Detection and Diagnosis of Important Virus and Virus-like Diseases in Commercial Citrus Orchards in Some Tropical Areas of Sudan

Abstract

This study represents the first comprehensive investigation on citrus virus and virus-like diseases in the main citrus growing areas in the central, eastern and northern Sudan. The occurrence, distribution, and incidence of several economically important virus, viroid and prokaryotic diseases were recorded based on characteristic symptoms observed during extensive field surveys performed during 2003-2006 in the main citrus groves in six states occupying a vast geographical area extending from the Blue Nile in Singa area to far north along the River Nile to Merowe, in addition to Kassala State in the east of Sudan. Citrus tristeza virus (CTV) and two common viroids, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) were detected and their identity were established using serodiagnosis for CTV only, and molecular techniques including genome cloning and sequencing for both classes of the pathogens. The present study represents the first record of detection and identification of CTV and the authentication of the presence of CEVd and HSVd. The field survey detected the wide distribution of several economically important virus and virus-like diseases in commercial citrus varieties based on visual characteristic external symptoms on foliage and fruits and on diagnostic internal symptoms on bark and wood at the budunion region. Apparently, most of the common citrus virus and virus-like diseases were prevalent in the surveyed citrus groves. The frequently observed diseases with high incidence levels included cachexia caused by HSVd (59%), citrus tristeza caused by CTV (37.5%), gummy bark (35.8%), psorosis (29.2%), and Kassala disease (25.8%). Other diseases such as stubborn, concave gum, Citrus variegation virus (CVV), Citrus exocortis viroid (CEVd), pop corn, witches' broom (WBDOL) and Vein enation virus (VEV) diseases showed lower incidence levels of 16.3%, 7.8%, 2.5%, 1.0%, 0.83, 0.3%, 0.3%, respectively. Because of sour orange rootstock, the infection of 4 CEVd is latent and could only be detected using the molecular techniques. CEVd was detected in asymptomatic citrus trees in Northern and Khartoum States and recorded an infection level of 24.4% based on these techniques. Mixed infection with CTV, CEVd, HSVd and others was not uncommon and encountered naturally under field condition based on visual observation and confirmed by serological and molecular techniques. The results indicated that tristeza is prevalent naturally in all the surveyed areas infecting orange, mandarin, grapefruit and lime. The virus infection is responsible for quick decline, death of citrus trees and severe yield losses. Tristeza has been found more widely spread in the northern States along the River Nile comprising Khartoum, River Nile and Northern States (45%-65%) compared to southern states (Gezira, Sinnar) and Kassala State with incidence ranging between 10% and 30%. Using direct tissue blot immunoassay (DTBIA), CTV was detected in 50% of the printed samples originating from suspected trees, mainly from orange trees, but was collected from different orchards. In other cases also mandarin, lime and grapefruit trees reacted positively in this serological assay. With a lower efficiency, however, the double antibody sandwich ELISA (DAS-ELISA) also successfully detected the virus in few fresh samples but not from dried citrus materials. The detection of CTV was further substantiated by a nested RT-PCR approach. Two-thirds of the test samples collected from mandarin, sweet orange, grapefruit, and lime trees reacted positive and a fragment size of 132 bp using PIN and PEX primer sets were obtained. Cloning and sequencing of these fragments further confirmed the identity and hence the first authentication of natural occurrence of CTV in commercial citrus orchards in Sudan. The multiple sequence alignments of Sudanese CTV isolates sequences with selected CTV isolates previously reported using Clustal X program, revealed the presence of three nucleotide sequence groups depending on the maximum sequence identity. 5 The sequence analysis of the first group showed 96.1- 97.4% nucleotide sequence identity to (AF01623), a CTV strain from California causing severe stem pitting symptoms. The second group was identical to the reference sequence (DSMZ PV-0332 from Israel) and showed 99% identity with CTV strains T30 (AF260651), T36 (U 16304) from Florida and with CTV-Qaha strain (AY340974) from Egypt. The third group consisting of only one Sudanese isolate that showed 94% identity to CTV strain T318A from Spain that causes severe stem pitting in sweet orange but demonstrated a lower nucleotide identity (92.3%-93.5%) with other CTV from the database. The RT-PCR and DTBIA techniques appeared to be more sensitive, reliable and rapid in detection of CTV compared to DAS-ELISA using polyclonal antibody. Therefore, DTBIA using monoclonal antibodies can equally be proposed for quarantine use and budwood certification program in the country. This study also verified the occurrence of citrus viroids, established their identities and characterized, as a first record in Sudan, the two common viroids CEVd and HSVd. A band of 370 bp corresponding to the full-length genome of CEVd was detected in total RNA preparations from some asymptomatic citrus species. While a full-length genome of Hop stunt viroid (HSVd) (300 bp), the causal organism of cachexia disease, was obtained from leaf tissues of symptomatic mandarin trees as well as from some asymptomatic citrus species (sweet orange and grapefruit) upon RT-PCR analysis with specific viroid primers. Cloning and Sequencing of the produced fragments confirmed the positive identity of the Sudanese isolates of CEVd and HSVd. The nucleotide sequence analysis of CEVd indicated a genome size of 369-371bp long circular RNAs, with sequence conservation between 93.8% -100%. While the nucleotide sequence analysis of HSVd revealed a genome size of 301-303 bp long circular RNAs, with sequence conservation between 93.7%-100%. The most stable secondary structure, in terms of energy, was predicted for all the 6 Sudanese CEVd and HSVd sequences at a folding temperature of 37°C and minimum energy of -183.4 kcal/mol using MFold software. They were all classical rod-like structure. One RY motif proximal to the right terminus of the CEVd secondary structure, which is typical of the Genus Pospiviroid, is present in all Sudanese CEVd-isolates. The nucleotide composition of the different Sudanese CEVd and HSVd-isolates appeared to have a highly base-paired and heat stable low molecular weight molecules, as indicated by a high G+C content (above 60.2% for CEVd and above 55.8% for HSVd) and a high G+C/A+U ratio (above 1.51and 1.26 for CEVd and HSVd, respectively). These highly base paired and heat stable low molecular weight structures are suggestive of viroidlike organisms. The motif discriminating cachexia and non-cachexia sequences of HSVd were identified by the presence of five nucleotide differences that affecting both the upper and the lower strands of the V domain. HSVd- Sudan 23 (River Nile State) and HSVd- Sudan 26 (Northern State) were found containing the characteristic nucleotide composition of cachexia inducing variant (CVd-IIb) in the variable region of the genome, while the other isolates were more similar to the CVd-IIa variant (Non-cachexia inducing variants) of HSVd. Phylogenetic analyses of the Sudanese HSVd-isolates using Neibourhood Joining Bootstrap Method were carried out together with 32 previously reported HSVd sequences from different molecular databases. A phylogenetic tree of all sequences, including the Sudanese sequences showed that the six Sudanese HSVd isolates were included in the citrus-type. Concluding perspectives emerging from the present study were outlined and consequently some future prospectives, which are envisaged to promote citriculture in Sudan, were suggested.