Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14␣-demethylase), through increased expression of the CYP51 gene, or through increased expression of efflux pumps. We found that CYP51 expression in DMI-resistant (DMI R ) isolates of the cherry leaf spot pathogen Blumeriella jaapii was increased 5-to 12-fold compared to that in DMI-sensitive (DMI S ) isolates. Analysis of sequences upstream of CYP51 in 59 DMI R isolates revealed that various forms of a truncated non-long terminal direct repeat long interspersed nuclear element retrotransposon were present in all instances. Similar inserts upstream of CYP51 were not present in any of 22 DMI S isolates examined.Sterol demethylation inhibitor (DMI) fungicides are the largest and most important group of antifungal agents and are used widely in both agriculture and medicine. These fungicides inhibit the sterol C-14 ␣-demethylation of 24-methylenedihydrolanosterol, a precursor of the cell membrane component ergosterol in fungi (4). DMIs were introduced for plant disease management in the 1970s. Since the 1980s, clearly defined field resistance to DMIs has been reported for powdery mildews of barley, cucumber, and grape (4,12,27) and reduced sensitivity to DMIs has been reported for several other fungal pathogens, including the apple scab pathogen Venturia inaequalis (21, 24).Molecular mechanisms leading to DMI resistance in several important human and plant fungal pathogens have been studied intensively (reviewed in references 11 and 23). Common mechanisms of DMI resistance include (i) mutations in the DMI target enzyme, 14␣-demethylase (CYP51), leading to a decreased affinity of DMIs to the target protein (3, 9, 10); (ii) overexpression or increased copy number of the CYP51 gene, leading to increased production of the target enzyme (16,25,30); and (iii) overexpression of ATP-binding cassette (ABC) transporters encoding efflux pumps (15,17,34). Additionally, some unknown mechanisms (4, 28, 32) may confer DMI resistance.The cherry leaf spot pathogen, Blumeriella jaapii, is a major pathogen of tart cherry that is only effectively controlled through regular fungicide applications. DMI fungicides have been used intensively for leaf spot management in Michigan since 1989, and we have noted a decline in control with DMIs in research plots and commercial orchards (G. W. Sundin, unpublished). The objective of this study was to characterize the molecular mechanism of DMI resistance in B. jaapii. Our hypothesis was that the resistance mechanism in this fungal pathogen would be similar to mechanisms previously reported in other fungi, and thus our work focused on isolating and analyzing the B. jaapii CYP51 gene and flanking sequences. Additional knowledge of DMI resistance mechanisms will increase our understanding of the evolution of fungicide resistance, could result in the development of a detection met...

The effect of solar UV-B radiation on the population dynamics and composition of the culturable bacterial community from peanut (Arachis hypogeae L.) was examined in field studies using plants grown under UV-B؊transmitting (UV-B؉) or UV-B؊excluding (UV-B؊) plastic filters. Our data demonstrate that solar UV-B selection alters phyllosphere bacterial community composition and that UV tolerance is a prevalent phenotype late in the season. The total bacterial population size was not affected by either UV-B treatment. However, isolates from the UV-B؉ plots (n ‫؍‬ 368) were significantly more UV tolerant than those from the UV-B؊ (n ‫؍‬ 363) plots. UV sensitivity was determined as the minimal inhibitory dose of UV that resulted in an inhibition of growth compared to the growth of a nonirradiated control. The difference in minimal inhibitory doses among bacterial isolates from UV-B؉ and UV-B؊ treatments was mainly partitioned among nonpigmented isolates, with pigmented isolates as a group being characterized as UV tolerant. A large increase in UV tolerance was observed within isolate groups collected late (89 and 96 days after planting) in the season. Identification of 200 late-season isolates indicated that the predominant UV-tolerant members of this group were Bacillus coagulans, Clavibacter michiganensis, and Curtobacterium flaccumfaciens. We selected C. michiganensis as a model UV-tolerant epiphyte to study if cell survival on UV-irradiated peanut leaves was increased relative to UV survival in vitro. The results showed an enhancement in the survival of C. michiganensis G7.1, especially following high UV-C doses (300 and 375 J m ؊2 ), that was evident between 24 and 96 h after inoculation. A dramatic increase in the in planta/in vitro survival ratio was observed over the entire 96-h experiment period for C. michiganensis T5

> Abstract The short-term population dynamics of the culturable bacterial community from field-grown peanut (Arachis hypogeae L.) was analyzed over three 2-day periods. As in other phyllosphere studies, significant numbers of pigmented organisms were detected, suggesting the importance of pigmentation in the colonization of this habitat. Isolates were grouped according to pigmentation (orange, pink, yellow, nonpigmented), and the sensitivity of each isolate in the collection (n = 617) to ultraviolet radiation (UVR) was determined as the minimal inhibitory dose (MIDC) of UVR that resulted in an inhibition of growth compared to an unirradiated control. The majority of isolates recovered (56.1%) had an MIDC equal to or exceeding that of Pseudomonas syringae 8B48, a known UV-tolerant strain. Among pigmentation groups, the mean MIDC of pink- and orange-pigmented isolates was significantly greater than that of yellow- or nonpigmented isolates at each sampling time of day. Identification of 213 of the isolates using fatty acid methyl ester analysis indicated that a large proportion of the isolates were gram-positive, with Bacillus spp. alone accounting for 35.7% of the total. The genus Curtobacterium contained the largest percentage of highly UVR-tolerant strains. Nonpigmented mutants of four Curtobacterium strains were selected following ethyl methane sulfonate mutagenesis; these nonpigmented mutants were significantly altered in survival following irradiation with UV-A wavelengths. The strategy of avoidance of UVR through colonization of the abaxial leaf surface was evaluated on three separate occasions by leaf imprint sampling. Only 3 of 120 leaves (2.5%) contained larger bacterial populations on the adaxial surface, indicating that colonization of the abaxial leaf surface is important to phyllosphere survival. Our results indicate that tolerance to UVR is a common phenotype among phyllosphere bacteria, suggesting that solar radiation has a strong influence on the microbial ecology of the phyllosphere.http://link.springer-ny.com/link/service/journals/00248/bibs/38n1p27.html

Phenotypic mechanisms that enhance bacterial UVR survival typically include pigmentation and DNA repair mechanisms which provide protection from UVA and UVB wavelengths, respectively. In this study, we examined the contribution of pigmentation to field survival in Clavibacter michiganensis and evaluated differences in population dynamics and leaf colonization strategies. Two C. michiganensis pigment-deficient mutants were significantly reduced in UVA radiation survival in vitro; one of these mutants also exhibited reduced field populations on peanut when compared to the wild-type strain over the course of replicate 25-day experiments. The UVR-tolerant C. michiganensis strains G7.1 and G11.1 maintained larger epiphytic field populations on peanut compared to the UVR-sensitive C. michiganensis T5.1. Epiphytic field populations of C. michiganensis utilized the strategy of solar UVR avoidance during leaf colonization resulting in increased strain survival on leaves after UVC irradiation. These results further demonstrate the importance of UVR tolerance in the ability of bacterial strains to maintain population size in the phyllosphere. However, an examination of several bacterial species from the peanut phyllosphere and a collection of environmental Pseudomonas spp. revealed that sensitivity to UVA and UVC radiation was correlated in some but not all of these bacteria. These results underscore a need to further understand the biological effects of different solar wavelength groups on microbial ecology.