Joseph D. Postman

Identification, detection and transmission of a new vitivirus from Mentha

SummaryMentha × gracilis ‘Variegata’ is an ornamental clone with a phenotype caused by virus infection. Several clones were ordered from mail-order nurseries in an attempt to identify a virus consistently associated with symptoms. One of these clones did not exhibit typical ‘Variegata’ symptoms, and steps were taken to identify any agents causing the ‘off-type’ symptoms. One of the viruses identified in the atypical ‘Variegata’ clone is a previously unknown virus, a member of the family Flexiviridae. Sequence and phylogenetic analysis indicate that the virus, designated as mint virus-2, is related to members of the species Grapevine virus A, Grapevine virus B and Heracleum latent virus, placing it in the genus Vitivirus. A detection protocol for the virus has been developed, and the mint aphid (Ovatus crataegarius) was able to transmit the virus in the presence of a helper virus but not from single infected plants.

Phylogeny and evolutionary histories of Pyrus L. revealed by phylogenetic trees and networks based on data from multiple DNA sequences

Reconstructing the phylogeny of Pyrus has been difficult due to the wide distribution of the genus and lack of informative data. In this study, we collected 110 accessions representing 25 Pyrus species and constructed both phylogenetic trees and phylogenetic networks based on multiple DNA sequence datasets. Phylogenetic trees based on both cpDNA and nuclear LFY2int2-N (LN) data resulted in poor resolution, especially, only five primary species were monophyletic in the LN tree. A phylogenetic network of LN suggested that reticulation caused by hybridization is one of the major evolutionary processes for Pyrus species. Polytomies of the gene trees and star-like structure of cpDNA networks suggested rapid radiation is another major evolutionary process, especially for the occidental species. Pyrus calleryana and P. regelii were the earliest diverged Pyrus species. Two North African species, P. cordata, P. spinosa and P. betulaefolia were descendent of primitive stock Pyrus species and still share some common molecular characters. Southwestern China, where a large number of P. pashia populations are found, is probably the most important diversification center of Pyrus. More accessions and nuclear genes are needed for further understanding the evolutionary histories of Pyrus.

Emerging Fruit Crops

Hundreds of fruit species with commercial potential are currently in a status of low economic importance. Some, such as quince, pomegranate, and figs, have been cultivated for thousands of years. Others have only been locally collected and consumed from wild populations of the fruit. The development of these underappreciated crops depends on a range of factors including the cultivation limitations, yields, uses of the fruit, and marketing potential. Although initially many crops are developed using selections from the wild, as they are developed, breeding programs work toward improving the crop for both production and quality. This chapter examines nine emerging crops chosen among hundreds of potential crops which are currently showing much promise as commercial crops. These include five tree fruits, namely, pawpaw, quince, mayhaw, pomegranate, and fig, and four berry crops, namely, blue honeysuckle, elder, goji, and ‘ōhelo.

Nuclear and chloroplast microsatellite markers to assess genetic diversity and evolution in hazelnut species, hybrids and cultivars

The US Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon, preserves more than 800 accessions of hazelnut (Corylus), including C. avellana cultivars and representatives of 10 other recognized shrub and tree species. Characterization and study of genetic diversity in this collection require cross-transferable markers, such as trinucleotide microsatellite or simple sequence repeat (SSR) markers and universal chloroplast SSR markers. We developed new SSR markers and evaluated 114 Corylus accessions representing 11 species and 44 interspecific hybrids. Eight of 23 SSRs generated easy-to-score alleles in all species and seven were highly polymorphic. For those seven, the average heterozygosity was moderate at 0.49, and mean allele number, genetic diversity and polymorphism information index were high at 11.71, 0.79 and 0.76, respectively. The three most polymorphic SSRs were CaC-C008, CaC-C040 and CaC-C118. Neighbor-joining (NJ) clustering and structure analysis agreed with classical taxonomic analysis and supported inclusion of C. maxima within the large polymorphic species, C. avellana. Analysis also indicated that C. californica is a distinct species rather than a botanical variety of C. cornuta. Six universal cpSSRs were polymorphic in Corylus and generated 21 distinct chlorotypes with an average of 3 alleles per locus. Diversity at these cpSSRs was high and ranged from 0.33 to 0.64, with an average of 0.54. Incongruence in NJ topologies between the nuclear and chloroplast markers could be attributed to chloroplast capture related to hybridization during the ancestral diversification of the genus, or to homoplasy. The phylogeographical relationships among the 21 chlorotypes in the 11 Corylus species support Asia as a refugium where several hazelnut lineages survived during glaciation and from which they continued to evolve after dispersal from Asia through the Mediterranean to Europe, and across the Atlantic and/or the Bering land bridge to North America.

Nomenclature and genetic relationships of apples and pears from Terceira Island

Heritage apple (Malus domestica Borkh. hybrids) and pear (Pyrus communis L. hybrid) trees grow in villages throughout Terceira Island, Azores, Portugal. Some of these pears have different names but similar morphology. The objective of this study was to determine synonymy, homology, and phylogeny of apples and pears from Terceira and to examine potential relationships of the island pears with standard apples and pears of Portuguese or American descent. Nine apple microsatellite markers were used to determine genetic relationships. Distance- and parsimony-based cluster analysis grouped these genotypes into separate apple and pear clades. The Terceira apples were divided into two clades: the maçā and the reineta-reinette. Among the 17 heritage apple genotypes, seven unique accessions were identified and four groups of synonyms, or possibly clones, were detected including: ‘Reineta Agosto’ and ‘Reineta Verde’ from Altares; ‘Reineta Castanha’ and ‘Reineta Verde Miuda’; ‘Maçā Pêra,’ ‘Maçā Calhau’, ‘Pêro Branco’ from Salga and from Terra-Chā and ‘Maçā Marmelo’; and the five genotypes ‘Maçā Sao Joao’, ‘Malápio Rosa’, ‘Maçā Gaspar’, ‘Maçā Branca’ and ‘Maçā Pato’. In addition, two homonyms were detected. ‘Pêro Vermelho’ from Terra Chā was a separate genotype from a tree from Doze Ribeiras of the same name, but Pêro Branco from Terra Chā appears to be a clone that can be distinguished by an additional allele at CH1F07a from a tree with that name from Salga. One pair of apple clones, ‘Reineta Agosto’ and ‘Reineta Verde’ from Altares appear to be derived from an unreduced gamete of ‘Golden Delicious.’ Another apple genotype ‘Maçā Acida’ could be a sibling of the ‘Maçā Pêra’ clonal group. Other tested standard apples from the US genebank were unrelated to Terceira genotypes. Of the seven heritage pears, five unique genotypes and one pair of synonyms were detected. ‘Pêra Papo Pintassilgo’ from Raminho and ‘Pêra Vermelha’ from the nursery of Serviço de Desenvolvimento Agario da Terceira (SDAT) were synonyms. ‘Passans du Portugal’ was related to ‘Pêra Cabaca’ but other standard pears from the US genebank were unrelated to Terceira genotypes. Future studies will include additional apple and pear cultivars from other Islands of the Azores and continental Portugal, and wild Asian species to further explore genetic relationships.

Mint virus X: a novel potexvirus associated with symptoms in ‘Variegata’ mint

Summary.Mentha × gracilis ‘Variegata’, an ornamental mint clone first described about 200 years ago, exhibits virus-like vein banding symptoms. Double-stranded RNA and virion isolations revealed the presence of three viruses in a ‘Variegata’ plant. Cloning and sequencing disclosed that one of the viruses was a previously unidentified species with similarities to members of the Flexiviridae family, designated as Mint virus X (MVX). The complete nucleotide sequence of the virus was determined. Phylogenetic analysis divulged the close relationship of the virus with lily virus X and strawberry mild yellow edge virus, members of the Potexvirus genus. A reverse transcription-polymerase chain reaction protocol was developed and used for detection of MVX in other ‘Variegata’ plants. All clones tested, obtained from nurseries around the United States were infected with MVX, making the virus a possible causal agent of the variegated symptoms.

Virus infections reduce in vitro multiplication of 'Malling Landmark' raspberry.

SummaryVirus-infected plants are often symptomless and may be inadvertently used as explant sources in tissue culture research. Our objective was to determine the effect of virus infection on micropropagation. We studied the effects of single and multiple infections of three common raspberry viruses on the in vitro culture of ‘Malling Landmark’ red raspberry (Rubus idaeus L.). Virus-infected reaspberry plants were produced by leaf-graft inoculation from known-infected plants onto virus-free ‘Malling Landmark’. Single-virus source plants were infected with either tobacco streak ilarvirus (TSV), tomato ringspot nepovirus (TomRSV), or raspberry bushy dwarf idaeovirus (RBDV) and were free of other viruses as determined by enzyme-linked immunosorbent assay (ELISA) and bioassay. Virus-free, single, and multiple virus-infected ‘malling Landmark’ explants were initiated into culture and multiplied on Andersons medium with 8.9 μM N6-benzyladenine (BA). At the end of the multiplication tests, ELISA reconfirmed virus infections. In vitro multiplication of ‘Malling Landmark’ was significantly reduced by multiple infections, and multiplication of plants infected with all three viruses (RBDV+TomRSV+TSV) was less than half that of virus free cultures. Shoot height and morphology of in vitro cultures were not influenced by virus infection. The greenhouse stock plant with the three-virus infection was stunted and yellow compared to the control and the other infected plants.

Evidence of sympatric speciation of elderberry carlaviruses.

Five new carlaviruses infecting elderberry were characterized and tentatively named as elderberry virus A-E (ElVA-ElVE). Their genome organization is similar to that of other carlaviruses with size ranging from 8540 to 8628 nucleotides, excluding the polyadenylated tails. ElVA, ElVB and ElVD share a common ancestor as do ElVC and ElVE, indicating that speciation may be sympatric with all viruses having emerged in elderberry. Analyses of the carlavirus conserved domains indicate that the 2-oxoglutarate and Fe(II)-dependent oxygenase motifs are reliable indicators of virus phylogenetic classification with recombination playing a significant role in the evolution of the genus. A universal RT-PCR assay that detects all the elderberry carlaviruses and potentially other members of the genus has been developed. This tool can be used for research and regulatory purposes as elderberry cultivation is rapidly expanding to new areas where the viruses may be absent.

First report of clover yellow edge phytoplasma in Corylus (hazelnut).

During investigations into the cause of a stunt syndrome affecting cultivated European hazelnut trees (Corylus avellana L.) in Oregon, the clover yellow edge (CYE) phytoplasma was detected for the first time in this crop. The cause of hazelnut stunt syndrome (HSS) is unknown, but the disease has been transmitted by grafting and apparently has moved within orchards through root grafts (1). Severely affected trees persist for many years, but their nut production is greatly reduced. Previous attempts to detect viruses, bacteria, and other pathogens have been unsuccessful. HSS has been observed only in Oregon and already had been present for more than 10 years when it was first reported in 1970 (1). In June, 1999, leaf samples were collected from two affected and two apparently healthy (symptomless) hazelnut trees in a field plot at Oregon State University, Corvallis, and from a healthy greenhouse-grown tree. Leaf samples were sent to the USDA Beltsville, MD, laboratory, where they were assessed for phytoplasma infection, using nested polymerase chain reactions (PCRs). PCRs were primed by phytoplasma universal primer pairs P1/P7 and F2n/R2 (3) for amplification of phytoplasma 16S ribosomal (r) DNA (16S rRNA gene) sequences according to the procedures of Gunderson and Lee (2). Phytoplasma-characteristic 1.2-kbp DNA sequences were amplified from all field-tree samples. No DNA sequences were amplified from samples of the greenhouse-grown tree. Restriction fragment length polymorphism patterns of rDNA digested with AluI, KpnI, HhaI, HaeIII, HpaII, MseI, RsaI, and Sau3A1 endonucleases indicated that all diseased hazelnut trees as well as symptomless field trees were infected by a phytoplasma classified in group 16SrIII (peach X-disease group), subgroup B (III-B, type strain CYE phytoplasma). No phytoplasmas were detected in samples from the greenhouse-grown tree. Nucleotide sequences were determined for 16Sr DNA fragments amplified from the hazelnut CYE phytoplasma in nested PCRs primed with F2n/R2. The sequences were deposited in GenBank under Accession no. AF189288. Sequence similarity between 16Sr DNAs of the hazelnut CYE strain (CYE-Or) and the Canadian clover yellow edge strain (CYE-C, GenBank Accession no. AF175304) phytoplasma was 99.9%. Decline and yellows disorders of hazelnut in Germany and Italy have been associated with infections by apple proliferation, pear decline, and European stone fruit yellows phytoplasmas (4). These phytoplasmas are classified in 16Sr group X, the apple proliferation group of phytoplasmas. This is the first report of the CYE phytoplasma infecting Corylus. References: (1) H. R. Cameron. Plant Dis. Rep. 54:69, 1970. (2) D. E. Gunderson and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) R. Jomantiene et al. HortScience 33:1069, 1998. (4) C. Marcone et al. Plant Pathol. 45:857,1996.

Micropropagation of pear (Pyrus sp.).

Elements of micropropagation include establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets. The wide genetic variation in Pyrus makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced dormant shoots or from scions grafted onto seedling rootstocks to impose juvenility. Clean shoots are recovered after testing for contaminants at the initiation stage on ½ strength Murashige and Skoog 1962 medium (MS), at pH 6.9 for 1 week or by streaking on nutrient agar. Although pear species and cultivars are cultured on several well-known media, MS is the most commonly used. Our studies showed that multiplication and growth of shoots are best on Pear Medium with higher concentrations of calcium chloride, potassium phosphate, and magnesium sulfate than MS medium and 4.4 μM N(6) benzyladenine. Pear shoots are often recalcitrant to rooting; however, a 5 s dip in 10 mM indole-3-butyric acid or naphthalene acetic acid before planting on basal medium without plant growth regulators is effective for many genotypes. Pear shoots store well at 1-4°C, and can hold for as long as 4 years without reculture. Cryopreservation protocols are available for long-term storage of pear shoot tips. Acclimation of in vitro-rooted or micrografted shoots in a mist bed follows standard procedures.