Autophagy-Related Gene PlATG6a Is Involved in Mycelial Growth, Asexual Reproduction and Tolerance to Salt and Oxidative Stresses in Peronophythora litchii.

Autophagy is ubiquitously present in eukaryotes. During this process, intracellular proteins and some waste organelles are transported into lysosomes or vacuoles for degradation, which can be reused by the cell to guarantee normal cellular metabolism. However, the function of autophagy-related (ATG) proteins in oomycetes is rarely known. In this study, we identified an autophagy-related gene, PlATG6a, encoding a 514-amino-acid protein in Peronophythora litchii, which is the most destructive pathogen of litchi. The transcriptional level of PlATG6a was relatively higher in mycelium, sporangia, zoospores and cysts. We generated PlATG6a knockout mutants using CRISPR/Cas9 technology. The P. litchii Δplatg6a mutants were significantly impaired in autophagy and vegetative growth. We further found that the Δplatg6a mutants displayed decreased branches of sporangiophore, leading to impaired sporangium production. PlATG6a is also involved in resistance to oxidative and salt stresses, but not in sexual reproduction. The transcription of peroxidase-encoding genes was down-regulated in Δplatg6a mutants, which is likely responsible for hypersensitivity to oxidative stress. Compared with the wild-type strain, the Δplatg6a mutants showed reduced virulence when inoculated on the litchi leaves using mycelia plugs. Overall, these results suggest a critical role for PlATG6a in autophagy, vegetative growth, sporangium production, sporangiophore development, zoospore release, pathogenesis and tolerance to salt and oxidative stresses in P. litchii.

Screening a Natural Product-Inspired Library for Anti-Phytophthora Activities.

Phytophthora is a genus of microorganisms that cause devastating dieback and root-rot diseases in thousands of plant hosts worldwide. The economic impact of Phytophthora diseases on crops and native ecosystems is estimated to be billions of dollars per annum. These invasive pathogens are extremely difficult to control using existing chemical means, and the effectiveness of the few treatments available is being jeopardized by increasing rates of resistance. There is an urgent need to identify new chemical treatments that are effective against Phytophthora diseases. Natural products have long been regarded as "Nature's medicine chest", providing invaluable leads for developing front-line drugs and agrochemical agents. Here, we have screened a natural product-inspired library of 328 chemicals against two key Phytophthora species: Phytophthora cinnamomi and Phytophthora agathidicida. The library was initially screened for inhibition of zoospore germination. From these screens, we identified twenty-one hits that inhibited germination of one or both species. These hits were further tested in mycelial growth inhibition studies to determine their half-maximal inhibitory concentrations (IC50s). Four compounds had IC50 values of approximately 10 µM or less, and our best hit had IC50s of approximately 3 µM against both Phytophthora species tested. Overall, these hits may serve as promising leads for the development of new anti-Phytophthora agrochemicals.

Growth Inhibition of Phytopathogenic Fungi and Oomycetes by Basidiomycete Irpex lacteus and Identification of its Antimicrobial Extracellular Metabolites.

In dual culture confrontation assays, basidiomycete Irpex lacteus efficiently antagonized Fusarium spp., Colletotrichum spp., and Phytophthora spp. phytopathogenic strains, with growth inhibition percentages between 16.7-46.3%. Antibiosis assays evaluating the inhibitory effect of soluble extracellular metabolites indicated I. lacteus strain inhibited phytopathogens growth between 32.0-86.7%. Metabolites in the extracellular broth filtrate, identified by UPLC-QTOF mass spectrometer, included nine terpenes, two aldehydes, and derivatives of a polyketide, a quinazoline, and a xanthone, several of which had antifungal activity. I. lacteus strain and its extracellular metabolites might be valuable tools for phytopathogenic fungi and oomycete biocontrol of agricultural relevance.

Exploration of microbiome of medicinally important plants as biocontrol agents against Phytophthora parasitica.

In a preliminary plant-based microbiome study, diverse bacterial taxa were identified from different medicinal plants using 16S rRNA gene sequencing. Based on initial antimicrobial screening, eight (8) bacterial endophytes in six (6) different genera, Streptomyces, Pseudomonas, Enterobacter, Bacillus, Arthrobacter, and Delftia, from four important medicinal plants Dodonaea viscosa, Fagonia indica, Caralluma tuberculata, and Calendula arvensis were selected for further analyses. Antimicrobial assays revealed that Pseudomonas taiwanensis MOSEL-RD23 has strong anti-Phytophthora activity. Volatiles produced by P. taiwanensis MOSEL-RD23and Bacillus flexus MOSEL-MIC5 inhibited the growth of Phytophthora parasitica by more than 80%. Ethyl acetate extracts of Streptomyces alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, Enterobacter hormaechei MOSEL-FLS1, and Bacillus tequilensis MOSEL-FLS3, and Delftia lacustris MB322 displayed high potency against P. parasitica. All these bacterial extracts showed strong inhibition of more than 80% inhibition in vitro against P. parasitica at different concentrations (4-400 µg/mL). Bacterial extracts showing strong antimicrobial activity were selected for bioactivity-driven fractionation and showed anti-Phytophthoral activity in multiple fractions and different peaks observed in UV-Vis spectroscopy. In the detached-leaf assay against P. parasitica on tobacco, 1% ethyl acetate bacterial extract of S. alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, E. hormaechei MOSEL-FLS1, B. tequilensis MOSEL-FLS3, and D. lacustris MB322 reduced lesion sizes and lesion frequencies caused by P. parasitica by 68 to 81%. Overall, P. taiwanensis MOSEL-RD23 showed positive activities for all the assays. Analyzing the potential of bacterial endophytes as biological control agents can potentially lead to the formulation of broad-spectrum biopesticides for the sustainable production of crops.

The RPA190-pc gene participates in the regulation of metalaxyl sensitivity, pathogenicity and growth in Phytophthora capsici.

Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. In this study, a conserved domain RPOLA-N of the RPA190 gene of P. capsici (RPA190-pc) was identified from the P. capsici SD1-9 strain. The role of the RPA190-pc underlying the metalaxyl resistance of P. capsici was investigated. Three P. capsici mutants, two with downregulated RPA190-pc (SD1-9C-3 and C-4) expression and one showing upregulation (OESD1-9-1), were obtained by Polyethylene Glycol (PEG) mediated protoplast transformations of P. capsici SD1-9. Quantitative real-time reverse transcription PCR results showed that RPA190-pc was downregulated by more than 60% in SD1-9C-3/C-4 and upregulated 3-fold in OESD1-9-1 compared with that of the control strain SD1-9. Evaluation of the metalaxyl resistance of these three transformants showed that the EC50 values of metalaxyl against SD1-9C-3, SD1-9C-4, and OESD1-9-1 were 120.0 µg·mL-1, 24.4 µg·mL-1, and 15573.0 µg·mL-1, respectively, corresponding to 63.3% decrease, 92.5% decrease, and 47.7-fold increase relative to the EC50 value in SD1-9. Compared with SD1-9, the mycelia of transformants SD1-9C-3, SD1-9C-4, and OESD1-9-1 showed more branches and shorter branches; and the transformants had different pathogenicity to different hosts plants. The expression of the candidate gene RPA190-pc during 10 life-history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage, and it gradually increased with the increase of SD1 and SD1-9 infection time of pepper leaves, indicated that RPA190-pc may be related to the growth and pathogenicity of P. capsici. These results indicate that the expression of RPA190-pc is involved in the regulation of P. capsici resistance to metalaxyl.

Inhibition of the lemon brown rot causal agent Phytophthora citrophthora by low-toxicity compounds.

BACKGROUND: Phytophthora spp., soil-borne oomycetes, cause brown rot (BR) on postharvest lemons. The management of this disease is based on cultural practices and chemical control using inorganic salts of limited efficacy. In the search for new alternatives, the aim of this work was to evaluate the effect of low-toxicity compounds to inhibit the growth of P. citrophthora and to control BR disease on lemons. Sodium bicarbonate, potassium sorbate, polyhexamethylene guanidine, Ascophyllum nodosum extract and a formulation containing phosphite salts plus A. nodosum (P+An) were evaluated. RESULTS: All tested products inhibited mycelial growth, sporangia formation and zoospore germination of P. citrophthora in vitro. In postharvest applications on artificially inoculated lemons, only P+An exhibited a BR curative effect, with incidence reduction of around 60%. When this formulation was applied in field treatments, BR incidence was reduced by 40% on lemons harvested and inoculated up to 30 days post application. CONCLUSION: Our results demonstrate the in vitro direct anti-oomycete effect of low-toxicity compounds and the in vivo efficacy of P+An formulation to control BR, encouraging the incorporation of the latter in the management of citrus BR. © 2020 Society of Chemical Industry.

Plant roots employ cell-layer-specific programs to respond to pathogenic and beneficial microbes.

Plant roots are built of concentric cell layers that are thought to respond to microbial infections by employing specific, genetically defined programs. Yet, the functional impact of this radial organization remains elusive, particularly due to the lack of genome-wide techniques for monitoring expression at a cell-layer resolution. Here, cell-type-specific expression of tagged ribosomes enabled the isolation of ribosome-bound mRNA to obtain cell-layer translatomes (TRAP-seq, translating ribosome affinity purification and RNA sequencing). After inoculation with the vascular pathogen Verticillium longisporum, pathogenic oomycete Phytophthora parasitica, or mutualistic endophyte Serendipita indica, root cell-layer responses reflected the fundamentally different colonization strategies of these microbes. Notably, V. longisporum specifically suppressed the endodermal barrier, which restricts fungal progression, allowing microbial access to the root central cylinder. Moreover, localized biosynthesis of antimicrobial compounds and ethylene differed in response to pathogens and mutualists. These examples highlight the power of this resource to gain insights into root-microbe interactions and to develop strategies in crop improvement.

Antifungal activity of chitosan against Phytophthora infestans, the pathogen of potato late blight.

Phytophthora infestans, the pathogen of potato late blight which is a devastating disease of potatoes, causes stem and leaf rot, leading to significant economic losses. Chitosan is a naturally occurring polysaccharide with a broad spectrum of antimicrobial properties. However, the specific mechanism of chitosan on Phytophthora infestans has not been studied. In this study, we found that chitosan significantly inhibited the mycelial growth and spore germination of Phytophthora infestans in vitro, reduced the resistance of Phytophthora infestans to various adverse conditions, and it had synergistic effect with pesticides, making it a potential way to reduce the use of chemical pesticides. In addition, chitosan could induce resistance in potato pieces and leaves to Phytophthora infestans. Transcriptome analysis data showed that chitosan mainly affected cell growth of Phytophthora infestans, and most of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene ontology (GO) terms revolved in metabolic processes, cell membrane structure and function and ribosome biogenesis. Differentially expressed genes (DEGs) related to adverse stress and virulence were also discussed. On the whole, this study provided new ideas for the development of chitosan as an eco-friendly preparation for controlling potato late blight.

How Do Trichoderma Genus Fungi Win a Nutritional Competition Battle against Soft Fruit Pathogens? A Report on Niche Overlap Nutritional Potentiates.

We present a case study report into nutritional competition between Trichoderma spp. isolated from wild raspberries and fungal phytopathogenic isolates (Colletotrichum sp., Botrytis sp., Verticillium sp. and Phytophthora sp.), which infect soft fruit ecological plantations. The competition was evaluated on the basis of nutritional potentiates. Namely, these were consumption and growth, calculated on the basis of substrate utilization located on Biolog® Filamentous Fungi (FF) plates. The niche size, total niche overlap and Trichoderma spp. competitiveness indices along with the occurrence of a stressful metabolic situation towards substrates highlighted the unfolding step-by-step approach. Therefore, the Trichoderma spp. and pathogen niche characteristics were provided. As a result, the substrates in the presence of which Trichoderma spp. nutritionally outcompete pathogens were denoted. These were adonitol, D-arabitol, i-erythritol, glycerol, D-mannitol and D-sorbitol. These substrates may serve as additives in biopreparations of Trichoderma spp. dedicated to plantations contaminated by phytopathogens of the genera Colletotrichum sp., Botrytis sp., Verticillium sp. and Phytophthora sp.

Morphologic, molecular, and pathogenic characterization of Phytophthora palmivora isolates causing flower rot on azalea.

The objective of this work was to characterize two Phytophthora palmivora isolates causing floral blight and rot in azalea plants and to evaluate the pathogenicity of this oomycete pathogen on several plant species. Azalea plants with symptoms of flower blight and rot were obtained in the municipality of Holambra-SP. After an attempt of isolation, colonies with Phytophthora characteristics grown only on selective V8 medium. Molecular identification of the isolates was done by amplification and sequencing of ITS and COX2 regions. In the phylogenetic analysis, the azalea isolates clustered with reference isolates of P. palmivora. Morphological characteristics were similar to those described for P. palmivora. Isolates were inoculated in healthy azalea plants and caused leaf blight and floral rot. The pathogen was re-isolated from symptomatic plants completing Koch's postulates. In a host range test, the azalea isolates were able to cause lesions on leaves of vinca, snapdragon, basil, and tomato, and affected both leaves and flowers of geranium. Fruit rot was observed on tomato, potato, sweet pepper, scarlet eggplant, zucchini, cucumber, maroon cucumber, onion, apple, papaya, guava, and carrot. This is the first report of the species P. palmivora causing flower blight and rot in azalea plants in Brazil and probably in the world.

Vepris macrophylla (Baker) I. Verd Essential Oil: An Antifungal Agent against Phytopathogenic Fungi.

Rutaceae are widely used in ethnomedicine to treat infectious diseases in humans and plants. In this study, the antifungal activity of the Vepris macrophylla leaf essential oil (VEO) and its main components, citral and citronellol, was evaluated against six phytopathogenic fungi. In addition, the possible action of VEO on the synthesis of mycotoxins was evaluated as well. To determine the antifungal activity of VEO we used the agar dilution method and VEO showed inhibitory activity against all the tested fungi. In particular, VEO resulted to be fungicidal against Phytophthora cryptogea and Fusarium avenaceum. For all other fungi VEO exhibited fungistatic activity and the weakest effect was observed on Alternaria solani. Citral was very effective against P. cryptogea, F. avenaceum, F. poae and F. graminearum. On the other hand, citronellol showed good activity towards P. cryptogea and F. avenaceum and weaker activity towards F. poae and F. graminearum. It can be concluded that VEO can be considered a promising antifungal agent, especially against P. cryptogea and F. avenaceum, suggesting a possible use in the formulation of new selective and natural fungicides.

Diurnal effects on sporangium and zoospore production by Phytophthora ramorum on Rhododendron 'Cunningham's White'.

We evaluated sporangium and zoospore production by three isolates of Phytophthora ramorum on Rhododendron 'Cunningham's White' leaves under light and dark conditions at both variable and constant (14 C) temperature. P. ramorum-infected leaves were detached and placed in funnels inside of a 62-L plastic storage container located in a growth chamber. Cool mist was introduced to the container to create a high-humidity environment. Sporangia and zoospores were collected over a 4-day period by misting leaves with 5 mL of distilled water, which was collected in conical test tubes that also contained runoff from the misting. Spores were collected daily just before a 13-h light period and again just before an 11-h dark period. Sporangia and zoospores in the collection tubes were counted using a dissecting microscope following staining with lactoglycerin/aniline blue. Large differences in sporangium and zoospore numbers observed for the dark versus light periods were observed on days 2, 3, and 4. A diurnal effect has been observed for production of propagules of other oomycetes, but such effects have not been previously reported for P. ramorum. This information will help provide a better understanding of patterns of inoculum production by P. ramorum and resulting fluctuations in inoculum density that will influence sudden oak death epidemics in forest ecosystems in the United States and other countries where it occurs.

The high-affinity phosphodiesterase PcPdeH is involved in the polarized growth and pathogenicity of Phytophthora capsici.

The cAMP signaling pathway has been shown to be important in controlling morphological changes and pathogenicity in plant pathogens. In the present study, we identified PcPdeH, a gene encoding a high-affinity phosphodiesterase (PDE), which is a key regulator of the cAMP signaling pathway. To elucidate the function of PcPdeH, PcPdeH-knockout mutants were obtained using a type II CRISPR/Cas9 system in Phytophthora capsici. The knockout transformants of PcPdeH showed vegetative growth defects and abnormal cyst germination. Infection assays indicated that compared with the wild type, PcPdeH-knockout mutants showed significantly reduced virulence on pepper and tobacco leaves and exhibited increased (1.5-2-fold) cAMP levels relative to the wild-type and CK strains. Based on these phenotypic features, we propose that PcPdeH is crucial for vegetative growth, cyst germination and pathogenicity in P. capsici.

A secreted protein of 15 kDa plays an important role in Phytophthora palmivora development and pathogenicity.

Phytophthora palmivora is a destructive oomycete plant pathogen with a wide host range. So far, little is known about the factors governing its infection structure development and pathogenicity. From the culture filtrate of a P. palmivora strain isolated from papaya, we identified a secreted glycoprotein of 15 kDa, designated as Ppal15kDa, using liquid chromatography tandem mass spectrometry. Two gene variants, Ppal15kDaA and Ppal15kDaB were amplified from a P. palmivora papaya isolate. Transient expression of both variants in Nicotiana benthamiana by agroinfiltration enhanced P. palmivora infection. Six Ppal15kDa mutants with diverse mutations were generated via CRISPR/Cas9-mediated gene editing. All mutants were compromised in infectivity on N. benthamiana and papaya. Two mutants with all Ppal15kDa copies mutated almost completely lost pathogenicity. The pathogenicity of the other four containing at least one wild-type copy of Ppal15kDa was compromised at varying levels. The mutants were also affected in development as they produced smaller sporangia, shorter germ tubes, and fewer appressoria. The affected levels in development corresponded to the levels of reduction in pathogenicity, suggesting that Ppal15kDa plays an important role in normal development of P. palmivora infection structures. Consistent with its role in infection structure development and pathogenicity, Ppal15kDa was found to be highly induced during appressorium formation. In addition, Ppal15kDa homologs are broadly present in Phytophthora spp., but none were characterized. Altogether, this study identified a novel component involved in development and pathogenicity of P. palmivora and possibly other Phytophthora spp. known to contain a Ppal15kDa homolog.

Transcriptome analysis clarified genes involved in resistance to Phytophthora capsici in melon.

Phytophthora blight caused by Phytophthora capsici is a devastating disease for melon plant. However, the underlying resistance mechanisms are still poorly understood. In this study, the transcriptome differences between the resistant ZQK9 and susceptible E31 at 0, 3, and 5 days post-inoculation (dpi) were identified by RNA-seq. A total of 1,195 and 6,595 differentially expressed genes (DEGs) were identified in ZQK9 and E31, respectively. P. capsici infection triggered massive transcript changes in the inoculated tissues. Genes related to plant defense responses were activated, which was reflected by a lot of up-regulated DEGs involved in pathogenesis-related (PR) genes, hormones biosynthesis and signal transduction, secondary metabolites biosynthesis and cell wall modification in resistant ZQK9. The dataset generated in this study may provide a basis for identifying candidate resistant genes in melon against P. capsici and lay a foundation for further research on the molecular mechanisms.

Phytophthora capsici PcFtsZ2 Is Required for Asexual Development and Plant Infection.

In bacteria, FtsZ proteins form a Z ring that is the initial step preceding septal fission. FtsZ proteins enable the division of mitochondria in early eukaryotes and are present in some kingdoms but have been lost in animals, fungi, and plants. Here, we have identified two Phytophthora capsici ortholog genes of Escherichia coli FtsZs, designated PcFtsZ1 and PcFtsZ2. Overexpression of PcFtsZ2 in E. coli fully complemented the overexpression phenotype of EcFtsZ. In contrast, overexpression of PcFtsZ1 in E. coli had minimal impact on cell division and separation. Thus, we focused on evaluating the impact of altered expression of PcFtsZ2 in P. capsici, as it exhibited the strongest phenotype. PcFtsZ2 was expressed at the highest levels in mycelia, sporangia, and germinating cysts, as well as in late infection. PcFtsZ2 mis-expression lines showed aberrant asexual growth and development of P. capsici. Alterations in the expression of PcFtsZ2 changed the distribution of mitochondria in hyphae and sporangia and, also, affected the number, size, and shape of actin plaques. Silencing of PcFtsZ2 restrained growth and development of invasive structures, especially cysts and sporangia, substantially inhibiting the ability of transformants to cause blight lesions. In overexpressed transformant lines, cyst and sporangial germination rates were only half that of controls, but hyphal growth from direct germination of sporangia was more rapid than controls. These transformant lines were only slightly impaired in virulence relative to controls. This study emphasizes the essential role of the evolutionarily conserved FtsZ2 proteins in affecting cytoskeleton dynamics.

G protein α subunit suppresses sporangium formation through a serine/threonine protein kinase in Phytophthora sojae.

Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, ß, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1.

Short peptides secreted by Bacillus subtilis inhibit the growth of mold on fresh-cut pumpkin (Cucurbita pepo).

BACKGROUND: This study investigates the efficacy of short peptides secreted by Bacillus subtilis for fungal inhibition in fresh-cut pumpkin and for maintaining its shelf life. RESULTS: Low-molecular-weight filtrate (LC < 1000 Da) of B. subtilis culture (BC) significantly lowered the total number of molds on fresh-cut pumpkin compared with the untreated control and a BC group after storage. Low-molecular-weight filtrate prevented the deterioration of sensory quality in a pumpkin incision, and reduced pectinase activity. It also inhibited the growth of Phytophthora capsici and Penicillium chrysogenum, and the activity of ß-1,3-glucan synthase (GS) secreted by both molds. Fifty-seven GS-inhibiting peptides were screened from 95 LC peptides with two to five amino acid residues. The two most potent peptides, AWYW and HWWY, had strongly suppressive effects on the growth of P. capsici and P. chrysogenum. CONCLUSION: Our study demonstrated that short peptides present in B. subtilis culture can play an important role in the maintenance of fresh-cut pumpkin by suppressing fungal growth. © 2019 Society of Chemical Industry.

Hydrodynamic Shape Changes Underpin Nuclear Rerouting in Branched Hyphae of an Oomycete Pathogen.

Multinucleate fungi and oomycetes are phylogenetically distant but structurally similar. To address whether they share similar nuclear dynamics, we carried out time-lapse imaging of fluorescently labeled Phytophthora palmivora nuclei. Nuclei underwent coordinated bidirectional movements during plant infection. Within hyphal networks growing in planta or in axenic culture, nuclei either are dragged passively with the cytoplasm or actively become rerouted toward nucleus-depleted hyphal sections and often display a very stretched shape. Benomyl-induced depolymerization of microtubules reduced active movements and the occurrence of stretched nuclei. A centrosome protein localized at the leading end of stretched nuclei, suggesting that, as in fungi, astral microtubule-guided movements contribute to nuclear distribution within oomycete hyphae. The remarkable hydrodynamic shape adaptations of Phytophthora nuclei contrast with those in fungi and likely enable them to migrate over longer distances. Therefore, our work summarizes mechanisms which enable a near-equal nuclear distribution in an oomycete. We provide a basis for computational modeling of hydrodynamic nuclear deformation within branched tubular networks.IMPORTANCE Despite their fungal morphology, oomycetes constitute a distinct group of protists related to brown algae and diatoms. Many oomycetes are pathogens and cause diseases of plants, insects, mammals, and humans. Extensive efforts have been made to understand the molecular basis of oomycete infection, but durable protection against these pathogens is yet to be achieved. We use a plant-pathogenic oomycete to decipher a key physiological aspect of oomycete growth and infection. We show that oomycete nuclei travel actively and over long distances within hyphae and during infection. Such movements require microtubules anchored on the centrosome. Nuclei hydrodynamically adapt their shape to travel in or against the flow. In contrast, fungi lack a centrosome and have much less flexible nuclei. Our findings provide a basis for modeling of flexible nuclear shapes in branched hyphal networks and may help in finding hard-to-evade targets to develop specific antioomycete strategies and achieve durable crop disease protection.

Derivatization of Natural Compound ß-Pinene Enhances Its In Vitro Antifungal Activity against Plant Pathogens.

BACKGROUND: The development of new antifungal agents has always been a hot research topic in pesticide development. In this study, a series of derivatives of natural compound ß-pinene were prepared, and the antifungal activities of these derivatives were evaluated. The purpose of this work is to develop some novel molecules as promising new fungicides. METHODS: Through a variety of chemical reactions, ß-pinene was transformed into a series of ß-pinene-based derivatives containing amide moieties and acylthiourea moieties. The antifungal activities of these derivatives against five plant pathogens including Colletotrichum gloeosporioides, Fusarium proliferatum, Alternaria kikuchiana, Phomopsis sp. and Phytophthora capsici were tested; preliminary structure-activity relationship was discussed. RESULTS: Some derivatives exhibited moderate or significant antifungal activity due to the fusion of the amide moiety or the acylthiourea moiety with the pinane skeleton. The structure-activity relationship analysis showed that the fluorine atom and the strong electron withdrawing nitro group, or trifluoromethyl group on the benzene ring of the derivatives had a significant effect on the improvement of the antifungal activity against Colletotrichum gloeosporioides, Fusarium proliferatum, Alternaria kikuchiana and Phomopsis sp. Meanwhile, the introduction of an ethyl group at the meta-position on the benzene ring of the derivatives could improve the antifungal activity against Phytophthora capsici. Compounds 4e, 4h, 4q, 4r exhibited broad-spectrum antifungal activity against the tested strains. Compound 4o had significant antifungal activity against Phytophthora capsici (IC50 = 0.18 µmol/L). These derivatives were expected to be used as precursor molecules for novel pesticide development in further research.