Comparative RNA-seq analysis of resistant and susceptible banana genotypes reveals molecular mechanisms in response to banana bunchy top virus (BBTV) infection.

Bananas hold significant economic importance as an agricultural commodity, serving as a primary livelihood source, a favorite fruit, and a staple crop in various regions across the world. However, Banana bunchy top disease (BBTD), which is caused by banana bunchy top virus (BBTV), poses a considerable threat to banana cultivation. To understand the resistance mechanism and the interplay of host suitability factors in the presence of BBTV, we conducted RNA-seq-based comparative transcriptomics analysis on mock-inoculated and BBTV-inoculated samples from resistant (wild Musa balbisiana) and susceptible (Musa acuminata 'Lakatan') genotypes. We observed common patterns of expression for 62 differentially expressed genes (DEGs) in both genotypes, which represent the typical defense response of bananas to BBTV. Furthermore, we identified 99 DEGs exclusive to the 'Lakatan' banana cultivar, offering insights into the host factors and susceptibility mechanisms that facilitate successful BBTV infection. In parallel, we identified 151 DEGs unique to the wild M. balbisiana, shedding light on the multifaceted mechanisms of BBTV resistance, involving processes such as secondary metabolite biosynthesis, cell wall modification, and pathogen perception. Notably, our validation efforts via RT-qPCR confirmed the up-regulation of the glucuronoxylan 4-O-methyltransferase gene (14.28 fold-change increase), implicated in xylan modification and degradation. Furthermore, our experiments highlighted the potential recruitment of host's substrate adaptor ADO (30.31 fold-change increase) by BBTV, which may play a role in enhancing banana susceptibility to the viral pathogen. The DEGs identified in this work can be used as basis in designing associated gene markers for the precise integration of resistance genes in marker-assisted breeding programs. Furthermore, the findings can be applied to develop genome-edited banana cultivars targeting the resistance and susceptibility genes, thus developing novel cultivars that are resilient to important diseases.

Resistance of Musa balbisiana Accessions of the Philippines to Banana Bunchy Top Virus.

Banana bunchy top disease (BBTD) is caused by banana bunchy top virus (BBTV), the most important virus affecting banana. Currently, no cultivar or accession of banana has complete resistance to BBTD. A total of 36 wild Musa spp. accessions, including 34 Musa balbisiana and 2 M. acuminata subsp. errans ("Agutay"), were screened for resistance against BBTV. In greenhouse tests using viruliferous banana aphids (Pentalonia nigronervosa), all M. balbisiana accessions remained symptomless, and BBTV was not detected in any of these plants by PCR at 3 and 6 months postinoculation. In contrast, 100% disease incidence was recorded in M. acuminata subsp. errans and in cv. Lakatan susceptible control plants. The PCR-negative M. balbisiana plants were then transferred to a field with high BBTV inoculum pressure where they remained symptomless and PCR-negative for up to 5 years, while all cv. Lakatan developed BBTD. Wild M. balbisiana accessions showed a high level of resistance and possibly immunity to BBTV and are expected to provide a resource for conventional and marker-assisted breeding.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Monitoring the distribution of banana bunchy top virus in South Africa: a country-wide survey.

Banana bunchy top disease is the most devastating viral disease of bananas worldwide and is caused by banana bunchy top virus (BBTV). The disease is spread by the banana aphid Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) and through infected propagation material. In 2016, the virus was detected for the first time in an isolated area in the South Coast region of KwaZulu-Natal Province (KZN), South Africa. The aim of this study was to conduct surveys across all banana-producing regions in South Africa, viz. KwaZulu-Natal, Mpumalanga, and Limpopo provinces. Over 1700 plant and aphid samples were collected from commercial farms and rural households in the three provinces, and more-intense sampling was done in the affected KZN region. A BBTV-specific PCR targeting DNA-R (encoding the master replication initiation protein, M-Rep) was used to detect virus-infected samples, and amplicons of the expected size were sequenced. Comparative phylogenetic analysis showed that the South African BBTV isolates clustered within the Pacific Indian Oceans genomic group, which includes isolates from India and other regions in Africa, with a bootstrap value of 94%. To date, the virus has been identified only in the South Coast region of KwaZulu-Natal Province. Intense management strategies, including scouting, removal of infected plants, and control of aphids, have been implemented in areas where positive samples were identified to minimize the spread of the virus.

A newly emerging alphasatellite affects banana bunchy top virus replication, transcription, siRNA production and transmission by aphids.

Banana bunchy top virus (BBTV) is a six-component ssDNA virus (genus Babuvirus, family Nanoviridae) transmitted by aphids, infecting monocots (mainly species in the family Musaceae) and likely originating from South-East Asia where it is frequently associated with self-replicating alphasatellites. Illumina sequencing analysis of banana aphids and leaf samples from Africa revealed an alphasatellite that should be classified in a new genus, phylogenetically related to alphasatellites of nanoviruses infecting dicots. Alphasatellite DNA was encapsidated by BBTV coat protein and accumulated at high levels in plants and aphids, thereby reducing helper virus loads, altering relative abundance (formula) of viral genome components and interfering with virus transmission by aphids. BBTV and alphasatellite clones infected dicot Nicotiana benthamiana, followed by recovery and symptomless persistence of alphasatellite, and BBTV replication protein (Rep), but not alphasatellite Rep, induced leaf chlorosis. Transcriptome sequencing revealed 21, 22 and 24 nucleotide small interfering (si)RNAs covering both strands of the entire viral genome, monodirectional Pol II transcription units of viral mRNAs and pervasive transcription of each component and alphasatellite in both directions, likely generating double-stranded precursors of viral siRNAs. Consistent with the latter hypothesis, viral DNA formulas with and without alphasatellite resembled viral siRNA formulas but not mRNA formulas. Alphasatellite decreased transcription efficiency of DNA-N encoding a putative aphid transmission factor and increased relative siRNA production rates from Rep- and movement protein-encoding components. Alphasatellite itself spawned the most abundant siRNAs and had the lowest mRNA transcription rate. Collectively, following African invasion, BBTV got associated with an alphasatellite likely originating from a dicot plant and interfering with BBTV replication and transmission. Molecular analysis of virus-infected banana plants revealed new features of viral DNA transcription and siRNA biogenesis, both affected by alphasatellite. Costs and benefits of alphasatellite association with helper viruses are discussed.

Banana bunchy top virus genetic diversity in Pakistan and association of diversity with recombination in its genomes.

Banana Bunchy top virus (BBTV) is a multipartite circular single strand DNA virus that belongs to genus Babuvirus and family Nanoviridae. It causes significant crop losses worldwide and also in Pakistan. BBTV is present in Pakistan since 1988 however, till now only few (about twenty only) sequence of genomic components have been reported from the country. To have insights into current genetic diversity in Pakistan fifty-seven genomic components including five complete genomes (comprises of DNA-R, -U3, -S, -M, -C and -N components) were sequenced in this study. The genetic diversity analysis of populations from Pakistan showed that DNA-R is highly conserved followed by DNA-N, whereas DNA-U3 is highly diverse with the most diverse Common Region Stem-loop (CR-SL) in BBTV genome, a functional region, which previously been reported to have undergone recombination in Pakistani population. A Maximum Likelihood (ML) phylogenetic analysis of entire genomes of isolates by using sequence of all the components concatenated together with the reported genomes around the world revealed deeper insights about the origin of the disease in Pakistan. A comparison of the genetic diversity of Pakistani and entire BBTV populations around the world indicates that there exists a correlation between genetic diversity and recombination. Population genetics analysis indicated that the degree of selection pressure differs depending on the area and genomic component. A detailed analysis of recombination across various components and functional regions suggested that recombination is closely associated with the functional parts of BBTV genome showing high genetic diversity. Both genetic diversity and recombination analyses suggest that the CR-SL is a recombination hotspot in all BBTV genomes and among the six components DNA-U3 is the only recombined component that has extensively undergone inter and intragenomic recombination. Diversity analysis of recombinant regions results on average one and half fold increase and, in some cases up to four-fold increase due to recombination. These results suggest that recombination is significantly contributing to the genetic diversity of BBTV populations around the world.

Elimination of BBTV via a systemic in vitro electrotherapy approach.

Banana bunchy top virus (BBTV) is the most destructive etiological agent limiting banana cultivation areas globally. This study attempted BBTV elimination by traditional shoot-tip culture (control) and alternative shoot-tip + electrotherapy (treated) techniques. Shoot-tip culture from Musa acuminata cv. 'Grand Naine' infected sources were exposed to 100 mA electric current for different time intervals (20-60 min). Virus indexing (via PCR) and genetic fidelity (by ISSR assay) from the cultures were tested, alongside the physio-biochemical parameters. Exposure of electric current for less than 50 min was ineffective for BBTV elimination. Still, a rise in the duration from 50 min or more led to eradicating the virus from some explants. Elimination of BBTV was complete from 100 % of explants exposed to 100 mA for 60 min, as confirmed by lack of BBTV detection even at six months after acclimatization. In the control treatment, the maximum efficiency of BBTV elimination was 28 % after eight subcultures. On the other hand, improved survival % was observed in the treated culture. Moreover, homogenous ISSR patterns were there between the treated and the mother plant and similar physio-biochemical activities were seen in electro-exposed cultures and healthy ones. Thus, the study reports complete BBTV-elimination from banana with international compliances, for the first time, via electrotherapy while maintaining genomic template and biochemical stability.

Spatial parameters associated with the risk of banana bunchy top disease in smallholder systems.

The Banana Bunchy Top Disease (BBTD), caused by the Banana Bunchy Top Virus (BBTV) is the most important and devastating in many tropical countries. BBTD epidemiology has been little studied, mixed landscape smallholder systems. The relative risks associated with this disease vary between geographical areas and landscapes. This work analyzed the management and vegetation conditions in smallholder gardens to assess the factors linked to landscape-level BBTV transmission and management. Mapping was done in this study area which is in a BBTD-endemic region, involving farmers actively managing the disease, but with household-level decision making. A spatial scanning statistic was used to detect and identify spatial groups at the 5% significance threshold, and a Poisson regression model was used to explore propagation vectors and the effect of surrounding vegetation and crop diversity. Spatial groups with high relative risk were identified in three communities, Dangbo, Houéyogbé, and Adjarra. Significant associations emerged between the BBTD prevalence and some crop diversity, seed systems, and BBTD management linked factors. The identified factors form important candidate management options for the detailed assessment of landscape-scale BBTD management in smallholder communities.

ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Estimating a novel stochastic model for within-field disease dynamics of banana bunchy top virus via approximate Bayesian computation.

The Banana Bunchy Top Virus (BBTV) is one of the most economically important vector-borne banana diseases throughout the Asia-Pacific Basin and presents a significant challenge to the agricultural sector. Current models of BBTV are largely deterministic, limited by an incomplete understanding of interactions in complex natural systems, and the appropriate identification of parameters. A stochastic network-based Susceptible-Infected-Susceptible model has been created which simulates the spread of BBTV across the subsections of a banana plantation, parameterising nodal recovery, neighbouring and distant infectivity across summer and winter. Findings from posterior results achieved through Markov Chain Monte Carlo approach to approximate Bayesian computation suggest seasonality in all parameters, which are influenced by correlated changes in inspection accuracy, temperatures and aphid activity. This paper demonstrates how the model may be used for monitoring and forecasting of various disease management strategies to support policy-level decision making.

Independent modulation of individual genomic component transcription and a cis-acting element related to high transcriptional activity in a multipartite DNA virus.

BACKGROUND: The genome of Banana bunchy top virus (BBTV) consists of at least six circular, single-stranded DNA components of ~ 1 kb in length. Some BBTV isolates may also carry satellite DNA molecules that are not essential for BBTV infection. The relation between multipartite DNA virus replication and their transcriptional levels and the underlying mechanism remain unclear. RESULTS: To understand the coordinated replication and transcription of the multiple genomic components, the absolute amounts of each BBTV DNA component were measured by real-time PCR (qPCR), and their transcriptional levels were determined by RNAseq and reverse transcription-qPCR (qRT-PCR). Significant differences were found in the absolute amounts of individual BBTV genomic components. Transcriptional levels of each BBTV genomic component obtained from the RNAseq data matched closely to those obtained from qRT-PCR, but did not correspond to the absolute amount of each DNA component. The ratio of transcript over DNA copies ranged from 46.21 to 1059.44%, which was possibly regulated by the promoter region in the intergenic region of each component. To further determine this speculation, the promoter region of the DNA-S, -M or -N was constructed to the upstream of green fluorescent protein (GFP) gene for transient expression by agrobacterium-mediated transformation method. The qRT-PCR showed the highest transcriptional activity was promoted by DNA-N promoter, about 386.58% activity comparing with CaMV 35S promoter. Confocal microscopy observation showed that the intensity of green fluorescence was corresponding to that of qRT-PCR. CONCLUSIONS: Our data clearly showed that BBTV was able to control the transcriptional level of each DNA component independently by through the promoter sequences in the intergenic region. Moreover, a cis-acting element from DNA-N component had a high transcriptional activity.

Development of a gold-nano particle based novel dot immunobinding assay for rapid and sensitive detection of Banana bunchy top virus.

An improved gold nanoparticle based Dot immunobinding assay (DIBA) was developed for the detection of Banana bunchy top virus (BBTV), that is more efficient, sensitive, rapid and simpler than conventional DIBA and ELISA. Instead of enzyme conjugates, gold nanoparticles were used as reporters owing to their unique optical properties. Antibody was raised against expressed recombinant coat protein of BBTV. The gold nanoparticles were conjugated to primary / detection antibody raised following immunization with recombinant coat protein, making it highly specific for the virus. Gold nanoparticle conjugated primary antibody (GCPab) based DIBA developed in this study has a detection efficiency comparable to ELISA. The results of using this assay format for detection of BBTV in banana plants from four geographical regions of India are also presented in this report. The test could detect the virus at sap dilution up-to 10-2. Using this improved DIBA, any lab with basic amenities can perform indexing on large numbers of samples.

Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars.

Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.

Genetic diversity of banana bunchy top virus isolates from China.

Banana bunchy top virus (BBTV) (the genus Babuvirus, the family Nanoviridae) is a single-stranded circular DNA virus with a genome composed of six components designated as DNA-R, -U3, -S, -M, -C, and -N. This study analyzed the nucleotide identities of the DNA-R of 23 isolates from banana-producing provinces of China, including Guangdong, Hainan, Guangxi, and Yunnan. Results showed that the nucleotide identity of DNA-R was 72.3-100%. Phylogenetic analysis indicated that these BBTV isolates were clustered in different subgroups within the Asian group (AG). Sequence analysis of the five other components (DNA -U3, -S, -M, -C, and -N) of the five isolates from China confirmed the results established for DNA-R of these BBTV isolates. This study suggested that the variation of DNA-R from Chinese BBTV isolates was considerably higher than the variation of other AG isolates, but their genetic diversity was low.

Comparative analysis of common regions found in babuviruses and alphasatellite molecules.

Viruses in the genus Babuvirus have multi-component ssDNA genomes and often associate with alphasatellite molecules containing two common motifs, a common-region stem-loop (CR-SL) involved in initiation of rolling-circle replication and a common-region major (CR-M) motif involved in secondary-strand synthesis. We compared known babuvirus genome components and alphasatellite CR-SL and CR-M sequences, defining five divergent CR-SL sequence classes. We identified iterated sequence elements in babuvirus genome components that have particularly conserved sequences and spatial arrangements between known babuviruses.

A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters.

The first generation transgenic crops used strong constitutive promoters for transgene expression. However, tissue-specific expression is desirable for more precise targeting of transgenes. Moreover, piercing/sucking insects, which are generally resistant to insecticidal Bacillus thuringiensis (Bt) proteins, have emerged as a major pests since the introduction of transgenic crops expressing these toxins. Phloem-specific promoters isolated from Banana bunchy top virus (BBTV) were used for the expression of two insecticidal proteins, Hadronyche versuta (Blue Mountains funnel-web spider) neurotoxin (Hvt) and onion leaf lectin, in tobacco (Nicotiana tabacum). Here we demonstrate that transgenic plants expressing Hvt alone or in combination with onion leaf lectin are resistant to Phenacoccus solenopsis (cotton mealybug), Myzus persicae (green peach aphids) and Bemisia tabaci (silver leaf whitefly). The expression of both proteins under different phloem-specific promoters resulted in close to 100% mortality and provided more rapid protection than Hvt alone. Our results suggest the employment of the Hvt and onion leaf lectin transgenic constructs at the commercial level will reduce the use of chemical pesticides for control of hemipteran insect pests.

Low genetic diversity of Banana bunchy top virus, with a sub-regional pattern of variation, in Democratic Republic of Congo.

Banana bunchy top virus (BBTV), belonging to the genus Babuvirus, is the most devastating and widespread banana virus. Banana and plantain are major crops in terms of household income and food security in Democratic Republic of Congo (DRC). Despite the large area under banana and plantain cultivation in the country, before this study, the genetic characterization of BBTV isolates had only been undertaken for two provinces. In the study presented here, genetic variation in BBTV was assessed from 52 BBTV isolates collected in five out of 11 provinces in DRC (Bandundu, Bas-Congo, Katanga, Kinshasa and Kasaï Oriental) and in two provinces using sequences previously described in databases. Full genome sequencing of DNA-R components was performed, revealing low genetic variation (98-100 % nucleotide identity) among the BBTV isolates detected. The phylogenetic analyses showed that all the DRC isolates were clustered in the South Pacific clade of BBTV. Based on the coding region for the replication initiator protein, haplotype diversity was estimated to be 0.944 ± 0.013, with 30 haplotypes from 68 isolates in DRC. Such diversity shows a haplotype distribution mainly at the sub-regional level in DRC. In addition, the sequence determination from the whole genome of selected isolates confirmed low genetic variation among isolates from seven DRC provinces (97-100 % nucleotide identity). This study strengthened the hypothesis of a single BBTV introduction some time ago, followed by the spread of the virus in the country.

Identification and in silico characterisation of defective molecules associated with isolates of banana bunchy top virus.

Banana bunchy top virus (BBTV) is a multi-component single-stranded DNA virus. From 267 potentially infected Musa plants, 24 apparently 'defective' BBTV components have been identified. Interestingly, 23/24 of these defective molecules were apparently derived from DNA-R. All of the identified defective molecules had retained at least part of the CR-SL and CR-M but had insertions and/or deletions that in most cases resulted in open reading frame disruptions. Our detection of three monophyletic but diverse (and therefore likely circulating) defective DNA-R lineages suggests that, in many cases, defective DNA-R molecules might remain associated with BBTV genomes for prolonged periods.

Miniaturized paper-based gene sensor for rapid and sensitive identification of contagious plant virus.

Plant viruses cause significant production and economic losses in the agricultural industry worldwide. Rapid and early identification of contagious plant viruses is an essential prerequisite for the effective control of further spreading of infection. In this work, we describe a miniaturized paper-based gene sensor for the rapid and sensitive identification of a contagious plant virus. Our approach makes use of hybridization-mediated target capture based on a miniaturized lateral flow platform and gold nanoparticle colorimetric probes. The captured colorimetric probes on the test line and control line of the gene sensor produce characteristic red bands, enabling visual detection of the amplified products within minutes without the need for sophisticated instruments or the multiple incubation and washing steps performed in most other assays. Quantitative analysis is realized by recording the optical intensity of the test line. The sensor was used successfully for the identification of banana bunchy top virus (BBTV). The detection limit was 0.13 aM of gene segment, which is 10 times higher than that of electrophoresis and provides confirmation of the amplified products. We believe that this simple, rapid, and sensitive bioactive platform has great promise for warning against plant diseases in agricultural production.

Asymmetric patterns of reassortment and concerted evolution in Cardamom bushy dwarf virus.

Nanoviruses are single-stranded DNA (ssDNA) plant viruses which have multipartite genomes consisting of discrete, individually encapsidated components. This multipartite strategy may lead to high rates of reassortment, whereby entire genome components are exchanged among different strains. However, few studies have explored the extent to which reassortment shapes the genetic diversity of nanovirus populations. Here we present an extensive analysis of reassortment among 163 Cardamom bushy dwarf virus (CBDV; Nanoviridae family, Babuvirus genus) isolates collected in Northeast India. We also examined evidence of recombination, which is known to play a role in the evolutionary dynamics of nanovirus populations. By sequencing six discrete genome components for each isolate, we demonstrate that over 40% of the isolates display evidence of at least one reassortment event during their evolutionary histories. Nevertheless, a bias in the frequencies at which different genome components reassort was observed, with the DNA-M and DNA-N components being the most predisposed to reassortment. This may reflect variation in the ability of different genome components to function efficiently in a foreign genomic background. Comparisons of the common regions of different genome components revealed signatures of concerted evolution mediated by frequent inter-component homologous recombination. This process, which has previously been reported in nanoviruses and other multipartite ssDNA viruses, may allow proteins which initiate replication to maintain control over distinct genome components. Notably, DNA-N, one of the genome components most prone to reassortment, also exhibited the most frequent inter-component homologous recombination. This supports the idea that inter-component homologous recombination may promote the efficient replication of novel components which are introduced into a genome via reassortment.

Identification and characterization of a distinct banana bunchy top virus isolate of Pacific-Indian Oceans group from North-East India.

Banana bunch top virus (BBTV) is considered to be a serious threat to banana production. A new isolate of the virus (BBTV-Umiam) was identified and characterized from local banana mats growing in mid-hills of Meghalaya in North-East India. The complete nucleotide sequence analysis revealed the presence of six full-length ssDNA components (DNA R, DNA U3, DNA S, DNA M, DNA C and DNA N) sharing major common region (CR-M) and a stem-loop common region (CR-SL). BBTV-Umiam showed a unique deletion of 20 nucleotides in the intergenic region of DNA R, the absence of predicted open reading frame (ORF) in DNA U3 and probability for a small ORF in DNA U3 expecting functional evidence at transcriptional level. Phylogenetic analysis based on 88 complete nucleotide sequence of BBTV DNA R available in GenBank generated two broad clusters of Pacific-Indian Oceans (PIO) and South-East Asian (SEA) groups including BBTV-Umiam within PIO cluster. However, BBTV-Umiam was identified as the most distinct member of the PIO group with 100% bootstrap support. This was further supported by the phylogenetic grouping of each genomic component of BBTV-Umiam at the distant end of PIO group during clustering of 21 complete BBTV sequences. BBTV-Umiam shared relatively less nucleotide identity with PIO group for each genomic component (85.0-95.4%) and corresponding ORF (93.8-97.5%) than that of earlier PIO isolates (91.5-99.6% and 96.0-99.3%, respectively). Recombination analysis revealed two intra-component and five inter-component recombination events in BBTV-Umiam, but none of them was unique. Moreover, the isolate was identified as major parental sequence for intra-component recombination event spanning the replication-associated protein encoding region in Tongan BBTV DNA R. The current study indicated differential evolution of BBTV in North-East India (Meghalaya). The natural occurrence of hybrids of Musa balbisiana and M. acuminata in this geographically isolated region could be the contributing factor in accumulating genetic distinctiveness in BBTV-Umiam which need further characterization.