小孢拟盘多毛孢PM-1菌株的液体发酵及除草活性物质的分离

前期研究发现分离自毛竹的小孢拟盘多毛孢Pestalotiopsis microspora PM-1菌株具有较强的除草活性,为了明确其产生除草活性物质的最佳液体发酵条件,对培养基种类及成分、培养温度、培养时间和培养方式进行了探讨,并利用薄层层析(thin-layer chromatography, TLC)和高效液相色谱(high performance liquid chromatography, HPLC)对除草活性物质进行了分离。小孢拟盘多毛孢在含硫酸亚铁的PD培养基中,30 ℃黑暗静止培养15天,得到的粗提物除草活性最强;除草活性物质经TLC分离获得7个条带,其中条带6具有较强的除草活性,HPLC分析发现该条带含有7个组分,其中组分3对马唐的活性较强,其保留时间为46.3 min。研究结果表明,利用最佳液体发酵条件对PM-1菌株进行发酵培养,可分离获得除草活性化合物——组分3。     

岗梅枝枯病病原鉴定

为明确引起岗梅Ilex asprella枝枯病的病原,对从广东梅州采集的岗梅枝枯病病部分离的致病菌株Gangmei 2进行了形态学观察,并运用MEGA-X构建了病原菌株rDNA内转录间隔区(internal transcribed spacer, ITS)的系统发育树。结果显示:病原菌的形态学特征与拟盘多毛孢属Pestalotiopsis真菌相似,在系统发育树中该菌株与小孢拟盘多毛孢菌Pestalotiopsis microspora在同一分支上。因此,初步将岗梅枝枯病病原菌鉴定为小孢拟盘多毛孢菌P.microspora。     

杨盘二孢激发子的分离及稳定性研究

 从杨盘二孢的培养滤液和菌丝中获得2种激发子粗提物,分别测定其糖和蛋白质的含量,发现滤液激发子粗提物(CFE)糖和蛋白质的含量分别为41.07和40mg/mL,菌丝激发子粗提物(CME)糖和蛋白质含量分别为48.07和55mg/mL。滤液激发子粗提物对温度不敏感而对碱性条件敏感;菌丝激发子粗提物对酸碱不敏感而对温度敏感。用Sephadex G-100柱层析的方法初步纯化2种激发子,并且菌丝激发子粗提物过柱后得到2个活性组分J14和J25;滤液激发子粗提物过柱后获得2个活性组分L9和L16。将4个组分进行烟草叶片过敏反应和I-895杨酶活性的诱导,结果表明,菌丝激发子强活性物质集中在J14组分;而滤液激发子强活性物质集中在L9组分。     

马尾松梢枯病的研究

由松壳色单隔孢(Diplodia pinea(Desm.)Kickx)引起的马尾松梢枯病在我国广东省首次发现。马尾松梢枯病的主要症状有梢枯、嫩梢弯曲、溃疡、流脂、叶枯,甚至全株死亡。病原菌的特征是分生孢子双孢,褐色,大小为28.5～39.9×14～17微米。松壳色单隔孢能侵染马尾松(Pinus massoniana)、湿地松(P.elliotii)、加勒比松(P.caribaea)和火炬松(P.taeda)。病原菌在受病的针叶、枝条和球果上越冬。每年3～5月第一次发病,7～8月第二次发病。当年生嫩梢未老化之前,病原菌能从伤口侵入或从嫩叶、嫩梢直接侵入。嫩梢老化以后,只能从伤口侵入。在自然条件下,病害主要发生在嫩梢伸长的后期,很少发生在嫩梢伸长的前期。在病树枯枝、枯叶上,松壳色单隔孢和多毛孢菌(Pestalotia sp.)之间存在生存竞争的关系,经接种试验证明后者对马尾松没有侵染能力,可利用它进行生物防治的研究。     

拟康宁木霉Hailin菌株对马尾松幼苗的促生和防病作用

采用菌株形态学和r DNA-ITS序列比对分析的方法进行了Hailin菌株的鉴定,采用平皿对峙法研究了Hailin菌株对尖孢镰孢菌(Fusarium oxysporum)的抑菌效果,测定了接种尖孢镰孢菌和Hailin菌株后对马尾松(Pinus massoniana Lamb)幼苗的发病率和生长指标的影响。经形态学、分子鉴定和进化树分析,菌株Hailin为拟康宁木霉(Trichoderma koningiopsis)。Hailin菌株与尖孢镰刀菌平皿对峙培养5d后,对其抑制率为77.78%;蘸根针刺微伤处理接种Hailin菌株30 d后,对马尾松幼苗立枯病的防治率为58.3%;接种Hailin菌株与不接种的马尾松幼苗相比,其株高、湿重、根长分别提高了3.86%、78.74%和13.2%。Hailin菌株抑制尖孢镰孢菌的作用机制包括竞争作用、重寄生作用以及通过产生代谢产物抑制病原菌的生长。其中,Hailin菌株通过非挥发性代谢产物和挥发性代谢产物对尖孢镰孢菌菌丝生长的抑制率分别为97.80%和77.80%。     

污斑新拟盘多毛孢为害蓝莓的首次报道及致病力测定

类拟盘多毛孢真菌(Pestalotiopsis-like fungi)是危害蓝莓的一类重要病原菌。实验室前期对比发现有8株分离自蓝莓病样的类拟盘多毛孢菌单独聚成一支，并且与危害蓝莓的类拟盘多毛孢已知种相互区分，表明这8株菌有可能是危害蓝莓的新纪录种或新种。为明确这8株菌的种群分类地位，本研究对供试菌株进行形态学鉴定及系统发育分析，结果表明它们隶属于污斑新拟盘多毛孢Neopestalotiopsis foedans,这是该菌危害蓝莓在国内外的首次报道。在此基础上，进一步研究了该新纪录种不同菌株对辽宁省5个蓝莓主栽品种的致病能力，结果表明这8株菌的致病力具有明显分化，不同蓝莓品种均对强致病菌株较敏感，生产中应当加以重视。     

芝麻枯萎病菌毒素成分分析及其对芝麻幼苗的毒性

芝麻枯萎病(sesame Fusarium wilt,SFW)是由尖孢镰刀菌芝麻专化型[Fusarium oxysporum Schl.f.sp.sesami(Zap.),FOS]引起的一种土传真菌病害,严重影响着我国芝麻生产。为确定FOS是否产生毒素以及毒素成分,利用液相色谱和液质联用等技术分离并分析了FOS菌株培养滤液的主要组成物质。研究发现FOS能够产生4种特异物质,分别为镰刀菌酸(5-丁基吡啶甲酸,FA),及C_(10)H_(13)NO_4(FA+O_2)、C_(10)H_(13)NO_3(FA+O)和C_(10)H_(11)NO_2(FA-H_2)_3种镰刀菌酸类似物。在Richard培养基上强致病力和弱致病力FOS菌株均可获得毒素,不同致病力的菌株所产生4种物质的含量和比例存在差异。芝麻幼苗生长试验结果证明含有上述特异物质的FOS培养滤液能够对幼苗生长产生抑制作用;而随着FOS毒素浓度的增大,芝麻幼苗受毒害程度增大。但在含相同FA浓度的FOS培养滤液处理下,不同芝麻品种幼苗生长受抑制程度无显著差异。该结果为进一步研究FOS致病机理及芝麻-FOS互作奠定了技术和理论基础。     

澳洲坚果叶枯病病原鉴定及其防治药剂筛选

对云南澳洲坚果主产区叶枯病进行采样调查和病原菌分离,得到了216个菌落。其中,81.9%为拟盘多毛孢(Pestalotiopsis spp.),12.5%为炭疽菌,0.6%为拟茎点霉。致病性测定结果显示,拟盘多毛孢具有较强的致病性,为叶枯病主要病原菌。经形态学和分子生物学鉴定,澳洲坚果叶枯病主要病原菌为小孢拟盘多毛孢(Pestalotiopsis microspora)。室内药剂毒力测试表明,对澳洲坚果叶枯病病原菌抑制效果较好的是50%多菌灵可湿性粉剂、240 g/L戊唑醇悬浮剂、12.5%烯唑醇悬浮剂,其次为77%氢氧化铜可湿性粉剂、10%苯醚甲环唑悬浮剂、20%噻菌铜悬浮剂等。     

九种杀菌剂对新拟盘多毛孢病菌的室内毒力作用

新拟盘多毛孢叶斑病是在草莓上发现的一种新病害。本研究对采自北京昌平、山东诸城、云南保山及广东惠州等地草莓Fragaria × ananassa发病叶片进行了组织分离,结合形态特征和ITS系统发育分析对所分离获得的8株疑似拟盘多毛孢叶斑病病菌进行了鉴定,并采用菌丝生长速率法测定了病原菌对8种代表性杀菌剂及新型药剂——双苯菌胺的敏感性。结果表明:所分离得到的病原菌为新拟盘多毛孢Neopestalotiopsis clavispora;双苯菌胺对8株新拟盘多毛孢 N. clavispora的毒力最强,平均EC₅₀值为 (0.004 ± 0.002) μg/mL,可作为防治草莓叶斑病的首选药剂;其次为咪鲜胺、嘧菌酯、戊唑醇、氟环唑、苯醚甲环唑和腈菌唑,平均EC₅₀值在0.006~0.803 μg/mL之间;百菌清毒力较低,而代森锰锌对新拟盘多毛孢 N. clavispora几乎没有抑制作用。研究结果对合理使用杀菌剂防治草莓新病害——新拟盘多毛孢叶斑病提供参考。     

青岛市草莓根腐病病原菌分离及鉴定

草莓根腐病是草莓根部重要病害,已经成为草莓产业发展的瓶颈。2014年,对山东省青岛市几个主要草莓种植基地的草莓根腐病进行了调查,并在室内用组织分离法对病原菌进行了分离。根据菌落形态特征及rDNA-ITS序列对其进行了鉴定。结果表明,引起草莓根腐病的病原菌有棒形拟盘多毛孢菌(Pestalotiopsis clavispora)、胶孢炭疽菌(Colletotrichum gloeosporioides)。其中,棒形拟盘多毛孢菌引起草莓病害为首次报道。     

松材线虫自然侵染后对不同松树组织结构的影响

 利用滑走切片技术从显微水平研究松材线虫（Bursaphelenchus xylophilus）在自然状态下入侵,对马尾松（Pinus massoniana）和黑松（Pinus thunbergii）显微结构的影响。结果表明,马尾松和黑松的组织结构在感病初期即表现出病变特征,并随感病时间推移病变加剧。马尾松在感病初期皮层薄壁细胞开始木质化,伴有细胞破裂并融合成空腔;至感病中期周皮的栓内层细胞、皮层薄壁细胞以及韧皮部薄壁细胞均发生木质化,细胞内含物增多,甚至形成层细胞也发生一定程度的木质化现象,树脂道因周围细胞破裂而变形;至感病晚期,木材以外所有部分完全被木质化、大量细胞破裂。黑松发病症状与马尾松大致相同但发病时间稍晚,发病程度也较轻,这可能是因为两者对松材线虫病的抗性强弱有差异。同一时期的组织病理学特征在空间上也存有差异,这与松材线虫入侵的位置及其在树体内的分布有关。     

不同种松树对松树蜂毒素抗性的初步研究

松树蜂(Sirex noctilio Fabricius)是一种重要的国际检疫性蛀干害虫。目前在我国松树蜂潜在寄主范围内,不同树种对松树蜂毒素抗性之间的差异尚不明确。本文以室内栽培的2~3 a生油松、樟子松、红松、湿地松、马尾松及雪松这6种树种为供试寄主,培育3个月后分别接种毒素、毒素真菌混合液并测定每株枝条针叶的RGB值以及叶绿素含量。结果表明,形态上油松和樟子松幼苗接种毒素、毒素真菌混合液后一段时间内,均出现了比较明显的针叶枯黄、脱落的现象,且混合组长势萎蔫,脱落严重,对照组无明显变化。马尾松幼苗只有接种混合液后变化明显,雪松、红松、湿地松接种后长势强劲,长出新芽。从数值化的角度,油松和樟子松幼苗接种毒素、毒素和真菌混合液后,针叶的RGB值减小及叶绿素含量值显著降低。马尾松幼苗只有接种混合液后,RGB值和叶绿素含量才减小。雪松、红松、湿地松接种毒素以及毒素真菌混合液后,RGB值及叶绿素含量均没有明显变化。以上可以看出樟子松和油松对于松树蜂毒素、毒素真菌混合液的抗性较弱,马尾松对松树蜂毒素具有一定的抗性,对毒素真菌混合液的抗性较弱,红松、湿地松、雪松对两者都具有一定的抗性。该结果可为明确松树蜂在我国潜在的寄主范围及寄主选择性提供科学依据。     

几种松树松材线虫病木和健木pH值差异的研究

 对江苏、安徽2疫区的黑松、赤松、马尾松、火炬松、湿地松等5个树种松材线虫病Bursaphelenchus xylophilus的病、健木pH值差异性分析结果表明:感病品种黑松、赤松、马尾松病木pH值降低,且差异显著;较抗病的湿地松虽然也降低,但差异不显著;抗病性强的火炬松病木pH值反而高于健木,且差异显著。由此可见,不同松树品种松材线虫病的病、健木pH值的差异性也不同,对松材线虫越敏感和受害越严重的,其病木pH值降低的就越多,与健木pH值差异就越显著。该分析结果初步为我国松材线虫为害比较严重的几种主要松树品种,利用指示剂进行松材线虫病快速检测的可行性提供了一个依据。     

噻虫啉防治松褐天牛试验初报

林间用2种噻虫啉粉剂进行防治松褐天牛试验。结果表明,这2种粉剂对该害虫有较强的杀灭效果,而且持效期长达28d以上。在喷洒1%噻虫啉微胶囊粉剂的林区,平均每个诱捕器的成虫诱捕量为11.7头,诱木上平均成虫产卵刻槽数为10.66个/m^2;而在空白对照区,平均每个诱捕器的成虫诱捕量为38头,是喷药区的3.25倍,平均刻槽数达144.82个/m^2,是喷药区的13.59倍。施药的相对防治效果可达60%.     

粉质拟青霉侵染菜青虫致病过程的研究

应用电镜扫描技术观察了粉质拟青霉 990 5菌株对菜青虫的侵染过程 ,根据感病虫体外部症状进行病情分级 ,并应用石蜡切片法观察了不同感病程度菜青虫虫体内部的组织病变情况。结果表明粉质拟青霉菌丝侵入菜青虫血腔后首先侵入脂肪体细胞 ,并证实脂肪体是粉质拟青霉繁殖最多的部位。致死状态表现为 :1级 ,感病菜青虫只是体壁出现病变 ,2级 ,菜青虫已死亡 ,体内各组织均发生不同程度的病变。 3级 ,菜青虫体内几乎所有的组织器官都已解体。     

多裂骆驼蓬生物碱类物质抑菌杀虫活性研究

采用生长速率法和浸叶法研究多裂骆驼蓬中生物碱类物质对8种植物病原真菌和2种蚜虫的抑菌杀虫作用。结果表明,水溶性生物碱、脂溶性生物碱和总生物碱提取液对供试病原真菌均有不同程度的抑制作用,其中对小麦赤霉病菌、黄瓜霜霉病菌、辣椒疫霉病菌和小麦条锈病菌抑制作用明显,0.5 0 g/mL试液的抑菌率均在7 8%以上;对供试2种蚜虫有明显的触杀活性,其中0.5 0 g/mL水溶液性、脂溶性和总生物碱对麦二叉蚜的校正死亡率分别为3 9.6 4%、3 5.4 0%和4 4.7 0%,对桃蚜的校正死亡率分别为2 8.1 7%、2 5.8 3%和3 1.2 2%。生物碱类物质的抑菌杀虫活性大小顺序为:总生物碱>水溶性生物碱>脂溶性生物碱。     

琼胶寡糖激发坛紫菜对附生细菌的减少作用

以琼胶寡糖处理坛紫菜Porphyra haitanensis,激发其防御反应,坛紫菜在18℃生长和35℃热激时的病烂现象得到明显改善。寡糖激发的防御信号能够杀死附生在藻上的细菌,健康藻上约94.7%的附生菌群在加入100μg.mL-1琼胶寡糖1 h内被杀死,但过氧化氢酶可部分抑制这种作用。利用3种抗生素组合获得无菌坛紫菜体系,回染致病细菌后,约60.8%的回染细菌在寡糖激发后的3 h内被消灭。这种附生细菌的减少现象不是源于琼胶寡糖的直接抑菌作用,可能与琼胶寡糖引起坛紫菜H2O2的瞬时暴发有关。结果表明,琼胶寡糖能够作为坛紫菜的抗性激发子,通过诱导坛紫菜自身的防御反应来减少附生细菌的数量,从而保持藻体的健康生长。     

Prevalence and pathogenicity of spinach root pathogens of the genus Pythium in Sweden

Several pathogenic Pythium spp. were isolated from spinach roots in a 4-year disease survey in commercial spinach fields in southern Sweden. Heterothallic and zoospore-producing isolates belonging to the filamentous, non-inflated zoosporangia (F) group of van der Plaats-Niterink were most abundant, followed by P. sylvaticum, P. ultimum var. ultimum and P. heterothallicum. The heterothallic hyphal swelling (HS) group and P. tracheiphilum were found in a few plants. Two types of pathogenicity tests were performed, to measure the effects of seedling infection and of root infection of older plants. These tests showed P. ultimum var. ultimum to be the most severe spinach pathogen inducing pre- and post-emergence damping off as well as root rot of older plants. Also P. heterothallicum and P. tracheiphilum damaged both seedlings and older plants, Pythium sylvaticum, Pythium 'group HS' and Pythium 'group F'were pathogenic only to seedlings, P. ultimum var. ultimum and Pythium group F were isolated in significantly higher frequencies from spinach grown in the autumn season than in the spring. No clear relationships were found between Pythium prevalence and disease severity index of surveyed field plants, between Pythium prevalence and plant developmental stage, or between prevalence of Pythium and other pathogens isolated. This is the first report of P. heterothallicum and P. sylvaticum being pathogenic on spinach.     

Evaluation of Infection Processes and Resulting Disease Caused by Dendryphion penicillatum and Pleospora papaveracea on Papaver somniferum

ABSTRACT Two pathogenic fungi of opium poppy, Pleospora papaveracea and Dendryphion penicillatum, were isolated from field material in Beltsville, MD. The processes of infection by these two fungi were studied to determine the optimal environmental conditions for infection. Both fungi formed appressoria capable of penetrating directly through the plant epidermal layer. Of the two fungi, P. papaveracea was more aggressive, causing more rapid necrosis. Appressorial formation by P. papaveracea occurred as early as 4 h after application of a conidial suspension to poppy leaves. P. papaveracea formed more appressoria than did D. penicillatum, especially at cool temperatures (7 to 13 degrees C). In greenhouse studies, P. papaveracea caused more damage to opium poppy than did D. penicillatum when applied in 10% unrefined corn oil. In the field, P. papaveracea was more consistent in its effects on opium poppy from a local seed source designated Indian Grocery. P. papaveracea caused higher disease ratings, more stem lesions, and equal or greater yield losses than did D. penicillatum on Indian Grocery. The late-maturing opium poppy variety White Cloud was severely damaged by disease, regardless of formulation or fungal treatment. P. papaveracea was the predominant fungus isolated from poppy seed capsules and the only fungus reisolated from the field the following year. These studies provide a better understanding of the infection process and the differences between these two pathogenic fungi and will be beneficial for the development of the fungi as biological control agents.     

Influence of bean rhizosphere on the biological properties and phosphorus fractionation in the calcareous soils amended with municipal sewage sludge

The biological and chemical conditions of the rhizosphere are known to considerably differ from those of the bulk soil, as a consequence of a range of processes that are induced either directly by the activity of plant roots or indirectly by the stimulation of microbial population and activity in the rhizosphere. Information about phosphorus(P) fractionation in the rhizosphere soils amended with municipal sewage sludge(MSS) is limited. were We carried out greenhouse experiments using a rhizobox in order to evaluate the effects of bean rhizosphere on the various inorganic P(Pi) fractions, organic P(Po), P in particulate fraction(PF-P), Olsen-P, dissolved organic C(DOC), microbial biomass P(MBP) and alkaline phosphatase(ALP) enzyme in 10 calcareous soils amended with MSS(10 g MSS was added to 1 kg soil). Non-occluded P, occluded P, calcium phosphate and residual P were also quantitated. The results showed that DOC, MBP and ALP activity strongly increased and PF-P and Olsen-P decreased in the rhizosphere soils compared with in the bulk soils(P<0.05). The contents of non-occluded P, occluded P and residual-P fractions in the rhizosphere soils were lower than in the bulk soils, while the contents of calcium phosphate and organic P in the rhizosphere soils were higher than in the bulk soils. Simple correlation coefficients showed that P uptake had positive relationship with non-occluded P, occluded P, calcium phosphate fractions and PF-P in the rhizosphere soils. The results suggest that the short-term application of MSS to the calcareous soils may increase Po and calcium phosphate fractions in the rhizosphere soils, and calcium phosphate fraction is potentially available to crops.