Detection and Characterization of Benzimidazole Resistance in California Populations of Colletotrichum cereale.

Colletotrichum cereale is the causal agent of turfgrass anthracnose, which has become a serious problem on annual bluegrass (Poa annua) and creeping bentgrass (Agrostis palustris) golf course putting greens. Thiophanate-methyl is a benzimidazole (methyl benzimidazole carbamate [MBC]) fungicide used for the management of anthracnose. In this study, we examined 481 isolates from 10 California populations to determine the presence and frequency of MBC resistance. An in vitro methodology was developed to construct a baseline sensitivity distribution using 60 isolates from an unexposed population (TCGC). The 50% effective dose (ED50) values for the baseline sensitivity distribution for thiophanate-methyl ranged from 0.14 to 2.3 µg/ml with a mean of 0.75 µg/ml. For 60 isolates assayed from an exposed population (AHCC), 57 isolates were not responsive to in vitro concentrations of thiophanate-methyl of up to 30 µg/ml. Isolates nonresponsive to thiophanate-methyl were not responsive to benomyl in vitro. Two isolates nonresponsive in vitro to thiophanate-methyl or benomyl were not controlled in vivo on annual bluegrass plants treated preventively with either fungicide at 11 mg/ml, confirming the results of the in vitro testing. The remaining 361 isolates from eight populations were tested using the single discriminatory dose of thiophanate-methyl at 10 µg/ml. A high proportion (>90%) of isolates from six of the populations were resistant to thiophanate-methyl, indicating the presence of practical resistance at these locations. To determine the molecular mechanism of MBC resistance, the two ß-tubulin genes, TUB1 and TUB2, of 12 resistant and 6 sensitive isolates were amplified and sequenced, revealing a glutamic acid to lysine substitution at position 198 of TUB2 that was present in all resistant isolates. This work confirms the presence of MBC resistance in C. cereale populations from California and presents methods and information that can be used to manage resistance to the MBC fungicides and improve anthracnose management programs.

Sensitivity Distributions of California Populations of Colletotrichum cereale to the DMI Fungicides Propiconazole, Myclobutanil, Tebuconazole, and Triadimefon.

The baseline sensitivity of a California population of Colletotrichum cereale (turfgrass anthracnose) to the sterol demethylation inhibitor (DMI) fungicide propiconazole was determined using an in vitro assay with known reproducibility. The 50% effective dose (ED50) values for propiconazole for a nonexposed, baseline population ranged from 0.025 to 0.35 µg/ml with a mean of 0.14 µg/ml. Examination of two DMI-exposed populations indicated an approximate increase of 6.5× in mean ED50 values. In vivo testing of two isolates with ED50 values of propiconazole at 0.15 and 0.90 µg/ml indicated reduced control for the less sensitive isolate by propiconazole at rates ≤38 µg/ml. It was determined that single discriminatory dose testing in vitro with propiconazole at 0.50 µg/ml could differentiate sensitive and resistant isolates. Using this dose, six additional populations were tested and DMI-exposed populations were found to be three to nine times less sensitive compared with the baseline population. Two populations were assayed for sensitivity to myclobutanil, tebuconazole, and triadimefon. Mean ED50 values for a nonexposed population were 0.72, 0.082, and 5.6 µg/ml, respectively; for a DMI-exposed population, mean ED50 values were 3.8, 0.35, and 18 µg/ml, respectively. This work provides information on the development of DMI resistance in populations of C. cereale in California and methodologies for future resistance monitoring for this pathogen.

Occurrence and Distribution of QoI-Resistant Isolates of Colletotrichum cereale from Annual Bluegrass in California.

Turfgrass anthracnose, caused by Colletotrichum cereale (ex. Colletotrichum graminicola), is an important disease of turf used on golf course putting greens. Recent management of the disease has become increasingly difficult, partly due to the possible development of practical resistance to the QoI fungicides. In all, 558 single-conidia isolates of C. cereale were collected from 10 California golf courses, 8 of which had been exposed to QoI fungicides and 2 where no fungicides had been used. Isolates were tested using a mycelial expansion assay on azoxystrobinamended media. For the two nonexposed populations, in vitro 50% effective dose (ED50) values ranged from 0.0060 to 0.089 µg/ml. All isolates from the exposed populations could not be fully inhibited by doses of azoxystrobin as high as 8.0 µg/ml. A subset of these isolates were tested in vitro with the QoI fungicides pyraclostrobin and trifloxystrobin and found to be similar in response, indicating that these isolates were fully cross-resistant to all three fungicides. In greenhouse pot experiments, three isolates nonresponsive to QoI fungicides in vitro were not controlled by label rates of the fungicides. Spore germination assays also were examined; for 10 isolates identified as sensitive by mycelial expansion assays, ED50 values for axoystrobin ranged from 0.0040 to 0.0047 µg/ml; for 25 isolates identified as QoI-resistant, 93 to 100% of the conidia germinated at azoxystrobin concentrations as high as 8.0 µg/ml relative to the nonamended check treatments. Mitochondrial cytochrome b genes from a subset of 15 isolates (12 resistant and 3 sensitive) were partially cloned and sequenced; all resistant isolates had an alanine substitution that corresponded to position 143 of the gene product. These results indicate that QoI resistance is present in California populations of C. cereale and is contributing to the difficulty in controlling this disease.

(2,3,4,4-Tetramethylcyclopentyl)methyl acetate, a sex pheromone from the obscure mealybug: first example of a new structural class of monoterpenes.

The sex pheromone of the obscure mealybug, Pseudococcus viburni, consists of (1R*,2R*,3S*)-(2,3,4,4-tetramethylcyclopentyl)methyl acetate, the first example of a new monoterpenoid structural motif in which the two isoprene units forming the carbon skeleton are joined by 2'-2 and 3'-4 connections rather than the usual 1'-4, head-to-tail connections. This highly irregular terpenoid structure, and the irregular terpenoid structures of related mealybug species, suggest that these insects may have unique terpenoid biosynthetic pathways.

Allelochemical potential of Callicarpa acuminata.

The allelochemical potential of Callicarpa acuminata (Verbenaceae) was investigated by using a biodirected fractionation study as part of a long-term project to search for bioactive compounds among the rich biodiversity of plant communities in the Ecological Reserve El Eden, Quintana Roo, Mexico. Aqueous leachate, chloroform-methanol extract, and chromatographic fractions of the leaves of C. acuminata inhibited the root growth of test plants (23-70%). Some of these treatments caused a moderate inhibition of the radial growth of two phytopathogenic fungi, Helminthosporium longirostratum and Alternaria solani (18-31%). The chloroform-methanol (1:1) extract prepared from the leaves rendered five compounds: isopimaric acid (1), a mixture of two diterpenols [sandaracopimaradien-19-ol (3) and akhdarenol (4)], alpha-amyrin (5), and the flavone salvigenin (6)]. The phytotoxicity exhibited by several fractions and the full extract almost disappeared when pure compounds were evaluated on the test plants, suggesting a synergistic or additive effect. Compounds (4), (5), and the semisynthetic derivative isopimaric acid methyl ether (2) had antifeedant effects on Leptinotarsa decemlineata. Compound 5 was most toxic to this insect, followed by (2), (4), and (6) with moderate to low toxicity. No correlation was found between antifeedant and toxic effects on this insect, suggesting that different modes of action were involved. All the test compounds were cytotoxic to insect Sf9 cells while (6), (4), and (1) also affected mammalian Chinese Hamster Ovary (CHO) cells. Compound 5 showed the strongest selectivity against insect cells. This study contributes to the knowledge of the defensive chemistry and added value of C. acuminata.