Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos

Summary Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 g·ml^–1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene^– vector and two from the cp gene⁺ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene⁺ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.Journal Series no. 3757 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Relationship between inoculum density and vector phenology on the incidence of potato virus Y in tobacco

The epidemiology of potato virus Y (PVY) in the tobacco crop, Nicotiana tabacum, was examined in the context of the seasonal abundance of aphid vectors, rate of disease progress, and disease gradient from a known virus source. The spring potato crop, Solanum tuberosum, was suspected of being the main source of inoculum; therefore, varying numbers of infected potato plants were used as the inoculum source in different test plots. A 3-wk lag phase was present in all disease progress curves prior to an exponential increase in disease incidence. The relatively low numbers of aphid vectors, primarily transient species, alighting on the crop during the lag phase were responsible for the primary spread of PVY from potato to tobacco. The arrival of large numbers of colonising aphid vectors, Myzus persicae, presumably from the harvested potatoes, coincided with the exponential increase in PVY incidence in tobacco. The initial number of potato plants infected with PVY was positively correlated with the final disease incidence, rate of disease progress, and the magnitude of radial dispersion of PVY into the tobacco. Aphid vector pressure was not a significant variable in the differences in spatial and temporal characteristics of PVY epidemics among test plots.     

Spread of zucchini yellow mosaic potyvirus in squash in Hungary

The temporal and spatial distribution of zucchini yellow mosaic potyvirus (ZYMV) was studied in a 3000‐m² zucchini squash field. The first infected plants were found 4 weeks after the field was exposed to virus source plants. The infection increased to nearly 74% by the end of the study. Alate aphids were active from the beginning of the study and 43 species were trapped in the field. Flights of vector species Acyrthosiphon pisum and Myzus persicae peaked during the fourth week which resulted in high virus incidence 4 weeks later. There was a significant correlation between the number of vectors caught in yellow pan traps and the number of infected plants in the field. In laboratory studies evaluating 11 aphid species, Aphis pomi de Geer was identified as a new vector species of ZYMV. Although this aphid was not caught in our field studies, it may be an important contributor in other areas where cucurbits are grown in close proximity to apple or other hosts of this aphid.     

Stabilization effects of spatial aggregation of vectors in plant disease systems

Stabilization effects of spatial aggregation of vectors were examined in insect-borne plant disease systems by constructing a model that describes the yearly dynamics of rice stripe virus disease transmitted by the small brown planthopperLaodelphax striatellus (Fallén). Two transmission paths between vectors were considered: vertical transmission from parents through eggs, and horizontal transmission from infected plants by acquisition feeding. In this model, a paddy field was divided into quadrats and horizontal transmission was assumed to occur within each quadrat. A negative binomial distribution was used to describe the frequency distribution of vectors per quadrat. The parameters of the model were estimated using field data collected in Ibaraki Prefecture, Japan. The model showed that (1) the disease cannot invade into an epidemiological system if the mean crowding of vectors is less than a critical value, (2) the proportion of infected vectors is maintained at about 30% irrespective of the vector density if vectors are highly aggregated, and (3) the proportion of infected plants is maintained at a low level irrespective of the vector density if vectors are highly aggregated. It was also shown that these stabilization effects of aggregation in this epidemiological system come from a mechanism that is common to other systems such as single-species systems and competition systems.     

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.     

Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus

Transgenic Carica papaya plants (cv. Sunset, R0 clone 55-1) carrying the coat protein gene of papaya ringspot virus (strain HA 5-1) remained symptomless and ELISA-negative for 24 months after inoculation with Hawaiian strains of papaya ringspot virus under field conditions. Non-transgenic and transgenic control plants lacking the coat protein gene developed disease symptoms within one month after manual inoculation or within four months when natural aphid populations were the inoculum vectors. Mean trunk diameter was significantly greater in cloned 55-1 plants compared with virus-infected controls (14.7 cm versus 9.3 cm after 18 months). Fruit brix, plant morphology, and fertility of 55-1 plants were all normal, and no pleiotropic effects of the coat protein gene were observed. These results indicate that pathogen-derived resistance can provide effective protection against a viral disease over a significant portion of the crop cycle of a perennial species.     

Measures of spatial heterogeneity for species occurrence or disease incidence with finite-counts

We propose two types of indices with finite-count correction to measure the spatial heterogeneity of binary characteristics of organisms, such as occurrence or non-occurrence of organisms and infected or non-infected plants. We consider the following two examples: plant occurrence in a grassland community, and yellow dwarf disease infection in a rice field. For the grassland community, N quadrats comprising n cells of equal area, were set at random sites in a grassland, and the occurrence of a given species A in each of n cells was recorded. For disease infection, N quadrats, each consisting of n rice plants, were set at random sites in a paddy field, and the number of plants infected with yellow dwarf virus in each quadrat were counted. In these examples, since the number of cells in a quadrat is finite, neither occurrence nor incidence increase infinitely, unlike the number of aphids on a maize leaf. The first category of index belongs to the mean : variance ratio type. The estimated index value for occurrence (or incidence) is the same as that for non-occurrence (or non-incidence). The second category belongs to the k-type of a negative binomial distribution. If some random plants die or recover from the disease then the expected value of the second type of index does not change. For n, the current indices approach the mean : variance ratio and the inverse of k in a negative binomial distribution, respectively. This indicates that these indices are suitable for binary data sets.     

Temporal and spatial spread of Lettuce mosaic virus in lettuce crops in central Spain: factors involved in Lettuce mosaic virus epidemics

Lettuce mosaic virus (LMV) is transmitted by aphid vectors in a nonpersistent manner as well as by seeds. The virus causes severe disease outbreaks in commercial lettuce crops in several regions of Spain. The temporal and spatial patterns of spread of LMV were studied in autumn 2002 in the central region of Spain. Symptomatic lettuce (var. Cazorla) plant samples were collected weekly, first at the seedling stage from the greenhouse nursery and later outdoors after transplantation. The exact position of symptomatic plants sampled in the field was recorded and then material was tested by enzyme‐linked immunosorbent assay to assess virus infection. Cumulative spatial data for infected plants at different growth stages were analysed using spatial analysis by distance indices. For temporal analysis, the monomolecular, Gompertz, logistic and exponential models were evaluated for goodness of fit to the entire set of disease progress data obtained. The results indicated that the disease progress curve of LMV epidemics in the selected area is best described by a Gompertz model and that the epidemic follows a polycyclic disease progression. Our data suggest that secondary cycle of spread occurs when noncolonising aphid species land on the primary infected plants (probably coming from infected seed) and move to adjacent plants before leaving the crop. The role of weeds growing close to lettuce fields as potential inoculum sources of virus and the aphid species most likely involved in the transmission of LMV were also identified.     

Analysing spatial patterns of spread of Lettuce necrotic yellows virus and lettuce big-vein disease in lettuce field plantings

Spatial patterns of spread of lettuce big‐vein disease (LBVD) and Lettuce necrotic yellows virus (LNYV) were examined in two plantings each consisting of two blocks of lettuce. LBVD came from planting land infested with viruliferous Olpidium brassicae resting spores, while LNYV was introduced by aphid vectors from external sources consisting of LNYV‐infected sowthistle (Sonchus oleraceus) weeds. Clustering of LBVD was obvious in an area where the soil was heavily infested with only sporadic occurrence elsewhere. There was a steep decline in LNYV incidence over distance from a concentrated external weed source, with clustering of LNYV‐infected plants at the crop edge closest to it. There was no evidence of secondary spread with LBVD or LNYV.     

Seedling dynamics of <Emphasis Type="Italic">Acer mono</Emphasis> and <Emphasis Type="Italic">Fagus crenata</Emphasis>: an environmental filter limiting their adult distributions

Spatial pattern of a biotic population may change over time as its component individuals grow or die out, but whether this is the case for desert annual plants is largely unknown. Here we examined seasonal changes in spatial patterns of two annuals, Eriogonum abertianum and Haplopappus gracilis, in initial (winter) and final (summer) densities. The density was measured as the number of individuals from 384 permanent quadrats (each 0.5 m × 0.5 m) in the Chihuahuan Desert near Portal, Arizona, USA. We used three probability distributions (binomial, Poisson, and negative binomial or NB) that represent three basic spatial patterns (regular, random, and clumped) to fit the observed frequency distributions of densities of the two annuals. Both species showed clear clumped patterns as characterized by the NB and had similar inverse J-shaped frequency distribution curves in two density categories. Also, both species displayed a reduced degree of aggregation from winter to summer after the spring drought (massive die-off), as indicated by the increased k-parameter of the NB and decreased values of another NB parameter p, variance/mean ratio, Lloyd’s Index of Patchiness, and David and Moore’s Index of Clumping. Further, we hypothesized that while the NB (i.e., Poisson-logarithmic) well fits the distribution of individuals per quadrat, its components, the Poisson and logarithmic, may describe the distributions of clumps per quadrat and of individuals per clump, respectively. We thus obtained the means and variances for (1) individuals per quadrat, (2) clumps per quadrat, and (3) individuals per clump. The results showed that the decrease of the density from winter to summer for each plant resulted from the decrease of individuals per clump, rather than from the decrease of clumps per quadrat. The great similarities between the two annuals indicate that our observed temporal changes in spatial patterns may be common among desert annual plants.     

Transformation of Dendrobium orchid using particle bombardment of protocorms

Transformed dendrobium orchids (Dendrobium x Jaquelyn Thomas hybrids) were recovered from protocorms bombarded by particles coated with the plasmid pGA482GG/cpPRV4, which contains the plant expressible Nos-NPT II and papaya ringspot virus (PRV) coat protein (CP) genes. Approximately 280 protocorms from four crosses were bombarded and potentially transformed tissues were identified by growth and green color on half-strength Murashige and Skoog medium supplemented with 2% sucrose and 50–100 mg 1^–1 kanamycin sulfate. Kanamycin concentrations that prevented growth of nontransformed tissues could not be used for long-term selection because such levels suppressed the regeneration of potentially transformed tissues. PCR and restriction analysis 21 months after treatment found 13 of 13 plants from two crosses, which appeared kanamycin-tolerant, to contain the Nos-NPT II gene, while only one of these plants carried the vector-linked PRV CP-gene. These results support use of particle bombardment for transformation of this important ornamental monocot.     

Epidemiology of an aphid nontransmissible potyvirus in fields of nontransgenic and coat protein transgenic squash

Spread of the aphid nontransmissible Zucchini yellow mosaicvirus virus (ZYMV) strain MV was monitored over two consecutive years in field plots of nontransgenic and transgenic squash expressing the coat protein (CP) gene of the aphid transmissible strain FL of Watermelon mosaic virus (WMV). The experimental approach was to mechanically inoculate plants with ZYMV strain MV and to assess subsequent transmissions, assumed to be vectored by aphids, of this strain to nonmechanically inoculated plants. Strain MV was distinguished from other ZYMV isolates by a threonine at position 10 of the CP or by a distinct electrophoretic pattern of a Nla IV-digested genomic cDNA fragment generated by RT-PCR. ZYMV strain MV was not detected in fields of nontransgenic plants, but was apparently aphid transmitted to 77 of 3,700 plants (2%) in transgenic fields. Despite the availability of numerous test plants and conditions of high disease pressure but low selection pressure, an epidemic of ZYMV strain MV did not develop in fields of transgenic plants. In contrast, the aphid transmissible ZYMV strain NY was aphid-transmitted to 99% (446/450) of transgenic plants under similar conditions. The relevance of these results in assessing environmental risks of transgenic plants expressing CP transgenes is discussed.     

Aphid behavioral responses to virus‐infected plants are similar despite divergent fitness effects

We compared the settling preferences and reproductive potential of an oligophagous herbivore, the pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), in response to pea plants, Pisum sativum L. cv. ‘Aragorn’ (Fabaceae), infected with two persistently transmitted viruses, Pea enation mosaic virus (PEMV) and Bean leaf roll virus (BLRV), that differ in their distribution within an infected plant. Aphids preferentially oriented toward and settled on plants infected with PEMV or BLRV in comparison with sham‐inoculated plants (plants exposed to herbivory by uninfected aphids), but aphids did not discriminate between plants infected with the two viruses. Analysis of plant volatiles indicated that plants inoculated with either virus had significantly higher green leaf volatile‐to‐monoterpene ratios. Time until reproductive maturity was marginally influenced by plant infection status, with a trend toward earlier nymph production on infected plants. There were consistent age‐specific effects of plant infection status on aphid fecundity: reproduction was significantly enhanced for aphids on BLRV‐infected plants across most time intervals, though mean aphid fecundity did not differ between sham and PEMV‐infected plants. There was no clear pattern of age‐specific survivorship; however, mean aphid lifespan was reduced on plants infected with PEMV. Our results are consistent with predictions of the host manipulation hypothesis, extended to include plant viruses: non‐viruliferous A. pisum preferentially orient to virus‐infected host plants, potentially facilitating pathogen transmission. These studies extend the scope of the host manipulation hypothesis by demonstrating that divergent fitness effects on vectors arise relative to the mode of virus transmission.     

Spatial distribution of species diversity indices and their correlation with plot size

In a physiognomically uniform Leucobryo-Pineteum phytocoenose the spatial pattern of point diversity was determined and the effect of quadrat size on the assessment of alpha diversity (in Whittaker's, 1977, sense) was analysed. In both cases the Shannon index of total species diversity and the evenness index were used to measure diversity. A contagious spatial pattern of point diversity and a high variation of point diversity values, as well as a strong non-linear dependence of H and J values on quadrat size, and also a decrease in H and J variation with an increase in quadrat size were found.Nomenclature follows Ehrendorfer (1973). Liste der Gefässpflanzen Mitteleuropas.     

Transmissibility of Broad bean wilt virus 1 by aphids: influence of virus accumulation in plants,virus genotype and aphid species

Broad bean wilt virus 1 (BBWV‐1) is transmitted by several aphid species in a non‐persistent manner. Transmission efficiency by vectors is a key factor for understanding virus epidemiology and applying disease control measures based on limiting virus spread. We evaluated the transmission rates of two genetically divergent BBWV‐1 isolates (PV‐132 from USA and Ben from Spain) infecting broad bean (Vicia faba L.) by isofemale lines of nine aphid species from eight different genera collected in Spain. Our analyses showed that: (a) the virus concentration in the source plant was a key factor in BBWV‐1 transmissibility; (b) The Spanish isolate Ben was transmitted more efficiently than the American isolate PV‐132 by most aphid species, but this was only due to the higher accumulation of Ben in plants, as both isolates had similar transmissibility after adjusting virus concentration and (c) The transmission rate varied greatly between the different aphid species.     

A grid‐based method for sampling and analysing spatially ambiguous plants

Abstract. Spatial data can provide much information about the interrelations of plants and the relationship between individuals and the environment. Spatially ambiguous plants, i.e. plants without readily identifiable loci, and plants that are profusely abundant, present non‐trivial impediments to the collection and analysis of vegetation data derived from standard spatial sampling techniques. Sampling with grids of presence/absence quadrats can ameliorate much of this difficulty. Our analysis of 10 fully‐mapped grassland plots demonstrates the applicability of the grid‐based approach which revealed spatial dependence at a much lower sampling effort than mapping each plant. Ripley's K‐function, a test commonly used for point patterns, was effective for pattern analysis on the grids and the gridded quadrat technique was an effective tool for quantifying spatial patterns. The addition of spatial pattern measures should allow for better comparisons of vegetation structure between sites, instead of sole reliance on species composition data.     

Comparative Epidemiology of Three Virus Diseases on Zucchini Squash

Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus – type W (PRSV‐W) and Zucchini lethal chlorosis virus (ZLCV) cause important diseases on zucchini squash crops in Brazil. ZYMV and PRSV‐W belong to the genus Potyvirus and are transmitted by aphids, whereas ZLCV belongs to Tospovirus and is transmitted by the thrips Frankliniella zucchini. These three viruses may occur simultaneously in the field, and the epidemiology of the corresponding diseases may be determined by interactions among viruses, hosts and vectors. In this work, the progress of the diseases caused by these viruses was studied over a temporal and geographic range for three planting seasons (PS). For the lethal chlorosis (ZLCV), a monomolecular model was found to be the best fit for the data, though only during the third PS. For data collected during the first two PS, the Gompertz model was found to fit the data best. The spatial distribution of disease indicated disease aggregation at the end of the crop cycle. For the yellow mosaic (ZYMV), the model that best fit in the 1st PS was the logistic and in the 2nd and 3rd PS was monomolecular. The spatial pattern of the disease was random when the disease incidence was low but aggregated when the disease incidence was high. The common mosaic (PRSV‐W) showed the lowest incidence in all three PS. An exponential model was the best fit for data collected during all PS, and the spatial pattern of the disease was random. Interactions among the three viruses apparently did not result in changes in the epidemiology of the diseases. Removal of sources of inoculum and planting at an unfavourable time for reproduction of virus vectors are the two main measures recommended for the control of these diseases. The use of insecticide is indicated only for the control of the F. zucchini.     

Field studies on the spread of African cassava mosaic

The spread of African cassava mosaic disease (ACMV) into healthy cassava fields was recorded at weekly intervals. In addition, 21 yellow water traps were placed in one field and the number of whiteflies caught was recorded twice a week. The number of Bemisia spp. feeding on cassava was also estimated. The results indicate that the pattern of disease spread is related to the pattern of infestation with Bemisia. Airborne whiteflies carried by the south-west prevailing wind alighted preferentially on cassava plants along the upwind edges (south and west borders) of the plantings. The pattern of incidence of mosaic disease resembled that of whiteflies. Along the SW-NE diagonal, there was a gradient of disease incidence with a maximum at the SW corner block. Similar gradients occurred in three different fields and they were maintained throughout the 6-month study, although gradually flattening with time. There were indications that the reservoirs both of the virus and of the vectors were located some distance upwind from the experimental fields.     

Papaya Ring Spot Virus: An Understanding of a Severe Positive-Sense Single Stranded RNA Viral Disease and Its Management

Viral diseases have been studied in-depth for reducing quality, yield, health and longevity of the fruit, to highlight the economic losses. Positive-sense single-stranded RNA viruses are more devastating among all viruses that infect fruit trees. One of the best examples is papaya ringspot virus (PRSV). It belongs to the genus Potyvirus and it is limited to cause diseases on the family Chenopodiaceae, Cucurbitaceae and Caricaceae. This virus has a serious threat to the production of papaya, which is famous for its high nutritional and pharmaceutical values. The plant parts such as leaves, latex, seeds, fruits, bark, peel and roots may contain the biological compound that can be isolated and used in pharmaceutical industries as a disease control. Viral disease symptoms consist of vein clearing and yellowing of young leaves. Distinctive ring spot patterns with concentric rings and spots on fruit reduce its quality and taste. The virus has two major strains P and W. The former cause disease in papaya while the later one in papaya. Virion comprises 94.4% protein, including a 36 kDa coat protein which is a component responsible for a non-persistent transmission through aphids, and 5.5% nucleic acid. Cross protection, development of transgenic crops, exploring the resistant sources and induction of pathogen derived resistance have been recorded as effective management of PRSV. Along with these practices reduced aphid population through insecticides and plant extracts have been found ecofriendly approaches to minimize the disease incidence. Adoption of transgenic crops is a big challenge for the success of disease resistant papaya crops. The aim of this review is to understand the genomic nature of PRSV, detection methods and the different advanced control methods. This review article will be helpful in developing the best management strategies for controlling PRSV.     

Spatial and temporal spread of Citrus tristeza virus and its aphid vectors in the North western area of Morocco

First report of Citrus tristeza virus (CTV,Closterovirus) in Morocco datesback to 1961 in collections of citrus varieties. An exhaustive survey of citrus in the north of the country in 2009 revealed that CTV was spread all over the citrus production area. We attempted to evaluate the relative contribution of different aphid species in the spread of CTV disease in a Citrus reticulata orchard at the Loukkous region during 2 years (2012 and 2013). The overall CTV incidence estimated in the experimental site increased from 17.8% in 2012 to 31.15% in 2013. The most abundant aphid species colonising clementine trees was Aphis spiraecola and A. gossypii. Both aphid species reached their maximum peaks during the spring season. The rate of viruliferous aphids, estimated by real‐time RT‐PCR of single aphid, revealed that 35.4% of winged A. gossypii and 28.8% of winged A. spiraecola were viruliferous, confirming a high inoculum pressure in the area surrounding the experimental site. The aphid species Toxoptera citricida, which is able to transmit the aggressive isolates of CTV, was not found in the Loukkous region. The study of the spatial distribution of the CTV showed that in general, the disease was randomly distributed in the field. Overall, the results seem to indicate that A. spiraecola may be considered as the major aphid species contributing to CTV spread in our experimental conditions. The prevalence of mild strains in the region and the high level of aphid flight activity could explain the rapid evolution of CTV incidence in the experimental area.