A sporulation medium forDiscula destructiva,the dogwood anthracnose fungus

A growth medium incorporating finely ground oak or maple leaf tissue was developed for the purpose of increasing in vitro conidium production by the dogwood anthracnose pathogen,Discula destructiva.     

An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells

Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in?vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low, suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as?a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication, nor did the DDR marks colocalize with latent episomes. Rather, a transient period of EBV-induced hyperproliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoprotein EBNA3C was required to attenuate the EBV-induced DDR. We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR that is attenuated by viral latency products to induce cell immortalization.     

Hyphal growth of Phytophtora cinnamomi on pine callus tissue

A procedure was developed which demonstrates the expression of differential resistance in pine callus tissues to the fungal pathogen Phytophthora cinnamomi Rands. Callus tissues were maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D and inoculated with hyphae of P. cinnamomi at 26°C in the dark. The number of intracellular hyphae was used as an index of resistance. Loblolly and loblolly × shortleaf pine hybrids were determined to be more resistant to infection and invasion by the fungus than were shortleaf and Virginia pine.Abbreviations (AL) loblolly pine—Alabama - (PL) South Carolina - (AS) shortleaf pine—Alabama - (CS) Georgia - (AV) Virginia pine—Alabama - (H1) loblolly × shortleaf pine hybrids—14–42 × 6-I-43 - (H2) I-523 × 6-D-8     

Micropropagation of shortleaf,Virginia and loblolly x shortleaf pine hybrids via organogenesis

Protocols were developed for the micropropagation of shortleaf pine (Pinus echinata Mill.), loblolly (P. taeda L.) x shortleaf pine hybrids, and Virginia pine (P. virginiana Mill.). For meristematic tissue induction, modified Gresshoff & Doy (GD) medium with a high concentration of benzyladenine (BA) and short pulse treatment was best for loblolly x shortleaf hybrids whereas a lower concentration of BA and longer pulse treatment was best for shortleaf and Virginia pines. Shoot growth rate for all species was generally slower on Schenk & Hildebrandt medium than on GD medium. Addition of activated charcoal improved shoot growth of shortleaf pine but not of Virginia pine or the loblolly x shortleaf hybrids. Separation of shoots was beneficial before placing in the advanced growth medium. Both GD and Litvay's media produced good advanced shoot growth, especially following the addition of 0.5% activated charcoal. Individual shoot heights of 2–3 cm and 8–12 weeks of age after separation from the cluster were best for rooting. Root induction declined rapidly thereafter. Modified GD medium with 0.5 mg 1^-1 -naphthaleneacetic acid plus 1.0 mg 1^-1 3-indolebutyric acid and 20 g 1^-1 sucrose was best for root induction for all species except shortleaf pine. Addition of activated charcoal produced better root systems. Too high a light intensity resulted in a lowered frequency of rooting. A large number of plantlets was produced.     

Cooperative Cross-Bridge Activation of Thin Filaments Contributes to the Frank-Starling Mechanism in Cardiac Muscle

Myosin cross-bridges play an important role in the regulation of thin-filament activation in cardiac muscle. To test the hypothesis that sarcomere length (SL) modulation of thin-filament activation by strong-binding cross-bridges underlies the Frank-Starling mechanism, we inhibited force and strong cross-bridge binding to intermediate levels with sodium vanadate (Vi). Force and stiffness varied proportionately with [Ca²⁺] and [Vi]. Increasing [Vi] (decreased force) reduced the pCa₅₀ of force-[Ca²⁺] relations at 2.3 and 2.0 μm SL, with little effect on slope (n_H). When maximum force was inhibited to ∼40%, the effects of SL on force were diminished at lower [Ca²⁺], whereas at higher [Ca²⁺] (pCa < 5.6) the relative influence of SL on force increased. In contrast, force inhibition to ∼20% significantly reduced the sensitivity of force-[Ca²⁺] relations to changes in both SL and myofilament lattice spacing. Strong cross-bridge binding cooperatively induced changes in cardiac troponin C structure, as measured by dichroism of 5′ iodoacetamido-tetramethylrhodamine-labeled cardiac troponin C. This apparent cooperativity was reduced at shorter SL. These data emphasize that SL and/or myofilament lattice spacing modulation of the cross-bridge component of cardiac thin-filament activation contributes to the Frank-Starling mechanism.     

A membrane technique for producing protoplasts ofCryphonectria parasitica

A method for the formation and regeneration of protoplasts of several strains of the chestnut blight fungus,Cryphonectria parasitica, is presented. The procedure utillizes cellophane membranes for growth and employs centrifugation for separation of protoplasts from hyphal fragments. Yields averaged 8.04×10⁶ protoplasts per membrane. Regeneration frequencies were 40–50% with a soft-agar overlay. These protoplasts are suitable for use in experiments designed to determine the role of dsRNA in hypovirulence ofC. parasitica.     

Common Multiple dsRNAs Are Present in Populations of the Fungus Discula destructiva Originating from Widely Separated Geographic Locations

DsRNAs were detected in 85/108 isolates of Discula destructiva, the cause of dogwood anthracnose, collected in South Carolina, Idaho, and Alabama. The eastern isolates contained a greater diversity of dsRNA than did Idaho isolates, but most isolates, irrespective of state of origin, contained two small bands (ca. 1.5–2.5 kb) with sequence homology indicated by Northern hybridization. Differences in the banding patterns suggest that genetic diversity of dsRNA in D. destructiva is generated rapidly and that D. destructiva can be simultaneously infected by multiple dsRNA viruses. Received: 19 June 2000 / Accepted: 16 August 2000     

In vitro detection of Cornus florida callus insensitive to toxic metabolites of Discula destructiva

Summary Dogwood anthracnose, caused by the fungus Discula destructiva Redlin, is a severe disease of flowering dogwood (Cornus florida L.) and Pacific dogwood (C. nuttallii Aud.). Disease control is inadequate in nurseries and landscapes and absent in the forest, and resistant cultivars are not commercially available. The ability to select tissues insensitive to culture filtrates from D. destructiva in vitro offers a novel and important approach for the selection of dogwood genotypes that are resistant to or tolerant of this devastating fungus. Embryo-derived dogwood callus cultures were established on Murashige and Skoog medium amended with benzyladenine (BA) and either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA). Selection for insensitivity to D. destructiva metabolites was done by placement of individual cultures on media amended with progressively higher concentrations of a partially purified culture filtrate (PPCF) containing lowmolecular-weight compounds. Following this selection process, cultures were challenged in a dose-response format with PPCF to determine whether the sensitivity of the callus to the culture filtrate had changed. During the selection period, the fresh weight of callus grown on medium containing 2,4-D and amended with PPCF was always less than that of callus grown on medium amended with the same concentration of potato-dextrose broth (PDB, negative control). Fresh weight of callus was greater on medium containing NAA amended with PPCF than on medium with the same concentration of PDB. Callus selected in the presence of NAA showed decreased sensitivity to toxic metabolites at higher concentrations of culture filtrate. The in vitro system described may assist in the identification of disease-resistant germplasm important to the long-term survival of flowering dogwood.     

News & Notes: Double-Stranded RNA in Isolates of Discula destructiva from the Pacific Northwestern United States and British Columbia,Canada

Twenty-nine isolates of Discula destructiva Redlin from the Pacific northwestern United States and British Columbia, Canada were extracted for the presence of double-stranded RNA by use of phenol-chloroform and CF-11 chromatography. Seven/29 were positive. Agarose-gel banding profiles of the western isolates were different from those found previously in 80/80 isolates from the eastern U.S. These results suggest that isolates of D. destructiva responsible for dogwood anthracnose in Washington, Oregon, Idaho, and British Columbia may have an origin different from that of the eastern isolates. Received: 26 July 1996 / Accepted: 7 August 1996