可可毛色二孢全基因组分泌蛋白的预测及分析

【目的】可可毛色二孢（Lasiodiplodia theobromae）是一种世界性分布的重要植物病原真菌,可引起严重的葡萄溃疡病（Botryosphaeria dieback）,影响果木品质并造成巨大的经济损失。本研究预测并分析可可毛色二孢基因组范围内的分泌蛋白,并明确其基本特征,为该病菌分泌蛋白致病机理的研究打下基础。【方法】依据已公布的可可毛色二孢全基因组序列,利用信号肽预测软件SignalP v5.0、跨膜结构分析软件TMHMM v2.0、细胞器定位分析软件ProtComp v9.0、GPI锚定预测软件big-PI Fungal Predictor和亚细胞器定位分析软件TargetP v2.0生物信息学软件对该菌中的典型分泌蛋白进行筛选。对分泌蛋白N端信号肽的长度、氨基酸使用频率及其切割位点进行统计分析。依据蛋白序列的同源性,应用BLASTP程序对分泌组蛋白进行功能注释分析,预测其生物学功能。采用蔗糖酶缺陷的酵母分泌系统,对所选分泌蛋白的信号肽进行活性检测。利用qRT-PCR方法检测所选分泌蛋白基因在可可毛色二孢侵染葡萄中的表达情况。【结果】在可可毛色二孢全基因组编码蛋白中共筛选获得552个潜在的具有典型信号肽的分泌蛋白,占全基因组预测蛋白总数的4.3%,其编码蛋白长度集中于101—400 aa。信号肽统计分析表明,其信号肽长度以18—20 aa的序列最为集中,信号肽长度为20 aa的蛋白数量最多。信号肽中使用频率最高的氨基酸为丙氨酸;非极性、疏水的氨基酸使用频率最高,占氨基酸总数的60.2%。其信号肽的-3至-1位置上的氨基酸相对保守,切割位点属于A-X-A类型,可被Sp I型信号肽酶识别并切割。336个分泌蛋白具有功能注释,其功能较多集中于细胞壁降解有关的酶类以及致病相关蛋白,并且这些蛋白在分子量、等电点、脂肪族氨基酸指数等方面均存在差异。通过蔗糖酶缺陷的酵母分泌系统证实,挑选的9个分泌蛋白信号肽均具有分泌活性。qRT-PCR检测结果表明,所选分泌蛋白基因在该病菌侵染初期的表达发生变化。【结论】利用生物信息学分析技术从可可毛色二孢全基因组中共预测获得552个经典分泌蛋白。其信号肽氨基酸长度分布广泛,氨基酸组成中非极性、疏水的氨基酸使用频率最高。功能注释主要集中在细胞壁组分降解相关的酶类、致病侵染相关的坏死诱导相关蛋白以及几丁质结合蛋白等。     

葡萄VvSUC12基因启动子的克隆及上游调控因子鉴定

枝干病害葡萄溃疡病近年来严重限制了葡萄产业发展。研究表明葡萄蔗糖转运蛋白参与寄主植物和病原菌的互作过程。为解析蔗糖转运蛋白VvSUC12在葡萄免疫反应过程中的功能,本研究克隆了VvSUC12基因翻译起始位点上游1 500 bp的启动子区,通过生物信息学分析发现该区域包含4个Dof转录因子结合序列(A/TAAAG)及多种激素调节与防御相关的顺式元件。实时荧光定量分析显示,接种可可毛色二孢菌显著诱导VvSUC12和VvDof19基因的表达。通过酵母单杂交实验筛选得到与VvSUC12启动子互作的转录因子VvDof19。酵母双杂交实验证实VvDof19具有自激活活性;进一步通过烟草瞬时表达发现Dof19-GFP的相对GUS活性约为对照GFP的9倍,表明转录因子VvDof19能够激活VvSUC12基因的表达。研究结果为深入研究蔗糖转运蛋白在葡萄免疫反应中的功能奠定基础。     

国外松枯梢病的松色二孢菌9个菌株生物学特性的研究

对来自不同发病程度，不同地点的国外松枯梢病松色二孢菌的９个菌株的生产，产孢等生物学特性进行了研究。结果表明，各菌株生长的最适温度为２５－３０℃，ＰＨ值为６－７，相对湿度为９０％以上；９２４５、Ｈ２１二菌株对湿度在培养其中加入１‰的硼对各菌株的生长和产孢有不同程度的抑制作用，尤以抑制产孢最明显，表现出硼对国外枯病的发生，发展有一定的控制作用。     

Effects Of Thinning In a Water-Limited Holm Oak Forest

ABSTRACT

A natural holm oak forest was selectively thinned to test thinning as a tool to reduce tree mortality, increase productivity, and reverse the recent regression of the dominant species (Quercus ilex) induced by climate change. Thinning increased aboveground productivity and reduced stem mortality in this Mediterranean forest during four years just after thinning, contributing to the maintenance of forest functioning under changing climatic conditions. Q. ilex was the only species positively affected by the thinning: stem growth increased for all stem sizes, and mortality was significantly lower in thinned plots. On the contrary, mortality rates of Phillyrea latifolia and Arbutus unedo were not significantly lower. Stem growth increased for P. latifolia only in the smallest stem-size class. Our results highlight the suitability of selective thinning for improving the forest productivity and ensuring the conservation of Mediterranean coppices. Other benefits of selective thinning, such as a decrease in the risk of fire dispersion and an increase in the water supply for human populations, are also discussed.     

Ophiostomatoid fungi associated with bark beetles onAbies veitchii in wave-regenerated forests

Relationships between tree mortality and bark beetle infestation onAbies veitchii at the wave-regenerated forest in Mt. Asahi, Okuchichibu area were investigated. Most of the firs with green needles and newly developed current year’s shoots in the dieback zone were heavily infested by bark beetles before the death of the trees. After heavy infestation of beetles, about half of the infested firs died within the year, and the other half died in the next year. When the species composition of bark beetles and associated ophiostomatoid fungi were investigated in Mt. Asahi and also at a typical wave-regenerated forest in Mt. Shimagare, Yatsugatake area,Cryphalus montanus andC. piceae were dominant beetle species for Mt. Asahi and Mt. Shimagare, respectively.Ophiostoma subalpinum andO. europhioides were dominant fungal species at both wave-generated forests. BecauseO. subalpinum was detected more frequently from deeper areas of sapwood thanO. europhioides, it was suggested that the fungal species may accelerate the death of stressed firs in wave-regenerated forests. Contribution No. 169, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

核桃枝枯病病原菌鉴定及其生物学特性

In May 2018,a new shoot dieback disease was found in several walnut orchards in Rizhao,Shandong Province.The fungal pathogen was obtained on PDA plate from diseased walnut shoots using a tissue isolation method.The purified fungal isolates formed white to grey colonies on PDA plate and three single spore isolates SDWa1-SDWa3 were obtained.Based on morphological characteristics and analysis of rDNA internal transcribed spacer (ITS),calmodulin (CAL),translation elongation factor 1-α (TEF1) and β-tubulin (TUB) sequences,the fungal isolates were identified as Diaporthe nobilis,a new pathogen on walnut.The test of Koch's rule confirmed that D.nobilis was the pathogen of walnut shoot dieback.To determine optimal growing conditions for the pathogen,its biological characteristics test was conducted.The results showed that the optimal growth temperature and pH value were at 25℃ and pH 5-9,respectively.The optimum carbon source was glucose,and favourable nitrogen source was peptone.     

甘肃省河西走廊沙棘枝枯病发生初报

为明确甘肃省河西走廊沙棘枝枯病的发生与为害情况,采用定点、定期观察和大面积调查相结合的方法,对河西地区沙棘上发生的枝枯病进行系统调查与分析。结果表明,该病主要为害枝干,严重时造成枝干枯死或树体死亡。经调查,气候条件是引起该病害发生流行的主要诱因,其次是树势衰弱。通常春季气温回升早、降雨早,发病早而且严重,降雨越多,发病越严重;一般沙棘枝枯病发病适温为25℃左右,温度低于20℃和高于30℃都不利于病害的发生。新老沙棘混栽林、管理粗放、结果量大,发病严重。另外,沙棘枝枯病发生严重会造成树皮伤口多而深,为沙棘干缩病菌的侵入提供了有利途径,因此,一般沙棘枝枯病发生严重的树林干缩病发生相对也比较严重。经调查研究分析,初步确定沙棘枝枯病是沙棘树上新发生的一种枝枯病,病害的发生总体呈逐年加重的趋势。     

桉树焦枯病对桉树生长量的损失估计研究

按照桉树焦枯病不同发病等级调查桉树各因子生长量,分析不同病级对桉树胸径、树高和材积生长的影响。结果表明,当发病等级达到Ⅳ级时对胸径、Ⅲ级时对树高、Ⅱ级时对材积的生长已经造成极显著影响。根据不同发病等级与损失率的关系进行回归分析,建立了胸径、树高和材积的损失率估测模型,该模型估测结果与调查实测数据相对误差1.32%(精度为98.68%)。以此模型估算福建省桉树人工林焦枯病危害,材积年损失率23.53%,年经济损失0.518亿元。     

Dieback intensity but not functional and taxonomic diversity indices predict forest productivity in different management conditions: Evidence from a semi-arid oak forest ecosystem

The relationships between different aspects of diversity(taxonomic,structural and functional)and the aboveground biomass(AGB)as a major component of global carbon balance have been studied extensively but rarely under the simultaneous influence of forest dieback and management.In this study,we investigate the relationships between taxonomic,functional and structural diversity of woody species(trees and shrubs)and AGB along a gradient of dieback intensity(low,moderate,high and no dieback as control)under two contrasted management conditions(protection by central government vs.traditional management by natives)in a semi-arid oak(Quereus brantii Lindl.)forest ecosystem.AGB was estimated and taxonomic diversity,community weighted average(CWM)and functional divergence indices were produced.We found that the aerial biomass was significantly higher in the intensively used area(14.57(±1.60)t/hm²)than in the protected area(8.70(±1.05)t/hm²)due to persistence of some large trees but with decreasing values along the dieback intensity gradient in both areas.CWM of height(H),leaf nitrogen content(LNC)and leaf dry matter content(LDMC)were also higher in the traditional managed area than in the protected area.In contrast,in the protected area,the woody species diversity was higher and the inter-specific competition was more intense,explaining a reduced H,biomass and LDMC.Contrary to the results of CWM,none of the functional diversity traits(FDvar)was affected by dieback intensity and only FDvar values of LNC,leaf phosphorus content(LPC)and LDMC were influenced by management.We also found significantly positive linear relationships of AGB with CWM and FDvar indices in the protected area,and with taxonomic and structural diversity indices in the traditional managed area.These results emphasize that along a dieback intensity gradient,the leaf functional traits are efficient predictors in estimating the AGB in protected forests,while taxonomic and structural indices provide better results in forests under a high human pressure.Finally,species identity of the dominant species(i.e.,Brant’s oak)proves to be the main driver of AGB,supporting the selection effect hypothesis.