Antifungal mode of action of triforine

Rapidly growing mycelia of Aspergillus fumigatus treated with 10 μg/ml triforine (N,N′-bis-(1-formamido-2,2,2-trichloroethyl)-piperazine) showed little or no inhibition in dry weight increase prior to 2 h. By 2.5–3 h, triforine inhibited dry weight increase by 85%. The effects of triforine on protein, DNA, and RNA syntheses corresponded to the effect on dry weight increase both in time of onset and magnitude. Neither glucose nor acetate oxidation were inhibited by triforine.Ergosterol synthesis was almost completely inhibited by triforine even in the first hour after treatment. Inhibition of ergosterol synthesis was accompanied by an accumulation of the ergosterol precursors 24-methylenedihydrolanosterol, obtusifoliol, and 14α-methyl-Δ^{8, 24 (28)}-ergostadienol. Mycelia treated with 5 μg/ml of triarimol (α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol) also accumulated the same sterols as well as a fourth sterol believed to be Δ^{5, 7}-ergostadienol.Identification of 4,4-dimethyl-Δ^{8, 24 (28)}-ergostadienol in untreated mycelia indicates that the C-14 methyl group is the first methyl group removed in the biosynthesis of ergosterol by A. fumigatus. The lack of detectable quantities of 4,4-dimethyl-Δ^{8, 24 (28)}-ergostadienol in triforine or triarimol-treated mycelia and the accumulation of C-14 methylated sterols in treated mycelia suggests that both fungicides inhibit sterol C-14 demethylation. The accumulation of Δ^{5, 7}-ergostadienol in triarimol-treated mycelia further implies that triarimol also inhibits the introduction of the sterol C-22(23) double bond.Two strains of Cladosporium cucumerinum tolerant to triforine and triarimol were also tolerant to the fungicide S-1358 (N-3-pyridyl-S-n-butyl-S′-p-t-butylbenzyl imidodithiocarbonate).     

Pheromone-based communication disruption ofAdoxophyes orana on peach using the new RAK 3+4 dispensers and their effect on development of fruit rot diseases

The effectiveness of the pheromone-based communication disruption method was examined against the summerfruit tortrix,Adoxophyes orana F.v.R. (Lepidoptera: Tortricidae), a pest of peach trees, using the new RAK 3+4 dispenser (BASF). NoA. orana males were captured in pheromone traps inside the experimental orchards, which were saturated with the RAK 3+4 dispensers. The percent of damaged leaves was practically zero, while the level of damaged fruits was 0–6% in pheromone-treated orchards. The percentage of fruit rot caused byMonilinia laxa was lower in pheromone-based communication disruption orchards than in the control. It was concluded that the RAK 3+4 dispenser could be used againstA. orana as an economical and environmentally friendly method.     

Age-related resistance to Pseudomonas syringae pv. tomato is associated with the transition to flowering in Arabidopsis and is effective against Peronospora parasitica

As plants mature it has been observed that some become more resistant to normally virulent pathogens. The ability to manifest the Age-Related Resistance (ARR) response in Arabidopsis to Pseudomonas syringae pathovars tomato (Pst) coincided with the transition to flowering in plants both delayed and accelerated in the transition to flowering. ARR was also associated with a change in PR-1 gene expression, such that young plants expressed PR-1 abundantly at 3 days post inoculation (dpi) while mature plants expressed much less. The Arabidopsis ARR response requires SA accumulation via isochorismate synthase (ICS1) [24]. ICS1 was expressed one dpi with virulent and avirulent Pst in both young and mature plants. The ARR response was also effective versus avirulent Pst providing an additional 4-fold limitation in bacterial growth. Arabidopsis ARR was found to be ineffective against two necrotrophs, Erwinia carotovora subspecies carotovora (bacterium) and Botrytis cinerea (fungus) and one obligate biotroph, Erysiphe cichoracearum (fungus). However, mature wild type, SA-deficient sid2 and NahG plants supported little growth of the obligate biotrophic oomycete, Peronospora parasitica. Therefore ARR to P. parasitica appears to be SA-independent, however the level of ARR resistance was somewhat reduced in these mutants in some experiments. Thus, there may be numerous defence pathways that contribute to adult plant resistance in Arabidopsis.     

Apoplastic redox metabolism: Synergistic phenolic oxidation and a novel oxidative burst

The plant apoplast is an important mediator of communication between the cell cytoplasm and its surroundings. Plant cell suspensions offer a convenient model system to gain insight into apoplastic physiology. Here, we describe a novel phenomenon that took place when two naturally occurring phenolics were added together to either soybean or tobacco cell suspensions. Acetosyringone (AS) and/or hydroxyacetophenone (HAP), phenolics found in the extracellular/apoplast of tobacco cells, were added to soybean or tobacco cell suspensions undergoing an oxidative burst. Individually, AS appeared to be utilized as a typical peroxidase substrate to scavenge hydrogen peroxide, while HAP was utilized at a much lower rate. However, when added together the rate of utilization of both phenolics increased and surprisingly resulted in the production of hydrogen peroxide. We have further characterized this novel phenomenon in suspension cells. This study demonstrates that certain phenolics in plants can cause co-oxidation which, as in animals, could alter the structure and bioactivity of surrounding phenolics.     

Comparison of a baiting method and PCR for the detection of Phytophthora fragariae var. rubi in certified raspberry stocks*

In winter 2002/2003, a total of 136 root samples from 57 different raspberry stocks in Scotland were examined for the presence of raspberry root rot caused by the fungus‐like pathogen Phytophthora fragariae var. rubi. All stocks had been planted as propagation material entered at different grades in the Scottish certification scheme or had applied for plant passports. For detection, a modified ‘Duncan bait test’ was compared to a nested PCR method. The two tests identified the same infected stocks: PCR detected 10 positive samples from four different stocks, while the bait test picked up two additional positive samples coming from the same four stocks. The two tests had a similar level of reliability in this examination and a recommendation for one or the other depends mainly on the technical equipment and skills available in the laboratory.     

Antitumor Activity of Mycobacterial Cell Wall‐DNA Complex (MCC) Against Canine Urinary Bladder Transitional Cell Carcinoma Cells

Introduction:  Mycobacterial cell wall‐DNA complex (MCC) is a bifunctional anticancer agent that induces cancer cell apoptosis and stimulates cytokine synthesis by immune cells. Intravesical MCC is currently being evaluated in humans with high‐grade urinary bladder cancer. Evaluation of MCC in dogs with transitional cell carcinoma (TCC) will allow mechanistic studies in a natural animal model of TCC, and a potentially beneficial therapy for dogs with this cancer. In this study, we have determined the anticancer activity of MCC against canine TCC cells in vitro .
Methods:  Canine TCC cells (K9TCC cell line) were incubated with MCC (0.05–100 μg/ml, 0.5–72 hours). Cellular proliferation was measured by MTT reduction. Cell cycle was analyzed by flow cytometry with propidium iodide. Apoptosis was identified by flow cytometry using anti‐active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies. Apoptosis‐inducing activity of 100 μg/ml MCC in combination with piroxicam (0.1–1.0 uM) was evaluated.
Results:  MCC inhibited K9TCC cell proliferation in a concentration‐dependent manner (maximal activity – 45% at 100 μg/ml MCC) in association with the presence of activated caspase‐3 and cleaved PARP. Inhibition of proliferation and apoptosis‐inducing activities of MCC were independent of cell cycle phase. A thirty‐minute exposure of MCC was sufficient for optimal activity. Piroxicam (0.5 uM) enhanced apoptosis‐inducing activity of MCC.
Conclusions:  MCC induces apoptosis in K9TCC cells. This activity is potentiated by piroxicam. Following positive results in vitro , in vivo studies have been initiated. One dog, treated to date, has had a minor reduction in tumor volume following the first course of treatment with no treatment‐related toxicity.     

Estimation of daily and total lactation milk yield of Chios ewes from single morning or evening records

Linear models were developed and evaluated for the estimation of daily (24 h) and total lactation milk yield of dairy sheep from single morning (am) or evening (pm) milking records. A total of 22,908 individual test-day milk records of 5110 ewes of the Chios breed, raised in 46 flocks, were used. Depending on the model, different daily milk yield estimates were derived for each ewe, accounting for lactation number, stage of lactation, season of previous lambing and interval between successive milkings. Daily milk yield was also estimated from doubling the single am or pm record. Actual and estimated yields were compared using root mean square errors (RMSEs), mean absolute differences, an accuracy parameter defined as the ratio of the actual yield variance over the sum of the variances of actual yield and absolute difference, and the product moment correlation between estimated and actual yield. Results were validated on independent datasets. Linear models resulted in less biased and more accurate estimates of the daily milk yield than simply doubling the am or pm record. Root mean square errors decreased by 7-37% and the mean absolute difference was reduced by 1-4% of the average daily yield. Higher accuracy and correlation were obtained from linear model application than doubling. Total lactation milk yield was predicted based on actual daily yield and compared to predictions based on estimated daily yield from am or pm records, as well as directly on single milking records. Root mean square errors obtained when daily yield had been estimated with linear models were 26-35% lower compared to doubling the am or pm yield and 0-13% lower compared to estimating the total lactation yield directly from single milking records. Linear model application also resulted in lower mean absolute difference and higher accuracy and correlation than doubling the am or pm record. Recording the yield of a single milking (am or pm) instead of both can benefit milk recording by reducing its cost and increasing farmer participation. In this context, linear models developed in the present study can be used for the accurate estimation of daily (24 h) and total lactation milk yield from single milking records.