Penyakit Kuning pada Wortel (Daucus carota L.) yang Berasosiasi dengan Fitoplasma di Jawa Barat.

Abstract

Carrot (Daucus carota L.) is in the family Umbelliferae grown for its edible root. Carrots are widely cultivated in the highlands with loose soil type that rich of humus. It is an important vegetable plant in Indonesia and is widely consumed because it is rich in beta-carotene (A vitamin). Carrot cultivation in Indonesia was initially concentrated in West Java, such as in Cipanas (Bogor) and Lembang (Bandung), but currently it has spread to Central and East Java and outside Java due to its higher demand. One of the problems in carrot cultivation is pathogen infections that cause plant diseases and can potentially reduce yield quality and productivity. Diseases infecting carrot plants in Indonesia caused by viruses, bacteria, fungi and nematodes have been widely reported, but until now there have been no reported diseases caused by phytoplasma. Infection of phytoplasma has been reported to cause significant yield loss in some countries. Therefore, it is necessary to conduct research to determine the status of phytoplasma infecting carrot in Indonesia. Research was carried out with the following objectives: (1) Conducting observation and survey to confirm the presence of yellowing disease and leafhoppers in carrot growing areas in West Java; (2) Studying the morphological, histological and molecular properties of phytoplasmas associated with yellow disease in carrot in West Java; (3) Studying the potential of leafhoppers species Orosius argentatus and Balclutha incisa as the vector of yellow disease in carrot. Research activity began with a field survey on carrot plantations, followed by identification and characterization of phytoplasma in the laboratory and transmission assay of phytoplasma through leafhoppers in a screenhouse. During the survey in Bogor, Cianjur and Bandung, yellowing symptoms and the presence of leafhoppers were observed. Plant samples from the field were brought to the laboratory for identification of phytoplasma. The identification method consisted of microscopic observation using a transmission electron microscope (TEM), molecular detection using nested-polymerase chain reaction (nested-PCR), followed by cloning of phytoplasma DNA. Planthopper samples from the field were brought to the laboratory for identification based on morphological characters and the molecular identification of phytoplasmas associated with planthoppers. Furthermore, sequence analysis was carried out based on the 16S rRNA gene using the BLASTn, phylogenetic analysis, and in silico RFLP. The incidence of yellow disease in carrots is low, ranging from 6.22 - 7.87% in Bogor, 7.47% in Cianjur, and 5.97% in Bandung. Five species of leafhopper were found in carrot fields showing yellow symptoms in Bogor and Cianjur, i.e. B. incisa, Cicadulina bipunctata, Empoascanara indica, Exitianus indicus, O. argentatus and one species of planthopper, Sogatella furcifera. Molecular detection using nested-PCR confirmed the association of phytoplasma with yellow symptom in carrot and 4 planthopper species. Observations using TEM showed that there were abnormalities in the shape and integrity of cells in carrot leaf tissue with yellow symptoms compared to healthy plant cells. In addition, phytoplasma cells were found in the phloem tissue of symptomatic plant leaves with a rounded (spherical) but not fixed (pleomorphic) shape measuring about 0.2-0.5 m. Chlorophyl content of symptomatic leaves decreases 0.106%, but carbohydrate content as starch and reducing sugar content increases 3.79% and 0.25%, respectively when compared to healthy leaves. Based on the nucleotide sequence analysis of the 16S rRNA gene, 2 groups of 16Sr phytoplasma were identified from carrot and planthopper species, i.e. 16SrI and 16SrII. Phytoplasma associated with yellow disease in carrots in Bogor had the highest similarity of 99.1% with the phytoplasma of the 16SrII-Peanut witches'- broom phytoplasma group (accession number L33765), while the phytoplasma from Cianjur had the highest similarity of 95.6% with the phytoplasma of the 16SrII-Cactus witches '-broom phytoplasma group (accession number EU099572), and phytoplasma from Bandung have similarities with group 16SrI-Polish tomato phyllody phytoplasma (accession number EU402598). Further phylogenetic analysis showed that phytoplasma in carrot from Bogor and Cianjur had a close relationship with phytoplasma group 16SrII-D in carrot from Saudi Arabia and India, respectively; while the phytoplasma in carrot from Bandung had a closed relationship with phytoplasma group 16SrI in carrot from Peru, Scotland, Serbia, United States (Texas and Wisconsin), Lithuania, and Cuba. Phytoplasma group 16SrI-Onion yellows phytoplasma is known to be associated with the planthopper C. bipunctata found around the carrot growing area in Bogor. Phytoplasma group 16SrII- Ca. Phytoplasma aurantifolia is known to be associated with planthoppers B. incisa and S. furcifera from Bogor and S. furcifera from Cianjur; in addition, phytoplasma group 16SrII-Cactus witches'-broom is known to be associated with B. incisa and O. argentatus from Cianjur. Adhesins is known as proteins that play an important role in helping the interaction between types of phytoplasmas and their insect vectors. Specific DNA bands of the adhesins gene were successfully amplified using PCR method. Furthermore, nucleotide sequence analysis confirmed that Onion yellows phytoplasma adhesins gene were detected in C. bipunctata from Bogor and Aster yellow phytoplasma in O. argentatus from Cianjur. These results proved that each of the planthopper species is an insect vector for both types of phytoplasma. Further characterization of phytoplasma groups that were identified from carrot plants was carried out using the RFLP in silico method. Three types of restriction enzymes, namely MseI (T'TA_A), RsaI (GT'AC), and HinfI (G'AnT_C) were used in this study and resulted in different DNA band cutting patterns among phytoplasma isolates associated with carrots with yellow symptoms and 4 species of leafhoppers. Two phytoplasma groups, i.e. the 16SrII and 16SrI groups were identified based on the analysis of the RFLP in silico test; this is the same groups as identified earlier by sequence analysis. Transmission of phytoplasma using leafhoppers B. incisa and O. argentatus showed that the two species could play an effective role in transmitting phytoplasma. The incubation period for phytoplasma transmission using B. incisa and O. argentatus was 26 days and 28 days, respectively, with the disease incidence reaching 80%. Transmission of phytoplasma through both species of leafhoppers causes symptoms of chlorosis on the leaves, stunted plant growth, small plant leaves, small petiole with many branches. Based on the research that has been done, it is confirmed that the association of phytoplasma group 16SrII and 16SrI with yellow disease in carrot and leafhoppers found in carrot growing areas. This report is the first in Indonesia, so the spread and distribution of yellow disease in other carrot growing areas needs to be investigated. Determination of the status of yellow disease in carrot in Indonesia is necessary in determining recommendations or policies for its control strategies.

Keywords: disease incidence, leafhoppers, nested-PCR, phylogenetic analysis, 16Sr RNA gene.