Margaret R. Pooler

Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis

By cloning and sequencing specific randomly amplified polymorphic DNA (RAPD) products, we have developed pairs of PCR primers that can be used to detect Xylella fastidiosa in general, and X. fastidiosa that cause citrus variegated chlorosis (CVC) specifically. We also identified a CVC-specific region of the X. fastidiosa genome that contains a 28-nucleotide insertion, and single base changes that distinguish CVC and grape X. fastidiosa strains. When using RAPD products to develop specific PCR primers, we found it most efficient to screen for size differences among RAPD products rather than presence/absence of a specific RAPD band.

Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data

Genetic relationships among 11 Xylella fastidiosa strains isolated from mulberry, almond, ragweed, grape, plum, elm, and citrus were determined by random amplified polymorphic DNA (RAPD). Twenty-two 10-base primers amplified a total of 77 discrete polymorphic bands. Phenetic analysis based on a similarity matrix corresponded well with previous reports on X. fastidiosa RFLP-based similarity relationships, indicating that RAPD-PCR amplification products can be used as a reliable indicator of genetic distance in X. fastidiosa. Cladistic analysis suggests the existence of five groups of X. fastidiosa: the citrus group, the plum-elm group, the grape-ragweed group, the almond group, and the mulberry group.

Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction

A sensitive and specific assay for detecting Xylella fastidiosa in potential insect vectors was developed. This assay involves immunomagnetic separation of the bacteria from the insect, followed by a two‐step, nested polymerase chain reaction (PCR) amplification using previously developed oligonucleotide primers specific to X. fastidiosa. A total of 347 leafhoppers representing 16 species were captured and sampled from American elm (Ulmus americana L.) trees growing in a nursery where bacterial leaf scorch caused by X. fastidiosa occurs. Two of these leafhopper species, Graphocephala coccinea and G. versuta, regularly tested positive for X. fastidiosa using this technique. These insects are therefore potential vectors of X. fastidiosa. Using immunocapture and nested PCR, it was possible to detect as few as five bacteria per sample.

INTERSPECIFIC HYBRIDIZATIONS BETWEEN THE NATIVE BITTERSWEET, CELASTRUS SCANDENS, AND THE INTRODUCED INVASIVE SPECIES, C. ORBICULATUS

Abstract Although many surveys of invasive plants have been conducted, relatively little research has been conducted on the biology of hybridization between invasive and native species. We hypothesized that interspecific hybridizations between the native bittersweet (Celastrus scandens L.) and the invasive introduced species (C. orbiculatus Thunb.) may be more vigorous and have less seed dormancy than C. scandens seedlings. To test this hypothesis, we performed controlled pollinations using C. scandens as the female parent and C. scandens or C. orbiculatus as the male parent. Although both the interspecific and intraspecific pollinations resulted in a comparable percentage of germinating seedlings, the seedlings from the interspecific crosses had less seed dormancy and were more vigorous than the intraspecific seedlings. These results indicate that the decline of the American bittersweet may be due in part to interspecific hybridizations with the invasive introduced species, and that the distinct genetic identity of C. scandens may be threatened.

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae).

Premise of the study: Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. Methods and Results: cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and sequenced using the Illumina MiSeq system. Approximately 11.9 million base pairs of sequence data were examined and 845 genic-SSRs were identified, including 469 dinucleotide, 360 trinucleotide, seven tetranucleotide, one pentanucleotide, and eight hexanucleotide repeats. Primer pairs were designed for 71 selectively chosen genic-SSRs containing trinucleotide repeat motifs and were used to amplify the corresponding loci in 18 diverse boxwood accessions. Twenty-three primer pairs amplified polymorphic loci, with two to 10 alleles per locus. Conclusions: These novel polymorphic genic-SSR markers will aid in evaluating genetic diversity of boxwood germplasm and allow verification of hybrids and cultivars for breeding programs.

MICROPROPAGATION OF CHINESE REDBUD (CERCIS YUNNANENSIS) THROUGH AXILLARY BUD BREAKING AND INDUCTION OF ADVENTITIOUS SHOOTS FROM LEAF PIECES

SummaryFactors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.

Genetic Diversity among Accessions of the Endangered Box Huckleberry (Gaylussacia Brachycera) Based on Aflp Markers

Abstract The box huckleberry (Gaylussacia brachycera) is a slow-growing, dwarf evergreen groundcover that is native to eight states in the Eastern United States. It is a rare plant with conservation status of rare to critically imperiled. Genetic relationships among 24 accessions of G. brachycera were determined using 66 polymorphic AFLP markers from 8 primer pairs. Accessions collected in western Virginia were the most distantly related to the other accessions, while accessions from Kentucky were the most variable. The information gained from this study will be useful to guide decisions regarding conservation, preservation, breeding, and re-introduction of this species.

Use of Mycelium and Detached Leaves in Bioassays for Assessing Resistance to Boxwood Blight

Boxwood blight caused by Calonectria pseudonaviculata is a newly emergent disease of boxwood (Buxus spp. L.) in the United States that causes leaf drop, stem lesions, and plant death. A rapid and reliable laboratory assay that enables screening hundreds of boxwood genotypes for resistance to boxwood blight is needed to enable breeding and selection of resistant cultivars. Using eight boxwood cultivars with differing susceptibilities, we examined parameters for a screening assay comparing whole plant inoculation with detached leaf inoculation, use of mycelium versus spores as the inoculum, comparison of times of the year for inoculation, and comparison of two leaf inoculation methods. Inoculation of detached leaves gave comparable results to inoculation of whole plants when compared across genotypes, although the detached leaf assay resulted in greater percentages of symptom expression. The time of year of plant inoculation (spring, summer, or winter) did not affect the relative expression of symptoms among the most resistant and susceptible genotypes. Inoculating plants with mycelium was as effective as spore inoculation for causing disease symptoms and allowed us to distinguish the more resistant genotypes, yet mycelium inoculation was much easier to prepare in large quantities for multiple assays.

Genetic diversity of Magnolia ashei characterized by SSR markers

The Ashe magnolia (Magnolia ashei) is a deciduous small tree most noted for its large 1–2 foot long leaves and fragrant creamy white flowers. Although the species is adapted to and used in landscapes in many parts of the U.S., it is endemic only to Northwest Florida where it is limited to ten counties growing on undisturbed bluffs and ravine banks. The populations are highly fragmented and are threatened by degradation of habitat, leading the species to be listed as endangered in the state of Florida. SSR markers were developed to determine the genetic diversity of wild populations of M. ashei in order to guide long-term conservation strategies. 18 marker loci identified a total of 82 alleles that were used to characterize allelic diversity of M. ashei from 11 wild populations, 14 cultivated sources, five accessions of M. macrophylla, and three interspecific hybrids. Results indicated a higher than expected level of heterozygosity within populations, and a clear distinction between Eastern and Western populations; conservation efforts should therefore focus on maintaining these distinct groups in corresponding ex situ seed orchards to counteract pressures due to overcollection, pollution, and loss of habitat due to development. Clustering of individuals was similar using several analytical methods, indicating that despite relatively small sample sizes, our analysis is a valid reflection of the diversity among and relationships between these populations.

Clonal Fidelity in Large Colonies of Gaylussacia brachycera Gray (Box Huckleberry) Assessed by DNA Fingerprinting

Abstract Gaylussacia brachycera (box huckleberry) is a slow-growing, dwarf evergreen member of the family Ericaceae that is native to eight states in the eastern United States. It is a rare plant with conservation status in several states of critically imperiled (S1). Botanists have been intrigued by this enigmatic native plant since it was discovered in 1796 in Virginia. One of the mysteries of this species is whether plants in a colony arose from different genotypes or are clonal. The species reproduces primarily by means of underground runners and appears to be self-sterile, so sexual reproduction within isolated colonies could be limited. Using molecular markers, we tested samples taken from three of the best-known colonies in Pennsylvania and one in Tennessee. Based on 104 polymorphic markers, we found that one of the Pennsylvania colonies contained two genotypes among 11 samples tested; one Pennsylvania colony contained three genotypes among five samples tested; and the other two colonies exhibited no variation among the 8–10 samples tested. This study represents the first time that molecular markers have been used in a systematic assay to determine the existence of variation among individuals within a colony of box huckleberry.