海南柑橘黄龙病发生分布调查及病原种类鉴定

本研究在海南全省范围进行了柑橘黄龙病的发生分布调查和病原种类鉴定,共采集了14个市县62处柑橘种植点的不同柑橘品种样品1227份。用柑橘黄龙病菌亚洲种和非洲种rpIA-rpIJ基因片段的通用检测引物A2/J5和柑橘黄龙病菌美洲种16S rRNA基因片段的特异检测引物GB1/GB3,对样品进行PCR扩增,发现海南柑橘黄龙病的病原为柑橘黄龙病菌亚洲种,检测样品中阳性率为79.01%,未发现柑橘黄龙病菌非洲种和美洲种。结果发现,柑橘黄龙病已分布在海南的琼中、澄迈、儋州、琼海等8个市县。同时选取海南澄迈、儋州、琼海等4个市县有代表性的甜橙、柚子、柠檬感染黄龙病的样品,扩增样品的rpIA-rpIJ基因A2/J5片段,进行序列比对和系统进化分析。序列比对结果发现,海南各地不同品种中的柑橘黄龙病菌rpIA-rpIJ基因A2/J5片段序列一致性为99.87%,序列十分保守。系统进化分析表明,海南各地不同品种的柑橘黄龙病菌都与黄龙病菌亚洲种聚集为同一进化分枝,与黄龙病菌非洲种和美洲种分属不同的进化分枝。本研究明确了海南柑橘黄龙病的发生分布情况和病原种类,对柑橘黄龙病的防控具有重要的理论意义。     

黄龙病对柑橘叶际微生物组的影响

为明确柑橘叶际微生物组对黄龙病发生的响应规律，采用荧光定量PCR技术检测采集柑橘叶片感染黄龙病菌的情况，通过扩增子测序分析法对PCR检测结果为阴性和阳性叶片的微生物群落进行比较，并通过相关性分析和网络分析对黄龙病菌和叶际微生物的相关性进行解析。结果显示，夏季采集的柑橘叶片中黄龙病菌的检出率为7.5%，而到秋季黄龙病菌的检出率上升到32.3%。黄龙病的发生显著改变了柑橘叶际细菌群落的组成和结构，对于细菌类群的丰度也有一定影响；而对叶际真菌群落的组成和结构无显著影响。黄龙病菌与叶际细菌存在普遍的负相关关系，正相关关系相对较少但相关性更强。黄龙病菌可能通过与厌氧棍状菌Anaerotruncus sp.、梭菌Clostridiales sp.、假单胞菌Pseudomonas sp.、鞘脂单胞菌Sphingomonas sp.、毛螺菌Lachnospiraceae sp.和链球菌Streptococcus sp.的负向互作对柑橘叶际整个细菌群落产生负影响。表明黄龙病发生对柑橘叶际真菌和细菌群落的影响规律不同，同时黄龙病菌可能通过与几种主要细菌的负向互作来实现对叶际微生物组的影响。     

柑橘黄龙病PCR检测技术研究

以柑橘黄龙病病原亚洲菌系β-操纵子的特异引物fA2/rJ5进行PCR扩增,依据是否有目标DNA片段的产生来检测柑橘黄龙病菌的存在与否。该检测技术,具有快速、简便、灵敏等特点,可用于柑橘黄龙病的早期诊断,对控制该病害的传播具有重要意义。     

田间柑橘植株不同部位黄龙病菌的PCR检测及发病原因分析

［目的］了解黄龙病菌在柑橘植株不同部位的分布,为深入研究病菌在植株体内的扩散情况奠定基础;明确病害的发生原因为有效防控该病害提供借鉴。［方法］ 调查浙江省台州市柑橘黄龙病（huanglongbing, HLB）的发生情况,通过常规和巢式PCR,检测了发病情形不同的两个果园内柑橘病株不同部位及不同植株中的黄龙病菌,并对其发病原因进行分析。［结果］ 发现一果园内病株的无症状叶片、有症状叶片和枝条中均含有黄龙病菌,而其周围植株不含病菌;另一果园内病株的有症状叶片、枝条、主干和砧木中均含有黄龙病菌,而其周围植株也含菌。［结论］分析认为这两种果园内柑橘植株发病原因不同,一种可能为通过携带黄龙病菌的柑橘木虱所感染,另一种可能为嫁接过程中通过带菌的接穗感染。     

土壤施硅对小麦和麦蚜的影响

为明确土壤施硅对小麦蚜虫的防控效果，本研究在小麦种植前分别配施0（CK）、40、80和120 kg/hm²四个浓度的硅肥，测量施硅后小麦生育期、生物量、叶面积、株高、产量指标以及荻草谷网蚜Sitobion miscanthi和禾谷缢管蚜Rhopalosiphum padi发生量。结果表明，施硅对小麦株高和穗数无显著影响，但80 kg/hm²和120 kg/hm²浓度施硅处理后小麦叶面积分别为1 786.03 mm²和2 033.54 mm²，生物量分别为133.68 g和134.50 g，小麦穗粒数分别为35.61粒和35.92粒，小麦千粒重分别为45.34 g和45.59 g，小麦产量分别为9 684.45 kg/hm²和9 835.05 kg/hm²，均显著高于对照，且小麦成熟期较对照提前3 d；80 kg/hm²和120 kg/hm²浓度施硅处理后禾谷缢管蚜发生量分别为90头和86头，均显著低于对照；40、80和120 kg/hm²浓度施硅处理后荻草谷网蚜发生量分别为128、117和105头，均显著低于对照。因此，田间土壤施硅80 kg/hm²和120 kg/hm²可以明显改善小麦农艺性状，提高小麦产量，并能减少麦蚜发生量。     

螺虫乙酯防治柑橘木虱药效试验

柑橘木虱是柑橘黄龙病菌的传播媒介。为控制柑橘黄龙病的为害与传播,进行了螺虫乙酯防控柑橘木虱的试验。结果表明,24%螺虫乙酯SC 4 000倍液对柑橘木虱的药后3 d防效为74.24%~87.04%;药后14 d为91.82%~92.62%;药后28 d为39.08%~88.19%,内吸传导性强,防效较好且持效期长,是防治柑橘木虱的有效药剂。     

湖南省永州地区柑橘慢衰病与黄龙病病原鉴定及分布调查

为明确湖南省柑橘黄化症状与柑橘慢衰病和柑橘黄龙病的相关性,应用形态学和分子生物学方法对湖南省永州地区表现黄化症状的柑橘进行了两种病原的鉴定及分布调查。结果表明,造成该地区柑橘黄化现象的主要原因为柑橘半穿刺线虫和黄龙病菌对柑橘的侵染。所检样本中,柑橘慢衰病平均发生率为82.1%,土壤中的半穿刺线虫群体密度最高达到3 077条/100mL。永州地区柑橘黄龙病病菌属于韧皮部杆菌属类细菌亚洲种,平均检出率为64.3%。柑橘半穿刺线虫和黄龙病菌在柑橘园存在混合侵染现象,混合侵染率为53.6%。柑橘半穿刺线虫在永州地区柑橘产区分布广泛,是造成柑橘黄化症状的重要病因,重视并有效防控柑橘半穿刺线虫已迫在眉睫。     

柑橘黄龙病菌效应蛋白与寄主互作研究进展与展望

柑橘黄龙病(citrus Huanglongbing, HLB)是柑橘生产上的毁灭性病害, 其病原是韧皮部杆菌属的革兰氏阴性难养细菌, 我国发生的黄龙病病原为韧皮部杆菌亚洲种(‘Candidatus Liberibacter asiaticus’, CLas)。由于黄龙病菌未能实现纯培养, 难以进行传统的分子和遗传操作, 导致其致病机理研究滞后。近年来, 有关黄龙病菌效应蛋白的筛选鉴定及其与寄主靶标互作机制成为研究热点。本文重点介绍了黄龙病菌分泌系统特征、效应蛋白与寄主互作机制及抗病种质创制, 展望了该领域未来的研究方向。     

柑橘黄龙病的传播介体-柑橘木虱在广东果园的发生调查

柑橘木虱是重要害虫也是柑橘黄龙病的传播介体,因此调查木虱发生动态及其携带黄龙病菌的情况对指导黄龙病的防控有重要意义。本研究调查了广东多个果园的黄龙病发病情况,观察了柑橘木虱的生物学特性,统计了柑橘木虱在不同月份的发生数量,检测了木虱携带黄龙病菌情况。结果发现:失管果园中,黄龙病发病严重且木虱数量大,已成为散播病害的重要源头,而与失管果园保持一定距离且加强木虱防控可减轻黄龙病危害;还发现:广东地区6月份柑橘木虱种群已有一定数量,7、8月,其种群数量再次攀升,到了9、10月份,种群数量保持在高位。11、12月间,种群数量快速下降。最后根据本研究的调查结果,提出了一些加强黄龙病防控的措施。     

柑橘溃疡病菌的侵染过程及噻森铜对溃疡病的防治效果

为高效精准防治柑橘溃疡病,本研究对不同成熟度柑橘叶片接种溃疡病菌Xanthomonas citri subsp.citri后的发病情况进行观察,并在显微镜下观察溃疡病菌侵染叶片的过程,并对不同浓度、不同施用方法下噻森铜的室内防治效果和田间防治效果进行测定,对施用噻森铜后土壤、叶片和果实中的残留进行测定。结果表明,溃疡病菌可侵染柑橘嫩叶、幼叶和未成熟叶,其在叶片上附着2~4 d既已进入寄主组织内部。15 mL/株和20 mL/株噻森铜灌根15 d接种溃疡病菌的防治效果显著,而噻森铜10 mL/株灌根的防治效果略差,对照植株严重发病。萌芽前15~20 d噻森铜灌根浓度为60、75和90 mL/株时,每株病叶数分别为3.9、3.9和3.7个,每株病斑数分别为8.4、8.5和8.5个,病果率分别为9.4%、11.5%和9.6%,3个浓度处理之间差异不显著,但均显著低于对照。萌芽前喷施 500倍噻森铜处理的每株病斑数为16.1个,与灌根60 mL/株噻森铜处理的每株病斑数差异不显著,但均显著低于对照;喷施噻森铜处理的病果率为16.8%,显著高于灌根60 mL/株噻森铜处理的,且两者均显著低于对照。萌芽前噻森铜不同施用方法的土壤、叶片和果实中铜离子含量均与常规管理的无显著差异。总之,萌芽前灌根或喷雾噻森铜可以克服生产上因萌芽不整齐和雨水带来的不利影响,进而有效防治柑橘溃疡病。     

Characterisation of the fungal population in citrus packing houses

The aim of this work was the characterisation of the environmental and superficial mycoflora of equipment and facilities of two citrus packing houses in Sao Paulo State, Brazil, in 2004 and 2005. One of the packing houses packed fruit for the export market (municipality of Matao), the other for the domestic market (municipality of Engenheiro Coelho). The study also identified the presence of isolates of Penicillium spp. resistant to thiabendazole and imazalil fungicides in packing houses. The environmental mycoflora was sampled according to the gravimetric method, using Petri dishes containing potato dextrose agar medium opened for 2 min. The superficial mycoflora on equipment and facilities was sampled with Rodac plates. The mycoflora in the environment and on surfaces of the packing houses in Matao were 12.3 and 52.3 cfu/plate, respectively, while these populations for the Engenheiro Coelho packing house were 46.3 and 68.2 cfu/plate, respectively. Cladosporium and Penicillium were the most prevalent genera of fungi. The contamination levels of clean zones in the packing houses (washing of fruits, packing table, boxes and containers) was not substantially lower than the contamination in dirty zones (reception of fruits and first selection). The percentage of P. digitatum isolates in Matao that was resistant to thiabendazole and imazalil was 25.9 and 1.5 in the environment and 30.1 and 16.0 on packing house surfaces, respectively. In Engenheiro Coelho, percentage of resistance to these fungicides was 51.9 and 0.1 in the environment and 39.2 and 0.9 on packing house surfaces, respectively.     

Modelling the progress of Asiatic citrus canker on Tahiti lime in relation to temperature and leaf wetness

The combined effect of temperature (15°C, 20°C, 25°C, 30°C, 35°C, 40°C and 42°C) and leaf wetness duration (0, 4, 8 12, 16, 20 and 24 h) on infection and development of Asiatic citrus canker (Xanthomonas citri subsp. citri) on Tahiti lime plant was examined in growth chambers. No disease developed at 42°C and zero hours of leaf wetness. Periods of leaf wetness as short as 4 h were sufficient for citrus canker infection. However, a longer leaf duration wetness (24 h) did not result in much increase in the incidence of citrus canker, but led to twice the number of lesions and four times the disease severity. Temperature was the greatest factor influencing disease development. At optimum temperatures (25–35°C), there was 100% disease incidence. Maximum disease development was observed at 30–35°C, with up to a 12-fold increase in lesion density, a 10-fold increase in lesion size and a 60-fold increase in disease severity.     

The citrus nursery site approval program for burrowing nematode and its beneficial effect on the citrus industry of Florida

The nursery site approval program for spreading decline of citrus, caused by the burrowing nematode Radopholus similis ¹ , is evaluated after 33 years of application in Florida (US). The economic benefits of the site approval program have far outweighed the costs engendered by the program. A site approval model is provided for use worldwide by regulatory agencies if similar pest problems are encountered.     

九里香挥发物对柑橘木虱的引诱效果及混配筛选试验

为明确九里香Murraya exotica挥发物对柑橘木虱Diaphorina citri的引诱效果,筛选田间诱杀柑橘木虱的高效混配物组合,采用Y-型嗅觉仪室内测试柑橘木虱对10种九里香挥发物的行为反应,并测定挥发物混剂对柑橘木虱引诱的增效作用以及九里香挥发物混剂对柑橘木虱的田间诱杀效果。试验结果表明,β-石竹烯、D-柠檬烯和芳樟醇对柑橘木虱均有显著的引诱效果,引诱率分别为80.00%、83.33%和73.33%,显著高于其它7种挥发物;混剂B(β-石竹烯∶D-柠檬烯∶芳樟醇=3∶3∶1)对柑橘木虱的引诱率达到88.89%,显著优于其它混剂,增效作用较好;田间悬挂黄色诱虫瓶15 d后,混剂B平均诱集到的柑橘木虱数量为23.3头/瓶,显著优于其它混剂,为矿物油对照的33倍。     

Fenpropimorph: a promising fungicide for postharvest diseases in citrus fruits

Sour rot, green mold and blue mold are postharvest diseases of citrus fruit. Benzimidazole and imidazole fungicides control molds, and guazatine controls sour rot, but strains ofPenicillium spp. resistant to these chemicals have been reported. Aqueous formulations of fenpropimorph, a morpholine fungicide, are very active in controlling fungi attacking postharvest citrus fruits. The fungicidal effect of the chemical in the coating wax was investigated in combined treatments with guazatine in dip or spray applications on various citrus fruit varieties. Fenpropimorph alone was as good as or a better curative and protective treatment than the current commercial treatments applied in packinghouses in Israel for decay control.     

柑橘喷施叶面肥对柑橘木虱､柑橘潜叶蛾和炭疽病发生的影响

柑橘生产上叶面肥的使用很普遍?为了评估喷施叶面肥对柑橘新梢期的主要害虫柑橘木虱Diaphorina citri和柑橘潜叶蛾Phyllocnistis citrella发生?为害的影响, 本文分别在养虫笼和网室条件下调查了以上2种害虫对喷不同叶面肥(尿素?复合氨基酸和葡萄糖)砂糖橘苗木的选择和为害, 以及柑橘炭疽病发生情况?试验结果表明:从木虱成虫选择性和产卵两个方面来看, 不同喷施处理的砂糖橘对木虱的吸引作用表现为尿素>复合氨基酸>葡萄糖>清水对照, 其中尿素和复合氨基酸处理吸引作用显著高于对照?喷施3种叶面肥后潜叶蛾为害程度均显著高于对照, 以葡萄糖处理的为害率和受害指数最高?另外, 尿素和复合氨基酸处理苗木的柑橘炭疽病病叶率和病情指数显著高于对照, 复合氨基酸处理病叶率显著高于葡萄糖?说明喷尿素?复合氨基酸叶面肥对柑橘木虱有较强的吸引作用, 同时加重柑橘炭疽病?另外, 与对照相比, 喷施3种叶面肥均能加重潜叶蛾的为害, 尤其是葡萄糖?     

基于DNA条形码技术的湖南省柑橘主产区实蝇幼虫分子鉴定

为明确湖南省柑橘主产区的实蝇入侵为害现状,从该省7个市(自治州)22个地点收集柑橘蛆果中的幼虫,利用DNA条形码技术对其进行分子鉴定,并以DNA条形码序列作为分子标记探究橘大实蝇Bactrocera minax的20个中国地理种群(湖南省6个组群共16个种群、其它3省市4个种群)以及1个印度地理种群间的亲缘地理关系,分析橘大实蝇在我国的遗传进化关系。结果表明,仅采集自邵阳市城步苗族自治县柑橘蛆果中的5头幼虫被鉴定为蜜柑大实蝇B. tsuneonis,其余21个地点采集的595头幼虫均被鉴定为橘大实蝇。21个橘大实蝇地理种群的平均单倍型多样性为0.75,核苷酸多样性为0.0032,核苷酸差异数为2.13,中国所有地理种群均具有较高的遗传多样性;单倍型网络进化图显示湖南、重庆、贵州种群共享的单倍型H3为原始单倍型,表明其为比较原始的种群;AMOVA分析结果显示种群内个体间遗传变异占总体变异的59.04%,是遗传变异的主要来源;遗传分化结果表明湖南省6个组群间均出现了中度至高度的遗传分化,FST在0.0521～0.7795之间。表明DNA条形码技术可用于蜜柑大实蝇和橘大实蝇幼虫的分子鉴定及其种群遗传进化分析。     

LncRNA在柑橘木虱与黄龙病病原菌互作中的差异表达分析及验证

为明确柑橘黄龙病的唯一自然传播媒介——柑橘木虱Diaphorina citri的长链非编码RNA(long non-coding RNA,lncRNA)是否参与调控黄龙病病原菌Candidatus Liberibacter asiaticus(CLas)的侵染及复制,采用生物信息学预测及PCR扩增方法进行lncRNA的预测、特征分析、验证及差异表达分析。结果显示,柑橘木虱的13个转录组RNA-Seq数据中共有10 192个lncRNA基因,对应15 747条lncRNA转录本;与蛋白质编码基因相比,柑橘木虱lncRNA具有更少的外显子数量和更短的转录本长度;随机选取的10条lncRNA基因中,有7条lncRNA基因在无菌柑橘木虱广州品系或赣州品系中有表达,其中1条lncRNA基因TCONS_00034665在无菌广州品系和无菌赣州品系中存在差异表达;带菌和无菌柑橘木虱成虫中预测获得2个差异表达的lncRNA基因TCONS_00096118和TCONS_00234564,实时荧光定量PCR验证发现TCONS_00234564与预测结果一致,在带菌柑橘木虱成虫中高表达。表明lncRNA参与了黄龙病病原菌与寄主柑橘木虱的互作。     

Soil application of imidacloprid and related SAR-inducing compounds produces effective and persistent control of citrus canker

Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced season-long control of citrus canker caused by Xanthomonas citri sbsp. citri. Imidacloprid is a neo-nicotinoid that breaks down in planta into 6-chloronicotinic acid, a compound closely related to the systemic acquired resistance (SAR) inducer isonicotinic acid. Potted Swingle citrumelo seedlings (Citrus paradisi × Poncirus trifoliata) were treated with imidacloprid and the SAR inducers, isonicotinic acid, and acibenzolar-s-methyl as soil drenches or with acibenzolar-s-methyl as a foliar spray 1week prior to inoculation of immature leaves with X. citri sbsp. citri. Seedlings were re-inoculated four times over a 24-week period. SAR induction was confirmed by expression of the PR-2 gene (β-1,3 glucanase). Soil drenches of imidacloprid, isonicotinic acid, and acibenzolar-s-methyl induced a high and persistent up-regulation of PR-2 gene expression and reduced the number of canker lesions for up to 24 weeks compared to 4 weeks for foliar acibenzolar-s-methyl. Soil applied inducers of SAR reduced canker lesions up to 70% compared with the untreated inoculated plants. Lesions on leaves were small, necrotic, and flat compared to pustular lesions on inoculated untreated plants. Populations of X. citri sbsp. citri per leaf were reduced 1–3 log units in soil-treated plants compared to inoculated untreated plants.