Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

A Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)-ELISA, triple antibody sandwich (TAS)-ELISA, antigen coated plate (ACP)-ELISA, and protein-A coated antibody sandwich (PAS)-ELISA. TAS-ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP-ELISA and PAS-ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV-infected plants from asymptomatic plant samples than did TAS-ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS-ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV-indexing of field samples, tissue sampling should be done during the rainy season when most BSV-infected plants express severe symptoms.     

Evaluation of micropropagated plantain and banana (Musa spp.) for banana streak badnavirus incidence under field and screenhouse conditions in Nigeria

Between 1991 to 1996, more than 50 Musa hybrids and 10 landraces were evaluated under field and screenhouse conditions for virus symptoms resembling those caused by banana streak badnavirus (BSV). The symptoms included chlorotic streaks, leaf deformation, stunting, cigar leaf death, distortion of the peduncle, bunch or fruits, and internal pseudostem necrosis. Immunosorbent electron microscopy (ISEM) of randomly selected plants with one or more of these symptoms confirmed the presence of BSV particles in 15 tropical Musa plantain hybrids (TMPx) and five Musa landraces. Under both field and screenhouse conditions, the incidence of symptomatic plants in the hybrids was significantly higher than in the landraces. The hybrids also generally had a higher concentration of BSV antigens, as determined by enzyme-linked immunosorbent assay (ELISA). By contrast, most BSV-infected landraces were symptomless and had very low or undetectable amounts of BSV antigens. There was a significant variation in incidence of symptomatic plants between genotypes, experiments and year of observation. These results are discussed in relation to the higher natural BSV incidence observed on some Musa hybrids as compared with their parental genotypes.     

Recovery from Cucumber Mosaic Virus Infection for ‘Calwonder’ Bell Pepper Plants Does not Counter Negative Impacts on Plant Growth

Cucumber mosaic virus (CMV) accumulation in leaves and stems of infected bell pepper plants at specific symptom stages was evaluated with an emphasis on the transition from full infection to recovery from Cucumber mosaic disease. Four symptom phases occurred in successive order, designated chlorosis (leaves 6–8), mosaic (leaves 9–11/12), leaf distortion (first series of leaves on secondary and tertiary branches) and recovery (progressive recovery with newly emerging leaves in tertiary and younger branches). In situ detection of CMV in leaf tissues revealed widespread occurrence in leaves expressing chlorosis and mosaic symptoms but reduced, localized occurrence in leaves in the recovery phase. Similarly, CMV accumulated to high levels throughout stems expressing chlorosis and mosaic symptoms but with dramatically reduced levels for plants in the recovery symptom phase. Stunting of internodes occurred at all locations above the inoculated leaves by the first expression of systemic symptoms, suggesting an impact on stem growth in response to initial virus invasion of young developing tissues of the stem. Despite the recovery from CMV infection, plant growth was negatively impacted early in the infection process and remained so through the course of the experiment.     

Detection of maize streak virus antigens over time in different parts of maize plants of a sensitive and a so-called tolerant cultivar by ELISA

Maize streak virus (MSV) capsid antigens were detected over time in different parts of maize plants of a sensitive (INRA508) and a so-called tolerant (“tolerant”) cultivar (IRAT297) using a direct or an indirect double antibody sandwich ELISA. Based on three types of experiments, it was shown that the antigens were distributed in the plant according to the age of the tissues. When the virus was inoculated on a particular leaf of 18-day old plants with infective Cicadulina mbila, only the young leaves above the inoculated one were positive by ELISA but not the older ones below. The antigens could not be detected in the inoculated leaf. At day 3 after inoculation, the antigens were detected in the sheath and/or in the whorl of the third leaf above the inoculated one but not in the oldest part of the leaf, the unfolded lamina. Plants of the sensitive cultivar were inoculated at 9 days with C. mbila deposited in the whorl. At 23 h after inoculation, the antigens were detected in the sheath but not in the whorl which was found to be positive only at 32 h. On the basis of these results, a hypothesis of the mode of virus infection is proposed. Our results contribute to a better understanding of the relationship between the age of the plant at inoculation and yield loss as well as secondary infection. By transmission tests with C. mbila, it was shown that virus could only be acquired from leaves exhibiting symptoms. Virus concentrations were measured in plant samples by ELISA using a range of dilutions of purified virus. The virus concentrations were higher in the sensitive than in the “tolerant” cultivar, but no difference in antigen distribution was observed between the two cultivars. The “tolerant” cultivar appeared to be resistant to virus multiplication.     

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.     

Incidence and infection rate of Maize streak virus disease by Cicadulina triangular on maize plants and its distribution from the lowest diseased leaf under tropical conditions

Several programmes have been initiated for the development of maize varieties with resistance traits of Maize streak virus (MSV) by International Institute of Tropical Agriculture (IITA), Ibadan, and have been released to farmers and research scientists. Therefore, a survey was conducted in five states in the south west of Nigeria (Oyo, Ogun, Ondo, Ekiti and Osun) during the raining planting season to determine the incidence of MSV disease by visual examination and sero-diagnostic screening of symptomatic plants. The determination of infection rate of MSV disease by Cicadulina triangular on maize plant and its distribution from the lowest diseased leaf was also studied. The mean MSV disease incidence observed in these states was 35.95% which confirms the presence of MSV in the south west of Nigeria. Sero-diagnostic screening of virus-induced symptomatic leaf samples indicated that out of the 250 leaves sampled per state, 24.4% tested positive for MSV in Oyo, 25.6% in Ondo, 34% in Ogun, 19.6% in Ekiti and 38.8% in Osun. In two-week-old plants, symptoms developed on the leaves that were emerging at the time of inoculation, while in six-week-old plants, symptoms developed on the leaves directly below the emerging leaves irrespective of the number of C. triangular used. These suggest that the lowermost leaf with symptoms of the disease indicates the growth stage at which a plant was infected. There was a relationship between symptom expression and plant age which could be very effective when carrying out surveys to gather information for epidemiological studies. In addition, the 10 varieties of maize inoculated with MSV through C. triangular transmission showed no significant difference in disease severity over time irrespective of the number of C. triangular used.     

Biopriming of micropropagated banana plants at pre- or post-BBTV inoculation stage with rhizosphere and endophytic bacteria determines their ability to induce systemic resistance against BBTV in cultivar Grand Naine

ABSTRACT

Rhizospheric and endophytic bacteria isolated from the roots and corms of banana were tested to find out their efficiency in controlling against banana bunchy top virus (BBTV). Bioformulations of mixtures of endophytic Bacillus pumilus and B. subtilis isolated from banana cv. Grand Naine and rhizobacterial isolate Pseudomonas fluorescens (Pf1) were found to be effective in increasing the growth and physiological parameters such as pseudostem girth and height, number of leaves, phyllochron, and leaf area in biohardened plants under greenhouse study. The consortia of bioformulation mixture of B. pumilus, B. subtilis, and P. fluorescens I showed 61.62% disease reduction over control. The defence enzymes such as peroxidase (POX), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and total phenol were induced to an elevated level in biohardened plants. The applications of bioformulations to plants led to delay the symptom expression for 63.75 to 70.50 days compared to control after challenge inoculation with the virus in 34–67% of plants that exhibited the symptoms till 150 DAI. However, biohardening of plants with the same combinations of bacteria three days after BBTV inoculation led to express the symptoms 29.16 to 36.71 days and there was a significant decrease in plant growth parameters. Biopriming prior to BBTV infection has attributed to the enhanced plant growth and resistance against BBTV whereas, the same treatments after virus inoculation did not induce resistance. This study has proved that the time of application of consortia of bio-inoculants determines their effect of induced resistance to BBTV in micropropagated plants.     

Age-related Resistance in Bell Pepper to Cucumber mosaic virus

We demonstrated the occurrence of mature plant resistance in Capsicum annuum‘Early Calwonder’ to Cucumber mosaic virus (CMV) under greenhouse conditions. When Early Calwonder plants were sown at 10 day intervals and transplanted to 10‐cm square pots, three distinct plant sizes were identified that were designated small, medium and large. Trials conducted during each season showed that CMV accumulated in inoculated leaves of all plants of each size category. All small plants (with the exception of the winter trial) developed a systemic infection that included accumulation of CMV in uninoculated leaves and severe systemic symptoms. Medium plants had a range of responses that included no systemic infection to detection of CMV in uninoculated leaves with the systemically infected plants being either symptomless or expressing only mild symptoms. None of the large plants contained detectable amounts of CMV in uninoculated leaves or developed symptoms. When plants were challenged by inoculation of leaves positioned at different locations along the stem or different numbers of leaves were inoculated, large plants continued to accumulate CMV in inoculated leaves but no systemic infection was observed. When systemic infection of large plants did occur, e.g. when CMV‐infected pepper was used as a source of inoculum, virus accumulation in uninoculated leaves was relatively low and plants remained symptomless. A time‐course study of CMV accumulation in inoculated leaves revealed no difference between small and large plants. Analyses to examine movement of CMV into the petiole of inoculated leaves and throughout the stem showed a range in the extent of infection. While all large plants contained CMV in inoculated leaves, some had no detectable amounts of virus beyond the leaf blade, whereas others contained virus throughout the length of the stem but with limited accumulation relative to controls.     

Infection with Phloem Limited Abutilon Mosaic Virus Causes Localized Carbohydrate Accumulation in Leaves of Abutilon striatum: Relationships to Symptom Development and Effects on Chlorophyll Fluorescence Quenching During Photosynthetic Induction

Abstract: Infection with phloem limited Abutilon Mosaic Virus caused localized carbohydrate accumulation (high levels of starch, sucrose, and hexoses) in leaves of Abutilon striatum during early symptom development. In mature leaves with attenuated symptoms, tissues showing faint vein-clearing had markedly higher carbohydrate contents than uniformly green areas of the same leaf. A similar pattern of carbohydrate accumulation was found in pale-green mosaics in mature leaves with overt symptoms when compared to green-islands of the same leaf, but overnight carbohydrate loses were comparable to controls. Because leaves with attenuated symptoms showed no further symptom development whereas the pale-green mosaics became yellow and eventually necrotic in leaves with overt symptoms, it seems unlikely that carbohydrate accumulation following impaired translocation was responsible for symptom expression. High carbohydrate status in leaves with attenuated symptoms had little effect on nonphotochemical quenching during early stages of photosynthetic induction. In leaves with overt symptoms, areas of high carbohydrate status with pale-green mosaics showed markedly slower nonphotochemical quenching. Early symptom areas of young leaves, and advanced symptom areas of mature leaves had low starch contents but were otherwise similar to controls in carbohydrate status. Impaired nonphotochemical quenching in these tissues tended to reflect the state of symptom development, rather than carbohydrate status. Plants with overt symptoms grew about half as fast as plants with attenuated symptoms.     

Rapid Detection of Banana Streak Virus by Loop‐mediated Isothermal Amplification Assay in South China

Banana streak virus (BSV) is a significant constraint to banana production and genetic improvement. It is necessary to develop and use BSV detection strategies that are both reliable and sensitive for the management of the virus. A loop‐mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of BSV. Four primers matching a total of six sequences of the conserved ORF III polyprotein genes were synthesized for developing a specific and sensitive LAMP for DNA extracts from field‐infected banana plants. LAMP assay could detect as low as 1 pg/μl template DNA. Test results of all field samples collected from different regions of South China showed that LAMP is more sensitive than PCR. This relatively simple and sensitive technique showed excellent potential with field‐collected samples and for routine screening of tissue culture materials in South China.     

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.     

Resistance of Pepper Lines to the Movement of Cucumber Mosaic Virus

The multiplication and the migration of cucumber mosaic virus (CMV) were studied in greenhouse conditions in one susceptible ‘Yolo wonder’ and two resistant ‘Milord’ and ‘Vania’ pepper varieties. DAS-ELISA tests have revealed that the virus is replicated in inoculated leaves of the resistant varieties as high as in the susceptible variety. In the susceptible variety ‘Yolo wonder’, CMV migrated from the leaf lamina to the petiole two days after inoculation and it became systemic three days later regardless the season. In ‘Milord’ the virus migrated from the leaf lamina to the petiole five days after inoculation and it became systemic during the winter 16 days after inoculation. Whereas plants of the same genotype were not infected systemically during the summer. In ‘Vania’, during the two seasons, CMV spread from the blade to the petiole five days after inoculation, but the virus was not detected beyond the inoculated leaf. These results show that ‘Milord’ and ‘Vania’ are resistant to CMV migration. Therefore, the resistance to CMV migration is affected by plant genotype and temperature. The study of effect of pepper plant phenology on infection has revealed that resistance to CMV migration is also affected by the development stage of the plants.     

Effect of banana bunchy top virus infection on morphology and growth characteristics of banana

Field experiments were conducted in Oahu, Hawaii, to investigate the effects of banana bunchy top virus (BBTV) infection on growth and morphology of banana ( Musa acuminata ). The time interval between aphid inoculation of BBTV and the initial appearance of disease symptoms (i.e. incubation period) was also determined. Plants infected with BBTV showed a significant reduction in petiole size (i.e. length and distance), plant canopy and height, leaf area, pseudostem diameter and chlorophyll content compared with control plants. Growth differences between virus-infected and control plants were not observed until 40–50 days after the plants were inoculated with viruliferous aphids. Other growth parameters such as petiole width and leaf production were not statistically different between infected and control plants. The incubation period of banana bunchy top disease or appearance of symptoms ranged from 25 to 85 days after aphid inoculation. However, PCR assays provided earlier detection of BBTV in banana plants compared with visual symptoms.     

Cultivar-specific impairment of strawberry growth,photosynthesis, carbohydrate and nitrogen accumulation by ozone

In a 2-year study, fruiting plants of strawberry (Fragaria × ananassa Duch.) cv. ‘Korona’ and ‘Elsanta’ were exposed for 2 months to 78 ppb ozone on average or filtered air without ozone in controlled environment chambers. Plant growth, photosynthesis, carbohydrate accumulation, and macronutrient concentrations were investigated in order to demonstrate cultivar-specific differences in the ozone sensitivity of ‘Korona’ and ‘Elsanta’ on the whole plant level. Moreover, the hypothesis was tested whether properties of the root system in strawberry were involved in ozone tolerance, for example, the roots’ ability to store or make available carbohydrates and their capacity to secure plants’ supply with nitrogen during a stress situation. In strawberry, ozone reduced leaf area by reducing leaf number. Moreover, specific leaf area (SLA) and relative leaf water content were reduced. Net photosynthesis was only slightly impaired, but activity of Rubisco and chlorophyll content in older leaves of cv. ‘Elsanta’ were significantly reduced. The most important, indirect impairment of photosynthesis was the reduction of plants’ total leaf area, which resulted in a decrease in plant biomass. The reduction of root biomass, the root/shoot ratio, and also the distribution of carbohydrates indicated a partitioning priority of the shoot at expense of the root system. Cultivar ‘Elsanta’ was characterized by significantly lower carbohydrate levels in ozone-exposed leaves, whereas levels remained fairly stable in ‘Korona’ leaves. In addition, nitrogen concentrations in leaves and roots decreased significantly in ‘Elsanta’, not in ‘Korona’. The reduced nitrogen concentration in leaves may be related with the more distinct reduction in Rubisco activity and chlorophyll content in older leaves of ‘Elsanta’.     

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.     

Influence of nitrogen on the production of hypericins by St. John’s wort

The effect of nitrogen supply on the production of ‘hypericins’ (hypericin and pseudohypericin) in leaves of St. John’s wort (Hypericum perforatum L.) was examined with plants grown in sand culture and soil. In sand culture, 56-d growth of St. John’s wort plants with decreased nitrogen levels resulted in increased production of hypericins in leaves. A short-term low nitrogen stress in sand culture also resulted in increased production of leaf hypericins. While growth in a low nitrogen-containing soil resulted in elevated levels of hypericins, their production was decreased by supplementation of the soil with additional nitrogen. Increased production of hypericins in St. John’s wort leaves did not require the nitrogen supply to be decreased to levels that resulted in nitrogen deficiency symptoms. Moreover, alteration in the production of leaf hypericins occurring with changes in nitrogen supply did not alter the concentration ratio of pseudohypericin and hypericin. Increased production of leaf hypericins was not associated with any significant changes in the number of dark glands on the leaves and only a weak correlation was observed between leaf dark gland number and levels of leaf hypericins. These results are discussed in terms of the biochemistry of naphthodianthrone production by St. John’s wort plants and implications for growth environment effects during cultivated growth of this medicinal plant.     

The Partial Characterization of a Badnavirus Infecting the Greater Asiatic or Water Yam (Dioscorea alata)

A bacilliform virus from Dioscorea alata, designated Dioscorea alata bacilliform virus (DaBV), from Barbados and West Africa and from other Dioscorea spp. from West African, Carribean, Asian and South American countries, has been characterized. The virus was transmitted by the mealybug, Planococcus citri and by mechanical transmission of partially purified preparations to several Dioscorea spp. DaBV was serologically related to a distinct bacilliform virus from Dioscorea bulbifera, to one isolate of sugarcane bacilliform badnavirus and two isolates of banana streak badnavirus (BSV) but was not related to another isolate of BSV or to Kalanchoe top spotting or cacao swollen shoot badnaviruses. The coat protein of DaBV was about 56 kDa and the nucleic acid was double-stranded DNA of about 7.5 kbp, part of which showed distant homology with other badnaviruses. Thus, DaBV is a distinct hitherto uncharacterized badnavirus.     

Flavescence dorée phytoplasma deregulates stomatal control of photosynthesis in Vitis vinifera

Flavescence dorée (FD) is among the major grapevine diseases causing high management costs; curative methods against FD are unavailable. In FD‐infected plants, decrease in photosynthesis is usually recorded, but deregulation in stomatal control of leaf gas exchange during FD infection and recovery is unknown. We measured the seasonal time course of gas exchange rates in two cultivars (‘Barbera’ and ‘Nebbiolo’) during the term of 1 year when grapevines experienced a water stress and another with no drought, with difference in gas exchange rates in response to FD infection and recovery as assessed by symptom observation and phytoplasma detection through PCR analysis. Chlorophyll fluorescence was also evaluated at the time of maximum symptom severity in ‘Barbera’, the cultivar showing the most severe stress response to FD infection, causing the highest damage in vineyards of north‐western Italy. In FD‐infected plants, net photosynthesis and transpiration gradually decreased during the season, more during the no drought year than during drought. During recovery, healthy (PCR negative) plants infected 2 years before, but not those infected an year before, regained the gas exchange performances to the level as measured before infection. The relationships between stomatal conductance and the residual leaf intercellular CO₂ concentration (c_i) discriminated healthy versus FD‐infected and recovered plants; at the same c_i, FD‐infected leaves had higher non‐photochemical quenching than healthy ones. We conclude that metabolic, not stomatal, leaf gas exchange limitation in FD‐infected and recovered grapevines is the basis of plant response to FD disease. In addition, we also suggest that such response is dependent upon water stress, by showing that water stress superimposes on FD infection in terms of stomatal and metabolic non‐stomatal limitations to carbon assimilation.