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A single nuclear gene controls nonsporulation in a novel isolate of the Dutch elm disease fungus Ophiostoma ulmi (Buism.) Nannf. This has been clearly demonstrated through segregation of the nonsporulating phenotype-in meiotic products recovered from crosses between a mutant nonsporulating isolate (WRB2-1) and wild-type sporulating isolates, between F1 progeny and their parents, and between F1 progeny. All crosses between nonsporulating and sporulating isolates yielded a 1:1 ratio for these two phenotypes in the meiotic products, whereas all crossings between isolates of the same phenotype produced meiotic products of that phenotype. The genetic stability of the nonsporulating phenotype was clearly shown when no disease symptoms were observed following artificial inoculation of the nonsporulating progeny into white elm, Ulmus americana L. Exposure to trifluoperazine, a calmodulin inhibitor, did not shift the nonsporulating isolates to the yeast phase, which supports our findings that nonsporulation is under genetic control rather than metabolic control. Key words: nonsporulation, Ophiostoma ulmi, mutant, single nuclear gene, meiotic products.

A test of comparative in vitro cerato-ulmin wilt toxin production in the aggressive and non-aggressive subgroups of the Dutch elm disease pathogen Ophiostoma ulmi was carried out by turbidity and ELISA methods. Ten non-aggressive, ten EAN aggressive and ten NAN aggressive isolates were tested from a range of geographical sources. In liquid shake cultures the non-aggressive isolates produced the greatest and the NAN aggressives the least mean biomass. Despite considerable variation in cerato-ulmin production by individual isolates in three separate experiments, both the turbidity and ELISA methods showed a clear separation of the non-aggressive and aggressive subgroups. Non-aggressive isolates produced little or no cerato-ulmin (ELISA range of means 0–56.0 ng/ml) and EAN and NAN aggressive isolates moderate to high levels (EAN 1.6–89.0 × 104 ng/ml and NAN 0.2–300 × 104 ng/ml). In the aggressive isolates no correlation was detected between cerato-ulmin production and either biomass or pathogenicity to clonal Commelin elm. The role of cerato-ulmin in the pathogenicity of O. ulmi is discussed.

The unique wilt toxin cerato-ulmin has been documented, covering its initial discovery and sequential experimental data to support its instrumental significance in the development of Dutch elm disease. Characterization of cerato-ulmin in view of its pathological implications for host specificity has brought up points that strongly suggest that cerato-ulmin is involved in Ophiostoma ulmi pathogenesis: 1) cerato-ulmin is produced only by O. ulmi, the Dutch elm disease pathogen, 2) cerato-ulmin causes symptoms of toxicity both external and internal that are similar to those of Dutch elm disease, 3) the toxicity of cerato-ulmin is selective and is highest with hosts of the pathogen, 4) high levels of cerato-ulmin apparently enhance the aggressiveness of O. ulmi, 5) cerato-ulmin affects host physiology, largely in a host specific manner, by increasing respiration rates and plasma membrane permeability; similar symptoms are observed in hosts affected by Dutch elm disease, in accordance with host susceptibility to Dutch elm disease, 6) in vitro production of cerato-ulmin is correlated with pathogenicities of O. ulmi wild-type isolates and, 7) cerato-ulmin is produced in vivo at relatively early stages in the development of external symptoms of Dutch elm disease.

The presence of cerato-ulmin (CU) in the synnema head fluid (HF) of Ceratocystis ulmi was shown by the results of light and scanning electron microscope observation and by demonstration of the assembling and disassembling of structures characteristic of CU in the HF. Further, through the combined application of acrylamide gel electrophoresis and double diffusion techniques, CU was detected as the major protein in the HF.

A practical apparatus was assembled and through its use a technique was developed to disrupt microquantities of Ceratocystis ulmi spores. spores were placed in a microfuge tube with buffer and glass beads. The microfuge tube was sealed and placed inside a holding tube and then subjected to rapid vibrations over a Vortex mixer. By this technique 80–90% of the spores were disrupted and it was possible to extract a high-quality ribosomal RNA fraction from the supernatant fluid of the disrupted spore sample. The technique has also proved applicable to spores of fungi other than C. ulmi .

Microstructures found in shake cultures of Ceratocystis ulmi , the fungus causing Dutch elm disease, have been isolated by a novel technique using the effect of bubbling gas through the culture filtrate.