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。     

基于近红外技术柑橘黄龙病田间快速检测方法研究

柑橘黄龙病在感病植株和健康植株之间传播速度快,因此快速检测柑橘黄龙病对其防治十分关键。本文研究了近红外技术快速检测柑橘黄龙病的方法。采用PLS-LDA建立的模型对未参与建模的样品进行了检测,结果表明,该模型检测的准确率与普通PCR检测的结果符合率达到100%,假阳性率小于1%。该技术具有检测周期短、无污染等优点,可用于田间黄龙病的快速检测。     

海南省柑橘主产区黄龙病和病毒病的发生危害情况调研初报

柑橘是我国南方重要的水果之一,柑橘黄龙病和柑橘病毒病已经对我国柑橘的安全生产造成了严重威胁。为了解柑橘黄龙病和病毒病对海南柑橘产业的危害情况,本研究通过荧光定量PCR(Quantitative PCR)和反转录PCR(RT-PCR)分别对随机采集自海南柑橘主产区澄迈县3个福橙种植园和琼中县4个绿橙种植园共计109个样品进行了柑橘黄龙病和柑橘5种主要病毒病的检测。结果表明:澄迈县福橙标准化种植示范基地和琼中县营根镇绿橙种植园2个新植橘园的柑橘黄龙病发病较轻,检出率仅为8%和11%,而其他5个橘园的柑橘黄龙病检出率均在75%以上,严重的达100%。除此之外,7个橘园均未检测到柑橘褪绿矮缩病毒;澄迈县和琼中县的衰退病毒病检出率分别在52.9%和77.8%以上,黄脉病毒病在83.3%和90%以上。澄迈县还检测到碎叶病毒病的存在,检出率在38.1%以上,除红湖农场外还存在裂皮病毒病,检出率在11.8%以上。琼中县的绿橙种植园3还存在裂皮病毒病,检出率为43.8%。综上所述,柑橘黄龙病和病毒病已经严重威胁到海南省福橙和绿橙主产区的柑橘生产安全。加强对海南柑橘苗木安全生产的同时,亟需采取有效的田间综合防控措施,以促进海南柑橘产业的健康发展。     

不同柑橘品种对柑橘黄龙病抗性试验

通过对芸香科12个柑橘品种人为嫁接携带柑橘黄龙病的接穗,观察接种后苗木的症状表现,测试不同柑橘品种对柑橘黄龙病的抗性和耐病性。结果表明,胡柚、金柑、佛手较为抗病,椪柑、瓯柑等较易感病。     

柑橘黄龙病常规PCR检测技术研究与初步应用

柑橘黄龙病是世界范围具有毁灭性危害的柑橘细菌性病害,在我国大部分柑橘产区发生,严重制约了我国柑橘产业的发展。本文根据黄龙病病原物亚洲韧皮杆菌核糖体16SrRNA基因设计了1对PCR引物HLBF468/HLBR877,并以此为基础建立了常规PCR反应体系,确定了检测体系的特异性和灵敏度。结果表明该体系的检测灵敏度比先前报道的常规PCR方法有了明显提高。利用建立的PCR体系完成了对广东和广西两省区果园柑橘黄龙病的抽样检测。本研究建立的常规PCR方法可以作为一种简便、准确、灵敏的检测技术应用于柑橘黄龙病的早期诊断。     

柑橘黄龙病PCR检测引物比较研究

柑橘黄龙病潜伏期长、尚无可用于大田的有效治疗药剂,快速、准确的早期检测是防控柑橘黄龙病的关键。PCR检测是目前柑橘黄龙病最常用的早期检测方法。为提高柑橘黄龙病PCR检测的准确性和检出率,本研究依据已测序的黄龙病菌全基因组序列对检测引物OI2c进行了改进（标记为OI2c-gj）,将其与其他常用的7对PCR检测引物进行了特异性和灵敏度的筛选和比较。结果表明,8对黄龙病PCR检测引物中,特异性较好的引物为OI1/OI2c-gj、Las606/LSS、P400F/P400R、A2/J5、16SF/16SR、primer1/primer2;灵敏度较好的引物从高到低依次为：OI1/OI2c-gj=Las606/LSS>P400F/P400R=A2/J5>16SF/16SR>primer1/primer2。综合比较引物特异性和灵敏度,本研究改进的引物OI1/OI2c-gj以及Las606/LSS、P400F/P400R、A2/J5对柑橘黄龙病检测有较好的准确性和检出率,建议用于柑橘黄龙病的早期检测。     

柑橘黄龙病菌核酸检测技术研究进展

候选韧皮部杆菌（Candidatus Liberibacter spp.）是一类通过虫媒传播，寄生于韧皮部的革兰氏阴性细菌，引起柑橘上的毁灭性病害——黄龙病（Huanglongbing,HLB）。柑橘感染黄龙病后在3～5年内便会衰亡或丧失结果能力，目前尚无有效防治药剂，果园若不及时在感染初期铲除病树，病害会在柑橘木虱的活动下迅速蔓延至整个果园，乃至摧毁整个地区的柑橘产业，造成不可估量的损失。培育无毒苗木、尽早发现和彻底铲除病树是防控柑橘黄龙病的关键，因此建立快速、精准的检测方法是防控黄龙病的基础。本文主要综述了柑橘黄龙病各种核酸检测方法及其优缺点，以期对建立更加灵敏、准确、高效的检测技术提供参考。     

江西省大余县柑橘黄龙病菌PCR检测

采用PCR扩增和核苷酸序列测定技术,对分别采自江西省大余县青龙镇郭屋坝果园和丰顺果园的各5份疑似柑橘黄龙病样品进行病原检测。结果表明:2个青龙郭屋坝果园样品中检测到柑橘黄龙病菌,分别命名为DY-LAS01和DY-LAS02,其他8份样品中则未检测到该病菌。此结果表明大余县已有柑橘黄龙病发生,希望引起当地有关部门的高度重视。     

富川县柑橘黄龙病综合治理集成技术应用与推广

柑橘黄龙病主要是因从疫区调运或种植带毒苗木并由昆虫媒介柑橘木虱传播扩散。通过实施柑橘黄龙病综合防控项目,在项目基地采取集成技术对其进行综合防控,以项目带动集成技术大面积推广应用,全面提升柑橘黄龙病防控水平,保护和促进柑橘产业稳步发展。     

柑橘黄龙病症状与“Candidatus Liberibacter asiaticus”PCR检测结果的相关性分析

柑橘黄龙病症状较为复杂,且因寄主品种、生长期、病程等因素而异。利用PCR检测其病原菌"Candidatus Liberibacter spp."是目前柑橘黄龙病诊断的可靠方法之一。分析柑橘黄龙病症状与PCR检测结果的相关性有助于提高黄龙病的田间诊断准确率。本研究结果表明,与PCR检测相比,根据症状诊断柑橘黄龙病具有较高的假阳性率(8.20%)和假阴性率(50%);通过分析1 839个样品的症状与病原PCR检测结果发现,表现为斑驳型黄化、均匀黄化和"绿岛"这3种叶部症状以及"红鼻子果"和畸形果这2种果实症状的PCR病原检出率高;具有斑驳和黄化、黄化和"绿岛"、"绿岛"和花叶等复合症状样品的PCR检测"Ca.L.asiaticus"的阳性率最高;直径小于1 cm的幼果中的"Ca.L.asiaticus"检测稳定性低。这些结果为更准确地通过症状诊断柑橘黄龙病提供了科学依据。     

Anatomical distribution of abnormally high levels of starch in HLB-affected Valencia orange trees

The citrus disease Huanglongbing (HLB or citrus greening) is characterized, among other symptoms, by extraordinary levels of starch accumulation in leaves. This condition denotes imbalances in carbohydrate source sink relationship which in turn may have direct implications in the overall health of HLB-trees and in future strategies to manage the disease. Using light, scanning, and transmission electron microscopy we investigated the extent of carbohydrate partitioning imbalances throughout the tree. In all aerial tissues, starch accumulation in HLB-affected trees far exceeded that of HLB-negative control trees. Starch accumulated extensively in photosynthetic cells as well as phloem elements and vascular parenchyma in leaves and petioles. In stems, starch was commonly observed in xylem parenchyma and in the phelloderm of HLB-affected trees but absent from control samples. In contrast, roots from HLB-affected trees were depleted of starch whereas roots from control trees contain substantial starch deposits. The data supports the notion that the substantial changes in carbohydrate partitioning observed throughout the citrus tree may not only be a result of HLB infection, but in itself, a cause for the rapid decline and death of infected trees.     

A phytoplasma closely related to the pigeon pea witches'-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of S?o Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.     

Preliminary research on soil conditioner mediated citrus Huanglongbing mitigation in the field in Guangdong, China

Huanglongbing (HLB, yellow shoot disease) is devastating citrus production worldwide. No effective control measure is currently available. In China, management of HLB through nutrient applications was used in the past and the interest has been renewed recently. In this study the effects of a soil conditioner (SC) on HLB-affected mandarin citrus (Citrus reticulate Blanco) were evaluated in four orchards (three with cultivar Shatangju and one with cultivar Chuntianju) in Guangdong Province, China. We observed that HLB trees showed more vigorous growth with lower titres of “Candidatus Liberibacter asiaticus”, the putative pathogen of HLB, than the untreated trees at 10 months post treatment (MPT) with SC. “Ca. L. asiaticus” titres in younger trees (2-year) were significantly lower than those in older trees (8-years old) (P?=?0.005). Comparison of SC treatments using 4-year-old Chuntianju showed that the “Ca. L. asiaticus” titres were significantly lower in new leaves with 7 MPT (94.51 % reduction or 2.59 times lower) than those with 1 or 4 MPT (P?=?0.002). The P, N, K, Mn, and organic matters contents in the treated orchard soils were all significantly higher than those in the non-treated soil tested at 2 MPT (p?<?0.05). Effects of SC treatments on fruit yield and quality were also significant. Our preliminary results showed that SC treatment could have various levels of positive influence on HLB-affected mandarin trees.     

Detection of 'Candidatus Liberibacter asiaticus' in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of 'Ca. L. asiaticus' in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of 'Ca. L. asiaticus' in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.     

Widespread occurrence of “Candidatus liberibacter africanus subspecies capensis” in Calodendrum capense in South Africa

Recent studies in citrus orchards confirmed that Citrus Greening, a heat sensitive citrus disease, similar to Huanglongbing (HLB), is associated with the presence of ??Candidatus Liberibacter africanus?? (Laf) in South Africa. Neither ??Candidatus Liberibacter asiaticus?? (Las), associated with HLB, ??Candidatus Liberibacter americanus??, nor ??Candidatus Liberibacter africanus ssp. capensis?? (LafC), previously detected in the Western Cape, South Africa on an indigenous Rutaceous species, Calodendrum capense (L. f.) Thunb. (Cape Chestnut), were detected in citrus. The current study aims to determine the potential role of C. capense in the epidemiology of Citrus Greening in South Africa and whether LafC poses a risk to citriculture. A total of 278?C. capense samples were collected throughout South Africa and tested for Liberibacters using real-time PCR. While LafC was found in 100 samples, distributed from all areas where collected, no evidence of Laf infection in any sample was found . The identity of the LafC present was confirmed by sequencing the amplicon derived from conventional PCR of the ?-operon of the ribosomal protein gene region of the first 17 infected trees found and of a representative sample from each district. The Liberibacter status of 44?C. capense and 272 citrus (Midnight Valencia) trees growing in close proximity to each other for over 15?years was determined. Out of 44?C. capense specimens, 43 were infected with LafC, but none of the citrus trees were infected with LafC. Based on the results of this it appears that natural spread of LafC to citrus does not occur.     

Antifeedant and sublethal effects of imidacloprid on Asian citrus psyllid,Diaphorina citri

BACKGROUND: Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, transmits the causal bacteria of the devastating citrus disease huanglongbing (HLB). Because of the variation in spatial and temporal uptake and systemic distribution of imidacloprid applied to citrus trees and its degradation over time in citrus trees, ACP adults and nymphs are exposed to concentrations that may not cause immediate mortality but rather sublethal effects. The objective of this laboratory study was to determine the effects of sublethal concentrations of imidacloprid on ACP life stages. RESULTS: Feeding by ACP adults and nymphs on plants treated daily with a sublethal concentration (0.1 µg mL^?1) of imidacloprid significantly decreased adult longevity (8 days), fecundity (33%) and fertility (6%), as well as nymph survival (12%) and developmental rate compared with untreated controls. The magnitude of these negative effects was directly related to exposure duration and concentration. Furthermore, ACP adults that fed on citrus leaves treated systemically with lethal and sublethal concentrations of imidacloprid excreted significantly less honeydew (7–94%) compared with controls in a concentration‐dependent manner suggesting antifeedant activity of imidacloprid. CONCLUSIONS: Sublethal concentrations of imidacloprid negatively affect development, reproduction, survival and longevity of ACP, which likely contributes to population reductions over time. Also, reduced feeding by ACP adults on plants treated with sublethal concentrations of imidacloprid may potentially decrease the capacity of ACP to successfully acquire and transmit the HLB causal pathogen. Copyright © 2009 Society of Chemical Industry     

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts,rootstocks and the Asian citrus psyllid

The use of proper management strategies for citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las) and transmitted by Asian citrus psyllid (ACP) (Diaphorina citri), is a priority issue. HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP populations. Here, dynamic populations of Las in different citrus hosts and each instar of ACP were studied, together with the seasonal growth and distribution of Las in different tissues, using conventional and TaqMan real‐time PCR. Different levels of susceptibility/tolerance to HLB were seen, resulting in different degrees of symptom severity and growth effects on hosts or rootstocks. Troyer citrange, Swingle citrumelo and wood apple were highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis of Las, mature and old leaves contained high concentrations in cool temperatures in autumn and spring. Las was detected earlier through psyllid transmission than through graft inoculation, and the amounts of Las (AOL) varied in different hosts. Thus, different AOL (10⁴–10⁷ copy numbers μL^?1) and Las‐carrying percentages (LCP; 40–53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP of psyllids were not affected by increasing the acquisition‐access time. The present study has significant implications for disease ecology. The combination of early detection, use of suitable rootstocks and constraint of psyllid populations could achieve better management of HLB disease.     

Tissue‐print and squash real‐time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR. This simplified method was validated and could detect HLB‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.     

Sequence and evolutionary analysis of ribosomal DNA from Huanglongbing (HLB) isolates of Western India

Citrus greening (Huanglongbing, HLB) is a widespread and economically important citrus disease all over the world. The disease is caused by a phloem-limited fastidious gram negative bacterium, “Candidatus Liberibacter spp.” which belongs to the alpha-proteobacteria group classified on the basis of its 16SrDNA sequence. Although the pathogen has been classified under three distinct groups, viz. Asian, African and American isolates, nothing is known about the status and the molecular variabilities among the Indian HLB isolates collected from different citrus cultivars grown in India. Five different HLB isolates showing variable symptoms based on their severity of infection on different citrus, viz. Mosambi, Rangpur lime, Cleopatra mandarin, acid lime and rough lemon, were studied by PCR amplification, sequence and evolutionary analysis of their 16S and 16S/23S rDNA intergenic regions. Analysis of the 16S/23S rDNA intergenic region separated all five Indian isolates from existing African isolates but failed to differentiate among Asian, American and Indian isolates. However, further analysis of complete 16S rDNA clearly indicated that Indian isolates fall within the Asian HLB group. Overall, our results suggest that all the five Indian HLB isolates taken for the current analysis belong to the Candidatus Liberibacter asiaticus strain, which showed distinct sequence variabilities and produced noticeable symptoms on the citrus trees. These results provide a robust framework for understanding how differences in pathogenicity among various HLB isolates may be related to evolutionary history.