Viroids are single-stranded, circular RNA molecules (234-406 nt) that infect a wide range of crop species and cause economic losses in agriculture worldwide. They are characterized by the existence of a population of sequence variants, attributed to the low fidelity of RNA polymerases involved in their transcription, resulting in high mutation rates. Therefore, these biological entities exist as quasispecies. This feature allows them to replicate within a wide range of host plants, both monocots and dicots. Viroid hosts include economically important crops such as tomato, citrus, and fruit trees such as peach and avocado. Given the high risk of introducing viroids to viroid disease-free countries, these pathogens have been quarantined globally. As discussed herein, Mexico represents a geographical landscape of viroids linked to their origin and comprises considerable biodiversity. The biological features of viroid species endemic to Mexico are highlighted in this communication. In addition, we report the phylogenetic relationships among viroid and viroid strains, their economic impact, geographical distribution, and epidemiological features, including a broad host range and possible long-distance, seed, or insect-mediated transmission. In summary, this review could be helpful for a better understanding of the biology of viroid diseases and future programs on control of movement and spread to avoid economic losses in agricultural industries.

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Mexico: A Landscape of Viroid Origin and Epidemiological Relevance of Endemic Species

Citrus viroid infection is emerging as a serious threat because of its efficient systemic movement within the host plant and its quick spread due to contaminated pruning tools. A survey was conducted to investigate the primary distribution and molecular characterization of Citrus bent leaf viroid (CBLVd) and its variants in different citrus cultivars. A total of 154 symptomatic citrus samples were collected and detected by RT‒PCR with newly designed specific primers with the incidence of 36.33%. During biological indexing study on Etrog citron, expressions of reduced leaf size, yellowing with a light green pattern, and bending were observed. Amplified products were sequenced and analyzed using a nucleotide BLAST search, which showed 98% homology with other CBLVd isolates. The results of the phylogenetic tree analysis showed the presence of two main groups (A and B), with the predominant variants of CBLVd, i.e., CVd-I-LSS (Citrus viroid Low Sequence Similarity) sequences, clustering in subgroup A1 along with newly detected CVd-I-LSS from Palestinian sweet lime (Citrus limettioides), which has been identified as a new host of CVd-I-LSS in Pakistan. Further analysis of the sequences in subgroup A1 showed that the variant of CVd-I-LSS infecting citrus cultivars had a close relationship with isolates reported from China, Japan, and Iran, which may have resulted from the exchange of planting material. This study also unveiled the variability in nucleotide sequences of CBLVd, which made it unable to be detected by old primers. The results of this study indicate that the widespread presence of divergent variants of CBLVd is a major concern for the citrus industry in Pakistan and other countries where virulent isolates of CBLVd are prevalent. These findings suggest the need for future research on effective management and quarantine measures to stop the spread of CBLVd.

Molecular characterization of divergent isolates of Citrus bent leaf viroid (CBLVd) from citrus cultivars of Punjab, Pakistan

Citrus has been promoted in the Lao People’s Democratic Republic (Lao PDR) as a poverty reduction strategy for at least two decades. However, citrus trees have been in widespread decline for no less than ten years. Since 2010, the authors have observed symptoms on citrus trees consistent with the bacterial disease huanglongbing (HLB). These symptoms included asymmetric leaf mottle, small lopsided fruit, poor fruit production and tree decline. The authors then initiated a long-term study on the occurrence of HLB in southern Lao PDR. Samples of leaf mid-ribs were collected from citrus trees in orchards, nurseries, and backyards across four provinces: Champasak, Sekong, Salavan, and Savannakhet. The presence of ‘Candidatus Liberibacter asiaticus’, the putative causal agent of the Asiatic form of HLB, was confirmed in 59 of 109 samples collected in all four provinces. The Asian citrus psyllid, the vector of ‘Ca. L. asiaticus’, was also observed on citrus trees and tested positive for the pathogen. The implications of these findings for citrus production in Lao PDR are discussed.&nbsp 

Investigating the Impact of Huanglongbing in Citrus in Southern Lao PDR

Development of a multiplex RT-qPCR assay to detect citrus apscaviroids and its use in citrus viroid surveys in Australia

A 328-nucleotide variant of citrus bent leaf viroid (CBLVd) was characterized by citrus varieties in Malaysia. After the first report in Malaysia, the emerging CBLVd was detected in five citrus species, namely Citrofortunella microcarpa, Citrus aurantifolia, Citrus hystrix, Citrus maxima, and Citrus sinensis. CBLVd was detected in 23 out of 133 symptomatic samples through RT-PCR. Sequence analysis of the RT-PCR amplicons from this study showed 99–100% sequence identity to the reference CBLVd Jp isolate and CBLVd isolates reported in Malaysia. Inoculation of sap, obtained from a CBLVd positive sample, into 6-month old healthy C. microcarpa seedlings showed symptoms of slight leaf bending, reduced leaf size of matured leaves, and mild mosaic between 4 to 6 months after inoculation. Moreover, the observed symptoms of chlorosis, midvein necrosis, leaf rolling, and smalling of leaves in calamondin, C. microcarpa (Bunge) Wijnands, were not reported in earlier studies and opened a new avenue for the study of symptomology. The mechanical transmissibility of CBLVd in the inoculated seedlings was reconfirmed by RT-PCR assay and sequencing. Based on the results, the sequence similarity of CBLVd isolates from different areas of Malaysia showed no significant difference among each other and the reference isolate. The CBLVd is mechanically transmissible and could produce variable symptoms in different hosts.

Molecular and biological characterization of citrus bent leaf viroid from Malaysia.

Citrus tristeza virus was detected for the first time in the Lao People’s Democratic Republic. Samples were collected from citrus trees across the southern provinces for testing in Australia. RNA was extracted and tested using conventional and real-time reverse transcription polymerase chain reactions with the virus detected in 12 of 59 samples tested. Viral identities were confirmed by sequencing. Additional confirmation was obtained by an enzyme-linked immunosorbent assay. The implications of the presence of this virus for citrus production in Lao are discussed briefly.

First report of citrus tristeza virus in Lao PDR

Citrus exocortis viroid, citrus bent leaf viroid, hop stunt viroid and citrus dwarfing viroid were detected for the first time in Lao PDR. Samples were collected from citrus trees across southern Lao PDR for laboratory testing in Australia. RNA was extracted and amplified using quantitative reverse transcription polymerase chain reaction (qRT-PCR); viroid identities were confirmed by sequencing.

First report of citrus exocortis viroid, citrus bent leaf viroid, hop stunt viroid and citrus dwarfing viroid in Lao PDR

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First report of citrus tristeza virus in Lao PDR

First report of citrus exocortis viroid, citrus bent leaf viroid, hop stunt viroid and citrus dwarfing viroid in Lao PDR