Molecular characterization of divergent isolates of Citrus bent leaf viroid (CBLVd) from citrus cultivars of Punjab, Pakistan (original) (raw)
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Identification and primary distribution of Citrus viroid V in citrus in Punjab, Pakistan
Background Citrus plants are prone to infection by different viroids which deteriorate their vigor and production. Citrus viroid V (CVd-V) is among the six citrus viroids, belongs to genus Apscaviroid (family Pospiviroidae) which induces symptoms of mild necrotic lesions on branches and cracks on trunk portion. Methods and results A survey was conducted to evaluate the prevalence of CVd-V in core and non-core citrus cultivated areas of Punjab, Pakistan. A total of 154 samples from different citrus cultivars were tested for CVd-V infection by RT-PCR. The results revealed 66.66% disease incidence of CVd-V. Citrus cultivars Palestinia Sweet lime, Roy Ruby, Olinda Valencia, Kaghzi lime, and Dancy were identified as new citrus hosts of CVd-V for the first time from Pakistan. The viroid infection was confirmed by biological indexing on indicator host Etrog citron. The reported primers used for the detection of CVd-V did not amplify, rather showed non-specific amplification, which led to the designing of new primers. Whereas, new back-to-back designed primers (CVd-V AF1/CVd-V AR1) detected CVd-V successfully and obtained an expected amplified product of CVd-V with 294 bp. Sequencing analysis confirmed the new host of CVd-V showing 98-100% nucleotide sequence homology with those reported previously from other countries while 100% sequence homology to the isolates reported from Pakistan. Based on phylogenetic analysis using all CVd-V sequences in GenBank, two main CVd-V groups (I and II) were identified, and newly identified isolates during this study fall in the group I. Conclusion The study revealed that there are some changes in the nucleotide sequences of CVd-V which made difficult for their detection using reported primers. All isolates of Pakistan showed high sequence homology with other isolates of CVd-V from Iran and USA whereas; the isolates from China, Japan, Tunisia, and Africa are distantly related. It is evident that CVd-V is spreading in all citrus cultivars in Pakistan.
2022
1. Background Citrus plants are prone to infection by different viroids which deteriorate their vigor and production. Citrus viroid V (CVd-V) is among the six citrus viroids, belongs to genus Apscaviroid (family Pospiviroidae) which induces symptoms of mild necrotic lesions and cracks. II. Methods and ResultsA survey was conducted to evaluate the prevalence of CVd-V in core and non-core citrus cultivated areas of Punjab, Pakistan. A total of 154 samples from different citrus cultivars were tested for CVd-V infection by RT-PCR. The results revealed 66.66% disease incidence of CVd-V. Citrus cultivars Palastinia Sweet lime, Roy Ruby, Olenda Valencia, Kaghzi lime, and Dancy were identified as new citrus hosts of CVd-V for the first time from Pakistan. The viroid infection was confirmed by biological indexing on indicator host Etrog citron. The reported primers used for the detection of CVd-V did not amplified, rather showed non-specific amplification, which led to the designing of new p...
Characterization and Sap Transmission of Citrus Bent Leaf Viroid in Malaysia
A 328 nucleotide (nt) variant of Citrus bent leaf viroid (CBLVd) was characterized from citrus varieties in Malaysia showing leaf bending, stunting and midvein necrosis. CBLVd was detected by RT-PCR assay using CBLVd specific primers in 12 out of 90 samples, collected from six different areas in Malaysia. The viroid was present in five species of citrus namely Citrofortunella microcarpa, Citrus aurantifolia, C. hystrix, C. maxima and C. sinensis. Sequence analysis of the isolates obtained from this study showed 99-100% sequence identity to CBLVd Jp isolate (AB006734). Inoculation of sap obtained from a CBLVd positive C. aurantifolia, inoculated into six months old C. microcarpa seedlings showed the symptoms leaf bending, reduced leaf size of matured leaves and mild mosaic between 4 to 6 months after inoculation. The presence of CBLVd in the inoculated seedlings were confirmed by RT-PCR assay and sequencing.Author SummaryThe authors during a limited survey collected the citrus sample...
Characterization of citrus HSVd isolates
Archives of Virology, 2004
Seven citrus isolates of Hop stunt viroid (HSVd) were subjected to retrotranscription and DNA amplification (RT-PCR), cloning and sequencing. Single stranded polymorphism (SSCP) analysis demonstrated the existence of variability among and within cachexia inducing sources of HSVd. The electrophoretic profiles of SSCP appeared to be able to discriminate between non-cachexia and cachexia sources of HSVd. Sequence analysis demonstrated that the variable (V) domain was very conserved among the cachexia variants. Five nucleotide differences, affecting both the upper (3 nucleotides) and the lower (2 nucleotides) strands of the V domain, were identified as a motif discriminating cachexia and non-cachexia sequences. These five nucleotides affect the organization of a short helical region and two flanking loops of the V domain probably modifying the three-dimensional geometry of the molecule. The stability of the minimum free energy rod-like conformation of the cachexia sequences is lower than the noncachexia. Information regarding the host effect on the evolution and variability of viroid quasispecies is also provided.
Agriculture and Biology Journal of North America, 2010
Two extensive surveys and laboratory work were conducted to determine the occurrence of citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd), the causal agent of cachexia (CVd-IIb) disease in the main citrus growing areas in three states in the northern part of Sudan, viz, Northern, River Nile and Khartoum. For CEVd, all, but one of the examined citrus trees failed to show typical exocortis symptoms. The only symptomatic orange tree encountered in the survey displayed the characteristic CEVd symptoms including tree stunting and bark cracking or bark splitting of the rootstock. While for the cachexia disease, most of the commercial citrus trees were found to be symptomless carriers except mandarin. Symptomatic mandarin trees which were grafted on sour orange rootstock displayed heavy gum impregnation (bark gumming), wood staining and deep bark cracking, in addition to severe stem pitting, bark pegging, twig dieback and yellowing of leaves. Pitting and gum development were usually encountered near the budunion but could spread to other sensitive portions of the infected tree. However, using viroid specific primers and reverse transcription polymerase chain reaction (RT-PCR) approach, both viroids were shown to exist in total RNA preparations from symptomatic and asymptomatic citrus species. Bands of 370 bp and 300 bp corresponding to the full length genomes of CEVd and CVd-IIb, respectively, were detected. The test was positive in 54 for CEVd and 20 for CVd-IIb out of 200 random samples tested from orange, grapefruit and mandarin. The results indicated that a considerably high percentage of citrus species in the northern part of Sudan, particularly in Khartoum State, were symptomless carriers of both CEVd and CVd-IIb. Being rapid, sensitive and in most cases reliable, the molecular approach could be considered indispensable for viroid detection and in testing programs to produce certified viroid-free planting materials.
ASSESSMENT OF CITRUS GREENING DISEASE INCIDENCE AN
ASSESSMENT OF CITRUS GREENING DISEASE INCIDENCE AND SEVERITY IN SARGODHA, PAKISTAN: A MOLECULAR CHARACTERIZATION STUDY, 2024
In terms of productivity, potential for spread, and area covered, citrus is regarded as one of the major fruit crops. Citrus fruits account for around 37% of all fruit produced, with mandarins and oranges making up nearly 21% of this total. These days, citrus greening disease (CGD) is a major concern. Citrus decline is a complicated issue that causes orchard output to gradually drop until it eventually results in unproductive orchards. Citrus greening, tristeza, and gradual decline are the most prevalent illnesses that cause citrus decline. This study's goal was to determine how common citrus greening illnesses are across Sargodha's citrus-growing tehsils and kinds. Data on prevalence, incidence, and severity were collected in Sargodha's several tehsils, including Shahpur, Bhalwal, Kot-Momin, and Sargodha. Using particular primer sets A2/J5 and OI1/OI2C, the pathogen was characterized from representative citrus greening disease samples. According to the results of the Citrus Greening Disease, Bhalwal had the highest incidence rate (53.33%). Shahpur had 50% of the highest disease severity, whereas Bhalwal had 25% of the lowest disease severity. Twenty symptomatic grape fruit samples, ten fruiter samples, fifteen kinnow samples, five musambi samples, and ten sweet orange samples had their DNA isolated using the CTAB method. The pathogen DNA was recovered from a sweet orange sample that exhibits definite signs of greening and kinnow. Musambi samples were also amplified, and the amplified PCR product was sequenced to examine genetic heterogeneity in several citrus cultivars. To check for variations that would be useful for managing diseases in the future, a phylogenetic tree was generated as part of the phylogenetic analysis process.
No true resistance to Huanglongbing (HLB), a citrus disease associated with infection of Candidatus Liberibacter asiaticus (CLas), is found within commercial citrus cultivars, though trifoliate orange ( Poncirus trifoliata ) has been described as resistant or tolerant. Through genotyping an intergeneric F 1 population by Genotyping-by-Sequencing, high-density SNP-based genetic maps were constructed separately for trifoliate orange and sweet orange ( Citrus sinensis ). Both genetic maps exhibited high synteny and high coverage of citrus genome. After exposure to intense HLB pressure for two years, Ct value of qPCR for CLas detection in leaves throughout ten time points during the next three years was above 35 in trifoliate oranges, under 28 in sweet oranges, and ranged from 24 to 38 and exhibited obvious segregation among progenies. Phenotypic data of percentage of healthy trees showed high correlation with the Ct value. By mapping the two traits at all time points, a total of nine c...
Incidence and Molecular Detection of Greening Disease in Two Citrus Cultivars in Sargodha, Pakistan
Sarhad Journal of Agriculture, 2021
C itrus is infected by several different pathogens, including fungi, prokaryotes, viruses, nematodes, and virus-like pathogens (Khan et al., 2016). Among these pathogens, the citrus greening disease is one of the most devastating and widely distributed pathogens in citrus orchards of significant citrus-growing areas of the world and Pakistan (Iftikhar et al., 2016; Saifullah et al., 2015). Citrus greening (CGD) disease is now alternatively termed as Huanglongbing (HLB) and is known with different names in different countries owing to their local languages. All the citrus cultivars are affected by this disease regardless of the rootstock. This graft transmissible disease can also be transmitted through an insect vector very efficiently (Graca, 1991; Batool et al., 2007). HLB being a century-old disease, probably originated at the end of the 19 th century in China (Batool et al., 2007; Bove, 2006). The greening disease is caused by gram-negative unculturable phloem limited bacterium (Murray and Schlifer, 1994) named Candidatus liberibacter. Jagoueix et al.
Molecular and Cellular Probes, 2013
The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ by five to six nucleotides located within the variable (V) domain referred to as the "cachexia expression motif". Sensitive hosts such as mandarin and its hybrids are seriously affected by cachexia disease. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological indexing on Parson's Special mandarin and/or direct nucleotide sequence analysis of amplicons from RT-PCR of HSVdinfected plants. Two independent high throughput assays to segregate HSVd variants by real-time RT-PCR and High-Resolution Melting Temperature (HRM) analysis were developed: one based on EVAGreen dye; the other based on TaqMan probes. Primers for both assays targeted three differentiating nucleotides in the V domain which separated HSVd variants into three clusters by distinct melting temperatures with a confidence level higher than 98%. The accuracy of the HRM assays were validated by nucleotide sequencing of representative samples within each HRM cluster and by testing 45 HSVd-infected field trees from California, Italy, Spain, Syria and Turkey. To our knowledge, this is the first report of a rapid and sensitive approach to detect and differentiate HSVd variants associated with different biological behaviors. Although, HSVd is found in several crops including citrus, cachexia variants are restricted to some citrus-growing areas, particularly the Mediterranean Region. Rapid diagnosis for cachexia and noncachexia variants is, thus, important for the management of HSVd in citrus and reduces the need for bioindexing and sequencing analysis.
Virusdisease, 2018
Citrus tristezavirus (CTV, genus Closterovirus) is one of the most serious pathogens responsible for huge loss of citrus trees worldwide. Four Indian CTV isolates, Kat1 (C. reticulata/Central India), D1 (C. sinensis/North India), B5 (Citrus limettoides/South India) and G28 (C. lemon/Northeast India) collected from different regions of India were characterized based on sequencing of 3 0 half genome (* 8.4 kb) comprising 10 open reading frames (ORFs2-11) and 3 0 UTR and the sequences were submitted to NCBI database as Acc. No KJ914662, HQ912022, HQ912023 and KJ914661, respectively. The present and previously reported Indian isolates Kpg3 and B165 were analyzed and compared with other Asian and international CTV isolates. The Indian CTV isolates had 92-99% nt identities among them. The phylogenetic analysis generated overall ten genogroups/lineages. Of them, all the Asian isolates fell into seven genogroups, whereas the Indian isolates into four. Indian isolates Kat1, D1 and Kpg3 grouped together, termed ''Kpg3Gr'', along with Florida severe isolate T3. The Indian isolates B5, and G28 were found to be two distinct and separate lineages, indicating that these isolates are two new CTV entities. Based on phylogenetic analysis, Kpg3Gr was identified as ''Indian VT'' subtype which is distinct from the Asian and the Western VT subtype within diversified VT genotype. The recombination detecting-program, RDP4 detected Indian isolates Kat1, B5, B165 and G28 as recombinants, where G28 as strong recombinant. The present study determined the occurrence of at least four CTV genotypes, B5 (distinct), B165 (T68 type) G28 (distinct) and Kpg3Gr in citrus growing regions of India.