Curent Situation of “Candidatus Liberibacter asiaticus” in Guangdong,China,Where Citrus Huanglongbing Was First Described

Citrus Huanglongbing （HLB,yellow shoot disease） was first observed in the coastal Chaoshan Plain of Guangdong Province,China,in the late 19th century based on descriptions of yellow shoot symptoms. “Candidatus Liberibacter asiaticus”has been considered as a putative pathogen associated with HLB since 1994.Information about the curent prevalence of this bacterium is important for HLB control in Guangdong and also provides useful reference for HLB study elsewhere.In 2007,we collected HLB symptomatic citrus samples from 16 cultivars in 12 prefecture cities,mostly in the north and west regions of Guangdong,where major citrus fruits are currently produced.Among the 359 samples collected,241 （67.1%） were positive for “Ca.L.asiaticus”,distributed in 15 out of the 16 cultivars from all 12 cities,indicating the widespread prevalence of “Ca.L.asiaticus” in Guangdong Province.The detection rates varied from 16.7 to 100% depending on location and cultivar.Lower detection rates were found in newer citrus cultivation cities among the previously less popular but now promoted cultivars.In reviewing the citrus management and pest control practice,we believe that infected nursery stocks play a key role in the current spread of “Ca.L.asiaticus”.

Distribution pattern and titer of Candidatus Liberibacter asiaticus in periwinkle(Catharanthus roseus)

Candidatus Liberibacter asiaticus （CaLas）, an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing （HLB） in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder （Cuscuta spp.）. To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.

A rapid multiplication system for'Candidatus Liberibacter asiaticus'through regeneration of axillary buds in vitro

‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘Changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

High genetic variation and recombination events in the vicinity of non-autonomous transposable elements from ‘Candidatus Liberibacter asiaticus'

Two miniature inverted-repeat transposable elements（MITEs）, MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus＇ known to be associated with citrus Huanglongbing（HLB, yellow shoot disease）. MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus＇ isolates from China, the United States and Brazil. Out of the 12 sequence types, three（T4, T5-2, T6） were reported for the first time. Recombination events were found in the two unique sequence types（T5-2 and T6） which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, S？o Paulo isolates and a few Florida isolates.

广佛手感染黄龙病叶果的症状及枝条的病原分布规律

2020年7—9月,在广东德庆县武垄镇田间调查广佛手黄龙病并采集疑似感病样品,利用实时荧光定量PCR对样品进行检测,对检测出有黄龙病菌(“Candidatus Liberibacter asiaticus”,CLas)的样品进行症状描述与系统分析,同时,对染病广佛手带果枝条(分为老叶、近果叶、嫩叶、果蒂和桔络5个部位)的病原时空分布进行分析,并以德庆县高良镇树龄一致的感染黄龙病贡柑枝条为对照进行比较分析。结果表明,感染黄龙病的广佛手叶片表现为典型斑驳、轻微斑驳、轻微黄化、均匀黄化、变细变小和革质等症状,以斑驳症状为主,存在无症状感染的现象,未发现缺锌状症状;感染黄龙病果实表现为变小、硬化、畸形、果蒂黄化、果实基部黄化等症状,存在无症状感染的现象,未发现“红鼻子果”症状。染病广佛手枝条的病原时空分布是动态变化的,但与贡柑不同,同时,随着果实成熟并未在果实桔络中大量富集;与贡柑相比,染病广佛手同一时期同一部位的CLas浓度普遍较低,说明广佛手可能具有一定的耐病性。

亚洲柑橘木虱与柑橘黄龙病菌互作的研究进展

亚洲柑橘木虱Diaphorina citri Kuwayama作为柑橘产业重要病害柑橘黄龙病的主要传播媒介,已经成为重点防治对象。该害虫与黄龙病之间的互作一直是相关研究的热点,本文就近年来该领域的研究进展做了一个总结,从亚洲柑橘木虱的获菌与传病机制、病原菌与柑橘木虱之间的互作以及病原菌感染寄主植物后对木虱的影响等方面进行了综述。期望为深入开展黄龙病相关研究、寻找防控新途径提供依据。

Evaluation of thermotherapy against Huanglongbing (citrus greening) in the greenhouse

Huanglongbing （HLB, or citrus greening） is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus （Las）. Few management options are available, aside from preventive measures such as removing infected plants, planting disease-free seedlings, and managing the insect vector. In this study, we assessed the efficacy of thermotherapy against HLB under controlled greenhouse conditions. A total of 60 two-year-old, graft-infected Citrus reticulata Blanco plants were used. The plants were randomly divided into three groups （45℃, 48℃, and untreated control）, with five plants/replicate （rep） and four reps/treatment. The treated plants were placed in phytotrons for a 4-h treatment session, repeated once per week for three consecutive weeks. Disease remission was observed eight weeks post-treatment. Real-time PCR assays revealed that Las titers in HLB-affected seedlings were significantly reduced in both 45 and 48℃ treatments four weeks after treatment, with the exception of eight plants. In contrast, Las titers in the untreated control plants increased significantly during the same period, with a maximum increase of 28-fold. Except for seven plants, Las titers in the new flushes of treated plants decreased more than 90% eight weeks after treatment. Las titers in mature leaves of treated plants decreased 56 and 60% in average at 45 and 48℃, respectively, eight weeks after treatment. The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment in both 45 and 48℃ treatments. Our results laid a good foundation for the further development of citrus free-disease seedling cultivation and Huanglongbing control in the field. The whole plants were replaced for scion or branch in previous as the research object in this study, and the expression of Huanglongbing symptoms combined with real-time polymerase chain reaction （PCR） were used to evaluate the effect of heat treatment in the greenhouse.

Beneficial horticultural responses from the application of solar thermotherapy to mature Huanglongbing-affected citrus trees

The detrimental effects of Huanglongbing(HLB)on citrus are well known and so is the need for effective methods to combat this disease.Solar thermotherapy(ST),one of the management methods to help alleviate some of the negative effects of HLB,is the process of heating trees to therapeutic temperatures by encompassing the tree within a plastic structure(Visqueen)to harness the sun’s natural energy.ST was applied to mature‘Valencia’citrus trees in three locations.Tree vigor,yield,and Candidatus Liberibacter asiaticus(Las)titer in leaves were monitored for two consecutive seasons post ST treatment,while fruit and juice quality were evaluated at the end of the second season.ST promoted an increase in canopy density in most groves tested and did not have a significant effect on fruit quantity,despite the prolonged exposure of the trees to increased temperatures.Moreover,Las titer was reduced in both leaves and juice processed from affected trees post ST.With respect to fruit quality,the ratio of total soluble solids to titratable acidity was higher post ST compared to controls as was the sucrose content of the juice in most treated groves,whereas limonin was higher in juice from control trees.Principal components analysis of aroma volatiles showed significant differences between juice from treated versus control trees with volatiles imparting top-note and freshness to orange juice,such as acetaldehyde,hexanal,Z-3-hexenol and linalool,being higher in juice fromtreated trees.Taste panels confirmed that flavor differences existed,ultimately showing that ST resulted in improved juice flavor in well-managed groves.Although the effects of ST on the trees appear temporary,such outcomes emphasize its utility when used as part of an integrated management strategy for HLB-affected citrus,imparting beneficial horticultural responses with minimal to positive effects on subsequent juice flavor.

Rapid DNA Extraction Methods for Direct-PCR Detection Citrus Huanglongbing

[ Objective] This study aimed to develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection. [ Method] From aspects of preparing procedure, prepare time and the quality of DNA, advantages and disadvantages of three sample preparation methods were compared, include two Direet-PCR extraction methods and one universal genomic DNA extraction kit method. In addition, PCR amplification effect on specific primers for 16S rDNA of ＂Candidatua Libefibacter asiaticus＂ （CLas） had also been evaluated. [ Result] The results showed that RT-qPCR detected CLas by using DNA obtained from one of Direct-PCR extraction method as templates. Under improved Direct-PCR extraction method, 16S rDNA of CLas could also be amplified by routine PCR. [ Conclusion] A simple and effective DNA extraction method of citrus Huanglongbing pathogen have been established, which provides technical supports for prepa- ration of large number of samples for detection of CLas.