Molecular Characterization of Banana streak virus Isolate from Musa Acuminata in China

Banana streak virus (BSV), a member of genus Badnavirus, is a causal agent of banana streak disease throughout the world. The genetic diversity of BSVs from different regions of banana plantations has previously been investigated, but there are relatively few reports of the genetic characteristic of episomal (non-integrated) BSV genomes isolated from China. Here, the complete genome, a total of 7722bp (GenBank accession number DQ092436), of an isolate of Banana streak virus (BSV) on cultivar Cavendish (BSAc...     

Vector transmission of Banana streak virus in the screenhouse in Uganda

Although mealybug transmission of Banana streak virus.(BSV) by Planococcus citri and Saccharicoccus sacchar has been demonstrated elsewhere, these mealybugs have not been identified on bananas in Uganda and their role and that of other agents in BSV transmission is not well documented. Insect samples were collected from banana farms in sites with low, moderate and high BSV infections in Uganda. Subsequently, live mealybugs and aphids were again collected and used in acquisition, retention and transmission tests, and BSV diagnosed using TAS‐ELISA. Dysmicoccus brevipes (pineapple mealybug), S. sacchari (sugarcane mealybug) and Pentalonia nigronervosa (banana aphid) were the most abundant insect species from banana fields sampled. Abundance of D. brevipes was positively and significantly correlated with BSV incidence unlike that of. P. nigronervosa. Transmission studies in the screenhouse showed that mealybugs acquired BSV one day after feeding on virus sources and approached optimum acquisition after the third day. Pineapple and sugarcane mealybugs retained BSV up to 5 days from the day of transfer from the virus source. BSV was first detected in the recipient banana plants 4 wk after transmission using pineapple mealybug and 6 wk after inoculation using sugarcane mealybug. Under screenhouse conditions, both mealybugs therefore appear to transmit BSV semipersistently.     

Detection of Banana streak virus in field samples of bananas from Uganda

Banana streak virus (BSV) is one of the major constraints to banana production in Uganda. To develop a diagnostic technique, 59 samples were taken from 30 farms at 14 locations across Uganda; a further three samples were taken from infector plants for BSV epidemiology experiments. BSV was found in 51 of the field samples and in the three infector plants. The possible variation of the virus was assessed by serology (ISEM and ELISA) using a broad‐spectrum antiserum and by PCR. Virus was poorly detected in many of the samples by serological tests even though other techniques showed its presence. Virus was detected in most samples by PCR with a degenerate primer set on extracted viral DNA and on immune‐captured (1C) or directly bound (DB) virus particles. The epidemiology experiment samples did not give a product with these degenerate primers but did with other primer sets. A diagnostic procedure was developed involving concentrating the virus in sap by polyethylene glycol precipitation followed by 1C‐ or DB‐PCR using a degenerate primer set which detected virus in most samples.     

Disease spread patterns of Banana streak virus in farmers, fields in Uganda

Three surveys were conducted to establish the disease spread patterns of Banana streak virus (BSV) in farmers, fields in Uganda. Transects were traced both across the fields and from infection foci within a field. BSV incidence in adjacent quadrats was also determined to quantify statistically the spatial relationships of infected plants in the fields. Severity assessment along transects across fields revealed clusters of plants with moderate to high severity and clusters of plants with no BSV or low severity. Symptom severity decreased away from foci of infection (b=?0.014; P=0.0081). Observed frequency of infected quadrat counts differed from corresponding expected frequency of infected quadrat counts (Poisson, s distribution, x²; P<0.01). BSV– infected plants, therefore, were aggregated in well‐established fields. Aggregation of infected plants in farmers, fields and the decrease of severity away from infection foci suggest the likely involvement of a slow moving vector in BSV transmission.     

The effect of Banana streak virus on the growth and yield of dessert bananas in tropical Australia

We have examined the effect of a strain of Banana streak virus (BSV‐Cav) on the growth and yield of dessert bananas (Musa AAA group, Cavendish subgroup cv. Williams) in north Queensland, Australia. Healthy and infected plants were compared in a replicated field experiment over plant and first ratoon crops. In both crops, symptom expression followed a similar pattern, increasing to a maximum near the estimated time of bunch initiation, then decreasing in the period prior to bunch emergence. There was no evidence of plant‐to‐plant spread of virus, but the rate of transmission through suckers was 100%. In the plant crop, the mean bunch weights of healthy and infected plants were not significantly different. However, BSV‐Cav infection resulted in an 18 day delay in harvest, causing a 6% reduction in yield per annum. In the ratoon crop, the mean bunch weight of infected plants was 7% less than that of healthy plants, and the interval between the harvest of plant and ratoon crops was delayed by 9 days, resulting in a 11% reduction in yield per annum. Also, the mean length of fruit from infected plants was 5% less than that of healthy plants, resulting in a smaller percentage of fruit in the extra large size category. We conclude that in horticulturally favourable conditions typical of the tropical Australian banana industry, the effects of BSV‐Cav infection on the growth and yield of Cavendish bananas are small.     

The host range of Tobacco streak virus in India and transmission by thrips

Tobacco streak virus (TSV) recently caused an epidemic in peanut (= groundnut, Arachis hypogaea) crops in Andhra Pradesh, India. In the epidemic area TSV occurred in many widely distributed weeds of which Parthenium hysterophorus probably plays a major role in its spread by thrips. Three thrips species, Megalurothrips usitatus, Frankliniella schultzei and Scirtothrips dorsalis were vectors in the presence of infected pollen. Of crop species, Helianthus annuus (sunflower) and Tagetes patula (marigold) could act as sources of inoculum. In limited tests, the virus was not seed‐transmitted in the peanut cultivar JL‐24 or in the sunflower hybrids KBSH‐41, ‐42, ‐44, and ‐50, MSFH‐17 and ZSH‐976. Strategies adopted to reduce the incidence of TSV are discussed.     

Marker-assisted breeding of <Emphasis Type="Italic">Musa balbisiana</Emphasis> genitors devoid of infectious endogenous Banana streak virus sequences

Breeding new interspecific banana hybrid varieties relies on the use of Musa acuminata and M. balbisiana parents. Unfortunately, infectious alleles of endogenous Banana streak virus (eBSV) sequences are present in the genome of Musa balbisiana genitors. Upon activation by biotic and abiotic stresses, these infectious eBSVs lead to spontaneous infections by several species of Banana streak virus in interspecific hybrids harboring both Musa acuminata and M. balbisiana genomes. Here we provide evidence that seedy M. balbisiana diploids display diverse eBSV allelic combinations and that some eBSVs differ structurally from those previously reported. We also show that segregation of infectious and non-infectious eBSV alleles can be achieved in seedy M. balbisiana diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful breeding of M. balbisiana diploid genitors devoid of all infectious eBSV alleles following self-pollination and on the potential of breeding additional M. balbisiana diploid genitors free of infectious eBSVs by crossing parents displaying complementary eBSV patterns. Our work paves the way to the safe use of M. balbisiana genitors for breeding banana interspecific hybrid varieties with no risk of activation of infectious eBSVs.     

A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus

Sequencing of plant nuclear genomes reveals the widespread presence of integrated viral sequences known as endogenous pararetroviruses (EPRVs). Banana is one of the three plant species known to harbor infectious EPRVs. Musa balbisiana carries integrated copies of Banana streak virus (BSV), which are infectious by releasing virions in interspecific hybrids. Here, we analyze the organization of the EPRV of BSV Goldfinger (BSGfV) present in the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) revealed by the study of Musa bacterial artificial chromosome resources and interspecific genetic cross. cv. PKW contains two similar EPRVs of BSGfV. Genotyping of these integrants and studies of their segregation pattern show an allelic insertion. Despite the fact that integrated BSGfV has undergone extensive rearrangement, both EPRVs contain the full-length viral genome. The high degree of sequence conservation between the integrated and episomal form of the virus indicates a recent integration event; however, only one allele is infectious. Analysis of BSGfV EPRV segregation among an F1 population from an interspecific genetic cross revealed that these EPRV sequences correspond to two alleles originating from a single integration event. We describe here for the first time the full genomic and genetic organization of the two EPRVs of BSGfV present in cv. PKW in response to the challenge facing both scientists and breeders to identify and generate genetic resources free from BSV. We discuss the consequences of this unique host-pathogen interaction in terms of genetic and genomic plant defenses versus strategies of infectious BSGfV EPRVs.     

Efficient Plant Genomic and Viral DNA Isolation from Mature Leaf Midrib of Banana (Musa spp)

The genomic DNA isolation from mature leaf midrib is a tough job, because of the abundance of polysaccharides and secondary metabolites, which interferes with DNA isolation as well as polymerase chain reaction (PCR) studies. The leaf midrib of 3^rd leaf from 3-moths old, ex-vitro developing banana [AAA, Dwarf Cavendish-Basrai (Sindhri banana)] plants (healthy and BBTV infected) was grinded in liquid N₂. Exact 0.3 g of leaf midrib powder was washed with washing buffer (100 mM Tris-Cl, 5 mM EDTA, 0.35 M sorbitol, 1% 2-mercaptoethanol) then homogenized in 0.8 ml of three different pre-heated (60°C) DNA isolation buffers. Supernatant was extracted through phenol: chloroform:isoamyl alcohol (25:24, v/v), chloroform: isoamyl alcohol (24:1, v/v) and finally with chloroform (100%) one by one. Maximum yields were ranged from 49.33 and 27.73 μg mg ^?1 DNA with impurities 5.67 and 5.87 μg mg^?1 through buffer I, while 45.77 and 25.53 μg mg^?1 DNA with 6.13 and 6.16 μg mg^?1 impurities through buffer III from healthy and infected plants respectively. Best one RAPD was observed in all the DNA samples isolated with different buffers, while viral amplification was good in DNA isolated with buffer I and II, when 10 (RAPD) and 25 ng DNA (C ₁ gene) was used as a template in a reaction of 25 μl. Meanwhile, buffer II is limited for viral DNA isolation while buffer I (1M Tris-Cl, 5M NaCl, 2 % cTAB, 50mM EDTA, 1 % PVP, 0.2 % 2-mercaptoethanol) has dual capacity for plant and virus DNA isolation. This described protocol is economic in terms of times, labor and cost.     

香蕉离体茎尖超低温保存研究

以香蕉(Musaspp.)试管苗为试材,对其离体茎尖小滴玻璃化法超低温保存的影响因素进行了研究。小滴玻璃化法和玻璃化法超低温保存后再生率的差异表明,香蕉更适合用小滴玻璃化法进行超低温保存。香蕉小滴玻璃化法超低温保存的方案如下:试管苗在60g/L蔗糖的MS培养基上培养1~2个月,剥离带有1~2片叶原基的茎尖,室温下装载30m in(可延长至4h),0℃下PVS2处理40~50m in。6个基因型的14个品种的再生率平均为46.9%。通过SSR分子标记检测,再生植株的遗传稳定性没有发生改变。该结果为香蕉种质资源的长期保存提供了理论依据和技术支撑。     

Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.     

Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and a new crystal structure of the ligase-adenylate intermediate

Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent DNA ligase known; it suffices for yeast cell growth in lieu of the essential yeast DNA ligase Cdc9. The Chlorella virus ligase–adenylate intermediate has an intrinsic nick sensing function and its DNA footprint extends 8–9 nt on the 3′-hydroxyl (3′-OH) side of the nick and 11–12 nt on the 5′-phosphate (5′-PO₄) side. Here we establish the minimal length requirements for ligatable 3′-OH and 5′-PO₄ strands at the nick (6 nt) and describe a new crystal structure of the ligase–adenylate in a state construed to reflect the configuration of the active site prior to nick recognition. Comparison with a previous structure of the ligase–adenylate bound to sulfate (a mimetic of the nick 5′-PO₄) suggests how the positions and contacts of the active site components and the bound adenylate are remodeled by DNA binding. We find that the minimal Chlorella virus ligase is capable of catalyzing non-homologous end-joining reactions in vivo in yeast, a process normally executed by the structurally more complex cellular Lig4 enzyme. Our results suggest a model of ligase evolution in which: (i) a small ‘pluripotent’ ligase is the progenitor of the much larger ligases found presently in eukaryotic cells and (ii) gene duplications, variations within the core ligase structure and the fusion of new domains to the core structure (affording new protein–protein interactions) led to the compartmentalization of eukaryotic ligase function, i.e. by enhancing some components of the functional repertoire of the ancestral ligase while disabling others.     

In vitro cloning and homestead cultivation of primitive Musa cultivars

Two primitive diploid Musa cultivars, Matti and Chemmatti from the extreme southern part of the Western Ghats were multiplied by in vitro culture of sucker-derived shoot apices. Decontaminated corm explants (1 cm x 1 cm) having shoot apex (approximately 0.3 cm) cultured for 1 month in Murashige and Skoog basal agar medium was cut vertically into eight segments and each segment having a part of shoot meristem was cultured in presence of 6-benzylaminopurine (BAP) and combinations of BAP and indole-3-acetic acid (IAA) or indole-3-butyricacid (IBA) to produce multiple shoots. After 12 weeks of culture, maximum number of shoots (32) in both the cultivars were produced in approximate 60% of the explants in presence of BAP and IAA each at 1.5 mg/l(-1) (Matti) and 40% of the explants in 2.5 mg/l(-1) of BAP and 1.5 mg/l(-1) of IAA (Chemmatti). Buds were formed from the base of the subcultured shoots and somewhat more number (34) of shoots were obtained in Matti than in Chemmatti (31) after 8 weeks. Difference in the concentration of cytokinin required for shoot initiation and multiplication, persistence of exudation through the subculture and red colouration of the early formed sheathing leaf bases in the shoots in Chemmatti indicated possible genotypic differences between the two cultivars. Multiple shoot proliferation achieved through five subcultures of the isolated shoots without any decline. Transfer of shoots (4-5 cm) into MS basal medium favoured rooting in 4 weeks and rooted plants (9 cm) were hardened and established (80-95%). Mericlones of Matti cultivated in homesteads produced bunches of uniform characters in 13 months.     

Natural host range,thrips and seed transmission of distinct Tobacco streak virus strains in Queensland,Australia

Diseases caused by Tobacco streak virus (TSV) have resulted in significant crop losses in sunflower and mung bean crops in Australia. Two genetically distinct strains from central Queensland, TSV‐parthenium and TSV‐crownbeard, have been previously described. They share only 81% total‐genome nucleotide sequence identity and have distinct major alternative hosts, Parthenium hysterophorus (parthenium) and Verbesina encelioides (crownbeard). We developed and used strain‐specific multiplex Polymerase chain reactions (PCRs) for the three RNA segments of TSV‐parthenium and TSV‐crownbeard to accurately characterise the strains naturally infecting 41 hosts species. Hosts included species from 11 plant families, including 12 species endemic to Australia. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV‐crownbeard. By contrast, crownbeard was both a natural host of, and experimentally infected by TSV‐parthenium but this infection combination resulted in non‐viable seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. TSV‐crownbeard was seed transmitted from naturally infected crownbeard at a rate of between 5% and 50% and was closely associated with the geographical distribution of crownbeard in central Queensland. TSV‐parthenium and TSV‐crownbeard were also seed transmitted in experimentally infected ageratum (Ageratum houstonianum) at rates of up to 40% and 27%, respectively. The related subgroup 1 ilarvirus, Ageratum latent virus, was also seed transmitted at a rate of 18% in ageratum which is its major alternative host. Thrips species Frankliniella schultzei and Microcephalothrips abdominalis were commonly found in flowers of TSV‐affected crops and nearby weed hosts. Both species readily transmitted TSV‐parthenium and TSV‐crownbeard. The results are discussed in terms of how two genetically and biologically distinct TSV strains have similar life cycle strategies in the same environment.     

Banana Xanthomonas wilt dynamics with mixed cultivars in a periodic environment Juliet

In this paper,we study a deterministic model with non-autonomous system for mixed cultivars to assess the effect of cultivar susceptibility and seasonal variation on banana Xanthomonas wilt(BXW)disease dynamics.A special case of two cultivars classified as highly susceptible for inflorescence infection(ABB)and less susceptible(AAA)cultivar is considered.The basic reproduction number corresponding to the non-autonomous sys­tem is derived and numerically computed to determine disease dynamics.Results showed that the disease dies out whenever the periodic basic reproduction number is less than unity and a periodic solution is obtained when it is greater than one.Results further showed that for both cultivars,the basic reproduction number increases with increas­ing values of the transmission rates and declines exponentially with increasing values of roguing rates.The critical roguing rate of ABB-genome cultivar was higher than that of AAA-genome cultivars.The peaks in disease prevalence indicate the importance of effective implementation of controls during the rainy season.We conclude that highly susceptible cultivars play an important role in the spread of BXW and control measures should be effectively implemented during the rainy season if BXW is to be eradicated.     

Identification of genetic markers linked to banana streak disease expression in inter-specific Musa hybrids

Recently-introduced inter-specific Musa hybrids, bred for improved yield and resistance to diseases, have been found to be widely infected with banana streak virus (BSV), the causal agent of banana streak disease (BSD). One hypothesis suggests: (1) that BSD occurrence in these inter-specific hybrids results from activation of BSV-Ol endogenous pararetrovirus sequences (EPRV) integrated into the Musa genome rather than from external sources of infection, and (2) that the process of genetic hybridisation may be one factor involved in triggering episomal expression of the BSV integrants. In order to test this hypothesis we carried out a genetic analysis of BSD incidence in a F1 triploid ( Musa AAB) population produced by inter-specific hybridisation between virus and disease-free diploid Musa balbisiana (BB) and tetraploid Musa acuminata (AAAA) parents. Half of the F1 progeny of this cross expressed BSV particles. Using PCR amplification to determine the presence or absence of BSV-Ol EPRVs, it was determined that this endogenous sequence was specific to the M. babisiana genome and occurred in a homozygous state. Using bulk segregant analysis, ten AFLP markers co-segregating with the absence and/or presence of BSV infection were identified in the M. balbisiana genome, but were absent from the M. acuminata genome. Seven of these markers segregated with the presence of a BSV particle and three with the absence of BSV particles. Analysis of the segregation of these markers using a test-cross configuration allowed the construction of a genetic map of the linkage group containing the locus associated with BSV infection in the F1 hybrid population. These data indicate that a genetic mechanism is involved in BSV appearance, and suggest that a monogenic allelic system confers the role of carrier to the M. balbisiana parent.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.