Xili Liu

Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance.

The potential for oxathiapiprolin resistance in Phytophthora capsici was evaluated. The baseline sensitivities of 175 isolates to oxathiapiprolin were initially determinated and found to conform to a unimodal curve with a mean EC50 value of 5.61 × 10-4 μg/ml. Twelve stable oxathiapiprolin-resistant mutants were generated by fungicide adaptation in two sensitive isolates, LP3 and HNJZ10. The fitness of the LP3-mutants was found to be similar to or better than that of the parental isolate LP3, while the HNJZ10-mutants were found to have lost the capacity to produce zoospores. Taken together these results suggest that the risk of P. capsici developing resistance to oxathiapiprolin is moderate. Comparison of the PcORP1 genes in the LP3-mutants and wild-type parental isolate, which encode the target protein of oxathiapiprolin, revealed that a heterozygous mutation caused the amino acid substitution G769W. Transformation and expression of the mutated PcORP1-769W allele in the sensitive wild-type isolate BYA5 confirmed that the mutation in PcORP1 was responsible for the observed oxathiapiprolin resistance. Finally diagnostic tests including As-PCR and CAPs were developed to detect the oxathiapiprolin resistance resulting from the G769W point mutation in field populations of P. capsici.

Photodegradation of the Isoxazolidine Fungicide SYP-Z048 in Aqueous Solution: Kinetics and Photoproducts

Previous research has demonstrated that 3-[5-(4-chlorophenyl)-2,3-dimethyl-3-isoxazolidinyl]pyridine (SYP-Z048), a newly developed nitrogen heterocycle substituted isoxazolidine compound, has good protective and curative activities against a wide range of fungal diseases of fruits and vegetables caused by Ascomycetes, Basidiomycetes, and Deuteromycetes. In this study, the photochemical behavior of SYP-Z048 was investigated in aqueous solution and in response to solar and low-pressure mercury ultraviolet (UV) lamp irradiation. SYP-Z048 photolysis was pH- and temperature-dependent and was described by a first-order degradation reaction. A total of 11 photoproducts were separated by high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) and were identified on the basis of (1)H and (13)C nuclear magnetic resonance (NMR) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) spectra. The photoproduct structures and kinetics suggested that the phototransformation of SYP-Z048 occurred via multiple reaction pathways that included the cleavage of the N-O bond in the isoxazolidine ring and the dechlorination of the benzene ring.

Inhibitory effect of bionic fungicide 2-allylphenol on Botrytis cinerea (Pers. ex Fr.) in vitro.

BACKGROUNDn2-Allylphenol is a registered fungicide in China to control fungal diseases on tomato, strawberry and apple. It is synthetic and structurally resembles the active ingredient ginkgol isolated from Ginkgo biloba L. bark. 2-Allylphenol has been used in China for 10 years. However, its biochemical mode of action remains unclear. An in vitro study was conducted on the biochemical mechanism of 2-allyphenol inhibiting Botrytis cinerea (Pers. ex Fr.).nnnRESULTSnThe inhibition was approximately 3 times stronger when the fungus was grown on non-fermentable source, glycerol, than that on a fermentable carbon source, glucose. Inhibition of B. cinerea and Magnaporthe oryzae (Hebert) Barr mycelial growth was markedly potentiated in the presence of salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial alternative oxidase. Furthermore, at 3 h after treatment with 2-allylphenol, oxygen consumption had recovered, but respiration was resistant to potassium cyanide and sensitive to SHAM, indicating that 2-allylphenol had the ability to induce cyanide-resistant respiration. The mycelium inhibited in the presence of 2-allylphenol grew vigorously after being transferred to a fungicide-free medium, indicating that 2-allylphenol is a fungistatic compound. Adenine nucleotide assay showed that 2-allylphenol depleted ATP content and decreased the energy charge values, which confirmed that 2-allylphenol is involved in the impairment of the ATP energy generation system.nnnCONCLUSIONnThese results suggested that 2-allylphenol induces cyanide-resistant respiration and causes ATP decrease, and inhibits respiration by an unidentified mechanism.

Activity of the novel fungicide oxathiapiprolin against plant-pathogenic oomycetes

BACKGROUNDnOxathiapiprolin was the first of the piperidinyl thiazole isoxazoline class of fungicides to be discovered and developed by DuPont in 2007. Although oxathiapiprolin has been reported to have high activity against plant-pathogenic oomycetes, such as Peronospora belbahrii, Phytophthora nicotianae and Ph. capsici, little is known about its effectiveness against other plant-pathogenic oomycetes and its protective and curative properties.nnnRESULTSnOxathiapiprolin exhibited substantial inhibitory activity against all of the plant-pathogenic oomycetes tested, with EC90 values ranging from 0.14 to 3.36 × 10(-3) µg mL(-1) , except the Pythium species Py. aphanidermatum and Py. deliense. Furthermore, doses as low as 10 µg mL(-1) were found to inhibit zoospore release and motility in Ph. capsici, while the mycelial development and sporangial production of Pseudoperonospora cubensis were restrained by an EC50 of 3.10 × 10(-4) and 5.17 × 10(-4) µg mL(-1) respectively. It was also found that oxathiapiprolin exhibited both protective and curative activity against the development of Ph. capsici infection in pepper plants under greenhouse conditions and in field tests.nnnCONCLUSIONnThe present study demonstrated that the novel fungicide oxathiapiprolin exhibits strong inhibitory activity against a range of agriculturally important plant-pathogenic oomycetes, including Phytophthora spp., Peronophythora litchii, Plasmopara viticola, Pe. parasitica, Ps. cubensis and Py. ultimum.

Assessing the risk for resistance and elucidating the genetics of Colletotrichum truncatum that is only sensitive to some DMI fungicides

The genus Colletotrichum contains a wide variety of important plant pathogens, and Colletotrichum truncatum is one of the most prevalent species of Colletotrichum on chili in China. Demethylation-inhibitor fungicides (DMIs) are currently registered chemical agents for the management of the anthracnose disease caused by Colletotrichum spp. To assess the risk for DMI resistance development, 112 C. truncatum isolates were collected from infected pepper in 13 regions of China. The sensitivity of C. truncatum isolates to five DMI fungicides was determined based on mycelial growth inhibition assay. C. truncatum was sensitive to prochloraz, epoxiconazole, and difenoconazole, but not to tebuconazole or myclobutanil. Baseline sensitivity using the 112 C. truncatum isolates was established for the first three effective DMIs. Prochloraz, epoxiconazole, and difenoconazole EC50 values were 0.053 ± 0.023, 1.956 ± 0.815, and 1.027 ± 0.644 μg/ml, respectively. Eleven stable DMI-resistant mutants all exhibited lower fitness levels than their wild-type parents, suggesting a low risk of DMI resistance in C. truncatum. By inducing gene expression, CtCYP51 expression increased slightly in the resistant mutants as compared to wild-types when exposed to DMI fungicides and thus contributed at least partially to resistance. Molecular docking with CYP51 structure models was used to explain differential sensitivity of the DMI fungicides in C. truncatum. Our results suggest that the M376L/H373N mutations in CYP51 changed the conformation of DMIs in the binding pocket. These changes prevented the formation of the Fe – N coordinate bond between the heme iron active site and tebuconazole or myclobutanil, and apparently contributed to tebuconazole and myclobutanil insensitivity of C. truncatum.

Metabolism of fungicide 2-allylphenol in Rhizoctonia cerealis.

2-Allylphenol is a biomimetic synthetic fungicide that mimics the compound ginkgol found in gingko fruit (Gingko biloba L.). This systemic fungicide can effectively suppress a wide range of plant diseases, including wheat sharp eyespot (Rhizoctonia cerealis). However, its degradation in environment after application is still unknown. To understand this fungicide degradation, major metabolites of 2-allylphenol in R. cerealis were examined. The parent and metabolites of 2-allylphenol were detected and quantified in the mycelia and liquid medium. Results showed that 2-allylphenol was metabolized and bio-transformed by R. cerealis, and four metabolites were found, including 2-(2-hydroxyphenyl) acetic acid (M1), 2-(2, 3-dihydroxypropyl) phenol (M2), 2-(2-hydroxypropyl)-phenol (M3) and 2-(3-hydroxypropyl)-phenol (M4). Based on the results, we propose that the biodegradation pathway is that 2-allylphenol is rapidly oxidized into metabolite M2 and hydrolyzed into M3 and M4, which formed M2, and carboxylation of M2 to 2-hydroxy-3-(2׳-hydroxyphenyl) propionic acid which undergo hydrolyzation and decarboxylation to form M1. 2-Allylphenol can be bio-transformed to new compounds by R. cerealis, suggesting the existence of microbe metabolic pathways for 2-allylphenol.

Pseudoperonospora cubensis in China: Its sensitivity to and control by oxathiapiprolin

The novel fungicide oxathiapiprolin has potential for the control of downy mildew of cucumber, which is caused by Pseudoperonospora cubensis. In this study, an in vitro bioassay with detached leaves was used to determine the baseline sensitivity to oxathiapiprolin for 77 Ps. cubensis isolates from 11 provinces in China. The baseline sensitivity curve was continuous, and the average EC50 value was 2.23u202f×u202f10-4u202fμgu202fml-1. In field trials, the control of downy mildew of cucumber was greater with oxathiapiprolin at 20 or 30u202fgu202fa.i.u202fha-1 than with dimethomorph at 262.5u202fgu202fa.i.u202fha-1. Oxathiapiprolin was taken up by cucumber roots and transported upwards to stems and leaves. The full length of PscORP1, the gene that encodes the target protein of oxathiapiprolin in Ps. cubensis, was sequenced for the first time. Our results suggested that oxathiapiprolin will be an excellent alternative fungicide for control of cucumber downy mildew. However, as Ps. cubensis is a high-risk pathogen, resistance development to oxathiapiprolin should be monitored and managed.

The novel fungicide SYP-14288 acts as an uncoupler against Phytophthora capsici

SYP-14288 is a novel fungicide developed by the Shenyang Research Institute of Chemical Industry in China. Although preliminary studies indicate that SYP-14288 is highly effective against 32 important plant pathogens belonging to a range of taxonomic groups, its mode of action remains unknown. In this study, we documented that SYP-14288 has excellent activity against all of the asexual life stages of the plant-pathogenic oomycete Phytophthora capsici, and is especially effective in blocking cyst germination and other life stages that require high energy consumption. In assays designed to determine the fungicides mode of action, addition of ATP reduced SYP-14288 inhibition of P. capsici, which suggested that SYP-14288 inhibits ATP synthesis of the pathogen. This inference was confirmed in that treatment with SYP-14288 sharply reduced the ATP content in P. capsici. The respiration rate of P. capsici was positively correlated with the concentration of SYP-14288 or of the fungicide fluazinam (an uncoupler of oxidative phosphorylation), but increases in respiration were greater with SYP-14288 than with fluazinam. These results indicate that SYP-14288 is a promising fungicide that functions as an uncoupler of oxidative phosphorylation.

Protocol of Phytophthora capsici Transformation Using the CRISPR-Cas9 System

Phytophthora capsici is an important plant pathogen, which causes significant economic losses on multiple vegetable crops worldwide. It is an ideal model pathogen to study the role of important genes, plant-pathogen interactions, and fungicide resistance mechanisms etc. due to its wide range of hosts and genetic diversity. A more efficient gene editing tool is required to do these studies. Here, we describe a detailed experimental procedure using the CRISPR-Cas9 system to edit genes of interest in P. capsici, which has been proven to be an accurate and efficient gene editing method in P. capsici.

Oxysterol-binding protein-related protein 2 is not essential for Phytophthora sojae based on CRISPR/Cas9 deletions: Role of PsORP2 in Phytophthora sojae

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are a large conserved family of lipid transfer proteins in eukaryotes. In oomycetes, some ORPs are the target of the novel oomycide oxathiapiprolin. By searching the Phytophthora sojae genome database, two ORP proteins, PsORP1 (Protein ID: 558498) and PsORP2 (Protein ID: 470921), were found. Here, we investigated the biological function of PsORP2. The expression level of PsORP2 was higher in germinated cysts and late infection than in other developmental stages. However, deletion of PsORP2 using CRISPR/Cas9 had no significant effect on sporangia production, zoospore production, cyst germination, oospore production, virulence or oxathiapiprolin sensitivity. PsORP1 also was not upregulated in ΔPsORP2 transformants. Collectively, our studies demonstrate that PsORP2 is not an essential protein for development or virulence in P. sojae under the conditions we tested.