Natural Infection of Murraya paniculata and Murraya sumatrana with CLas in Java.

The phloem-limited bacterium, 'Candidatus Liberibacter asiaticus' (CLas), is the putative causal pathogen of the severe Asiatic form of huanglongbing (citrus greening) and is most commonly transmitted by the Asiatic citrus psyllid (ACP), Diaphorina citri. CLas severely affects many Citrus species and hybrids and has been recorded in the Citrus relative, orange jasmine, Murraya paniculata (L.) Jack (syn. M. exotica L.). In this study, 13 accessions of three Murraya species (M. paniculata, M. sumatrana Roxb. and M. lucida (G.Forst.) Mabb,) and the Papuan form of a putative hybrid (M. omphalocarpa Hayata) were identified morphologically and molecularly based on sequence identity of the matK-5'trnK region of the chloroplast genome, and infection on these plants under field conditions was determined by PCR and qPCR on 2-4 occasions over 14 months. CLas was repeatedly detected in leaflet midribs by PCR and qPCR on four and three accessions of M. paniculata and M. sumatrana, respectively. It was not detected in leaflet midribs of single accessions of M. lucida and M. omphalocarpa. The species identification of the CLas-positive accessions was further confirmed using all the molecular taxonomic markers consisting of the six fragments of the maternally inherited chloroplast genome and part of the nuclear-encoded ITS region. The results indicated that natural infection of M. paniculata and M. sumatrana with CLas can occur in Java. This is the first demonstration of the natural infection of M. sumatrana with CLas. Further studies are required to determine if infections persist in the absence of D. citri.

Susceptibility of the Banana Inflorescence to Blood Disease.

The bacterium Ralstonia syzygii subsp. celebesensis causes Blood disease of banana, a vascular wilt of economic significance in Indonesia and Malaysia. Blood disease has expanded its geographic range in the last 20 years and is an emerging threat to Southeast Asian banana production. Many aspects of the disease cycle and biology are not well understood, including the ability of different parts of the female and male inflorescence of banana to act as infection courts. This study confirms that the banana varieties of Cavendish, and Kepok 'Kuning' are susceptible to Blood disease and that an inoculum concentration of 102 CFU/ml of R. syzygii subsp. celebesensis is adequate to initiate disease after pseudostem inoculation. Data show that infection occurs through both the male and female parts of a banana inflorescence and the rachis when snapped to remove the male bell. The infection courts are the female flowers, the male bell bract scar, the male bell flower cushion, the snapped rachis, and deflowered fingers. The location of these infection courts concurs with the dye studies demonstrating that dye externally applied to these plants parts enters the plant vascular system. Thus, the hypothesis is supported that infection of R. syzygii subsp. celebesensis occurs through open xylem vessels of the male and female parts of the banana inflorescence.

Transmission of Blood Disease in Banana.

Banana Blood disease is a bacterial wilt caused by Ralstonia syzygii subsp. celebesensis and is an economically important disease in Indonesia and Malaysia. Transmission of this pathogen is hypothesized to occur through insects mechanically transferring bacteria from diseased to healthy banana inflorescences and other pathways involving pruning tools, water movement, and root-to-root contact. This study demonstrates that the ooze from the infected male bell and the sap from various symptomatic plant parts are infective, and the cut surfaces of a bunch peduncle, petiole, corm, pseudostem, and the rachis act as infection courts for R. syzygii subsp. celebesensis. In addition, evidence is provided that R. syzygii subsp. celebesensis is highly tool transmissible, that the bacterium can be transferred from the roots of a diseased plant to the roots of a healthy plant and transferred from the mother plant to the sucker. We provide evidence that local dispersal of Blood disease occurs predominantly through mechanical transmission by insects, birds, bats, or human activities from diseased to healthy banana plants and that long-distance dispersal occurs through the movement of contaminated planting material. Disease management strategies to prevent crop losses associated with this emerging disease are discussed based on our findings.

Diagnostics of Banana Blood Disease.

Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis is a bacterial wilt disease that causes major yield losses of banana in Indonesia and peninsular Malaysia. The disease has significantly increased its geographic distribution in the past decade. Diagnostic methods are an important component of disease management in vegetatively propagated crops such as banana to constrain incursions of plant pathogens. Therefore, the objectives of this study were (i) to design and rigorously validate a novel banana Blood disease (BBD) real-time PCR assay with a high level of specificity and sensitivity of detection and (ii) to validate published PCR-based diagnostic methods targeting the intergenic region in the megaplasmid ("121 assay" with primer set 121) or the phage tail protein-coding sequence in the bacterial chromosome ("Kubota assay" and "BDB2400 assay" with primer set BDB2400). Assay validation included 339 samples (174 Blood disease bacteria, 51 bacteria associated with banana plants, 51 members of the Ralstonia solanacearum species complex, and 63 samples from symptomatic and healthy plant material). Validation parameters were analytical specificity (inclusivity and exclusivity), selectivity, limit of detection, accuracy, and ruggedness. The 121 assay and our newly developed BBD real-time PCR assay detected all R. syzygii subsp. celebesensis strains with no cross-specificity during validation. Two different PCR assays using the primer set BDB2400 lacked specificity and selectivity. This study reveals that our novel BBD real-time PCR assay and the conventional PCR 121 assay are reliable methods for Blood disease diagnostics, as they comply with all tested validation parameters.

Geographic Expansion of Banana Blood Disease in Southeast Asia.

Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis is a bacterial wilt causing significant crop losses in Indonesia and Malaysia. Disease symptoms include wilting of the plant and red-brown vascular staining, internal rot, and discoloration of green banana fruit. There is no known varietal resistance to this disease in the Musa genus, although variation in susceptibility has been observed, with the popular Indonesian cooking banana variety Kepok being highly susceptible. This study established the current geographic distribution of Blood disease in Indonesia and confirmed the pathogenicity of isolates by Koch's postulates. The long-distance distribution of the disease followed an arbitrary pattern indicative of human-assisted movement of infected banana materials. In contrast, local or short-distance spread radiated from a single infection source, indicative of dispersal by insects and possibly contaminated tools, water, or soil. The rapid expansion of its geographical range makes Blood disease an emerging threat to banana production in Southeast Asia and beyond.

Evolutionary Analysis of the Highly Conserved Insect Odorant Coreceptor (Orco) Revealed a Positive Selection Mode, Implying Functional Flexibility.

Odorant coreceptor (Orco) represents one of the essential genes in the insect olfactory system, which facilitates signal transduction and heterodimerization with different odorant receptors (Ors) in the insect antennal dendritic membrane. Evolutionary analysis by detecting positive selection is important to examine the functional flexibility of Orco that potentially supports insect survival. The maximum likelihood codon substitution model was applied using CODEML program as implemented in PAML ver 4.9e package across 59 Orco codon sequences available from GenBank. These sequences represented five major insect orders and two reproductive systems (holometabola and nonholometabola). In the site model that identified common ω values for Orco, it was clearly shown that Orco was under strong purifying selection, indicated by the ω value that was far from 1 (ω: 0.03). However, in to the branch model, positive selection was detected to be acting on Dipteran lineages, whereas in the branch-site model, several sites were under significant positive selection occurring in the following four clades: Coleoptera, Diptera, Lepidoptera, and Psocodea. The typical evolutionary mode acting on Orco was consistent with the entropy value [H(x)], confirming that 48.9% of the Orco site was under conservation (H(x) < 0.5), whereas 26.9% of the Orco sites was under high variation (H(x) > 1). These findings confirmed that Orco genes are generally highly conserved and can possibly be used for the manipulation of insect pest control programs. However, positive selection that acts on certain lineages suggested future adaptive evolutionary ability of Orco to anticipate flexible functions for successful olfactory processes.

Analysis of reduced and oxidized antioxidants in Hevea brasiliensis latex reveals new insights into the regulation of antioxidants in response to harvesting stress and tapping panel dryness.

Latex diagnosis (LD) is applied to optimize the natural rubber production and prevent tapping panel dryness (TPD), a physiological syndrome affecting latex production in Hevea brasiliensis. The reduced thiol content (RSH) is one of the biochemical parameters associated with the risk of TPD. However, RSH is difficult to interpret because of the influence of the environment. In order to better understand the regulation of antioxidants and to better interpret RSH, a key parameter of LD, this study analysed in latex both oxidised and reduced forms of ascorbic acid (AsA) and glutathione, and their cofactors as well as other latex diagnosis parameters in response to harvesting stress (tapping and ethephon stimulation) and TPD occurrence. The content of antioxidants in latex had a high variability among five rubber clones. The concentration in AsA was about ten times higher than GSH in laticifer, GSH accounting for about 50% of RSH. For short-term harvesting stress, RSH increased with tapping frequency and ethephon stimulation. TPD is associated with high latex viscosity and bursting of lysosomal particles called lutoids, as well as for several rubber clones with lower RSH and GSH contents. These results suggest that a high level of RSH shows the capacity of laticifer metabolism to cope with harvesting stress, while a drop in RSH is the sign of long stress related to lower metabolic activity and TPD occurrence. RSH remains an essential physiological parameter to prevent TPD when associated with reference data under low and high harvesting stress. This study paves the way to understand the role of AsA and GSH, and carry out genetic studies of antioxidants.

Corrigendum to "Analysis of reduced and oxidized antioxidants in Hevea brasiliensis latex reveals new insights into the regulation of antioxidants in response to harvesting stress and tapping panel dryness" [Heliyon 8 (7) (June 2022) Article e09840].

[This corrects the article DOI: 10.1016/j.heliyon.2022.e09840.].

Differentiation of "Candidatus Liberibacter asiaticus" isolates by variable-number tandem-repeat analysis.

Four highly polymorphic simple sequence repeat (SSR) loci were selected and used to differentiate 84 Japanese isolates of "Candidatus Liberibacter asiaticus." The Nei's 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.

RNA-seq data of tea mosquito bugs, Helopeltis bradyi, antennae.

Tea Mosquito Bug (TMB), Helopeltis bradyi (Hemiptera: Miridae) is one of the major pest infesting tea and cocoa plantations worldwide. Developing olfaction-based control methods was urges as an alternative to commonly used but non-environmental friendly chemical pesticides. However, the molecular mechanisms underlying TMB reception mechanism are still lacking. Here, we collected a pooled male and female TMB antennae for RNA extraction followed by sequencing using the BGISEQ-500 platform and de novo assembly. TMB antennae RNA-seq data yielded 32,142 unigenes with N50 and GC (%) were 2322 and 40.25; subsequently. The RNA-seq data are available in GenBank Sequence Read Archive (SRA) database with accession number SRR13327229. De novo transcriptome analysis had identified several genes involved in TMB odorant reception includes; 39 OBPs (odorant binding proteins), 10 CSPs (chemosensory proteins), 81 Ors (odorant receptors), 1 Orcos (co-receptors), 9 SNMPs (sensory neuron membrane proteins), 3 GRs (gustatory receptors) and 4 IRs (ionotropic receptors). Our study presents the first RNA seq for TMB antennae, which serve the primary molecular resources data, which will facilitate further research to develop olfaction-based control methods, potentially contribute to TMB management strategies.

Transcriptomic data of the Musa balbisiana cultivar Kepok inoculated with Ralstonia syzigii subsp. celebesensis and Ralstonia solanacearum.

The increasing production of banana is hampered by the spread of banana plant diseases, one of which is caused by a group of bacteria, including those of causing wilt diseases. In Indonesia, blood disease is one of the important banana wilt diseases since loss due to the infection can reach up to 50%. There are numerous publications on the pathogen identification causing banana blood disease based on the molecular approach, however to date, no detailed molecular data are available for the interaction of banana host plant against the pathogen. Here, we present three raw data sets of the total RNA-seq from the inoculated Musa balbisiana cutivar kepok (ABB genome) inoculated with Ralstonia syzigii subsp. celebesensis, Ralstonia solanacearum and mock. The data provide essential knowledge for differentiating the banana response against pathogen, reveal pathogenesis-related genes and gene functions in the plant system, and research development to design blood disease-resistance of banana as one of the management strategies. Raw reads of RNA-seq data can be found in NCBI's Sequence Read Archive (SRA) database with the accession number of SRR10547839 (RSC), SRR10547840 (RS), and SRR10547841 (Mock).

RNA-seq data of banana bunchy top virus (BBTV) viruliferous and non-viruliferous banana aphid (Pentalonia nigronervosa).

Banana bunchy top disease (BBT) is one of the most economically serious viral diseases of banana caused by banana bunchy top virus (BBTV: Nanoviridae: Babuvirus). BBTV is a circular, ssDNA virus which is suitable in the phloem tissue and currently only being transmitted by the banana aphid (Pentalonia nigronervosa) in a persistent, non-propagative, circulative manner. Interaction of BBTV and banana aphid had been studied in several ways, such as transmission and translocation of BBTV inside the banana aphid body at cellular level. However, the molecular mechanism underlying the interaction between BBTV and banana aphid have been poorly understood. Therefore, this transcriptomic study was conducted to obtain the raw data for differential genes expression study in BBTV viruliferous (Vr) and non-viruliferous (NVr) banana aphid. Here, we present two data sets of RNA seq raw reads which is available in GenBank Sequence Read Archive (SRA) database with accession number of SRX6918251 and SRX6918252 for the Vr and NVr banana aphid respectively.

Data on banana transcriptome in response to blood disease infection.

Blood disease of Banana (BDB) is one of the prevalent disease caused by Ralstonia syzygii subsp. celebesensis (Rsc) which cause substantial loss on banana production in Indonesia. To date, the genetic basis of plant defense mechanism caused by blood disease in banana is not available. As a matter of fact, the knowledge of global gene expression will provide important information on plant response to the pathogen infection. Data from transcriptomic analysis in response to blood disease infection from Musa acuminata cv. Mas Kirana (AA group), representing the A genome, and Musa balbisiana cv. Klutuk (BB group), representing the B genome, were firstly reported. The transcriptome data discussed in this publication are accessible through NCBI's Gene Expression Omnibus with GEO Series accession number GSE138749. These data provide the basis for further investigation on the global gene expression which is pivotal to understand the mechanism of disease resistance from two banana genomes in response to blood disease infection.

Evaluation of genetic diversity among 'Candidatus Liberibacter asiaticus' isolates collected in Southeast Asia.

The aim of this study was to investigate the genetic diversity and relationships among 'Candidatus Liberibacter asiaticus' isolates from different hosts and distinct geographical areas in Southeast Asia. Genetic diversity among 'Ca. Liberibacter asiaticus' was estimated by sequencing four well-characterized DNA fragments: the 16S ribosomal DNA (rDNA) and 16S/23S intergenic spacer regions; the outer membrane protein (omp) gene region; the trmU-tufB-secE-nusG-rplKAJL-rpoB region (gene cluster region); and the bacteriophage-type DNA polymerase region. The sequences of the 16S rDNA and 16S/23S intergenic spacer regions were identical among all 'Ca. Liberibacter asiaticus' isolates. In contrast, nucleotide substitutions were observed in both the omp gene and the gene cluster regions. However, extended bacteriophage-type DNA polymerase sequences acquired by thermal asymmetric interlaced polymerase chain reaction provided the most sequence diversity among isolates. Phylogenetic analysis of the bacteriophage-type DNA polymerase sequences revealed three clusters in the Southeast Asian 'Ca. Liberibacter asiaticus' population. All Indonesian 'Ca. Liberibacter asiaticus' isolates clustered in one group. The other clusters were not correlated with geographic distribution. The differences in genetic sequences did not reflect differences in the original citrus host (mandarin or pummelo). These results suggest that the bacteriophage-type DNA polymerase region would be useful for molecular differentiation between different Southeast Asian 'Ca. Liberibacter asiaticus' isolates.

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.

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.

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.