Citrus greening disease (HLB) on Citrus reticulata (Mandarin) caused by Candidatus Liberibacter asiaticus in Bangladesh
Introduction
Citrus greening (huanglongbing; HLB) is considered as one of the most devastating citrus diseases in the world [[1], [2], [3]]. It is caused by phloem-limited unculturable fastidious bacteria, Candidatus Liberibacter species. The bacteria live in the host plant's phloem tissues and impede the movement of nutrients [4]. Three species of bacteria have been identified for causing citrus greening, Candidatus Liberibacter asiaticus (Las) in Asia, Florida and Brazil, Candidatus Liberibacter americanus (Lam) in Brazil and Candidatus Liberibacter africanus (Laf) in Africa [1,5,6]. Among them Candidatus Liberibacter asiaticus is the most destructive and widespread in Asia as well as in the Western hemisphere [1,7]. It is transmitted and spread by propagation of infected citrus seedling through grafting method and sap-sucking insect vector Asian citrus psyllid (Diaphorina citri Kuwayama) [1,8]. These two reasons have made citrus greening disease extremely difficult to control in both nursery and orchard. Indian subcontinent has been a historic trade route since beginning, so the disease and its vector might have proliferated throughout Asia, Africa and Americas.
The disease has shown long latency period within plant body and it may take several years before an infected tree expresses peculiar disease symptoms [3]. Uneven distribution of pathogen in plant body and confusing disease symptoms make pathosystem more complicated [9]. Symptoms of citrus greening include yellowing of leaves with blotchy mottling that is not uniform across the leaf, small upright leaves with thickened leaf midribs and veins [1,10]. The leaves drop, shoots are stunted, and the branches gradually die as the disease progresses. Other symptoms are out of season flowering and fruiting, small and lopsided fruit with small dark aborted seeds, premature and excessive fruit drop [11]. The orange peel and pulp experiences an increase in secondary metabolites including hydroxycinnamic acids, limonin, nomilin, narirutin, and hesperidin. Resulting from these chemical changes, juice made from symptomatic fruit is described as distinctly bitter, sour, salty/umami, metallic, musty, and lacking in sweetness and fruity/orange flavor [2]. It is often perplexed with nutrient deficiencies (e.g. zinc, manganese, magnesium, iron or boron deficiency symptoms) [10,12,13]. This disease causes substantial economic losses in many parts of America, Asia and Africa by shortening the tree life and reducing productivity with poor quality colorless fruit left on the trees [1]. According to one estimate, the citrus production in Florida (USA) has decreased by 74% [14], which is responsible for 59% of the citrus supply to the overall USA [15]. Occurrence or prevalence of citrus greening has been reported in most of the major citrus producing areas of the world, mainly in America (mostly in Brazil, United States of America, and Mexico), China, India and South Africa [2]. In Arabian Peninsula, recent studies showed that Oman got lime infected severely with HLB in different parts of the country [16]. It is the major limiting factor for global citrus industries and is rapidly invading new areas. This disease was also prevalent in Bangladesh for long time [17,18].
Detection of Las is not easy due to its low concentration and irregular distribution in the citrus hosts [[19], [20], [21]]. This disease is difficult to be diagnosed by conventional procedures, such as electron microscopy of ultra-thin sections, bioassay on indicator plants, or ELISA, serological techniques, and dot-blot DNA hybridization [[22], [23], [24]]. A polymerase chain reaction (PCR) detection method was developed in 1996 [25,26], based on the amplification of 16S rDNA. This method has been extensively and efficiently used to detect Candidatus Liberibacter species in several countries where the disease is present. Hence, a new PCR detection method based on the amplification of ribosomal protein genes, that permits identification of both species directly by the size of the amplified DNA, was developed in 1999 [26,27]. This PCR method is as specific and at least as sensitive for the detection of Candidatus Liberibacter species present in Asia and Africa. In this study, the objective was to identify the pathogen causing citrus greening disease of Mandarin Orange (Citrus reticulata) in Bangladesh. Therefore, we described a PCR method based on the amplification of ribosomal protein genes of the rplKAJL-rpoBC operon (β-operon) which allows detection of the Candidatus Liberibacter asiaticus directly on the gels based on the size of the amplified DNA [27] and also done partial sequencing of amplified DNA for identification.
Section snippets
Plant sample collection through visual investigation
In August 2016, suspected leaves with characteristics of irregular blotchy chlorosis symptom or nutritional deficiency-like symptoms were collected from mandarin orange orchard. A total of eight symptomatic leaf samples were collected along with healthy leaf from major citrus growing areas of Bangladesh. Among the samples, each three samples were collected from Sylhet (Jaintapur) and Moulvibazar (Akbarpur) and two samples from Khagrachari were collected on the basis of visual investigation
Detection based on field inspection
Field detection of citrus greening was based on visual inspection based on the symptoms which includes yellowing of tree canopy, blotchy mottled leaves with small lopsided fruits with aborted seeds provide the best indications of greening infection. In case of nutrient deficiency, yellowing of leaves shows a regular pattern but Candidatus Liberibacter asiaticus infected leaves demonstrate an irregular, asymmetric pattern of yellowing which can be seen in Fig. 3. Sometimes asymptomatic leaves
Conclusion
The detection of Candidatus Liberibacter asiaticus is of considerable importance due to its wide-spread existence and its negative effects on quality and yield of citrus fruits and health of the citrus trees. Its detection is a milestone for citrus cultivation in Bangladesh. The present study ventilated the way of more investigation on Candidatus Liberibacter asiaticus and analysis of genetic diversity of its BD strains. These findings may facilitate in the immediate management technique or
Authorship contributions
This experimental work was generally split equally between all author, each one performing their fair share in both the experimental procedure and the report structure. More specifically, Mohammad Monirul Hasan Tipu: Conceptualization, Investigation, Methodology, Writing – original draft, Formal analysis, supervision, Validation, Writing – review & editing, Md. Mashiur Rahman: Conceptualization, Writing – review & editing, Resources, Software, Visualizaition, Md. Mynul Islam: Conceptualizaiton,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
Financial assistance from Citrus Development Project, BARI, Gazipur, Bangladesh is duly acknowledged. Authors acknowledge logistic support of authority of Laboratory of the Plant Bacteriology and Biotechnology, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh for providing laboratory facility.
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