基于纳米孔测序技术的柑橘黄龙病检测方法建立

柑橘黄龙病(Huanglongbing)是柑橘生产上最具毁灭性的病害,及时快速地进行早期检测和诊断是防控黄龙病的关键措施之一.本文利用掌上纳米孔测序仪MinION对携带黄龙病菌Candidatus Liberibacter asiaticus的DNA样品进行测序,并利用Blast、GraphMap、minimap2以及两种bwa的比对方法将测序结果比对到黄龙病菌基因组上,比对结果均匀的比对到黄龙病菌基因组上,并未发现严重的偏倚现象,验证了其测序结果的可靠性.本技术可弥补因柑橘木虱Diaphorina citri(Kuwayama)虫体过小或损坏难以进行光学识别的不足,并可同时检测虫体是否携带有黄龙病菌,对有黄龙病发生风险但尚未有黄龙病实际发生的柑橘种植区提供实时实地的监控与预警.     

难培养菌的多条带PCR产物产生原因分析

【目的】分析严重威胁柑橘产业发展的毁灭性病害——柑橘黄龙病的强致病性病原亚洲种“Candidatus Liberibacter asiaticus”的种群多样性研究中出现多条带PCR产物的原因,为难培养菌的分子生物学研究提供参考。【方法】通过使用PCR、聚丙烯酰胺凝胶电泳(PAGE)和测序相结合分析“Ca. L. asiaticus”基因组上2个短串联重复(Short tandem repeats,STR)基因的PCR产物。【结果】单个菌株可扩增获得多个PCR条带且其扩增产物多态性受寄主品种影响;这2个STR基因的PCR产物在菌株间呈明显的多态性;扩增所获得的序列可来自“Ca. L. asiaticus”本身,也可来自其寄主或内生菌。【结论】难培养菌的STR基因PCR产物多态性产生的主要原因是该基因内部的串联重复序列数目存在差异,但目的菌及外源物种(寄主或内生菌等)基因组的非特异性扩增也是影响其多态性的因素。     

Early detection surveillance for an emerging plant pathogen: a rule of thumb to predict prevalence at first discovery

Emerging plant pathogens are a significant problem for conservation and food security. Surveillance is often instigated in an attempt to detect an invading epidemic before it gets out of control. Yet in practice many epidemics are not discovered until already at a high prevalence, partly due to a lack of quantitative understanding of how surveillance effort and the dynamics of an invading epidemic relate. We test a simple rule of thumb to determine, for a surveillance programme taking a fixed number of samples at regular intervals, the distribution of the prevalence an epidemic will have reached on first discovery (discovery-prevalence) and its expectation E(q*). We show that E(q*) = r/(N/Δ), i.e. simply the rate of epidemic growth divided by the rate of sampling; where r is the epidemic growth rate, N is the sample size and Δ is the time between sampling rounds. We demonstrate the robustness of this rule of thumb using spatio-temporal epidemic models as well as data from real epidemics. Our work supports the view that, for the purposes of early detection surveillance, simple models can provide useful insights in apparently complex systems. The insight can inform decisions on surveillance resource allocation in plant health and has potential applicability to invasive species generally.     

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

广东省橘园黄龙病发生与危害的影响因素研究

【目的】柑橘黄龙病是世界柑橘生产上的毁灭性病害。探明广东省橘园黄龙病发生与危害的影响因素,可为该病的科学防控提供依据。【方法】在广东省惠州、肇庆、云浮、江门、阳江、清远、韶关等柑橘产区,调查了不同柑橘品种、不同气候和地理条件、不同种植模式和不同管理水平果园的黄龙病发病率,并以常规PCR和实时荧光定量PCR对样品进行了分子检测。【结果】不同柑橘栽培品种发病率:红江橙为78.05%,马水橘为76.04%,砂糖橘为73.20%,贡柑为69.30%,年橘为63.00%,沙田柚为58.68%,柠檬为56.96%;砧木品种发病率:酸橘为15.30%,枳壳为6.57%,柠檬为4.43%;低纬、高温、多台风地区果园的黄龙病发病率较高;山地果园的黄龙病发病率低于平地果园;连片种植果园的黄龙病发病率高于零散种植果园;管理不善的果园中黄龙病发病率高于管理较好的果园。【结论】除了自然因素(低纬、高温、多台风、平原地形)外,聚集种植和管理不善等因素也有利于黄龙病的发生与流行。因此,有针对性地安排果园布局和加强果园管理可以减轻黄龙病的危害。     

柑橘黄龙病隐症寄主九里香内生细菌分离及功能鉴定

【目的】分析黄龙病高发区的九里香植株叶及茎干中内生细菌,为寻找具有抗柑橘黄龙病的内生细菌奠定基础。【方法】利用平板培养法及基于16S rDNA的限制性酶切长度多态性(Restriction fragment length polymorphism,RFLP)序列分子鉴定法,对九里香植物内生细菌进行多样性分析。【结果】在兼性厌氧的生长环境下,从九里香植株中分离获得可培养内生细菌26株,分属于9个细菌属的14个种,其中肠杆菌属(Enterobacter sp.)(IF=19.23%)、芽胞杆菌属(Bacillus sp.)(IF=38.46%)为九里香可培养内生细菌的优势菌属。茎干中内生细菌丰度高于叶中内生菌丰度。建立了九里香内生细菌16S rDNA文库,对文库质量检测显示,该克隆文库的覆盖度(Coverage C)为94.97%,结合Rarefaction曲线分析,表明所构建的克隆文库是相对充分的。对文库中179个阳性克隆进行HaeШ、MspⅠ、RsaⅠ3种限制性内切酶分析,得到20个不同的操作分类单元(Operational taxonomic units,OTUs),其中沙雷氏菌属(Serratia sp.)为九里香植株中内生细菌的绝对优势菌属。测定了14株内生细菌的功能,其中9株菌能产生吲哚-3-乙酸(IAA);具有抗生素(phlD)合成能力的内生细菌有4株;结合nifH和NFb固氮培养基确定有3株内生细菌具有固氮能力;1株内生细菌具有ACC脱氨酶合成能力;8株内生细菌具有铁细胞合成能力;3株内生细菌具有淀粉水解能力;2株内生细菌显示强阳性的蛋白酶合成能力,4株内生细菌具有以上4种功能。【结论】九里香植株中内生细菌具有丰富的多样性,并且可能对九里香植株生长发育及抗生物和非生物胁迫有着重要的生理功能。     

Metabolomic analysis elucidates how shade conditions ameliorate the deleterious effects of greening (Huanglongbing) disease in citrus

Under tropical and subtropical environments, citrus leaves are exposed to excess sunlight, inducing photoinhibition. Huanglongbing (HLB, citrus greening), a devastating phloem-limited disease putatively caused by Candidatus Liberibacter asiaticus, exacerbates this challenge with additional photosynthetic loss and excessive starch accumulation. A combined metabolomics and physiological approach was used to elucidate whether shade alleviates the deleterious effects of HLB in field-grown citrus trees, and to understand the underlying metabolic mechanisms related to shade-induced morpho-physiological changes in citrus. Using metabolite profiling and multinomial logistic regression, we identified pivotal metabolites altered in response to shade. A core metabolic network associated with shade conditions was identified through pathway enrichment analysis and metabolite mapping. We measured physio-biochemical responses and growth and yield characteristics. With these, the relationships between metabolic network and the variables measured above were investigated. We found that moderate-shade alleviates sink limitation by preventing excessive starch accumulation and increasing foliar sucrose levels. Increased growth and fruit yield in shaded compared with non-shaded trees were associated with increased photosystem II efficiency and leaf carbon fixation pathway metabolites. Our study also shows that, in HLB-affected trees under shade, the signaling of plant hormones (auxins and cytokinins) and nitrogen supply were downregulated with reducing new shoot production likely due to diminished needs of cell damage repair and tissue regeneration under shade. Overall, our findings provide the first glimpse of the complex dynamics between cellular metabolites and leaf physiological functions in citrus HLB pathosystem under shade, and reveal the mechanistic basis of how shade ameliorates HLB disease.