We investigated the mechanism of resistance to demethylation inhibitors (DMI) in Penicillium digitatum by isolating the CYP51 gene, which encodes the target enzyme (P450 14DM ) of DMI, from three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of CYP51 in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced CYP51, including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased CYP51 expression. We also found that if the number of copies of the repeat was reduced to two, resistance and CYP51 expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances CYP51 expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.Demethylation inhibitors (DMIs) are widely used as fungicides in agriculture and medicine. In the 1970s, the development of resistance to DMI fungicides under practical conditions was thought to be unlikely (9, 16). However, in practice, DMI-resistant strains occur widely in several important plant and animal pathogens, such as Erysiphe graminis, Sphaerotheca fuliginea, Pyrenophora teres (12), and Candida albicans (11), causing acute problems in crop production and in the treatment of candidiasis of AIDS patients. Determining the mechanism of resistance is, therefore, quite important.We previously showed that an ATP-binding cassette (ABC) transporter gene, PMR1, is involved in DMI resistance in Penicillium digitatum by disrupting the PMR1 gene and concomitantly increasing sensitivity to DMI fungicides (21). PMR1 expression is strongly induced by fungicide treatment in both DMI-sensitive and DMI-resistant strains, but the constitutive expression level of PMR1 in the resistant strain was relatively higher than that in the sensitive strain (21). ABC transportermediated resistance to toxicants in yeast and human cells is a consequence of increased constitutive expression of the ABC transporter gene (3, 7), so we thought that the higher level of constitutive expression of the PMR1 gene could be responsible for the higher DMI resistance of the resistant strains. However, introduction of the PMR1 coding region under the control of a strong constitutive promoter, PgpdA, into a DMIsensitive strain had no observable effect on DMI resistance (H. Hamamoto, O. Nawata, K. Hasegawa, R. Nakaune, Y. J. Lee, Y. Makizumi, K. Akutsu, and T. Hibi, submitted for publication), suggesting that the constitutive expression level of PMR1 is not the determinative factor for DMI resistance. The coding sequences of this gene from three DMI-sensitive and three DMI-resistant strains were also identical. These results sugges...

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Bacterial fruit blotch of cucurbits is a destructive disease caused by Acidovorax avenae subsp. citrulli, which is a typical seedborne pathogen. In seed health testing for this disease, we have detected many strains of Acidovorax with some differences from A. avenae subsp. citrulli. Their 16S rRNA sequences were divided into six types. The most common sequence was completely consistent with that of A. avenae subsp. avenae originally isolated from rice. The other sequences were over 99% similar but not identical to those of A. avenae subsp. avenae and A. avenae subsp. citrulli. Some commercialized antibodies against A. avenae subsp. citrulli reacted with several of these strains. Some of these strains incited yellow spots or brownish water-soaked lesions mainly on young true leaves of cucumber and squash after spray inoculation. Histological observations showed that these strains entered the leaf tissues of cucurbit plants through stomata and multiplied in the intercellular spaces of parenchymatous tissues as well as in the vascular tissues. The amount of bacterial multiplication and spread in the tissues differed among the strains, presumably reflecting their ability to induce symptoms. These isolated strains are therefore different from A. avenae subsp. citrulli, and their potential threat to the cultivation of cucurbits is lower than that of A. avenae subsp. citrulli.

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