Distribution and genetic diversity of Coguvirus eburi in South African citrus and the development of a real-time RT-PCR assay for citrus-infecting coguviruses

Citrus virus A (CiVA), a novel negative-sense single-stranded RNA virus assigned to the species Coguvirus eburi in the genus Coguvirus, was detected in South Africa with the use of high-throughput sequencing (HTS) after its initial discovery in Italy. CiVA is closely related to citrus concave gum-associated virus (CCGaV), recently assigned to the species Citrus coguvirus. Disease association with CiVA is however incomplete. CiVA was detected in grapefruit (Citrus paradisi Macf.), sweet orange (C. sinensis (L.) Osb.) and clementine (C. reticulata Blanco) in South Africa and a survey to determine the distribution, symptom association and genetic diversity was conducted in three provinces and seven citrus production regions. The virus was detected in ‘Delta’ Valencia trees in six citrus production regions and a fruit rind symptom was often observed on CiVA-positive trees. Additionally, grapefruit showing symptoms of citrus impietratura disease were positive for CiVA. This virus was primarily detected in older orchards that were established prior to the application of shoot tip grafting for virus elimination in the South African Citrus Improvement Scheme. The three viral encoded genes of CiVA isolates from each cultivar and region were sequenced to investigate sequence diversity. Genetic differences were detected between the ‘Delta’ Valencia, grapefruit and clementine samples, with greater sequence variation observed with the nucleocapsid protein (NP) compared to the RNA-dependent RNA polymerase (RdRp) and the movement protein (MP). A real-time detection assay, targeting the RdRp, was developed to simultaneously detect citrus infecting coguviruses, CiVA and CCGaV, using a dual priming reverse primer to improve PCR specificity.

Short Communication: Variations of morphology, anatomy, and metabolite profiles of Citrus reticulata Blanco cv. Tawangmangu grafts produced by shoot tip grafting using several rootstocks

Nuryandani E, Susandarini R, Indrianto A, Nuringtyas RT, Fathima AM, Subandiyah S. 2020. Short Communication: Variations of morphology, anatomy, and metabolite profiles of Citrus reticulata Blanco cv. Tawangmangu grafts produced by shoot tip grafting using several rootstocks. Biodiversitas 21: 4671-4676. Cultivation of Citrus reticulata Blanco cv. Tawangmangu, one of leading mandarin cultivars in Indonesia, remains challenging due to the limitations of suitable rootstocks. The use of suitable rootstocks will greatly determine the success of grafting and affect the performance of the scion. This study aimed to evaluate the morphological, anatomical, and metabolic profiles of grafts between Tawangmangu and rootstocks from various species using shoot tip grafting. Observations of morphological, anatomical, and metabolite variation were carried out eight weeks after the beginning of the growth of the scions. The morphological characters were observed on leaves and stems of the scions. Anatomical data were obtained from the examination of longitudinal sections of grafts prepared using paraffin-embedded method. Analysis of metabolite profiles was performed using LC-MS. The results showed that for two of the three rootstocks used, C. ichangensis × C. reticulata cv. Austera (Japansche Citroen (JC)) and Citrus (Fortunella) japonica (Fortunella) scion growth was vigorous and resulted in a large number of leaves and long stems. Morphological variation was found in the leaf shape, as indicated by wider and wavy leaves of grafts with Fortunella, while narrower and straighter leaves were found in grafts with JC. The grafting onto the third rootstock, C. hystrix, induced dwarfing of the scions. Variation in the anatomical profiles was found in vascular vessels, and in exodermic and endodermic cell sizes. Differences in metabolic profiles of the grafts were found compared to scion and rootstocks. Results of this study provide an important contribution to the successful improvement of Tawangmangu.

Elimination of Indian Citrus Ringspot Virus in Kinnow by Using Phytoproteins with Shoot‐tip Grafting

The combined beneficial effect of shoot‐tip grafting with phytotherapy of different phytoproteins obtained from roots of Boerhaavia diffusa and leaves of Clerodendrum aculeatum to eliminate Indian citrus ringspot virus (ICRSV) from Kinnow is reported. The study also reports the effect of these phytoproteins on the growth and proliferation of Kinnow explants when nodal segments from infected mother plants (confirmed by RT‐PCR) cultured in MS containing different concentrations of aqueous extracts of these individual phytoproteins. Shoot‐tips from these nodal sprouts were grafted on Citrus jambhiri under in vitro condition. Phytoproteins from C. aculeatum were found utmost effective in respect of elimination of 50% virus followed by B. diffusa (40%). In respect of promotion of growth and proliferation of nodal explants, B. diffusa was found most effective followed by C. aculeatum. The plants were considered virus‐free when they showed absence of ICRSV in both DAC‐ELISA and RT‐PCR tests.Plant Tissue Cult. & Biotech. 27(1): 1-11, 2017 (June)

POTENSI PEMANFAATAN TEKNOLOGI EMBRIOGENESIS SOMATIK IN VITRO DALAM PERBANYAKAN MASSAL BENIH JERUK BEBAS PENYAKIT

Mata tempel yang digunakan sebagai batang atas benih sebar jeruk di Indonesia berasal dari tanaman induk di Blok Penggandaan Mata Tempel (BPMT). Tanaman induk tersebut merupakan turunan dari produk shoot tip grafting (STG) in vitro yang menghasilkan induk jeruk bebas penyakit dan identik dengan induknya. Dengan makin berkembangnya teknologi kultur in vitro, benih sebar yang sehat dan serupa induknya dapat dihasilkan melalui teknologi embriogenesis somatik (ES). ES merupakan suatu proses di mana sel-sel somatik (haploid maupun diploid) berkembang membentuk tumbuhan baru melalui tahap perkembangan embrio tanpa melalui fusi gamet. Banyak faktor yang memengaruhi keberhasilan proses tersebut, antara lain genotipe, jenis dan fase perkembangan eksplan, serta komposisi dan jenis media kultur. Tanaman jeruk hasil ES secara genetik identik dengan induknya, namun subkultur berulang dapat menyebabkan terjadinya variasi somaklonal. Teknologi ini juga efektif untuk mengeliminasi penyakit virus dan penyakit Huanglongbing (CVPD) yang terbawa induk. Namun adanya karakter juvenil pada produk tanamannya menghambat upaya penyediaan benih jeruk bebas penyakit di Indonesia secara cepat. Untuk mengatasinya dikembangkan teknik sambung embrio kotiledon atau planlet hasil ES pada batang bawah jeruk secara ex vitro. Dengan cara ini, tanaman yang dihasilkan dapat tumbuh, berbunga, dan berproduksi dengan rentang waktu normal di lapangan. Penggunaan teknologi ES diharapkan dapat mempercepat produksi massal benih jeruk bebas penyakit pada masa mendatang.