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Late blight caused by Phytophthora infestans is the most devastating disease of potato. Phytophthora infestans produces many secondary metabolites and effector proteins, involved in the pathogenesis, which compromise host defense mechanisms. Pectinesterase (PE) is a cell wall degrading enzyme secreted by P. infestans to infect the host. To examine the role of PE in P. infestans, 15 strains of P. infestans were isolated from infected potato leaves in Yunnan, China. We analyzed the biological effects of exogenously added PE on P. infestans and its activity and gene expression after infection of potato using quantitative real-time polymerase chain reaction (RT-PCR). It was found that PE significantly promotes the growth of P. infestans, increases the weight of mycelium and the number of sporangia, and promotes the sporangial germination. PE accelerated the infection process of P. infestans on potato. The pathogenicity of P. infestans was positively correlated with PE activity and gene expression. PE is a key to the virulence difference of potato late blight.

IntroductionSoft rot Pectobacteriaceae (SRP) bacteria are globally dispersed pathogens that cause significant economic loss in potato and other crops. Our understanding of the SRP species diversity has expanded in recent years due to advances and adoption of whole-genome sequence technologies. There are currently 34 recognized SRP species that belong to the Dickeya and Pectobacterium genera.MethodsWe used whole-genome sequencing based analysis to describe the current distribution and epidemiology of SRP isolated from diseased potato samples obtained from commercial potato cropping systems in the United States. Our primary objectives in the present study were to: (1) identify the species of these SRP isolates recovered from potato samples across 14 states in the US, (2) describe the variation among SRP isolates from various US locations and track their temporal changes, and (3) evaluate the evolutionary relationships among these SRP isolates to deduce their source. We collected 118 SRP strains from diseased potato plants and tubers in 14 states between 2015 and 2022.ResultsWe identified three Dickeya and eight Pectobacterium species from diseased potato samples. Dickeya dianthicola, Pectobacterium parmentieri, P. carotovorum, and P. versatile appeared to be the predominant species, constituting 83% of the isolates. Furthermore, all D. dianthicola strains studied here as well as 90% of US D. dianthicola isolates sequenced to date exhibit significant clonality.DiscussionThe prevalence of this specific group of D. dianthicola, temporally and geographically, aligns with the occurrence of blackleg and soft rot outbreaks in the northeastern US after 2014. The genomic diversity observed in P. parmentieri implies multiple introductions to the US from at least four distinct sources, earlier than the arrival of the predominant group of D. dianthicola. In contrast, P. carotovorum and P. versatile appear to be widespread, long-term endemic strains in the US.

SYP-14288 is a fungicide as an uncoupler of oxidative phosphorylation, which is effective in controlling fungal pathogens like Rhizoctonia solani. To determine whether R. solani can develop SYP-14288 resistance and possibly multi-drug resistance (MDR), an SYP-14288-resistant mutant of R. solani X19-7 was generated from wild-type strain X19, and the mechanism of resistance was studied through metabolic and genetic assays. From metabolites of R. solani treated with SYP-14288, three compounds including M1, M2, and M3 were identified according to UPLC-MS/MS analysis, and M1 accumulated faster than M2 and M3 in X19-7. When X19-7 was treated by glutathione-S-transferase (GST) inhibitor diethyl maleate (DEM) and SYP-14288 together, or by DEM plus one of tested fungicides that have different modes of action, a synergistic activity of resistance occurred, implying that GSTs promoted metabolic resistance against SYP-14288 and therefore led to MDR. By comparing RNA sequences between X19-7 and X19, six cytochrome P450s (P450s) and two GST genes were selected as a target, which showed a higher expression in X19-7 than X19 both before and after the exposure to SYP-14288. Furthermore, heterologous expression of P450 and GST genes in yeast was conducted to confirm genes involved in metabolic resistance. In results, the P450 gene AG1IA_05136 and GST gene AG1IA_07383 were related to fungal resistance to multiple fungicides including SYP-14288, fluazinam, chlorothalonil, and difenoconazole. It was the first report that metabolic resistance of R. solani to uncouplers was associated with P450 and GST genes.

Soil treatment with disinfectants has been used for controlling soilborne phytopathogens. Besides suppressing specific pathogens, how these disinfectants impact soil health, especially soil microbial communities, is yet to be systemically determined. The objectives of this study were to examine the effects of three representative disinfectants, including the dazomet fumigant, fenaminosulf fungicide, and kasugamycin antibiotic on chemical properties, enzymatic activities, and microbial communities in soil for cucumber cultivation. Results showed that 14 days after soil treatment with these chemicals, residual content of dazomet and kasugamycin quickly declined in soil and were undetectable, while fenaminosulf residues were found at 0.48 ± 0.01 mg/kg. Total nitrogen and total carbon increased in soil after dazomet treatment. Urease and sucrase activities were significantly restrained after disinfectant application. The disinfectants did not significantly change the taxon of predominant bacteria and fungi but altered the relative abundance and diversity of soil microbiome, as well as microbial interspecific relationships. Moreover, cucumber cultivation enhanced the overall soil microbial diversity and enzymatic activities, which diminished the difference of soil microbiome among four treatments. The difference in soil microbial diversity among the four treatments became smaller after planting cucumber. Thus, soil microbial communities were affected by soil disinfectants and gradually recovered by cucumber application.

The fungicide SYP-14288 has a high efficiency, low toxicity, and broad spectrum in inhibiting both fungi and oomycetes, but its mode of action (MoA) remains unclear on inhibiting fungi. In this study, the MoA was determined by analyzing the metabolism and respiratory activities of Rhizoctonia solani treated by SYP-14288. Wild-type strains and SYP-14288-resistant mutants of R. solani were incubated on potato dextrose agar amended with either SYP-14288 or one of select fungicides acting on fungal respiration, including complex I, II, and III inhibitors; uncouplers; and ATP synthase inhibitors. Mycelial growth was measured under fungicides treatments. ATP content was determined using an ATP assay kit, membrane potential of mitochondria was detected with the JC-1 kit, and respiratory rate was calculated based on the measurement of oxygen consumption of R. solani. A model of metabolic fingerprinting cluster was established to separate oxidation inhibitors and phosphorylation inhibitors. All the results together displayed a clear discrimination between oxidation inhibitors and phosphorylation inhibitors, and the latter inhibited ATP synthase production having or uncoupling activities. Based on the model, SYP-14288 was placed in phosphorylation inhibitor group, because it significantly reduced ATP content and membrane potential of mitochondria while increasing respiratory rate in R. solani. Therefore, the MoA of SYP-14288 on R. solani was confirmed to involve phosphorylation inhibition and possibly uncoupling activity.

Ceratocystis manginecans causes mango wilt with significant economic losses. In the infection court, cerato-platanin (CP) family proteins (CPPs) are believed to involve in pathogenesis but has not been determined in C. manginecans. To confirm this function, a CP protein (CmCP) of C. manginecans was characterized in this study. A protoplast of C. manginecans was prepared by treating its mycelia with driselase and lysing enzymes. The cmcp gene was edited using CRISPR/Cas-U6-1 expression vectors in 60% PEG and 50 μg/mL hygromycin B in the medium, resulting in mutants with cmcp deletion (Δcmcp). A complemented mutant (Δcmcp-C) was obtained by transforming cmcp to Δcmcp. Both Δcmcp and Δcmcp-C were characterized by comparing them with a wild-type strain on morphology, mycelial growth, conidial production and pathogenicity. Additionally, cmcp was transformed and expressed in Pichia pastoris, and the derived recombinant protein CmCP caused a severe necrosis on Nicotiana tabacum leaves. CmCP-treated plant leaves showed symptoms of hypersensitive response including electrolyte leakage, reactive oxygen species generation and overexpression of defense-related genes PR-1, PAD3, ERF1, HSR203J, and HIN1. All those results suggested that cmcp gene was required for the growth development of C. manginecans and functioned as a major pathogenicity factor in mango infection.

Pink rot (Phytophthora erythroseptica) of potato is a major concern in many potato production regions. The pathogen produces zoospores that serve as a primary inoculum for infection. To understand how the pink rot incidence is related to pathogen population, qualitative, and quantitative chemical analyses were conducted. It was demonstrated that P. erythroseptica zoospores required a minimal population of 103 zoospores/ml (threshold) for initiating germination and the subsequent infection; the percentage of zoosporic germination was positively correlated with the density of zoospores above the threshold. To elucidate the density-dependent behavior, zoospore exudate (ZE) was extracted from high-density (105/ml) zoospore suspension. Zoosporic inocula of P. erythroseptica at different concentrations were inoculated on potato tubers. Necrotic lesions were caused by inoculum with 100 zoospores per inoculation site; 5 zoospores per site did not cause lesions on the tuber. However, five zoospores did cause lesions when they were placed in ZE, suggesting ZE contained chemical compounds that regulate germination of zoospores. ZE was collected and analyzed using liquid chromatography mass spectroscopy (LC-MS). Results showed that the amino acid leucine was associated with zoosporic germination. Therefore, zoosporic germination and infection of P. erythroseptica were mediated by signaling molecules secreted from zoospores.

Potato common scab (PCS) is an economically important disease worldwide. In this study we demonstrated the possible role of Streptomyces violaceusniger AC12AB in controlling PCS. Isolates of Streptomyces scabies were obtained from CS infected tubers collected from Maine United States, which were confirmed by morphological and molecular analysis including 16S rRNA sequencing and RFLP analysis of amplified 16S-23S ITS. Pathogenicity assays related genes including txtAB, nec1, and tomA were also identified in all S. scabies strains through PCR reaction. An antagonistic bacterial strain was isolated from soil in Punjab and identified as S. violaceusniger AC12AB based on 16S rRNA sequencing analysis. Methanolic extract of S. violaceusniger AC12AB contained azalomycin RS-22A which was confirmed by 1H and 13C-NMR, 1H/1H-COSY, HMBC and HMQC techniques. S. violaceusniger AC12AB exhibited plant growth promotion attributes including Indole-3-acetic acid production with 17 μgmL-1 titers, siderophores production, nitrogen fixation and phosphates solubilization potential. When tubers were inoculated with S. violaceusniger AC12AB, significant (P < 0.05) PCS disease reduction up to 90% was observed in greenhouse and field trials, respectively. Likewise, S. violaceusniger AC12AB significantly (P < 0.05) increased potato crop up to 26.8% in field trial. Therefore, plant growth promoting S. violaceusniger AC12AB could provide a dual benefit by decreasing PCS disease severity and increasing potato yield as an effective and inexpensive alternative strategy to manage this disease.

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.