Phytophthora RxLR effector PcSnel4B promotes degradation of resistance protein AtRPS2.

Phytophthora capsici deploys effector proteins to manipulate host immunity and facilitate its colonization. However, the underlying mechanisms remain largely unclear. In this study, we demonstrated that a Sne-like (Snel) RxLR effector gene PcSnel4 is highly expressed at the early stages of P. capsici infection in Nicotiana benthamiana. Knocking out both alleles of PcSnel4 attenuated the virulence of P. capsici, while expression of PcSnel4 promoted its colonization in N. benthamiana. PcSnel4B could suppress the hypersensitive reaction (HR) induced by Avr3a-R3a and RESISTANCE TO PSEUDOMONAS SYRINGAE 2 (AtRPS2), but it did not suppress cell death elicited by Phytophthora infestin 1 (INF1) and Crinkler 4 (CRN4). COP9 signalosome 5 (CSN5) in N. benthamiana was identified as a host target of PcSnel4. Silencing NbCSN5 compromised the cell death induced by AtRPS2. PcSnel4B impaired the interaction and colocalization of Cullin1 (CUL1) and CSN5 in vivo. Expression of AtCUL1 promoted the degradation of AtRPS2 and disrupted HR, while AtCSN5a stabilized AtRPS2 and promoted HR, regardless of the expression of AtCUL1. PcSnel4 counteracted the effect of AtCSN5 and enhanced the degradation of AtRPS2, resulting in HR suppression. This study deciphered the underlying mechanism of PcSnel4-mediated suppression of HR induced by AtRPS2.

Two point mutations N771S and K847N in the VHA-a of Phytophthora litchii confer resistance to fluopimomide.

The phytopathogenic oomycete Phytophthora litchii is the culprit behind the devastating disease known as "litchi downy blight", which causes large losses in litchi production. Although fluopimomide exhibits strong inhibitory efficacy against P. litchii, the exact mechanism of resistance is still unknown. The sensitivity of 137 P. litchii isolates to fluopimomide was assessed, and it was discovered that the median effective concentration (EC50) of the fungicide had a unimodal frequency distribution with a mean value of 0.763 ± 0.922 µg/mL. Comparing the resistant mutants to the equivalent parental isolates, the resistance mutants' survival fitness was much lower. While there was no cross-resistance between fluopimomide and other oomycete inhibitors, there is a notable positive cross-resistance between fluopimomide and fluopicolide. According to the thorough investigation, P. litchii had a moderate chance of developing fluopimomide resistance. The point mutations N771S and K847N in the VHA-a of P. litchii (PlVHA-a) were present in the fluopimomide-resistant mutants, and the two point mutations in PlVHA-a conferring fluopimomide resistance were verified by site-directed mutagenesis in the sensitive P. capsici isolate BYA5 and molecular docking.

Ametoctradin resistance risk and its resistance-related point mutation in PsCytb of Phytophthora sojae confirmed using ectopic overexpression.

Ametoctradin is mainly used to treat plant oomycetes diseases, but the mechanism and resistance risk of ametoctradin in Phytophthora sojae remain unknown. This study determined the ametoctradin sensitivity of 106 P. sojae isolates and found that the frequency distribution of the median effective concentration (EC50) of ametoctradin was unimodal with a mean value of 0.1743 ± 0.0901 µg/mL. Furthermore, ametoctradin-resistant mutants had a substantially lower fitness index compared with that of wild-type isolates. Although ametoctradin did not show cross-resistance to other fungicides, negative cross-resistance to amisulbrom was found. In comparison to sensitive isolates, the control efficacy of ametoctradin to resistant mutants was lower, implying a low to moderate ametoctradin resistance risk in P. sojae. All ametoctradin-resistant mutants contained a S33L point mutation in PsCytb. A system with overexpression of PsCytb in the nucleus was established. When we ectopically overexpressed S33L-harboring PsCytb, P. sojae developed ametoctradin resistance. We hypothesized that the observed negative resistance between ametoctradin and amisulbrom could be attributed to conformational changes in the binding cavity of PsCytb at residues 33 and 220.

Characteristics of fluopicolide-resistance mutants in Phytophthora nicotianae, the pathogen causing black shank disease in tobacco.

Black shank, a devastating disease in tobacco production worldwide, is caused by the oomycete plant pathogen Phytophthora nicotianae. Fluopicolide is a pyridinylmethyl-benzamides fungicide with a unique mechanism of action and has been widely used for controlling a variety of oomycetes such as Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis, P. nicotianae and Bremia lactucae. However, the fluopicolide-resistance risk and molecular basis in P. nicotianae have not been reported. In this study, the sensitivity profile of 141 P. nicotianae strains to fluopicolide was determined, with a mean median effective concentration (EC50) value of 0.12 ± 0.06µg/mL. Five stable fluopicolide-resistant mutants of P. nicotianae were obtained by fungicide adaptation, and the compound fitness index of these resistant mutants were lower than that of their parental isolates. Additionally, cross-resistance tests indicated that the sensitivity of fluopicolide did not correlate with other oomycete fungicides, apart from fluopimomide. DNA sequencing revealed two point mutations, G765E and N769Y, in the PpVHA-a protein in the fluopicolide-resistant mutants. Transformation and expression of PpVHA-a genes carrying G765E and N769Y in the sensitive wild-type isolate confirmed that it was responsible for fluopicolide resistance. These results suggest that P. nicotianae has a low to medium resistance risk to fluopicolide in laboratory and that point mutations, G765E and N769Y, in PpVHA-a are associated with the observed fluopicolide resistance.

Sensitivity analysis and point mutations in BcSDHB confer cyclobutrifluram resistance in Botrytis cinerea from China.

Botrytis cinerea is one of the most destructive pathogens worldwide. It can damage over 200 crops, resulting in significant yield and quality losses. Cyclobutrifluram, a new generation of succinate dehydrogenase inhibitors, exhibits excellent inhibitory activity against B. cinerea. However, the baseline sensitivity and resistance of B. cinerea to cyclobutrifluram remains poorly understood. This study was designed to monitor the sensitivity frequency distribution, assess the resistance risk, and clarify the resistance mechanism of B. cinerea to cyclobutrifluram. The baseline sensitivity of B. cinerea isolates to cyclobutrifluram was 0.89 µg/mL. Cyclobutrifluram-resistant B. cinerea populations are present in the field. Six resistant B. cinerea isolates investigated in this study possessed enhanced compound fitness index compared to the sensitive isolates according to mycelial growth, mycelial dry weight, conidiation, conidial germination rate, and pathogenicity. Cyclobutrifluram exhibited no cross-resistance with tebuconazole, fludioxonil, cyprodinil, or iprodione. Sequence alignment revealed that BcSDHB from cyclobutrifluram-resistant B. cinerea isolates had three single substitutions (P225F, N230I, or H272R). Molecular docking verified that these mutations in BcSDHB conferred cyclobutrifluram resistance in B. cinerea. In conclusion, the resistance risk of B. cinerea to cyclobutrifluram is high, and the point mutations in BcSDHB (P225F, N230I, or H272R) confer cyclobutrifluram resistance in B. cinerea. This study provided important insights into cyclobutrifluram resistance in B. cinerea and offered valuable information for monitoring and managing cyclobutrifluram resistance in the future.

Resistance risk, resistance mechanism and the effect on DON production of a new SDHI fungicide cyclobutrifluram in Fusarium graminearum.

Fusarium head blight in wheat is caused by Fusarium graminearum, resulting in significant yield losses and grain contamination with deoxynivalenol (DON), which poses a potential threat to animal health. Cyclobutrifluram, a newly developed succinate dehydrogenase inhibitor, has shown excellent inhibition of Fusarium spp. However, the resistance risk of F. graminearum to cyclobutrifluram and the molecular mechanism of resistance have not been determined. In this study, we established the average EC50 of a range of F. graminearum isolates to cyclobutrifluram to be 0.0110 µg/mL. Six cyclobutrifluram-resistant mutants were obtained using fungicide adaptation. All mutants exhibited impaired fitness relative to their parental isolates. This was evident from measurements of mycelial growth, conidiation, conidial germination, virulence, and DON production. Interestingly, cyclobutrifluram did not seem to affect the DON production of either the sensitive isolates or the resistant mutants. Furthermore, a positive cross-resistance was observed between cyclobutrifluram and pydiflumetofen. These findings suggest that F. graminearum carries a moderate to high risk of developing resistance to cyclobutrifluram. Additionally, point mutations H248Y in FgSdhB and A73V in FgSdhC1 of F. graminearum were observed in the cyclobutrifluram-resistant mutants. Finally, an overexpression transformation assay and molecular docking indicated that FgSdhBH248Y or FgSdhC1A73V could confer resistance of F. graminearum to cyclobutrifluram.

Analysis of resistance risk and mechanism of the 14α-demethylation inhibitor ipconazole in Fusarium pseudograminearum.

Ipconazole is a broad-spectrum triazole fungicide that is highly effective against Fusarium pseudograminearum. However, its risk of developing resistance and mechanism are not well understood in F. pseudograminearum. Here, the sensitivities of 101 F. pseudograminearum isolates to ipconazole were investigated, and the average EC50 value was 0.1072 µg/mL. Seven mutants resistant to ipconazole were obtained by fungicide adaption, with all but one showing reduced fitness relative to the parental isolates. Cross-resistance was found between ipconazole and mefentrifluconazole and tebuconazole, but none between ipconazole and pydiflumetofen, carbendazim, fludioxonil, or phenamacril. In summary, these findings suggest that there is a low risk of F. pseudograminearum developing resistance to ipconazole. Additionally, a point mutation, G464S, was seen in FpCYP51B and overexpression of FpCYP51A, FpCYP51B and FpCYP51C was observed in ipconazole-resistant mutants. Assays, including transformation and molecular docking, indicated that G464S conferred ipconazole resistance in F. pseudograminearum.

Fungicide Resistance: Progress in Understanding Mechanism, Monitoring, and Management.

Fungicide treatments are often essential for maintaining healthy crops and to achieve reliable and high-quality yields. However, continued use of fungicides with the same modes of action can lead to development of fungicide resistance, which has emerged in various plant pathogens and is a serious threat to effective crop protection. Exploration of resistance mechanisms is critical for resistance monitoring and management. This brief review summarizes advances during the past five decades in understanding the molecular resistance mechanisms of plant pathogenic fungi and oomycetes to major classes of fungicides, including benzimidazoles, myosin inhibitors, sterol demethylation inhibitors, quinone outside inhibitors, succinate dehydrogenase inhibitors, anilinopyrimidines, carboxylic acid amides, and oxysterol-binding protein homolog inhibitors. Based on known resistance mechanisms, PCR- and loop-mediated isothermal amplification-based approaches have been developed to allow high-throughput monitoring and early/rapid detection of emerging resistance. Classical principles in fungicide resistance management are also summarized, including using different modes of action of fungicides, limiting the number of applications of the chemicals with site-specific modes of action, and avoidance of their eradicant use. Future studies on novel strategies of disease management, including development of epigenetics- and RNA-based fungicides, will provide valuable knowledge for management of fungicide resistance.

Three point mutations in AaCYP51 combined with induced overexpression of AaCYP51 conferred low-level resistance to mefentrifluconazole in Alternaria alternata.

Tomato early blight is a significant disease that causes substantial losses to tomato yield and quality. Mefentrifluconazole, an isopropanol-azole subgroup of triazole fungicides, has been registered in China for controlling various plant diseases, including tomato early blight, grape anthracnose, and apple brown spot. However, limited information is available on the mefentrifluconazole resistance risk and mechanism in plant pathogens. The sensitivity to mefentrifluconazole of 122 isolates of Alternaria alternata, one of the causal agents of tomato early blight, collected from different provinces in China, was evaluated. The results showed a unimodal curve for the sensitivity frequency, with an average EC50 of 0.306 µg/mL. Through fungicide adaption, six resistant mutants (N4, N5, T4, T5, NG1, and NG10) were obtained from three parental isolates, with a mutation frequency of 3.28 × 10-4 and resistance factors ranging between 19 and 147. The survival fitness of the resistant mutants, except for NG1, was significantly lower than that of their parental isolates. Positive cross-resistance was observed between mefentrifluconazole and difenoconazole or fenbuconazole, whereas no cross-resistance was found with three non-DMI fungicides. Furthermore, three distinct point mutations were detected in the AaCYP51 protein of the resistant mutants: I300S in T4 and T5; A303T in N4, NG1, and NG10; and A303V in N5. Compared to the parental isolates, the AaCYP51 gene was overexpressed in all six resistant mutants when treated with mefentrifluconazole. In summary, the resistance risk of A. alternata to mefentrifluconazole was low, and point mutations and overexpression of the AaCYP51 gene were identified as contributing factors to mefentrifluconazole resistance in A. alternata.

Two typical acyl-CoA-binding proteins (ACBPs) are required for the asexual development and virulence of Phytophthora sojae.

Being found in all eukaryotes investigated, acyl-CoA-binding proteins (ACBPs) participate in lipid metabolism via specifically binding acyl-CoA esters with high affinity. The structures and functions of ACBP family proteins have been extensively described in yeasts, fungi, plants and mammals, but not oomycetes. In the present study, seven ACBP genes named PsACBP1-7 were identified from the genome of Phytophthora sojae, an oomycete pathogen of soybean. CRISPR-Cas9 knockout mutants targeting PsACBP1 and PsACBP2 were created for phenotypic assays. PsACBP1 knockout led to defects in sporangia production and virulence. PsACBP2 knockout mutants exhibited impaired vegetative growth, zoospore production, cyst germination and virulence. Moreover, Nile red staining of PsACBP2 knockout and over-expression lines showed that PsACBP2 is involved in the formation of lipid bodies in P. sojae. Our results demonstrate that two ACBP genes are differently required for growth and development, and both are essential for virulence in P. sojae.

Resistance assessment of pyraoxystrobin in Magnaporthe oryzae and the detection of a point mutation in cyt b that confers resistance.

Pyraoxystrobin is a new QoI fungicide developed in China. The present study was aimed at determining the baseline sensitivity of M. oryzae to pyraoxystrobin and investigating the potential resistance risk and resistance mechanism of pyraoxystrobin in M. oryzae. The results showed that the mean EC50 of 109 M. oryzae isolates to pyraoxystrobin was 0.0094 µg/mL and the sensitivity exhibited a unimodal distribution. The established baseline sensitivity could provide critical data for monitoring sensitivity changes of M. oryzae to pyraoxystrobin in rice fields. The potential resistance risk was assessed by investigating the biological characteristics of the resistant mutants obtained by fungicide adaptation. The results indicated that the resistance risk of pyraoxystrobin in M. oryzae was medium to high with positive cross-resistance between pyraoxystrobin and azoxystrobin, but without cross resistance between pyraoxystrobin and carbendazim, isoprothiolane, and prochloraz. Further investigation revealed that the pyraoxystrobin-resistant mutants had a G143S mutation in the cyt b protein. Molecular docking confirmed that the G143S substitution conferred high resistance to pyraoxystrobin in M. oryzae. Collectively, the results of this study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for pyraoxystrobin.

Monitoring and characterization of prochloraz resistance in Fusarium fujikuroi in China.

Rice bakanae disease, caused by Fusarium fujikuroi, is a destructive seed-borne disease throughout the world. Prochloraz, a DMI (C-14α-demethylase inhibitor) fungicide, has been registered in China for >20 years. Prochloraz resistance in F. fujikuroi was severe in China with resistance frequencies of 34.56%, 45.33%, and 48.45% from 2019 to 2021. The fitness of prochloraz-resistant population was lower than that of sensitive population, with an average CFI of 2.86 × 106 and 4.56 × 106, respectively. No cross-resistance was detected between prochloraz and tebuconazole or hexaconazole, and the prochloraz-resistant isolates were still sensitive to fludioxonil, phenamacril, and pydiflumetofen. S312T mutation in Ffcyp51b or overexpression of Ffcyp51a and Ffcyp51b was detected in the highly resistant isolates. AS-PCR primers were designed to detect the prochloraz-resistant isolates with S312T mutation in the field. Resistant isolates carrying S312T mutation were the dominant group in prochloraz-resistant population with frequencies of 43.26%, 23.59%, and 71.20% from 2019 to 2021, which indicated that more attention should be paid to this genotype when monitoring and managing the prochloraz resistance in F. fujikuroi.

Characterization of Prochloraz Resistance in Fusarium fujikuroi from Heilongjiang Province in China.

Prochloraz is widely used to control rice bakanae disease caused by Fusarium fujikuroi. The current study was aimed at monitoring the development of F. fujikuroi resistance to prochloraz in the Heilongjiang Province and analyzing the fitness of F. fujikuroi strains with different resistance levels. The results indicated that most of the 89 F. fujikuroi strains collected from the Heilongjiang Province were resistant to prochloraz, with resistance frequency reaching 92.1%. To assess the field resistance risk of prochloraz, 21 F. fujikuroi strains with different resistance levels were selected to investigate their biological characteristics and assess their fitness. Mycelial growth, sporulation, and germination rates were significantly different among the tested strains. However, when grouped into two subpopulations, no significant difference was tested between prochloraz-resistant and prochloraz-sensitive strains. Pathogenicity assays revealed that the disease severity index of prochloraz-resistant strains was higher than that of prochloraz-sensitive strains. Cross-resistance assays showed no cross-resistance between prochloraz and five other fungicides, namely phenamacril, ipconazole, tebuconazole, carbendazim, and fluopyram. Ffcyp51A gene overexpression was observed in the prochloraz-resistant F. fujikuroi strains after exposure to prochloraz. Collectively, these results indicated that F. fujikuroi resistance against prochloraz was severe. Furthermore, prochloraz-resistant strains were highly fit and could potentially become a dominant population in rice fields, consequently resulting in yield loss.

Characterization of Colletotrichum spp. Sensitivity to Carbendazim for Isolates Causing Strawberry Anthracnose in China.

Strawberry anthracnose caused by Colletotrichum species is an important disease that may cause significant economic losses. Based on multilocus sequence analyses and morphological characteristics, 64 isolates from strawberry anthracnose samples collected from nine Chinese provinces and municipalities were identified as three species: Colletotrichum fructicola (29 isolates), Colletotrichum siamense (23 isolates), and Colletotrichum nymphaeae (12 isolates). Isolates of C. siamense showed strong aggressiveness to fruit and leaves. Isolates of C. fructicola showed strong aggressiveness to crowns. Isolates of C. nymphaeae were weakly or not pathogenic to fruit, leaves, or crowns. Sensitivity to carbendazim was determined for a total of 75 isolates, including 11 previously preserved. Two isolates of C. siamense were highly resistant (HR) and 21 were moderately resistant (MR). Nine isolates of C. fructicola were sensitive (S), 24 were HR, and four were MR. All 15 isolates of C. nymphaeae were insensitive, and their mycelial growth was not completely inhibited on potato dextrose agar amended with 500 µg/ml carbendazim. Beta-tubulin (TUB2) of representative isolates was amplified and sequenced, revealing a glutamic acid substituted by alanine at codon 198 in HR isolates of C. siamense and C. fructicola. MR isolates of C. siamense and C. fructicola had a point mutation at codon 200, causing a replacement of phenylalanine acid by tyrosine. No point mutation was detected at codons 50, 167, 198, 200, or 240 in TUB2 of C. nymphaeae insensitive isolates. Overall, this study revealed that C. fructicola was the dominant species causing anthracnose on strawberry and could improve the understanding of the management of fungicide resistance in Colletotrichum species on strawberry in China.

Survival Cost and Diverse Molecular Mechanisms of Magnaporthe oryzae Isolate Resistance to Epoxiconazole.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases on rice worldwide. Epoxiconazole is a 14α-demethylation inhibitor (DMI) with excellent control on rice blast; to date, no resistant isolates have been observed in the field. Four mutants resistant to epoxiconazole were generated from three parental isolates via fungicide adaptation. Resistance was stable after 10 weekly consecutive transfers on fungicide-free medium. Three parameters, including growth rate, sporulation in vitro, and aggressiveness, were significantly lower for mutants compared with their parental isolates, with the exception of the low-resistance isolate. Sporulation and aggressiveness were negatively correlated with effective concentration values for 50% inhibition of mycelial growth for parental isolates and mutants (P < 0.05). Cross-resistance was found between epoxiconazole and prochloraz (ρ = 0.863, P = 0.000) or difenoconazole (ρ = 0.861, P = 0.000). The resistance factor for mutants was positively correlated with the relative expression of MoCYP51A in epoxiconazole treatment (r = 0.977, P = 0.02). In addition, two putative amino acid substitutions in MoCYP51A were found in two resistant mutants: Y126F in the high-resistance mutant and I125L in the low-resistance mutant. Mutation Y126F reduced the affinity of MoCYP51A with epoxiconazole, whereas I125L was not in the binding pocket of epoxiconazole. No amino acid change or overexpression in MoCYP51B was found in any of the mutants studied. To our knowledge, this is the first study to report DMI resistance observed in M. oryzae. The survival cost of M. oryzae resistance to epoxiconazole might be the reason why DMI resistance has not yet emerged in field populations worldwide.

An FYVE-Domain-Containing Protein, PsFP1, Is Involved in Vegetative Growth, Oxidative Stress Response and Virulence of Phytophthora sojae.

Proteins that contain the FYVE zinc-finger domain are recruited to PtdIns3P-containing membranes, participating in numerous biological processes such as membrane trafficking, cytoskeletal regulation, and receptor signaling. However, the genome-wide distribution, evolution, and biological functions of FYVE-containing proteins are rarely reported for oomycetes. By genome mining of Phytophthora sojae, two proteins (PsFP1 and PsFP2) with a combination of the FYVE domain and the PX domain (a major phosphoinositide binding module) were found. To clarify the functions of PsFP1 and PsFP2, the CRISPR/Cas9-mediated gene replacement system was used to knock out the two genes respectively. Only heterozygous deletion mutants of PsFP1 were recovered, and the expression level of PsFP1 in the heterozygous knockout transformants was significantly down-regulated. These PsFP1 mutants showed a decrease in mycelial growth and pathogenicity and were more sensitive to hydrogen peroxide. These phenotypes were recovered to the level of wild-type by overexpression PsFP1 gene in the PsFP1 heterozygous knockout transformant. In contrast, deletion of PsFP2 had no significant effect on vegetative growth, asexual and sexual reproduction, pathogenicity, or oxidative stress sensitivity. PsFP1 was primarily localized in vesicle-like structures and both the FYVE and PX domains are important for its localization. Overall, our results indicate that PsFP1 plays an important role in the vegetative growth and virulence of P. sojae.

Mutations in ORP1 Conferring Oxathiapiprolin Resistance Confirmed by Genome Editing using CRISPR/Cas9 in Phytophthora capsici and P. sojae.

Oxathiapiprolin is a novel fungicide that was recently registered in a number of countries to control plant-pathogenic oomycetes such as Phytophthora capsici. In our previous study, point mutations G770V and G839W in oxysterol binding protein-related protein 1 (ORP1) were detected in oxathiapiprolin-resistant P. capsici isolates (PcORP1). Here, we used the CRISPR/Cas9 system to verify the effects of these two point mutations on P. capsici phenotypes. Transformants containing heterozygous G770V and G839W mutations in PcORP1 showed high levels of oxathiapiprolin resistance. The G770V transformants showed otherwise similar phenotypes compared with the wild-type isolate BYA5, including sporangia and zoospore production, cyst germination, and pathogenicity. However, two independent transformants with heterozygous G839W mutations in PcORP1 could not produce sporangia. Three transformants with an unexpected point mutation in PcORP1 (ΔN837) showed high oxathiapiprolin resistance, and either similar or significantly reduced fitness compared with BYA5. The same deletion (ΔN837) was confirmed to confer oxathiapiprolin resistance in P. sojae by using CRISPR/Cas9. These homozygous P. sojae mutants also showed either similar or strongly reduced fitness compared with the wild-type parent isolate P6497. These results improve our understanding of oxathiapiprolin resistance in Phytophthora spp., and will be useful for the development of novel oxysterol-binding protein homolog inhibitor fungicides.

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 2.23 × 10-4 µg ml-1. In field trials, the control of downy mildew of cucumber was greater with oxathiapiprolin at 20 or 30 g a.i. ha-1 than with dimethomorph at 262.5 g a.i. ha-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.

Proteomic profile of the plant-pathogenic oomycete Phytophthora capsici in response to the fungicide pyrimorph.

Pyrimorph is a novel fungicide from the carboxylic acid amide (CAA) family used to control plant-pathogenic oomycetes such as Phytophthora capsici. The proteomic response of P. capsici to pyrimorph was investigated using the iTRAQ technology to determine the target site of the fungicide and potential biomarker candidates of drug efficacy. A total of 1336 unique proteins were identified from the mycelium of wild-type P. capsici isolate (Hd3) and two pyrimorph-resistant mutants (R3-1 and R3-2) grown in the presence or absence of pyrimorph. Comparative analysis revealed that the three P. capsici isolates Hd3, R3-1, and R3-2 produced 163, 77, and 13 unique proteins, respectively, which exhibited altered levels of abundance in response to the pyrimorph treatment. Further investigations, using Cluster of Orthologous Groups of Proteins (COG) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 35 proteins related to the mode of action of pyrimorph against P. capsici and 62 proteins involved in the stress response of P. capsici to pyrimorph. Many of the proteins with altered expression were associated with glucose and energy metabolism. Biochemical analysis using d-[U-(14) C]glucose verified the proteomics data, suggesting that the major mode of action of pyrimorph in P. capsici is the inhibition of cell wall biosynthesis. These results also illustrate that proteomics approaches are useful tools for determining the pathways targeted by novel fungicides as well as for evaluating the tolerance of plant pathogens to environmental challenges, such as the presence of fungicides.

Characteristics of famoxadone-resistant mutants of Phytophthora litchii and their effect on lychee fruit quality.

Lychee downy blight (LDB), a common disease caused by the oomycete Phytophthora litchii, poses a significant threat to both pre- and post-harvest stages, leading to substantial economic losses. Famoxadone, a quinone outside inhibitor fungicide, was registered for controlling LDB in China in 2002. However, limited information is available regarding the risk, mechanism, and impact on lychee fruit quality associated with famoxadone resistance. In this study, we determined the sensitivity of 133 P. litchii isolates to famoxadone, yielding a mean EC50 value of 0.46 ± 0.21 µg/mL. Through fungicide adaption, we derived resistant mutants with M124I and Y131C substitutions in PlCyt b (Cytochrome b in P. litchii) from wild-type isolates. In vitro assessments revealed that the fitness of the resistant mutants was significantly lower compared to the parental isolates. These laboratory findings demonstrate a moderate resistance risk of P. litchii to famoxadone. Molecular docking analyses indicated that the M124I and Y131C alterations disrupted hydrogen bonds and weakened the binding energy between famoxadone and PlCyt b. This indicates that the M124I and Y131C changes do indeed confer famoxadone resistance in P. litchii. Infection caused by famoxadone-resistant mutants exhibited a decreased or comparable impact on the characteristic traits of lychee fruit compared to the sensitive isolate. For future detection of famoxadone-resistant strains, AS-PCR primers were designed based on the M124I substitution.