基于高通量测序及分离培养初步研究美国大豆中真菌多样性

中国是全球最大的大豆进口国,进境大豆所携带的病原真菌传入我国的风险极高。基于高通量测序技术对6批美国大豆真菌多样性进行分析,同时采用分离培养获得单一菌株,依据菌落形态、显微结构及分子技术进行鉴定。高通量测序结果显示,大豆中所有真菌共计5门15纲35目63科112属155种,主要为链格孢属(Alternaria)、球腔菌科(Mycosphaerellaceae)、小戴卫霉科(Davidiellaceae)、小囊菌科(Plectosphaerellaceae)、赤霉属(Gibberella)、附球霉属(Epicoccum)、间座壳属(Diaporthe)、隐球菌属(Cryptococcus)。分离培养结果显示,得到真菌共计40株10种,分别是大豆北方茎溃疡病菌(Diaporthe phaseolorum var.caulivora)、大豆南方茎溃疡病菌(Diaporthe phaseolorum var.meridionalis)、大豆拟茎点种腐病菌(Diaporthe longicolla/Phomopsis longicolla)、大豆炭腐病菌(Macrophomina phaseolina)、尖孢镰刀菌(Fusarium oxysporum)、菌核菌(Sclerotinia sclerotiorum)、镰刀菌(Fusarium sp.)、附球菌(Epicoccum sp.)、交链孢菌(Alternaria sp.)、小双胞腔菌(Didymella sp.)。     

拟康宁木霉Tk1的分离鉴定、拮抗作用及其生物学特性

为了分离利用高效植物病害生物防治菌株，从广东药用植物金丝皇菊根部分离获得一株木霉菌株Tk1。经过形态观察、rDNA-ITS和EF-1α序列分析明确其为拟康宁木霉Trichoderma koningiopsis。用平板对峙培养法测定，该菌株对金丝皇菊枯萎病菌（尖孢镰刀菌Fusarium oxysporum）和柑橘炭疽病菌（胶胞炭疽菌Colletotrichum gloeosporioides）的抑制率分别为68.5%和77.1%，且在后期能完全覆盖或部分覆盖病原真菌。其无菌发酵液对金丝皇菊枯萎病菌的抑制率为45.55%。该菌株营养生长和产孢的最适碳源分别为乳糖和葡萄糖，最适氮源是酵母膏；在pH 4~11的培养基上均可生长、产孢，最适pH为7；12 h交替光照条件下菌丝的生长速度最快，全光照条件下产孢数量最大；该菌株的致死温度为73℃处理10 min。     

马尾松叶表微生物多样性

为发掘对林木生长发育有利的优良微生物资源,并筛选适合叶表微生物的收集方法,以马尾松针叶为试验材料,分别用悬摇法和超声波法收集马尾松叶表微生物,用扩增子高通量测序技术、MUSCLE和Qiime软件研究马尾松叶表微生物的多样性。结果表明:扫描电镜观测结果显示,马尾松针叶表面定殖有大量微生物,包括真菌(菌丝及孢子)和细菌。扩增子高通量测序结果表明,马尾松叶表微生物物种丰富,包含细菌运算分类单位(OTUs)490个,真菌OTUs 1273个。马尾松叶表细菌以未分类的蓝细菌属(unidentified_Cyanobacteria)(36.53%)、未分类的拜叶林克氏菌属(unidentified_Beijerinckia)(28.60%)、鞘氨醇单胞菌属(Sphingomonas)(2.35%)为优势属;叶表真菌以枝孢属(Cladosporium)(2.45%)、拟盘多毛孢属(Pestalotiopsis)(0.92%)、无头孢菌属(Capnobotryella)(0.91%)为优势属。针对叶表细菌多样性的研究表明,悬摇法和超声波法均有较高的物种检出度;在叶表真菌多样性的研究中超声波法优于悬摇法,但超声波法样品间数据变异性较大,测定结果不稳定。     

四硫代碳酸钠对蔬菜根病的防治效果及对土壤生物学性状的影响

为明确四硫代碳酸钠(sodium tetrathiocarbonate,STTC)在蔬菜根病防治上的应用价值及制定应用技术,本研究通过室内瓶土熏蒸法,进行了STTC对病菌的室内毒力测定;用温室盆栽法,进行植前熏蒸处理,测定土壤中病菌数量的变化和对病害的防效,同时进行植后熏蒸处理,测定药剂处理点和根际处病菌数量的变化;通过土壤施药后随即移栽幼苗的方法,评价药剂对蔬菜幼苗的安全性;通过室内模拟田间熏蒸条件,测定药剂不同浓度和熏蒸时间对土壤中主要微生物类群数量和主要土壤酶活性的影响。结果显示STTC对辣椒疫霉病菌Phytophthora capsici、黄瓜枯萎病菌Fusarium oxysporumf.sp.cucumerinum、茄子黄萎病菌Verticillium dahliae、番茄立枯病菌Rhizoctonia solani和蔬菜菌核病菌Sclerotinia sclerotiorum均有较好的杀灭活性,LD50分别为3.956、4.241、5.222、5.457和6.197mg/kg;80g/m2 STTC移植前处理对辣椒疫霉病和黄瓜枯萎病的防效可分别达到91.01%和82.51%;浓度低于900μg/mL的STTC对当日熏蒸后移栽的3叶期辣椒、黄瓜、番茄、白菜和油菜幼苗均表现安全;在辣椒和黄瓜生长期穴施1 200μg/mL STTC能有效减少土壤中辣椒疫霉病菌和黄瓜枯萎病菌的数量;土壤中微生物类群数量监测结果表明,在0.1~50.0μg/g浓度范围内,STTC对土壤真菌有强烈抑制作用,对土壤细菌和放线菌为先抑制后促进。对土壤酶活性测定结果表明,当STTC用量为0.1、1.0、5.0、10.0和50.0μg/g时,对土壤脲酶和蔗糖酶的活性表现为先抑制后促进,对蛋白酶的活性表现为在STTC低浓度时活性增加,高浓度时为先抑制后增加。综合结果表明,STTC用于经济作物生产中的土壤熏蒸具有安全、有效和施用简便的特点,或可广泛使用。     

花绒寄甲人工繁育及应用研究

为解决花绒寄甲人工繁育技术的关键问题，进一步提高产量，开展了低温保存对花绒寄甲卵活力的影响、替代寄主选择、成虫人工饲料配方、室内生活史等研究，并在后期初步开展了花绒寄甲对桃红颈天牛的野外防效试验。结果表明，4℃条件下保存0～60 d对花绒寄甲卵孵化率的影响存在显著差异，不经低温处理的卵孵化率最高，可达70.87%，低温保存60 d后，孵化率降为65.81%；大麦虫可作为花绒寄甲幼虫的替代寄主，最佳接虫比为1:5，此时化蛹率达70.40%，羽化率达94.03%；黄粉虫蛹、大蜡螟幼虫无法繁育出花绒寄甲成虫；黄粉虫幼虫干粉、大麦虫幼虫干粉、无菌水、脯氨酸、羟氨酸、丝氨酸、胱氨酸、葡萄糖、亚油酸混合配制的饲料使花绒寄甲成虫存活率达96.70%，单雌产卵量达161.10粒，产卵期持续43 d，是成虫的最佳人工饲料；经室内繁育的花绒寄甲一年发生2代，成虫寿命2～3年，卵期平均8.64 d、幼虫期平均6.63 d、蛹期平均36.88 d。花绒寄甲可用于防治桃红颈天牛，野外最佳释放比为8:1～ 10:1（天敌:天牛），虫口减退率达72.70%。     

Microbial residues were increased by film mulching with manure amendment in a semiarid agroecosystem

Amino sugars, as a kind of microbial residue, are strongly associated with cycling of microbial-derived soil organic matter. However, responses of amino sugars to agricultural practices on the Loess Plateau in North-western China are poorly known. The objective was to evaluate effects of film mulching (no film mulching + NPK fertilizers, CK; film mulching + NPK fertilizers, PF; film mulching + NPK fertilizers + cow manure, FM) on accumulations of amino sugars in this region. FM significantly increased total amino sugar by 190.46 mg kg^?1 in 0–10 cm layer and 214.66 mg kg^?1 in 10–20 cm layer relative to CK, but PF significantly decreased it by 139.28 mg kg^?1 in 0–10 cm layer. Ratios of glucosamine to muramic acid were markedly decreased by 2.50 in 0–10 cm layer and 2.28 in 10–20 cm layer in FM than CK, suggesting a tendency of microbial residues pool shift towards bacterial residues in this agroecosystem. These results indicated film mulching alone was not benefitial to accumulation of amino sugar while organic manure contributed to the build-up of amino sugar partly due to manure contained microbial residues. The different patterns of amino sugars suggested significant changes in the quality of microbial-derived organic matter.     

切花菊‘白扇’开放式组培快繁体系的建立

以切花菊‘白扇’的带腋芽茎段为外植体,以次氯酸钠为表面消毒剂,确定最佳的消毒浓度及消毒时间;以山梨酸钾、次氯酸钠和代森锰锌为抑菌剂,确定开放式初代、继代和生根培养基中抑菌剂的最佳组合,建立开放式组培快繁体系。试验结果表明：外植体最佳消毒条件为0.1%（V/V）次氯酸钠消毒15 min;在初代培养基中,抑菌剂最佳组合为50 mg/L代森锰锌、0.01%（V/V）次氯酸钠和5 mg/L山梨酸钾,该组合的污染率较低,为36.7%。开放式培养中芽的生长情况、诱导率均无显著差异;在继代增殖和生根培养阶段,抑菌剂的最佳组合为40 mg/L代森锰锌、0.01%（V/V）次氯酸钠和5 mg/L山梨酸钾,芽的增殖系数和生根率较高,分别为5.93%、80.0%,芽生长良好,与常规组培无明显差异。     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.