Botrytis cinerea, the causal agent of the grey mould disease, developed resistance to multiple fungicides. However, the role of cell membrane in survival competition of B. cinerea upon quinone outside inhibitor (QoI) fungicide has not yet been elucidated. In this paper, the enhancement of cystamine, a transglutaminase inhibitor, on membrane integrity of B. cinerea was determined, and the effect of the enhancement on the sensitivity of B. cinerea to pyraclostrobin was investigated. The results showed that pyraclostrobin inhibited mycelial growth with EC50 as 1.122 and 3.042 μg/ml at 24 and 48 hr, respectively. In the treatment of 5 and 50 μg/ml pyraclostrobin, membrane integrity of B. cinerea was broken, causing high permeability, lipid peroxidation, flocculent and malformed surface with vague septum and abundant agglomerates inside and outside the mycelia. Cystamine even at 50 and 200 μg/ml had little inhibitory effect on mycelial growth. However, in presence of 50 or 200 μg/ml cystamine, the mycelia from pyraclostrobin treatment possessed a significantly reduced leakage, lower MDA content, and a revived fibrous and transparent surface. Meanwhile, SEM images showed that membrane integrity of the mycelia was significantly improved and the agglomerates were dramatically disappeared. Synergy assays further revealed that B. cinerea regained less sensitivity to pyraclostrobin inhibition. In conclusion, membrane integrity controls mycelia sensitivity and is required for survival competition of B. cinerea upon pyraclostrobin.
Pyraclostrobin (PYR) is a commonly used strobilurin fungicide, which inhibits mitochondrial respiration at the ubiquinol oxidation center site of the cytochrome bc1 complex. Little information is available regarding the crystal structure of PYR on its fungicidal effect. In this study, the crystal structures of eight PYRs (PYR-A to H) from different sources are determined by using high-resolution X-ray powder diffraction (XRPD) and model construction with the Pawley refinement module. The effects of PYRs on mycelium growth, the kinetics of mycelial growth, conidial germination, and tube elongation of conidia of Botrytis cinerea from tomato are compared. The level of organic acids in the mitochondrial tricarboxylic acid cycle of PYR-treated B. cinerea is analyzed. The results show that PYR-A to PYR-H have their own unique character of XRPD patterns, but the crystal morphology of eight PYRs presents in the triclinic crystal system and space group P1̅ . PYR-D with the eclipsed conformation and rational edge angles α (72.599°) and β (98.612°) in the crystal cell shows the highest inhibitory effect against mycelium growth with EC 50 as 3.383 μg mL −1 , the best time-dependent effects on the mycelium growth kinetics, and the strongest inhibition on tube elongation of conidia, whereas PYR-E with anticonformation is the worst. Moreover, a significant accumulation of fumarate, malate, and oxalate in the PYR-D-treated mycelium is observed. These findings reinforce the need for a definite crystal structure of PYR to limit usage and mitigate future selection pressure for gray mold management.
Insecticides are more broadly known
to affect insect cellular immunity,
but the components in hemocytes and their response to insecticide
stress are still unknown. In this paper, a method based on trifluoroacetic
acid extraction, followed by IC-CD/ESI-MS analysis, was developed
to simultaneously determine tricarboxylic acid (TCA) cycle metabolites
and anion components in hemocytes from Mythimna separata larvae. Validation gave excellent selectivity, recovery (88.7–107.6%),
linear correlation (r
2 > 0.9961), precision
(<3.89%), LOD (0.002–0.006 mg/L), LOQ (0.006–0.020
mg/L), and a short chromatographic run. The method was verified by
administration of 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl
3-(1,3-dioxoiso-indolin-2-yl) propanoate (QDP) or emamectin benzoate
(EMB) to hemocytes in vitro and larvae in vivo. TCA metabolites including
citrate, α-ketoglutarate, fumarate, malate, and oxaloacetate,
and anions including acetate, oxalate, chloride, carbonate, and sulfate
were identified and clearly separated. QDP and EMB showed a biphasic
dose effect on TCA metabolites, and the contrary hormesis paralleled
the different actions of QDP and EMB. The inhibition or improvement
of cellular immunity depended on the QDP concentration. In conclusion,
a highly sensitive, reliable, and robust method was developed, enabling
the monitoring of hemocyte immunity by the quantification of TCA metabolites
and anion components in minute hemocyte samples.
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