Management of plant virus and viroid diseases is a matter of vital importance and concern to the farmer, horticulturist, forester and gardener. It is well established that the virus and viroid diseases in different crops cause enormous losses in terms of quantity and quality of products (see Chapter-3 of Volume-I) (Sastry 2013b). Because of the serious losses they cause to agriculture and horticulture, virus/viroid diseases have acquired great importance in the realm of plant pathology and call for effective management measures against them. These diseases are not amenable to control by direct methods. Unlike fungicides and bactericides, no commercial viricides have yet been developed. However a number of indirect control measures for different crop plant-virus/viroid pathosystems suitable to varied agro ecosystems have been developed with an aim to avoid the pathogen or to minimize their impact on crop yields (Thresh 2003, 2006). As these diseases routinely cause systemic infections and have very effective methods of transmission, preventing their spread has become a complicated problem. Some diseases like Tobacco mosaic virus (TMV), Potato virus X (PVX), Cucumber green mottle virus (CGMMV), Citrus exocortis viroid (CEVd) and Potato spindle tuber viroid (PSTVd) are highly contagious and affect large number of hosts belonging to several families.

Management of Virus and Viroid Diseases of Crops in the Tropics

Citrus Greening Disease (CGD) was critically reviewed with respect to its history, origin, symptomatol - ogy, host range, pathogenic association, identification and detection, transmission and management. It is evident that the citrus disease has emerged as a potential serious threat to citrus production in all the citrus growing countries of the world including Pakistan. It is has been proved that in Pakistan the greening disease is the major cause of citrus decline; hence it is necessary to formulate definite planning and strategies.

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Citrus Greening Disease - A major cause of citrus decline in the world - A Review

Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing disease of lemon trees, reported in Pakistan, India, and more recently in Turkey and China CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus-induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses Contigs assembled from the Illumina sequenced short reads were used to construct the whole genome of Citrus yellow vein clearing virus (CYVCV), consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames The CYVCV genome organization and size resembled that of flexiviruses, and search for sequence homologies revealed that Indian citrus ringspot virus (ICRSV) (Mandarivirus, Alphaflexiviridae) is the most closely related virus However, CYVCV had an overall nucleotide sequence identity of ≈74% with ICRSV Although the two viruses were similar with regard to genome organization, viral particles, and herbaceous host range, CYVCV caused different symptoms in citrus and was serologically distinct from ICRSV Primer pairs were designed and used to detect the virus by conventional and quantitative reverse transcription-polymerase chain reaction on yellow vein clearing symptomatic field trees as well as graft- and mechanically inoculated host plants Collectively, these data suggest that CYVCV is the causal agent of yellow vein clearing disease and represents a new species in the genus Mandarivirus

https://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-06-12-0140-R

Identification and characterization of citrus yellow vein clearing virus, a putative new member of the genus Mandarivirus.

Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), a noncachexia variant of Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) were co-inoculated as two-, three-, four-, and five-viroid mixtures to Clementine trees grafted on trifoliate orange to evaluate their effect on symptom expression, tree growth, and fruit yield. Most trees infected with CEVd-containing viroid mixtures developed exocortis scaling symptoms, as did CEVd alone, whereas most trees infected with HSVd- or CVd-IV-containing mixtures developed bark-cracking symptoms. Trees infected with mixtures containing both CEVd and CVd-IV revealed the existence of antagonism between these two viroids in terms of the expected bark-scaling and cracking symptoms. Synergistic interactions also were identified in trees infected with certain viroid combinations that, in spite of lacking CEVd, expressed exocortis-like scaling symptoms. Viroid interactions also affected the expected response of trees in terms of vegetative growth and fruit yield. Trees infected with viroid combinations containing CEVd or CVd-III were smaller and produced less fruit than trees infected with mixtures not containing these viroids. Viroid interactions on scion circumference and cumulative fruit yield, in terms of additivity of their effects, were statistically confirmed using a factorial analysis of variance model with two mean estimation approaches. In single-viroid infections, CEVd, CVd-III, and, to a lesser extent, CBLVd consistently and significantly reduced tree size and fruit yield. Conversely, HSVd and CVd-IV slightly increased fruit yield and reduced scion circumference. Rare and not consistent significant interactions were detected with the five-, four-, and three-viroid combinations. Antagonistic interactions between CEVd and CVd-III or CBLVd and CVd-III were revealed over the years with consistent significance. The antagonistic interaction between CEVd and CVd-IV was highly significant over the years when additional viroids were present; however, this antagonism appeared much later in the case of an exclusive interaction. HSVd and CVd-IV showed a consistent and significant synergistic interaction on yield only when both viroids were exclusively present. These results demonstrate antagonistic or synergistic relationships between citrus viroids depending on the viroid mixtures present in the host.

https://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-96-0356

Interactions between citrus viroids affect symptom expression and field performance of clementine trees grafted on trifoliate orange.

Viroid dwarfing for high density citrus plantings.

In different citrus areas of Pakistan, lemon and sour orange trees show vein clearing frequently associated with leaf crinkling. Bark inoculation of different citrus species and varieties reproduced the symptoms on sour orange and Femminello Siracusano lemon, whereas Navelina ISA 315, Hamlin and Pineapple sweet orange showed only mild mottling and rings. Rough lemon, Ciaculli and Parson's Special mandarin, Marsh Seedless grapefruit, Mexican lime, alemow, Etrog Arizona 861-S1 citron and Dweet tangor were symptomless. Mechanical inoculation with scalpel blade failed to transmit the disease. DAS-ELISA tests for citrus variegation virus and citrus tristezavirus and electron microscopic observations for viral particles were negative. On the basis of symptoms and host range, it is suggested that yellow vein clearing might be a new graft transmissible disease. During an investigation on the sanitary state of citrus growing in different areas of Pakistan within the framework of a cooperative project between the Italian and Pakistani governments, widespread infectious variegation and ringspot-like symptoms were observed on lemon and sour orange. Since E LISA for citrus variegation virus (CVV) was negative in these trees, it was suspected to be a new disease (4). Later Bove (2) reported the same symptoms and called the disease "yellow vein clearing" (YVC). We report here on a preliminary investigation on YVC. MATERIALS AND METHODS Field survey. The investigation was conducted between 1987 and 1991 in four different citrus growing areas of Pakistan; Islamabad, Renala Kurd, Sahiwal and Sihala, noting the disease symptoms and the citrus species affected. Careful inspection of a collection of 15 lemon lines at the National Agricultural Research Council (NARC), Islamabad, was undertaken. Transmission trials. Two means of transmission were investigated, tissue grafting, and mechanical inoculation. The first trial was performed on ten Femminello Siracusano lemon trees obtained by shoot-tip-grafting, which were bud-inoculated in December 1989 with Eureka Cascade lemon field source and grown in the glasshouse at 18-32 C. For mechanical transmission, ten sour orange seedlings were inoculated in the spring of 1992 by making about 20 cuts in the bark using a scalpel that had repeatedly cut a young symptomatic twig of Femminello Siracusano lemon, and kept in the glasshouse at 18-32 C. Host range. Bark patches of Femminello Siracusano lemon were inserted in the trunks of eight one-yr old budlings of each of the following: Femminello Siracusano lemon, alemow, Ciaculli and Parson's Special mandarin, Marsh seedless grapefruit, rough lemon, Mexican lime, Etrog Arizona 861-S1 citron, Dweet tangor, Hamlin, Pineapple and Navelina ISA 315 sweet orange. Eight sour orange seedlings were also inoculated. Field sources of CVV and citrus psorosis were used as controls. Plants were kept in the glasshouse at 18-35 C. Electron microscopy (EM) and ELISA tests. Small pieces of lateral veins from symptomatic leaves of Fernminello Siracusano lemon were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer, pH 6.8 for 6 hr at room temperature followed by post fixation in 2% osmium tetroxide in the same buffer for 4 hr at room temperature. The material was then dehydrated in an ethanol series and embedded in Epon araldite. Ultrathin sections were cut and stained with uranyl acetate, and examined in a (Zeiss EM 109) transmission electron microscope. Leaf samples were assayed by the DAS-ELISA technique using a specific antisera for CVV (5) and CTV (1). The Other Virus Diseases 365 tests were repeated at three different times of the year (April, June and September).

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Yellow Vein Clearing of Lemon in Pakistan

Seedlings of trifoliate orange, Troyer citrange and sour orange, and trees of sweet orange and clementine grafted on the above rootstocks, have shown a different reduction in size when inoculated with two combinations of citrus viroids. Inocula used were: ARA, a source containing citrus exocortis viroid (CEVd) plus two additional citrus viroids (CVd), that induced severe stunting on trifoliate orange and citrange (as seedlings or rootstocks); CMC, a source having a viroid RNA CVd-I11 group, that induced mild dwarfing. With both viroids sources 5 yr after inoculation, no effect was observed on sour orange and fruits showed no differences in quality. More yield was noted in Moro sweet orange grafted on trifoliate orange and citrange inoculated both with ARA and CMC inocula than uninoculated trees. Electrophoretic profiles of extracts from different seedlings and trees showed no modification of the pattern of viroids during 5 yr of observations. Since Cohen (8) proposed the use of exocortis to obtain dwarfed trees, many studies have evaluated stunting effects of strains or sources of viroids on different stock-scion combinations (4,5,6,7,9,10,13,14). Recent studies have shown that many citrus viroids (CVd), distinct from CEVd, are present in commercial plantings often as a mixture. Some of them have been found separately or inoculated on susceptible species where they induce mild symptoms of exocortis (1, 11) or xyloporosis diseases (12). Their effects on commercial stock-scion combinations have been investigated by different authors (6, 12, 14). In this paper we report the results of the field evaluation of two inoculum sources to limit the tree size, and of the biochemical analysis to determine any change in viroid content during 5 yr of observation. Preliminary results have been published elsewhere (2). MATERIALS AND METHODS Six-month-old seedlings of trifoliate orange, Troyer citrange and sour orange, grown in the greenhouse, were planted in the field in the spring of 1983 at a spacing of 1 x 1 m. Twentyfour trees of each species were budded to Moro sweet orange and SRAl63 clementine in June, 1984. Moro scions were taken from virus-free nucellar trees, whereas clementine were from an old virus-free line from Corsica. The same number of seedlings of each stock were left ungrafted. Inoculation was done in the summer of 1984, by insertion of two pieces of bark into each trunk. Two different inoculum sources were used (eight trees per treatment). The ARA source was a clementine grafted on sour orange which induced a severe reaction on Etrog citron. Polyacrylamide gel electrophoresis (PAGE) revealed the presence of CEVd and two other bands belonging to the CVd-I1 and CVd-I11 groups (11). The CMC source was a clementine on alemow and gave a mild reaction on Etrog citron. Extracts of this source showed in PAGE a single band belongingto CVd-I11 group. Both sources were free from tristeza, psorosis and citrus variegation. Eight uninoculated trees were left as healthy controls. Field obsermations. Five years after inoculation. the trunk circumference above and below the budunion for budded trees and at 30 cm above the ground for seedlings and the yield were measured. The presence of symptoms was checked every year. Treatment means were compared using Duncan's multiple range test. Electrophoretic and hybridization tests. Nucleic acids were extracted from field samples collected each year in summer. The method of extraction and the electrophoretic and hybridizaViroids and Viroid Induced Diseases 231 TABLE 1 CIRCUMFERENCE OF SEEDLINGS, MORO SWEET ORANGE AND CLEMENTINE SRA 63 FIVE YEARS AFTER INOCULATION Rootstocksand inocula Seedlingsz Moro Clementiney stock scion stock scion

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Field Evaluation of Dwarfing Effect of Two Combinations of Citrus Viroids and Different Citrus Species

Author(s): Catara, A.; Azzaro, A.; Davino, M.; Grimaldi, V.; Hussain, M.; Saleem, M.; Mirza, M. S.

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Survey for Tristeza and Greening in Punjab (Pakistan)

Citrus production is worldwide threatened by Colletotrichum spp., causal agents of pre- and postharvest anthracnose. The recent limitation on the use of copper-based antimicrobials, due to its demonstrated noxious effect on the environment, makes the control of this pathogen difficult. Thus, alternative products able to reduce/phase out copper in organic citrus farming are needed. In this study, the efficacy of 11 commercial alternative products were evaluated in vitro, in growth chamber, in open field and in postharvest environments. In vitro, mineral fertilizers, basic substances, essential oils, plant defense stimulators and biological control agents were able to inhibit the mycelial growth with variable efficacy. On artificially infected citrus fruit, almost all tested products significantly reduced disease incidence and severity, but with lower efficacy than copper. The efficacy of mineral fertilizers-based Kiram and Vitibiosap 458 Plus, citrus essential oil-based Prev-Am Plus and chitosan-based Biorend was confirmed in open field trials, in naturally infected citrus fruits. In these trials Biorend was the best alternative product, significantly reducing disease incidence (71% DI reduction) with better results than copper (47.5%). Field treatments reduced the incidence and severity of the disease in postharvest conditions, especially in fruits field-treated three times. Overall, selected products tested in open field can represent a good alternative to copper compounds in the view of future limitation of its use.

/pdf/assessing-copper-alternative-products-for-the-control-of-pre-1ly14ki2.pdf

A. Azzaro

Papers

Yellow Vein Clearing of Lemon in Pakistan

Field Evaluation of Dwarfing Effect of Two Combinations of Citrus Viroids and Different Citrus Species

Survey for Tristeza and Greening in Punjab (Pakistan)

Assessing Copper-Alternative Products for the Control of Pre- and Postharvest Citrus Anthracnose