Occurrence of prophage and historical perspectives associated with the dissemination of huanglongbing in mainland China

Huanglongbing (HLB), associated with a non-culturable bacterium ‘Candidatus Liberibacter asiaticus’ (CLas), is a highly destructive citrus disease with a long but poorly documented history in China. No effective treatment for HLB is available. The identification of new prophages in abundant CLas genomic sequence data provides new insights into both the diversity of CLas strains and HLB management. In this study, CLas populations from nine provinces were surveyed for the presence of prophage. Two major prophage typing groups (PTGs) were discovered to be associated with two different altitude regions: strains of CLas in PTG1 from high altitude regions (HAR) mainly contained prophage Type 1 only or Types 1 and 3, whereas strains of CLas in PTG2 from low altitude regions (LAR) mainly contained prophage Type 2. The discovery of these CLas population patterns provides evidence for independent origins of HLB in HAR and LAR. Guangdong province is the generally recognized domestic region of origin for HLB and is primarily responsible for the dissemination of HLB in LAR through transport of seedlings. Both Yunnan and Sichuan provinces are the probable regions of origin for HLB in HAR. PTG2 was further divided into two subgroups: PTG2-1, found in Guangdong, Fujian and Guangxi and PTG2-2, found in Jiangxi, Zhejiang and Hunan. These regions and prophage types are correlated with early and late introductions of HLB in LAR. These molecular analyses were supported by studying the history of the dissemination of HLB in historical documents.     

广东梅州柑橘黄龙病和病毒病的发生调查及其黄龙病菌原噬菌体多样性

【目的】调查柑橘黄龙病（Huanglongbing,HLB）和病毒病在广东省梅州市柑橘主产区的危害情况,分析该地区柑橘黄龙病菌（CLas）的原噬菌体多样性。【方法】以16S rDNA为模板设计引物,利用实时荧光定量PCR（qPCR)检测梅州市不同抽检地在2017—2019年3年间柑橘黄龙病的发病情况;同时分别采用已报道的柑橘衰退病毒（Citrus tristeza virus,CTV）、柑橘裂皮病毒（Citrus exocortis viroid,CEVd）、柑橘褪绿矮缩病毒（Citrus chlorotic dwarf-associated virus,CCDaV）、柑橘碎叶病毒（Citrus tatter leaf virus,CTLV）和柑橘黄脉病毒（Citrus yellow vein clearing virus,CYVCV）的特异性检测引物,提取样品的RNA,反转后以样品的cDNA为模板,通过普通PCR的方法分析梅州市抽检地区柑橘病毒病的发生情况。此外,以基于2种原噬菌体类型（SC1和SC2）对应的超变异基因区域设计的2对引物（Lap-TJ-F/Lap-TJ-R1和Lap-TJ-F/Lap-TJ-R2）,运用普通PCR方法分析该地区CLas菌株原噬菌体的遗传多样性。【结果】除2018年9月梅州大浦顺兴公司蜜柚基地的抽检样品外,梅州市其他被检地区均发现CLas。总体来说,梅州市的脐橙CLas检出率为55.3%,蜜柚为61.5%,沙田柚为61.7%。其中2017年采集自平原县大拓镇的蜜柚和沙田柚样品CLas检出率为100%,兴宁市白马镇的蜜柚为100%,沙田柚为80%;其他抽检地区的样品CLas检出率在16.7%—83.3%。虽然梅州市部分地区的检出率较高,但其病原菌的含量并不高,大部分处于柑橘黄龙病发病初、中期,属可防可控阶段;此外,梅州市柑橘病毒的总检出率不高,CTV、CTLV和CYVCV为梅州的主要病毒,CEVd和CCDaV没有检测到。CTV、CTLV和CYVCV在脐橙上的检出率依次为78.9%、7.9%和21.1%,蜜柚为15.4%、25.0%和9.6%,沙田柚为6.4%、2.1%和4.3%。基于Lap-TJ-F/Lap-TJ-R1和Lap-TJ-F/Lap-TJ-R2来研究CLas原噬菌体多样性的PCR扩增条带共有4种类型（SC1-1、SC1-2、SC2-1和SC2-2）,经过PCR电泳和测序得出,来自梅州市脐橙和沙田柚的CLas菌株以SC2-1型为主,蜜柚上以SC1-1型为主。【结论】梅州柑橘产区的柑橘黄龙病目前属于可防可控阶段,其病原菌菌株的原噬菌体在不同品种上具有其独特性;由于在抽检时发现有柑橘病毒病的存在,因此在种植过程中需加强种苗监管,同时应采取有效措施对柑橘黄龙病和柑橘木虱（Diaphorina citri）进行绿色防控。     

Effects of feeding levels on growth performance,feed utilization,body composition and apparent digestibility coefficients of nutrients for juvenile Chinese sucker,Myxocyprinus asiaticus

An experiment was conducted to determine effects of feeding levels on growth performance, feed utilization, nutrient deposition, body composition and apparent digestibility coefficients (ADCs) of nutrients for juvenile Chinese sucker (initial weight, 11.77±0.22 g). Chinese sucker were fed a practical diet from 0% (starvation) to 4.0% (at 0.5% increments) body weight (bw) day^?1 for 8 weeks. The results showed that growth performance, feed utilization, nutrient deposition, body composition and ADCs of dry matter, protein and energy were significantly (P<0.05) affected by feeding levels. Survival was the lowest for the starvation group. Final mean body weight, growth rate, thermal‐unit growth coefficient (TGC) increased with feeding rate from 0% to 3.0% bw day^?1 (P<0.05) and showed no significant differences above the level (P>0.05). Feed conversion rate was significantly lower at a feeding level of 2.5% bw day^?1 than above and below the level (P<0.05). Protein efficiency ratio was markedly highest at the 2.5% bw day^?1 ration level (P<0.05). Fish fed at the feeding level (1.0% bw day^?1), which represented a maintenance ration (energy gain was less than 2.27 kJ fish^?1 day^?1), showed positive protein deposition but negative lipid deposition. This indicates that fish fed a maintenance ration mobilize body lipid reserve to support protein deposition. Lipid contents of whole body, muscle and liver increased with increasing feeding rates from 0.5% to 3.0% bw day^?1 and showed no significant differences above the level (P>0.05). Protein contents of whole‐body composition increased with feeding rate from 0.5 to 3.0% bw day^?1 (P<0.05) and showed no significant differences above the level (P>0.05), whereas muscle and liver remained relatively stable with the different ration amount with the exception of fish fed a ration of 0.5% bw day^?1, at which Chinese sucker possessed significantly lower body protein concentration (P<0.05). The ADCs of dry matter, protein and energy decreased with increasing feeding levels from 0.5% to 3.0% bw day^?1 and then remained relatively constant over the level. Based on the broken‐line regression analysis using WG data, the optimum and maintenance feeding levels for Chinese sucker were 3.10% bw day^?1 and 0.45% bw day^?1 respectively.     

链烷技术检测蝗虫食性方法的研究

为验证链烷技术测定蝗虫食性的精确性,确定蝗虫粪便中链烷的回收率。于2007年夏季在内蒙古太仆寺旗天然草地,用优势植物羊草(Leymus chinensis)、糙隐子草(Cleistegenes squarrosa)和克氏针茅(Stipa kylovii)各500g,饲喂360只成虫的亚洲小车蝗(Oedaleus asiaticus)。试验期内每天记录蝗虫实际牧草采食量、采食成分和排粪量,利用气相色谱分析牧草和粪样的链烷含量,应用链烷技术测定蝗虫的牧草采食比例,并与实际值进行比较。结果表明,3种牧草链烷模式存在种间差异;蝗虫粪便中链烷的回收率随链烷长度的增加而线性增加;蝗虫采食羊草、糙隐子草和克氏针茅比例的测定值与实际值存在显著正相关(P<0.05),相关系数分别为0.925 5、0.956 6和0.978 2。因此链烷技术可以精确测定典型草原蝗虫的食物组成。     

配合饲料高密度培育一龄胭脂鱼种的研究

研究了投喂配合饲料,在水泥池中高密度养殖胭脂鱼鱼种的技术,结果表明:胭脂鱼成活率90.06%,饵料系数2.086 0。并且得出脂胭鱼全长、体重关系式Y=0.044X2.604 5(R2=0.804 3)。     

土霉素对柑橘黄龙病的防治效果及对柑橘产量和品质的影响

为有效防控柑橘黄龙病，于2017年在佛罗里达大学柑橘研究与教育中心的奥本代尔市柑橘试验场进行田间试验筛选黄龙病的防治药剂及其浓度，测定注射土霉素后叶片中柑橘黄龙病菌亚洲种Cadidatus Liberibacter asiaticus、土霉素、淀粉含量、柑橘产量、出汁率、可溶性固形物含量和酸度，显微镜下观察注射土霉素后淀粉粒的分布。结果表明，浓度为1.6g/株土霉素处理180 d后柑橘叶片中黄龙病菌亚洲种含量减少幅度最大，为91.78%；注射浓度为1.6g/株土霉素后7d，叶片中土霉素含量达142.2 μg/kg，注射后11 d叶片中土霉素含量达到最大，为239.8 μg/kg，注射后45 d叶片中土霉素含量低至99.6 μg/kg以下；注射后7~90d，叶片中黄龙病菌亚洲种含量呈波浪形变化，注射90 d后叶片中黄龙病菌亚洲种含量一直处于增长趋势，叶片中黄龙病菌亚洲种含量总体随着土霉素含量的升高而降低；注射浓度为1.6g/株土霉素后，叶片中淀粉含量大幅度下降，60d时达到最低值，为3.3 μg/mm²，90d时达到峰值，为14.5 μg/mm²；单株产量为16.7 kg，与注射浓度为0.8 g/株土霉素处理差异不显著，但均显著高于其它2个处理；柑橘出汁率、可溶性固形物和酸度均与清水对照差异不显著。表明土霉素可有效抑制黄龙病菌亚洲种，减少柑橘叶片内淀粉含量，增加柑橘产量，但对果实品质未产生显著影响。     

草地蝗虫悬浮速度的研究

在内蒙古锡林郭勒盟镶黄旗蝗灾发生区,采集了严重危害草原的4龄期、5龄期及成虫的亚洲小车蝗,分别对它们的活体、死体的悬浮速度进行了测定,为草地蝗虫吸捕机的设计与改进提供了理论依据。     

重庆市胭脂鱼资源及保护现状

收集整理了重庆渔政网2007～2009年的胭脂鱼误捕信息,以及1997～2009年的《长江三峡生态环境公报》中有关胭脂鱼的资料,着重分析了胭脂鱼在重庆的分布区域、资源现状与保护现状等,提出了加强增殖放流效果研究、规范误捕记录、进行产卵场调查、增设保护区等建议。     

柑桔黄龙病的常规PCR及荧光定量PCR检测

【目的】为柑桔黄龙病的早期诊断和寄主体内病原菌的动态监测提供一种稳定、可靠的检验检疫技术。【方法】利用亚洲韧皮杆菌核糖体蛋白基因rplJ/rplL设计了2对PCR引物CQULA03F/CQULA03R、CQULA04F/CQULA04R和1条TaqMan探针CQULAP1，以此为基础建立了常规PCR和TaqMan探针法、SYBR Green I荧光染料法两种荧光定量PCR（共3种）反应体系；确定了3种体系各自的检测灵敏度、特异性和准确性，据此对3种体系进行了比较；从2004年7月到2005年5月还利用常规PCR和TaqMan探针荧光定量PCR体系完成了对柑桔黄龙病病原菌在寄主体内的周年变化的动态监测。【结果】两种荧光定量PCR方法的灵敏度比常规PCR高出至少2～3个数量级，而TaqMan探针法由于使用了杂交探针，其特异性尤其可靠，另外两种定量PCR较小的产物片段使得它们具有更好的稳定性，加上荧光定量PCR方法本身受污染可能性小、操作简便等固有优势，使得前者更适合于柑桔黄龙病的检测。【结论】本研究建立的2种荧光定量PCR方法可以为柑桔黄龙病的病害早期诊断以及柑桔黄龙病病原菌近缘种的甄别提供准确、灵敏、快速的检验检疫技术。     

温湿度对亚洲小车蝗飞行能力及主要能源物质利用的影响

本研究采用昆虫飞行数据微机采集系统(飞行磨)吊飞方法,测定了温、湿度对10日龄亚洲小车蝗(Oedaleus asiaticus)雌雄成虫的飞行能力及主要能源物质利用的影响。结果表明,在环境温度为28℃、相对湿度(RH)为60%时,10日龄亚洲小车蝗成虫表现出最优的飞行能力,单个个体的最大飞行时间、最大飞行距离和最大飞行速度分别可达1.62h、9.87 km和2.03 km·h~(-1)。在温度16℃以下或28℃以上,其飞行能力明显降低。在40%~80%RH时,成虫均能进行正常的飞行活动。环境温、湿度会显著影响亚洲小车蝗成虫飞行能源物质的消耗情况(P0.05)。在最适的温、湿度条件下,小车蝗飞行所需的能源物质(甘油三酯)最少,其飞行单位距离消耗的甘油三酯也最低,能源利用效率最高。较高或较低的温、湿度条件,能源物质的消耗都显著高于最适条件。飞行能源物质利用效率的不同是导致其在不同温、湿度下飞行能力产生差异的主要原因之一。