Siti Subandiyah

Characterization of the tufB-secE-nusG-rplKAJL-rpoB Gene Cluster of the Citrus Greening Organism and Detection by Loop-Mediated Isothermal Amplification

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to amplify the uncharacterized regions adjacent to the nusG-rplKAJL-rpoB gene cluster of citrus greening organism (GO) isolates from different locations in Japan and Indonesia. Conventional PCR was used to amplify the internal nusG-rplKAJL-rpoB gene cluster of these isolates, and the complete sequence of this 6.1-kb fragment was determined. Comparisons with other bacterial sequences showed that the fragment is the tufB-secE-nusG-rplKAJL-rpoB gene cluster. The organization of this gene cluster is similar to that of the homologous cluster found in Escherichia coli. Except for three nucleotide changes, the sequence was identical among Japanese and Indonesian isolates. A loop-mediated isothermal amplification (LAMP) assay based on the conserved sequence of the nusG-rplKAJL-rpoB gene cluster was developed for the detection of the GO. The LAMP product was rapidly detected on nylon membranes by staining with AzurB. LAMP could detect as low as about 300 copies of the nusG-rplKAJL-rpoB fragment of the Japanese and Indonesian isolates of GO. The LAMP-based detection method, which does not depend upon a thermal cycler and electrophoresis apparatus, will be useful for under-equipped laboratories, including those found in extension centers and quarantine offices.

Complex endosymbiotic microbiota of the citrus psyllid Diaphorina citri (Homoptera: Psylloidea).

Abstract We investigated the endosymbiotic microbiota of the citrus psyllid Diaphorina citri that vectors Liberobacter sp., the causative agent of citrus greening disease. Eubacterial 16S rDNA fragments were amplified by PCR and cloned from the whole DNA of D. citri. RFLP analysis showed that the PCR products contained five major sequence types. Nucleotide sequences of the five types were determined and subjected to molecular phylogenetic analysis. The first sequence was a member of the γ-Proteobacteria, closely related to mycetocyte symbionts of other psyllids. The second sequence belonged to the β-Proteobacteria, related to Oxalobacter and Herbaspirillum. The third sequence, belonging to the γ-Proteobacteria, showed a significant similarity to the sequences of Arsenophonus spp., known as endosymbionts of a triatomine bug and a parasitoid wasp. The fourth sequence was almost identical to the sequences of Liberobacter spp. in the α-Proteobacteria. The fifth sequence showed a high similarity to the sequences of Wolbachia spp. in the α-Proteobacteria. In situ hybridization experiments using specific oligonucleotide probes confirmed that the first and second sequences originated from the mycetocyte and syncytium symbionts, respectively. The presence/absence of the five endosymbiotic bacteria in adult individuals of D. citri from an Indonesian natural population was examined by diagnostic PCR analysis. The mycetocyte symbiont and the syncytium symbiont exhibited 100% infection, whereas the Arsenophonus, Liberobacter and Wolbachia showed partial infections at a rate of 83.3%, 45.2% and 76.2%, respectively.

Isolation and characterization of two entomopathogenic fungi attacking Diaphorina citri (Homoptera, Psylloidea) in Indonesia

In an attempt to suppress the propagation of citrus greening disease in Indonesia, we explored pathogens ofDiaphorina citri which vectors the disease. At two orange orchards, manyD. citri adults were found to be dead and covered with fungal mycelia. Two fungi,Paecilomyces fumosoroseus andHirsutella citriformis, were consistently isolated from the infected insects. Molecular phylogeny of their 18S rDNA sequences showed that they belong to the ascomycetous clade of the Clavicipitales/Hypocreales, which embraces many entomopathogenic fungi. When healthy adults ofD. citri were inoculated with conidia of theP. fumosoroseus, the insects died within 6 d.

Comparison of 16S rDNA and 16S/23S intergenic region sequences among citrus greening organisms in Asia

Polymerase chain reaction was used to amplify and sequence the 16S ribosomal RNA gene (rDNA) and 16S/23S intergenic region of several isolates of citrus greening organism (GO) from Japan, the Philippines, Indonesia, and Thailand. The sequences of 16S rDNA were identical among all the isolates studied, very similar to the published sequences of Thai (99.4 to 100% identity), Nepalese (100% identity), and Indian (98.8% identity) strains, and less similar to an African strain (97.5% identity). The sequences of the intergenic region between 16S and 23S rDNA were also identical among the isolates examined as well as the reported Nepalese and Thai isolates. They were close to the sequences of reported strains of India and China (99.2%) and apart from those of the African strain (85.5%). These results suggested that some isolates of GO from Japan, the Philippines, Indonesia, Thailand, and Nepal constitute one strain, which is similar to Indian and Chinese strains and distinct from the African strain.

Differentiation of “Candidatus Liberibacter asiaticus” Isolates by Variable-Number Tandem-Repeat Analysis

ABSTRACT Four highly polymorphic simple sequence repeat (SSR) loci were selected and used to differentiate 84 Japanese isolates of “Candidatus Liberibacter asiaticus.” The Neis measure of genetic diversity values for these four SSRs ranged from 0.60 to 0.86. The four SSR loci were also highly polymorphic in four isolates from Taiwan and 12 isolates from Indonesia.

EFFECT OF IRON DEFICIENCY ON ROOT FERRIC CHELATE REDUCTASE, PROTON EXTRUSION, BIOMASS PRODUCTION AND MINERAL ABSORPTION OF CITRUS ROOT STOCK ORANGE JASMINE (MURRAYA EXOTICA L.)

Three-week iron (Fe) deficiency stress experiments were conducted using two citrus root stocks, Fe-deficiency tolerant Orange Jasmine (OJ, Murraya exotica L.) and the sensitive Flying Dragon [FD, Poncirus trifoliata var. monstrosa (T. Ito) Swingle]. Root ferric chelate reductase activity and proton extrusion increased in OJ between 12 and 18 d of stress, whereas there was no change in FD. Dry weight of OJ roots increased in contrast to FD which decreased. The Mn content in OJ remained the same even under Fe stress. Zn content in OJ roots doubled while that of FD increased 4-folds. The shoot/root Fe accumulation ratio increased in OJ while it decreased in FD. OJ apparently has mechanisms for increasing root biomass, controlling Fe reutilization and regulating manganese (Mn) and zinc (Zn) absorption in response to Fe deficiency. These mechanisms could help maintain homeostasis under heavy metal stress, which would be useful for improved growth of economically important citrus species.

Interactions between plant nutrition and symptom expression in mandarin trees infected with the disease huanglongbing

A survey of Siem mandarin trees (Citrus reticulata Blanco) grown in different soil types and given different fertiliser regimes showed that applications of a foliar fertiliser reduced the symptom expression of trees infected with huanglongbing by ∼40%. In contrast, infected trees growing in a sandy soil, or in a clay-loam, did not differ in their level of symptom expression, despite differences in the fertility of the two soils. These data suggest that infection restricts either nutrient uptake or transport and, therefore, that foliar applied minerals may prolong tree life and reduce yield losses.

Ecology, Epidemiology and Disease Management of Ralstonia syzygii in Indonesia

Ralstonia solanacearum species complex phylotype IV strains, which have been primarily isolated from Indonesia, Australia, Japan, Korea, and Malaysia, have undergone recent taxonomic and nomenclatural changes to be placed in the species Ralstonia syzygii. This species contains three subspecies; Ralstonia syzygii subsp. syzygii, a pathogen causing Sumatra disease of clove trees in Indonesia, Ralstonia syzygii subsp. indonesiensis, the causal pathogen of bacterial wilt disease on a wide range of host plants, and Ralstonia syzygii subsp. celebesensis, the causal pathogen of blood disease on Musa spp. In Indonesia, these three subspecies have devastated the cultivation of susceptible host plants which have high economic value. Limited knowledge on the ecology and epidemiology of the diseases has hindered the development of effective control strategies. In this review, we provide insights into the ecology, epidemiology and disease control of these three subspecies of Ralstonia syzygii.

Identification of purple blotch pathogen of shallot by PCR using specific primer for Alternaria genus

Abstract Purple blotch (Alternaria porri), having symptom similar to stemphylium blight (Stemphylium vesicarium), is one of the important diseases that significantly lowered shallot yield in Indonesia. However, stemphylium blight has never been reported. Purple blotch pathogens from farmers’ crops in Bantul and experimental crop in Centre of Innovative Agricultural Technology Universitas Gadjah Mada (CIAT-UGM) Sleman Regencies were identified to observe whether Stemphylium sp. was found with A. porri. Thirteen isolates obtained showed variability in colony texture, colour and pigmentation. DNA fingerprinting through Polymerase Chain Reaction of BOX and Enterobacterial Repetitive Intergenic Consensus region dispersed those isolates into two main groups. Identification using Alternaria-specific primers, Dir1ITSAlt/Inv1ITSAlt revealed that 10 isolates were Stemphylium sp. and 3 isolates were Alternaria sp. A. porri was only identified from CIAT-UGM. This finding emphasise that the Alternaria-specific primer was able to amplify Stemphylium sp. Besides, stemphylium blight disease may have been occurred in farmers’ crops in Bantul.

Peningkatan Ketahanan Tanaman Pisang Kepok Kuning terhadap Penyakit Darah melalui Variasi Somaklonal dan Simbiosis Endofitik

One of the obstacles that was encountered in the banana cultivation is blood disease. Blood diseases is caused by Ralstonia solanacearum that is subsequently revised to become blood disease bacteria (BDB). Until now the control of banana blood disease is very difficult. Control of banana blood disease with chemical injections and soil treatment is not effective. This study was aimed to obtain Kepok Kuning cultivar banana seedlings which was resistant towards blood disease obtained from in vitro selection by using BDB growing filtrate and induced resistance by inoculation of antagonistic endophytic bacteria.The observation of Kepok Kuning banana explants treated with various concentrations of BDB growing filtrate showed that the percentage of living explants decreased to 83.33% when the BDB growing filtrate concentration increased to 15%. Treatment of banana explants with BDB growing filtrate also affected the number of roots, shoots, and leaves. Treatment with a single antagonistic endophytic bacteria suppressed the intensity of banana blood disease to 0% in comparison with the mixture of antagonistic endophytic bacteria if Kepok Kuning banana explants were not treated with BDB growing filtrate. When Kepok Kuning banana explants were treated with BDB growing filtrate, the intensity of banana blood disease suppressed to 0% after the high concentration of BDB growing filtrate and the antagonistic endophytic bacteria mixture were applied. Salah satu kendala yang dihadapi dalam usaha budidaya pisang adalah adanya penyakit darah. Penyakit darah disebabkan oleh bakteri Ralstonia solanacearumyang selanjutnya direvisimenjadi bakteri darah pisang (Blood Disease Bacteria orBDB). Sampai saat ini pengendalian penyakit darah pisang sangatsukar dilakukan. Pengendalian penyakit darah dengan suntikan bahan kimia dan perlakuan tanah tidak efektif untuk diaplikasikan. Penelitian ini bertujuan untukmendapatkan bibit pisang kultivar Kepok Kuning yang tahan penyakit darah yang diperoleh dariseleksi in vitro dengan menggunakan filtrat pertumbuhan BDB dan induksi ketahanan melalui inokulasi jasad renik endofitik yang bersifat antagonis. Hasil pengamatan terhadap eksplan pisang Kepok Kuning yang diperlakukan dengan berbagai konsentrasi filtrat BDB menunjukkan bahwa pada eksplan yang diperlakukan dengan filtrat pertumbuhan BDB, persentase tumbuhnya akan berkurang hingga 83,33% jika konsentrasifiltrat pertumbuhanBDBmencapai 15%. Selain itu perlakuan planlet pisang dengan filtrat pertumbuhan BDB akan mempengaruhi jumlah akar, tunas, dan daun. Perlakuan dengan jasad renik endofitik antagonis secara tunggal mampu menekan intensitas penyakit darah hingga 0% jika dibandingkan dengan perlakuan campuran apabila sebelumnya eksplan pisang Kepok Kuning tidak diperlakukan dengan filtrat pertumbuhan BDB. Apabila sebelumnya planlet pisangKepokKuning diperlakukan dengan filtrat pertumbuhan BDB maka mampu menekan intensitas penyakit darah hingga 0% jika konsentrasi filtrat pertumbuhanBDBsemakin tinggi dan diperlakuan campuran jasad renik endofitik.