Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary spreads worldwide and causes serious economic losses. Considering the development of fungicide resistance and chemical residues, it is urgently necessary to explore alternative fungicides. In this study, the activity of the natural product cinnamic acid was assessed. The EC₅₀ values for cinnamic acid inhibition of mycelial growth of 103 S. sclerotiorum strains ranged from 9.37 to 42.54 µg/ml with an average EC₅₀ value of 18.77 (±3.39) µg/ml. No cross-resistance was detected between cinnamic acid and the commonly used fungicides carbendazim or dimethachlon. After treatment with cinnamic acid, mycelia distorted with more branches, no sclerotia developed, and the oxalic acid content decreased, whereas cell membrane permeability increased significantly. In pot experiments, cinnamic acid at 2,000 µg/ml provided over 95% efficacy against both carbendazim-sensitive and carbendazim-resistant strains of S. sclerotiorum. The expression of the sclerotia development-correlated genes Sac1, Pac1, Smk1, and Pka1 decreased, whereas the Cna1 gene expression increased. Altogether, cinnamic acid shows potential to be a natural alternative to commercial fungicides or a lead compound to develop new fungicides for the control of Sclerotinia stem rot. The biological characteristics contribute to the understanding of the action mechanism of cinnamic acid against S. sclerotiorum.

Phytophthora blight of pepper caused by Phytophthora capsici Leonian is a destructive disease throughout the world. Cuminic acid, extracted from the seed of Cuminum cyminum L., belongs to the benzoic acid chemical class. In this study, the sensitivity and biochemical response of P. capsici to cuminic acid was determined. The mean EC₅₀ (50% effective concentration) values for cuminic acid in inhibiting mycelial growth and zoospore germination of the 54 studied P. capsici isolates were 14.54 ± 5.23 μg/mL and 6.97 ± 2.82 μg/mL, respectively. After treatment with cuminic acid, mycelial morphology, sporangium formation and mycelial respiration were significantly influenced; cell membrane permeability and DNA content increased markedly, but pyruvic acid content, adenosine triphosphate (ATP) content, and ATPase activity decreased compared with the untreated control. In pot experiments, cuminic acid exhibited both protective and curative activity. Importantly, POD and PAL activity of the pepper leaves increased after being treated with cuminic acid. These indicated that cuminic acid not only showed antifungal activity, but also could improve the defense capacity of the plants. All the results suggested that cuminic acid exhibits the potential to be developed as a new phytochemical fungicide, and this information increases our understanding of the mechanism of action of cuminic acid against Phytophthora capsici.

The point mutation at codon 200 (TTC→TAC, F200Y) confers moderate resistance to carbendazim in Sclerotinia sclerotiorum. This mutant genotype (F200Y) has been detected mainly by determining the minimum inhibitory concentration (MIC), which requires 3 to 5 days. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum. Specific LAMP primers were designed and concentrations of LAMP components were optimized. The optimal reaction conditions were 62 to 63°C for 45 min. The new LAMP assay requires no special equipment and is highly sensitive and specific (the i.e., it generated positive results with F200Y mutant genotype but generated negative results with other carbendazim-resistant mutants and with a variety of carbendazim-resistant mutants of Botrytis cinerea and Fusarium graminearum). Inclusion of the loop backward (LB) primer reduced the reaction time to 15 min. Results were identical with LAMP and MIC determinations. The advantages of the LB-accelerated LAMP assay for detection of the F200Y mutant genotype were demonstrated by assaying sclerotia produced on rape stems that were artificially inoculated in the field. The results indicated that the new LAMP assay represents an improved way to detect the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum.

In order to develop a novel biofungicide, the antifungal activity and action mode of cuminic acid from the seed of Cuminum cyminum L. against Fusarium oxysporum f. sp. niveum (FON) on watermelon was determined systematically. In this study, the median effective concentration (EC₅₀) value for cuminic acid in inhibiting mycelial growth of FON was 22.53 μg/mL. After treatment with cuminic acid, the mycelial morphology was seriously influenced; cell membrane permeability and glycerol content were increased markedly, but pigment and mycotoxin (mainly fusaric acid) were significantly decreased. Synthesis genes of bikaverin (Bike1, Bike2 and Bike3) and fusaric acid (FUB1, FUB2, FUB3 and FUB4) both were downregulated compared with the control, as confirmed by quantitative RT-PCR. In greenhouse experiments, cuminic acid at all concentrations displayed significant bioactivities against FON. Importantly, significant enhancement of activities of SOD, POD, CAT and decrease of MDA content were observed after in vivo cuminic acid treatment on watermelon leaves. These indicated that cuminic acid not only showed high antifungal activity, but also could enhance the self-defense system of the host plant. Above all, cuminic acid showed the potential as a biofungicide to control FON.