The complete sequence of a sugarcane mosaic virus isolate causing maize dwarf mosaic disease in China

The complete sequence of a potyvirus from maize in Zhejiang Province was determined. The RNA was 9596 nucleotides long, excluding the 3′-poly (A) tail, and there was a single long open reading frame (ORF) of 9192 nts encoding a 346.1 ku polyprotein. The polyprotein had substantial amino acid sequence homology with those encoded by the RNAs of a Chinese isolate of sorghum mosaic virus (SrMV-C) and a Bulgarian isolate of maize dwarf mosaic virus, but it was most closely related to sugarcane mosaic virus (SCMV) isolates, for which only partial sequences have been published. According to the published criteria for distinguishing potyviruses, the sequence reported here is clearly a strain of SCMV, but it also showed a surprisingly high amino acid homology with SrMV-C in the HC-Pro, P3 and Cl proteins.     

Genetic diversity and population structure of Sorghum mosaic virus infecting Saccharum spp. hybrids

Sugarcane mosaic disease is widespread in many countries and has been identified to be caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV). Viral surveys of SCMV, SrMV and SCSMV were performed from 104 leaf samples of Saccharum spp. hybrid growing in China and two leaf samples in Myanmar. Sorghum mosaic virus was a major causal agent for sugarcane mosaic disease in China whereby 72.1% (75/104) of samples had SrMV infection alone, 6.7% (7/104) were mixed with SCMV and 17.3% (18/104) were mixed with SCSMV. Sugarcane streak mosaic virus infection alone occurred in 3.8% (4/104) of samples, but no single infections were observed for SCMV. Two viruses (SrMV and SCSMV) were detected in sugarcane mosaic samples in Myanmar. Phylogenetic analysis revealed that all of the SrMV isolates were clustered into three major lineages encompassing six phylogroups/genotypes based on the CP sequences (825 nucleotides) of 113 Chinese and 2 Burmese isolates from this study and 73 isolates reported worldwide. Six clearly distinct SrMV phylogroups (G1–G6) were formed and shared 74.3–94.1% nucleotide identity and 84.7–98.1% amino acid identity of CP sequences. SrMV‐G5 was identified to be new distinct phylogroup that was restricted to the Fujian and Guangxi provinces. The unique SrMV‐G6 phylogroup only occurred in Yunnan province. Insertion/deletion mutations, negative selection and frequent gene flow are factors driving the genetic evolution and population structure of SrMV in China.     

The complete sequence of a sugarcane mosaic virus isolate causing maize dwarf mosaic disease in China

The complete sequence of a potyvirus from maize in Zhejiang Province was determined. The RNA was 9596 nucleotides long, excluding the 3'-poly (A) tail, and there was a single long open reading frame (ORF) of 9192 nts encoding a 346.1 ku polyprotein. The polyprotein had substantial amino acid sequence homology with those encoded by the RNAs of a Chinese isolate of sorghum mosaic virus (SrMV-C) and a Bulgarian isolate of maize dwarf mosaic virus, but it was most closely related to sugarcane mosaic virus (SCMV) isolates, for which only partial sequences have been published. According to the published criteria for distinguishing potyviruses, the sequence reported here is clearly a strain of SCMV, but it also showed a surprisingly high amino acid homology with SrMV-C in the HC-Pro, P3 and Cl proteins.     

Impact of serial thermotherapy on sugarcane mosaic virus titre and regeneration in sugarcane: Auswirkung serieller thermotherapie auf zuckerrohr-mosaikvirustiter und regeneration bei zuckerrohr

Serial thermotherapy with hot water involving different treatment series was followed in two sugarcane cultivars namely Co 740 and CoC 671 for assessing its effect on sugarcane mosaic virus (SCMV) titre in the regenerated plants. Observations on virus titre and sett regeneration were recorded at different intervals after planting the treated setts. The direct antigen coating (DAC) ELISA method was followed with the polyclonal antisera raised against the widespread local SCMV strain. All the thermo treatments affected the sett regeneration in both the cultivars. Although none of the treatment series produced virus free plants, the SCMV titre was comparatively reduced in the regenerated plants at an early phase (4 weeks after planting) in severe hot water treatment series but drastic reduction in sett germination was observed. However, the SCMV titre picked up later (8 weeks after planting) in all the treatment series. The early reduction in SCMV titre could be utilized for obtaining more virus free plants in meristem culture.     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

In vitro culture of sugarcane infected with maize streak virus (MSV)

Young buds and leaf tissues of sugarcane cv. R 574 (a Saccharum hybrid), healthy or infected with Maize Streak Virus (MSV) were cultured in vitro. The success rate of the cultures from infected material is lower than that from healthy material. The symptoms of the disease were displayed on most of the plantlets produced from buds but only on 6% of the plantlets regenerated from calli. The indirect double-antibody sandwich ELISA technique using a monoclonal antibody makes it possible to identify virus-infected plantlets. In vitro, the infection results in the reduced growth of shoots.     

Elimination of five viruses from sugarcane using in vitro culture of axillary buds and apical meristems

Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus elimination through meristem tissue culture originating from both apical and axillary shoots were compared. The average rates of regeneration and elongation from apical meristem tissues were 91 and 66%, respectively, with the virus-free rate among elongated shoots ranging from 61–92%. Mature axillary buds were cultivated in vitro to produce axillary shoots, from which meristem tissues were excised and cultured. These meristem tissues regenerated (77–100%) and elongated (55–88%) in culture medium at approximately the same rate as the apical meristems. The average virus elimination rate was 90% among elongated shoots derived from mature axillary buds. All five viruses can be eliminated by meristem tissue culture from both apical and axillary shoots using a standardized procedure. The overall average efficiency of virus-free plant production was 45 and 58% from apical and axillary shoots, respectively. There were no significant differences for shoot induction or virus elimination when the meristems were harvested from either the apical or the axillary shoots. This is the first report of SrMV or SCSMV elimination from sugarcane, as well as elimination of any mixed virus infections. This new method of harvesting meristems from axillary buds greatly expands the amount of material available for therapeutic treatments and thereby increases the probability of eliminating viruses from infected sugarcane.     

A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane

RNA interference (RNAi) is commonly used to produce virus tolerant transgenic plants. The objective of the current study was to generate transgenic sugarcane plants expressing a short hairpin RNAs (shRNA) targeting the coat protein (CP) gene of sugarcane mosaic virus (SCMV). Based on multiple sequence alignment, including genomic sequences of four SCMV strains, a conserved region of ~ 456 bp coat protein (CP) gene was selected as target gene and amplified through polymerase chain reaction (PCR). Subsequently, siRNAs2 and siRNA4 were engineered as stable short hairpin (shRNA) transgenes of 110 bp with stem and loop sequences derived from microRNA (sof-MIR168a; an active regulatory miRNA in sugarcane). These transgenes were cloned in independent RNAi constructs under the control of the polyubiquitin promoter. The RNAi constructs were delivered into two sugarcane cultivars ‘SPF-234 and NSG-311 in independent experiments using particle bombardment. Molecular identification through PCR and Southern blot revealed anti-SCMV positive transgenic lines. Upon mechanical inoculation of transgenic and non-transgenic sugarcane lines with SCMV, the degree of resistance was found variable among the two sugarcane cultivars. For sugarcane cultivar NSG-311, the mRNA expression of the CP–SCMV was reduced to 10% in shRNA2-transgenic lines and 80% in shRNA4-transgenic lines. In sugarcane cultivar SPF-234, the mRNA expression of the CP–SCMV was reduced to 20% in shRNA2-transgenic lines and 90% in shRNA4 transgenic lines, revealing that transgenic plants expressing shRNA4 were almost immune to SCMV infection.     

Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue‐cultured plantlets grown in vitro. Six mutants of strain XaFL07‐1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly‐β‐hydroxybutyrate than the wild‐type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non‐ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild‐type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.     

Field performance of micropropagated forestry species

Summary Micropropagated plants both from angiosperms (bamboo, birch, eucalyptus, tamarind, teak, willow) and gymnosperms (Douglas-fir, loblolly pine, Monterey pine, and redwood) have been established in the field. Plantlets were regenerated from juvenile explants (via adventitious or axillary buds) as well as explants from mature trees [apical and axillary buds (nodal segments)]. Plantlets regenerated from adventitious buds tend to show early maturation traits (Douglas fir, loblolly pine, Monterey pine). A population of selected clones showed superior performance and yield over seedlings derived from the same trees. Increased biomass production was obtained with plantlets derived from tissue culture ofEucalyptus spp. when compared to seedlings. No morphologic variation was observed in micropropagated plants. Plantlets derived from tissue culture grew very uniformly. Early flowering was observed with plantlets derived from tissue culture (tamarind, teak). Based on a presentation at an International Training Course on the Application of Biotechnology in Forest Trees held in Caracas, Venezuela, May 1991.     

Induced defence responses and protective effects on tomato against Xanthomonas vesicatoria by an aqueous extract from Solanum lycocarpum infected with Crinipellis perniciosa

This study investigated the induced defence responses and protective effects on susceptible tomato (Lycopersicon esculentum Mill.) against Xanthomonas vesicatoria (Doidge) by a heat-treated aqueous extract (VLA) from dry necrotic tissue of ‘Lobeira’ (Solanum lycocarpum St. Hil.) branches infected with the fungus Crinipellis perniciosa (Stahel) compared with acibenzolar-S-methyl (ASM), a commercial inducer of resistance. Plantlets were sprayed with VLA and ASM and challenged 4 days later with a virulent strain of X. vesicatoria, under greenhouse conditions. The disease severity, fresh weight of shoots, the activities of phenol peroxidase (POX), polyphenol oxidase (PPO), chitinase (CHI), phenylalanine ammonia-lyase (PAL), lignin deposition, and soluble phenolic contents were evaluated in the leaf tissues. Reduction of the bacterial spot severity was observed in plantlets treated with VLA which conferred 63% of the ASM protection. This protective effect and lesion reduction promoted by VLA were probably associated particularly with POX and PAL activities, lignin deposition on leaf tissues and, to a less extent, CHI activity.     

In vitro reactions of sugarcane (Saccharum SP.) plantlets inoculated with 2 strains of Xanthomonas albilineans (Ashby) Dowson

The symptoms of the leaf scald disease can be reproduced in vitro through the inoculation of sugarcane tissue culture plantlets. The pathogen is detected in the inoculated plantlet and is maintained at the surface of the base of the plantlets grown in vitro. Two strains of X. albilineans belonging to different serovars and lysovars reacted like pathotypes. The importance of the plant incubation temperature is clearly demonstrated. Further, in vitro the disease goes through the same phase of latency as in the field.     

Meristem culture and virus eradication in Alstroemeria

Stem apical meristems, rhizome apical meristems and rhizome axillary meristems excised from Alstroemeria plants were grown in vitro on modified Murashige and Skoog (MS) media containing different concentrations of gibberellic acid and 6-benzylaminopurine (BA). Plantlets developed from stem apical meristems never regenerated a rhizome and eventually died. The highest regeneration rate (74.1%) of plantlets with a rhizome was observed when rhizome axillary meristems were grown on modified MS medium containing M 8.9 of BA. Alstroemeria mosaic potyvirus (AlMV) could be eradicated from infected Alstroemeria plants through meristem culture. The rate of virus eradication was 73.7 and 14.7% for plantlets developing from explants measuring 0.7 mm and 2.0 mm, respectively. Greenhouse evaluation of virus-negative and AlMV-infected Alstroemeria plants showed that healthy plants produced more floral stems, more vegetative stems, longer floral stems and gave a higher fresh weight than infected plants.     

Photosynthesis in Sugarcane Varieties Infected with Strains of Sugarcane Mosaic Virus

Symptoms of sugarcane mosaic virus disease caused by strains B and D were accompanied by a reduction in the rate of photosynthesis per unit area; the severe symptoms of strain A and the mild symptoms of strain I produced rates no different from those of healthy plants of variety C.P. 31-588. All four strains decreased chlorophyll content and increased the amount of light transmitted by the leaf. Photosynthetic rates were reduced less than the reduction of chlorophyll would indicate. Symptoms moderated on older leaves, but rates of photosynthesis per dm² were less in older leaves of both healthy and diseased plants. Sugarcane varieties differed in their rates of photosynthesis in both healthy and in virus-infected plants; a significant reduction in photosynthetic rate by sugarcane mosaic occurred in three of the four varieties.     

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.     

Characteristics of carrot plant regenerated from two cell line selected as either ionic-Al tolerant cell or Al-phosphate utilizing cells

Plantlets of carrot (Daucus carota L.) were regenerated from two types of cell lines. One type was selected as ionic-Al tolerant (IAT) cells, while the second type featured Al-phosphate utilizing cells (IPG). Their tolerance characteristics were investigated. The plantlets from IAT were directly regenerated, whereas those from IPG were regenerated after somatic hybridization with wild-type cells previously inactivated with iodacetamide, because the IPG cells had completely lost the ability to regenerate naturally.The sexual progeny of IAT showed Al-tolerant properties, established by testing their root elongation in the presence of 500 µM Al ions. Most of the calli obtained from the somatic hybrids grew more rapidly than the wild-type cells when Al-phosphate was used as a sole source of phosphorus. Thus, we obtained two types of carrot plantlets, regenerated from IAT and IPG. Both possessed the tolerant characteristics as observed with the stress-selected cells.     

In vitro organogenesis and somatic embryogenesis from leaf explants of Leucosceptrum canum sm.

Plantlets were obtained from leaf explants of a Labiatae tree — Leucosceptrum canum Sm. using plant tissue culture techniques. Two types of calli proliferated from the leaf explants when grown on different media, one of which was amenable to somatic embryogenesis. Differentiation of the embryoids started from the fourth passage of culture and continued up to the seventh passage. The number of embryoids decreased with the age of the callus. The capacity of such embryoids to form entire plantlets was studied using different nutrient mileux. Embryoids formed plantlets on Murashige and Skoog's (MS) medium fortified with benzylaminopurine plus indolebutyric acid. Organogenesis was observed in shoot-buds derived from explants of in vitro regenerated plantlets on MS basal medium supplemented with benzylaminopurine. Culture regenerated plantlets were transferred to MS medium without sucrose and growth hormones; finally transferred to pots containing sterile vermiculite where they are growing.Abbreviations MS Murashige and Skoog's medium - 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - Kn kinetin - BAP benzylaminopurine - CW coconut water     

Transformation and regeneration of the self-incompatible species Nicotiana alata Link & Otto

A transformation and regeneration system has been developed for Nicotiana alata, a plant which is being intensively studied as a model of gametophytic self-incompatibility. Plantlets can be regenerated efficiently from seedling hypocotyls. Kanamycin-resistant, transformed plants have been obtained by cocultivation of regenerating hypocotyls with Agrobacterium tumefaciens strain LBA4404 containing a binary vector. The transformation frequency was low with <1% of tissue explants regenerating transformed plants. The transformed plants contained from one to three copies of the introduced DNA. In most cases, the kanamycin resistance phenotype was transmitted to the offspring as a normal Mendelian factor. In one unusual case, none of the offspring inherited the kanamycin resistance of the transformed maternal parent. This plant may have been chimeric or the kanamycin resistance gene may have been inactivated.     

Investigation into the ability of Gluconacetobacter sacchari to live as an endophyte in sugarcane

The relatively low numbers and sporadic pattern of incidence of the acetic acid bacterium Gluconacetobacter sacchari with the pink sugarcane mealybug (PSMB) Saccharicoccus sacchariCockerell (Homoptera: Pseudococcidae) over time and from different sugarcane-growing regions do not indicate that Glac. sacchari is a significant commensal of the PSMB, as has been previously proposed. This study was conducted to investigate the hypothesis that Glac. sacchari is, like its closest relative Glac. diazotrophicus, an endophyte of sugarcane (Saccharum officinarum L.). In this study, bothGlac. sacchari and Glac. diazotrophicus were isolated from internal sugarcane tissue, although the detection of both species was sporadic in all sugarcane-growing regions of Queensland tested. To confirm the ability of Glac. sacchari to live endophytically, an experiment was conducted in which the roots of micropropagated sugarcane plantlets were inoculated with Glac. sacchari, and the plantlets were subsequently examined for the presence of the bacterium in the stem cells. Pure cultures of Glac. sacchari were grown from homogenized surface sterilized sugarcane stems inoculated withGlac. sacchari.Electron microscopy was used to provide further conclusive evidence that Glac. sacchari lives as an endophyte in sugarcane. Scanning electron microscopy of (SEM) sugarcane plantlet stems revealed rod-shaped cells of Glac. sacchari within a transverse section of the plantlet stem cells. The numbers of bacterial cells inside the plant cell indicated a successful infection and colonization of the plant tissue. Using transmission electron microscopy, (TEM) bacterial cells were more difficult to find, due to their spatial separation. In our study, bacteria were mostly found singularly, or in groups of up to four cells inside intercellular spaces, although bacterial cells were occasionally found inside other cells.