免疫的白及1份,病情指数为0; 表现为高抗的白及4份,病情指数为1.7～4.7; 表现为抗病的白及6份,病情指数为5.6～9.4; 表现为中抗的白及5份,病情指数为12.7～18.3; 表现为感病的白及5份,病情指数为32.0～49.1; 表现为高感的白及2份,病情指数为62.2～70.7。综上认为,表现为免疫和高抗的5份白及(分别来自云南红河、广西恭城、广西百色、贵州遵义、湖北宜昌)病情指数低、抗锈病能力强,可推广应用或作为培育抗锈病优良种质的亲本材料。该研究结果为后续开展白及锈病的有效防治与致病机理研究提供了支撑。     

甘肃省一例苹果霉心病病原鉴定

【目的】对甘肃省天水市苹果霉心病病果中分离到的真菌进行病原鉴定。【方法】通过Koch’s法则证病,采用形态学和分子生物学方法对病菌进行种类鉴定。【结果】从甘肃省天水市苹果霉心病病果中分离得到一株淡灰褐色真菌,用该菌的分生孢子悬浮液接种苹果果实,可引起与自然发病相同的苹果霉心病症状。该菌的分生孢子具有不同的形态:产生于分生孢子盘上的分生孢子具3个隔膜,极少数4 5个隔膜,纺锤形或长椭圆形,淡褐色至褐色,基细胞色稍淡,附属丝缺,孢子大小为(12.95 20.42)μm×(4.98 7.97)μm[av.(16.75±1.89)μm×(6.47±0.86)μm];产生于丝状分生孢子梗及分生孢子堆上的分生孢子具2 9个隔膜,纺锤形、棒状或蠕虫状,初始色淡,渐呈褐色,(12.45 59.76)μm×(4.98 11.21)μm[av.(30.10±11.16)μm×(7.26±1.28)μm]。【结论】经形态学和分子生物学鉴定(GenBank登录号JF320818),将该病菌鉴定为Discostroma fuscellum(Berk.&Broome)Huhndorf。这是Discostroma fuscellum引起苹果霉心病在我国的首次报道。     

海南菠萝一种叶斑病病原菌的分离与鉴定及多基因序列分析

为明确菠萝叶斑病病原菌可可毛色二孢菌在海南省各市县的亲缘关系及遗传差异,从海南省的海口、澄迈、儋州、三亚、保亭等16个市县进行病样采集和病样分离,根据科赫氏法则鉴定,获得42株病原菌.观察形态特征,并基于多基因联合序列分析其遗传多样性.结果 表明,通过形态学鉴定和ITS与TUB2基因序列联合进化树分析发现,其中来自海口、澄迈、儋州、三亚、保亭等16个市县的16株代表病原菌均鉴定为可可毛色二孢菌(Lasiodiplodia theobromae),其分生孢子平均大小为(22.06～31.07) μm× (11.77～16.48) μm;通过ITS、TUB2、EF-1α、GAPDH、CHS-1和ACT基因拼接序列聚类分析,结果分为3个类群,海南岛的中部(儋州,昌江,白沙,五指山,万宁,琼海等地)聚为一个类群,中北部(屯昌,临高)聚为一个类群,西南部(东方,乐东,三亚等地)聚为一个类群.该结果说明来源于不同产地不同菠萝品种上的可可毛色二孢菌遗传多样性丰富,在海南省16个市县菠萝叶斑病菌中可可毛色二孢菌L.theobromae为优势种.     

利用细胞松弛素B分离柑橘及金柑植物微核的研究

采用细胞松弛素B(CB)处理结合蔗糖梯度超速离心方法分离柑橘和金柑亚原生质体的结果表明,柑橘和金柑原生质体的直径为20～30μm,采用蔗糖梯度超速离心分离亚原生质体,其适宜离心力是1400000×g;4',6-二脒基-2-苯基吲哚(DAPI)的染色效果最好,细胞核和细胞质可明显区分;愈伤组织经CB处理36h后再分离亚原生质体,可显著提高微原生质体的产量,分离的微原生质体直径为1.8～3.5μm,产量达到2～3×104个·g-1(FW)。     

广东省柑橘炭疽病病原菌的形态与分子鉴定

炭疽病是柑橘的主要真菌性病害之一。2007年春,广东省德庆县名优柑橘品种贡柑炭疽病暴发流行。为了明确该县及广东省其他地区柑橘炭疽病菌的种类,为防治提供依据,对采集自广东省6个地区柑橘属10个栽培品种上的炭疽病样本进行病原菌分离,共获得柑橘炭疽病菌单孢菌株75株,对其中10株代表性的菌株进行了种类鉴定。通过培养性状和形态学特征观测、核糖体DNA(rDNA)内转录间区(ITS)序列分析、ITS区特异性引物PCR检测和系统发育关系比较等方面的研究,结果表明:10个柑橘炭疽病菌菌株均为盘长孢状刺盘孢Colletotrichum gloeosporioides,未发现国际上其他国家报道的严重危害柑橘花器和幼果部位的柑橘花后落果病病原菌——尖刺盘孢C.acutatum。     

沙田柚黄龙病病原体的电镜观察与PCR检测

柑桔黄龙病可以侵染沙田柚而产生典型的‘斑驳’症状。我们通过电镜观察表明,带病沙田柚的病原体在形态,大小和构造上均与柑桔黄龙病病原体（类细菌）相似：PCR扩增也证明其病原体DNA与柑桔黄龙病病原体的PCR产物具有很高的同源性。     

Molecular systematics of citrus-associated Alternaria species

The causal agents of Alternaria brown spot of tangerines and tangerine hybrids, Alternaria leaf spot of rough lemon and Alternaria black rot of citrus historically have been referred to as Alternaria citri or A. alternata. Ten species of Alternaria recently were described among a set of isolates from leaf lesions on rough lemon (Citrus jambhiri) and tangelo (C. paradisi × C. reticulata), and none of these isolates was considered representative of A. alternata or A. citri. To test the hypothesis that these newly described morphological species are congruent with phylogenetic species, selected Alternaria brown spot and leaf spot isolates, citrus black rot isolates (post-harvest pathogens), isolates associated with healthy citrus tissue and reference species of Alternaria from noncitrus hosts were scored for sequence variation at five genomic regions and used to estimate phylogenies. These data included 432 bp from the 5' end of the mitochondrial ribosomal large subunit (mtLSU), 365 bp from the 5' end of the beta-tubulin gene, 464 bp of an endopolygalacturonase gene (endoPG) and 559 and 571 bp, respectively, of two anonymous genomic regions (OPA1-3 and OPA2-1). The mtLSU and beta-tubulin phylogenies clearly differentiated A. limicola, a large-spored species causing leaf spot of Mexican lime, from the small-spored isolates associated with citrus but were insufficiently variable to resolve evolutionary relationships among the small-spored isolates from citrus and other hosts. Sequence analysis of translation elongation factor alpha, calmodulin, actin, chitin synthase and 1, 3, 8-trihydroxynaphthalene reductase genes similarly failed to uncover significant variation among the small-spored isolates. Phylogenies estimated independently from endoPG, OPA1-3 and OPA2-1 data were congruent, and analysis of the combined data from these regions revealed nine clades, eight of which contained small-spored, citrus-associated isolates. Lineages inferred from analysis of the combined dataset were in general agreement with described morphospecies, however, three clades contained more than one morphological species and one morphospecies (A. citrimacularis) was polyphyletic. Citrus black rot isolates also were found to be members of more than a single lineage. The number of morphospecies associated with citrus exceeded that which could be supported under a phylogenetic species concept, and isolates in only five of nine phylogenetic lineages consistently were correlated with a specific host, disease or ecological niche on citrus. We advocate collapsing all small-spored, citrus-associated isolates of Alternaria into a single phylogenetic species, A. alternata.     

柑橘溃疡病及其微生物防治研究进展

柑橘是我国第一大水果,柑橘溃疡病是导致柑橘产量和品质下降的最具破坏性细菌性病害之一,给柑橘产业造成了巨大的经济损失,严重阻碍了柑橘产业的可持续发展。微生物防治柑橘溃疡病具有安全、环保、高效等优点,是当前研究的热点。本文主要概述了柑橘溃疡病特征及其病原菌分类、分布,全面分析了柑橘溃疡病病原菌主要致病机理和协助致病机理;系统梳理了柑橘溃疡病生防微生物的多样性;重点总结了微生物通过产生活性物质、诱导激活植物免疫防御系统等防治柑橘溃疡病的作用机制;最后,我们提出了柑橘溃疡病微生物防治面临的挑战以及未来可能的解决途径,以期为柑橘产业的健康发展提供参考。     

First Report of Black Leaf Spot Caused by Alternaria alternata on Ramie in China

In 2012 and 2013, black leaf spot disease was observed on ramie plants in Hunan and Hubei Provinces, China. In the field, the symptoms of this disease included dark green to black big spots on leaves, often resulting in upwardly curled leaf margins. The pathogen isolates were identified as Alternaria alternata (Fr.) Keissler on the basis of morphology and sequence similarity of 99–100% to the published data for internal transcribed spacer (ITS), and glyceraldehyde‐3‐phosphate dehydrogenase (gdp). To our knowledge, this is the first report of Alternaria leaf spot of ramie in China.     

化州柚查尔酮合成酶基因克隆与序列分析

利用CTAB-LiCl法提取高质量的化州柚总RNA,采用RT-PCR技术克隆查尔酮合成酶基因,获得广东道地药材化橘红资源化州柚的查尔酮合成酶基因。该基因编码区全长1176bp,编码391个氨基酸残基,与同样来源于柑橘属的查尔酮合成酶基因同源性高达98%。CTAB-LiCl法能提取高质量的化州柚总RNA,可以用于后续基因克隆和分析;克隆获得的查尔酮合成酶具有编码区,与同属植物相同基因具有高度序列同源性。     

Distribution ofGeotrichum candidum citrus race in citrus groves and non-citrus fields in Japan

The presence ofGeotrichum candidum citrus race, the citrus sour rot pathogen, was examined in soils of citrus groves and non-citrus fields of Japan. Soil samples were collected from 223 sites (118 sites in citrus groves, and 105 sites in fields cultivated with 33 species of non-citrus plants and in evergreen broad-leaved forest) in 11 main citrus-growing prefectures, and Hokkaido, a non-citrus-growing area. Of 236 soil samples from citrus groves, 95.76% containedG. candidum citrus race and 0.42% contained the non-citrus race; while of 210 samples from non-citrus fields, 62.85% and 4.76% contained the citrus race and the non-citrus race respectively. All of the citrus race isolates obtained either from citrus groves or non-citrus fields were pathogenic on lemon (Citrus limon) and satsuma mandarin (Citrus unshiu), but some of these isolates failed to infect orange (Citrus sinensis). The non-citrus races were pathogenic on ripe tomato fruit (Lycopersicon esculentum) and ripe muskmelon fruit (Cucumis melo var.reticulatus). Results indicated that citrus sour rot pathogen is widely distributed in citrus groves and non-citrus fields of diverse plant species in Japan.     

A TaqMan PCR Method for Routine Diagnosis of the Quarantine Fungus Guignardia citricarpa on Citrus Fruit

With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real‐time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real‐time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi‐copy rDNA gene. Co‐amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real‐time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real‐time PCR methods showed that the TaqMan method was more sensitive.     

Enzymatic differences between the endophyte Guignardia mangiferae (Botryosphaeriaceae) and the citrus pathogen G. citricarpa

The endophyte Guignardia mangiferae is closely related to G. citricarpa, the causal agent of citrus black spot; for many years these species had been confused with each other. The development of molecular analytical methods has allowed differentiation of the pathogen G. citricarpa from the endophyte G. mangiferae, but the physiological traits associated with pathogenicity were not described. We examined genetic and enzymatic characteristics of Guignardia spp strains; G. citricarpa produces significantly greater amounts of amylases, endoglucanases and pectinases, compared to G. mangiferae, suggesting that these enzymes could be key in the development of citrus black spot. Principal component analysis revealed pectinase production as the main enzymatic characteristic that distinguishes these Guignardia species. We quantified the activities of pectin lyase, pectin methylesterase and endopolygalacturonase; G. citricarpa and G. mangiferae were found to have significantly different pectin lyase and endopolygalacturonase activities. The pathogen G. citricarpa is more effective in pectin degradation. We concluded that there are significant physiological differences between the species G. citricarpa and G. mangiferae that could be associated with differences in pathogenicity for citrus plants.