Huanglongbing (HLB, aka citrus greening), one of the most devastating diseases of citrus, has wreaked havoc on the global citrus industry in recent decades. The culprit behind such a gloomy scenario is the phloem-limited bacteria “Candidatus Liberibacter asiaticus” (CLas), which are transmitted via psyllid. To date, there are no effective long-termcommercialized control measures for HLB, making it increasingly difficult to prevent the disease spread. To combat HLB effectively, introduction of multipronged management strategies towards controlling CLas population within the phloem system is deemed necessary. This article presents a comprehensive review of up-to-date scientific information about HLB, including currently available management practices and unprecedented challenges associated with the disease control. Additionally, a triangular disease management approach has been introduced targeting pathogen, host, and vector. Pathogen-targeting approaches include (i) inhibition of important proteins of CLas, (ii) use of the most efficient antimicrobial or immunity-inducing compounds to suppress the growth of CLas, and (iii) use of tools to suppress or kill the CLas. Approaches for targeting the host include (i) improvement of the host immune system, (ii) effective use of transgenic variety to build the host’s resistance against CLas, and (iii) induction of systemic acquired resistance. Strategies for targeting the vector include (i) chemical and biological control and (ii) eradication of HLB-affected trees. Finally, a hypothetical model for integrated disease management has been discussed to mitigate the HLB pandemic.

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the ‘Kinnow mandarin’, a commercial citrus crop cultivated in the northwest of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcription polymerase chain reaction (RT-PCR) that is time consuming. Here, we describe a novel, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. To standardize the RT-LAMP assay, four different primers were designed and tested to target the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. The ICRSV RT-LAMP assay developed in the present study is a simple, rapid, sensitive, specific technique. Moreover, the assay consists of only a single step and is more cost effective than existing methods. This is the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large-scale indexing of field samples in diagnostic laboratories, in nurseries, and for quarantine applications.

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of ‘Candidatus Liberibacter asiaticus’ (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of ‘Ca. L. asiaticus’ was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The ‘Ca. L. asiaticus’ isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of ‘Ca. L. asiaticus’ and CTV isolates and their association with citrus decline in Bhutan.