Phytopathology最新文献

The development of xylem embolism in 1-year-old stems of Japanese black pine (Pinus thunbergii) seedlings was monitored by compact magnetic resonance imaging (MRI) after inoculation with the pinewood nematode (Bursaphelenchus xylophilus). In parallel, the nematode distribution and population structure in the stems were examined by isolating the nematodes using the Baermann funnel technique. The vertical length and volume of massive embolisms in each seedling were strongly correlated with the maximum relative embolized area (REA) in stem cross-sections. Embolism development and nematode reproduction were not restricted to the inoculation site, as any portion of the stem could be the initial point of a population burst. The nematode population in the stem xylem was strongly correlated with the REA and with the circumferential proportion of cambial death in cross-sections monitored by MRI. The proportion of second-stage juveniles was also correlated with the REA in the xylem. In contrast, the nematode population in bark tissue was not correlated with either the REA or cambial death. These results suggested that nematode reproduction in the cambial zone is the key step in pine wilt disease, and second-stage juveniles were suggested to induce massive embolisms in the advanced stage of the disease.

Grape downy mildew, caused by Plasmopara viticola poses a threat to grape cultivation globally. Early detection of fungicide resistance is critical for effective management. This study aimed to assess the prevalence and distribution of mutations associated with resistance to Quinone oxide inhibitors (QoI, FRAC 11), Quinone inside inhibitors (QiIs, FRAC 21, cyazofamid), Carboxylic acid amides (CAA, FRAC 41), and Quinone inside and outside inhibitor, stigmatellin binding mode (QioSI, FRAC 45, ametoctradin) in P. viticola populations in the eastern United States and Canada; and evaluate whether these mutations are linked to fungicide resistance correlate with specific P. viticola clades. A total of 658 P. viticola samples were collected from commercial vineyards across different states and years in the eastern United States and Canada and sequenced for the PvCesA3 and cytb genes and the ITS1 region. Results showed P. viticola clades aestivalis, vinifera, and riparia were prevalent in the eastern United States and Canada. QoI resistance was widespread, with the A-143 resistant genotype prevalent in P. viticola clades aestivalis and vinifera. CAA resistance, associated with the G1105S mutation, was mainly identified in P. viticola clade aestivalis from Georgia, New York, and Ontario. A TaqMan-probe based assay was developed to detect G1105S mutation in P. viticola conferring CAA fungicide resistance. The TaqMan assay demonstrated sensitivity at even low DNA concentrations and specificity in distinguishing between sensitive and resistant genotypes. This study provides insights into geographic distribution of fungicide resistance in P. viticola populations and presents a reliable method for detecting CAA resistance in P. viticola.

Tea leaf spot caused by Lasiodiplodia theobromae is a newly discovered fungal disease in southwest China. Due to a lack of knowledge of its epidemiology and control strategies, the disease has a marked impact on tea yield and quality. Pyriofenone is a new fungicide belonging to the aryl phenyl ketone fungicide group, which has shown marked efficacy in controlling various fungal diseases. However, its mechanism of action is not yet understood. This study found that pyriofenone exhibits strong in vitro inhibitory activity against various phytopathogenic fungi. Specifically, it showed strong inhibitory activity against L. theobromae, with a half-maximal effective concentration (EC₅₀) value of 0.428 μg/ml determined by measuring the mycelial growth rate. Morphological observations, using optical, scanning electron, and transmission electron microscopy, revealed that pyriofenone induces morphological abnormalities in L. theobromae hyphae. At lower doses, the hyphae became swollen, the distance between septa decreased, and the hyphal growth rate slowed. At higher doses and longer exposures, the hyphae collapsed. Transcriptomic and bioinformatic analyses indicated that pyriofenone can affect the expression of genes related to membrane transporters. Homology modeling suggested that pyriofenone may bind to a candidate target protein of the major facilitator superfamily transporter, with a free binding energy of -7.1 kcal/mol. This study suggests that pyriofenone may potentially regulate the transport of metabolites in L. theobromae, thus affecting hyphal metabolism and interfering with hyphal growth. Pyriofenone exhibits in vitro inhibitory activity against various tea foliar pathogens and holds promise for future applications to the control of tea foliar diseases.

Rapid, field-deployable assays such as loop-mediated isothermal amplification (LAMP) are critical for detecting nursery and forest pathogens such as Phytophthora ramorum and P. kernoviae to prevent pathogen spread. We developed and validated four LAMP assays for genus-level detection of Phytophthora spp., species-level detection of P. kernoviae and P. ramorum, and lineage-level detection of the P. ramorum NA1 lineage. The cross-reactivity of the two species-specific LAMP assays was evaluated using a set of 18 Phytophthora spp. known to infect nursery crop hosts. The correct target species were detected by the species-level LAMP assays. The Phytophthora spp. LAMP assay was evaluated against 27 Phytophthora spp. and other bacterial and fungal pathogens and reacted with all the Phytophthora spp. evaluated but no other bacterial or fungal species. The limit of detection (LOD) of the P. kernoviae LAMP was 100 fg/µl, and the LOD of the P. ramorum LAMP assay was 1 pg/µl of DNA. The NA1 LAMP assay was tested against the NA1, NA2, EU1, and EU2 lineages of P. ramorum and was lineage-specific but had a higher LOD (100 pg/µl) than the species-specific LAMP assays. Both P. ramorum and P. kernoviae LAMP assays were highly precise (>0.94) in detecting the respective pathogens in symptomatic rhododendron leaves and co-inoculation experiments. The four LAMP assays were run in tandem on a microfluidic chip and smartphone platform and can be used in the field to detect and monitor spread of these regulatory Phytophthora spp. in forest and/or nursery settings.

Stenotaphrum secundatum is a premium turf grass in warm temperate and subtropical regions of the world and is the most important turf species in Australia based on the value of its production. A new disease called buffalo grass yellows (BGY) has become a problem on turf farms in Australia. We surveyed turf farms in New South Wales (NSW), Queensland (Qld) and Western Australia to determine whether panicum mosaic virus (PMV) and sugarcane mosaic virus (SCMV) were associated with BGY. PMV was only found on three farms, two located in the Hawkesbury Valley near Sydney, and a third at Echuca, about 800 km to the southwest of the former location. SCMV was more prevalent, present in all major cultivars and states surveyed. We analyzed phylogenetic relationships for SCMV and found that isolates infecting S. secundatum in Australia belonged to three clades. The first included Australian isolates typical of the population of viruses circulating in Digitaria didactyla. The second included a single NSW isolate from S. secundatum 'SS100' that grouped with otherwise American isolates of SCMV recorded in S. secundatum and Saccharum officinale from Florida, and Zea mays from Ohio. Finally, an isolate of SCMV from S. secundatum originating from a turf farm in southeast Qld grouped with viruses mostly infecting maize; this record is potentially the first maize-adapted strain of SCMV in Australia. Our study sheds light on the aetiology of the BGY disease syndrome and invasion history of PMV and SCMV in Australia.

Gray mold is an important disease of crops and is widespread, harmful, difficult to control, and prone to developing fungicide resistance. Screening new fungicides is an important step in controlling this disease. Hydroxychloroquine is an anti-inflammatory and anti-malarial agent, which has shown marked inhibitory activity against many fungi in medicine. This study evaluated the inhibitory activity of hydroxychloroquine against several phytopathogenic fungi, finding a half-maximal effective concentration of 113.82 μg/ml against the hyphal growth of Botrytis cinerea, with significant in-vivo curative effects of 92.37% or 78.37% for gray mold on detached tomato leaves or fruits at 10.0 or 200.0 mg/ml, respectively. Ultrastructural studies indicated that hydroxychloroquine induced collapse of hyphae, with a wrinkled surface, unclear organelle boundaries, and organelle disintegration. Transcriptomic assays revealed that hydroxychloroquine could affect the expression of metabolism-related genes. Molecular docking and molecular dynamics analyses indicated that hydroxychloroquine bound to glucose-methanol-choline oxidoreductase, with low free energy value of -11.4 kcal/mol. Cell membrane permeability assays and hyphal staining confirmed that hydroxychloroquine damaged the cell membrane, causing leakage of hyphal contents and disturbing cell function. Biochemical assays indicated that hydroxychloroquine reduced the concentration of soluble proteins and reducing sugars in the hyphae. In total, hydroxychloroquine disturbed amino acid metabolism, therefore inhibiting the production of biomacromolecules, damaging the cell membrane, and restraining the growth of hyphae, and hence inhibiting gray mold on tomato. This study will explore the use of medicine in the development of agricultural fungicides and their application in managing crop diseases, providing valuable background information.

Diseases that affect the vascular system or the pith are of great economic impact since they can rapidly destroy the affected plants, leading to complete loss in production. Fast and precise identification is thus important to inform containment and management, but many identification methods are slow because they are culture-dependent and they do not reach strain resolution. Here we used culture-independent long-read metagenomic sequencing of DNA extracted directly from stems of two tomato samples that displayed wilt symptoms. We obtained enough sequencing reads to assemble high quality metagenome-assembled genomes (MAGs) of Ralstonia solanacearum from one sample and of Pseudomonas corrugata from the other. The genome sequences allowed us to identify both pathogens to strain level using the genomerxiv platform, perform phylogenetic analyses, predict virulence genes, and infer antibiotic and copper resistance. In the case of R. solanacearum, it was straightforward to exclude the pathogen from being the Select Agent Race 3 biovar 2. Using the Branchwater tool, it was also possible to determine the world-wide distribution of both pathogen strains based on public metagenomic sequences. The entire analysis could have been completed within two days starting with sample acquisition. Steps necessary towards establishing metagenomic sequencing as a more routine approach in plant diseases clinics are discussed.

Meloidogyne enterolobii and M. incognita are major pests of sweetpotato. The ability of M. enterolobii to cause symptoms and reproduce on nematode-resistant cultivars threatens the sweetpotato industry. To evaluate the penetration, development, and reproduction of M. enterolobii and M. incognita on sweetpotato, a time-course study was conducted using the genotypes 'LA14-31' (resistant to M. enterolobii and intermediate-resistant to M. incognita), 'LA18-100' (susceptible to M. enterolobii and resistant to M. incognita), and 'LA19-65' (resistant to M. enterolobii and susceptible to M. incognita), with 'Beauregard' (susceptible to both species) and 'Jewel' (resistant to M. enterolobii and intermediate-resistant to M. incognita) as controls. Sweetpotato roots were collected at 7-, 9-, 11-, 13-, 21-, and 35-days post-inoculation (DPI), stained with acid fuchsin, and analyzed for nematode developmental stages. Nematode reproduction was evaluated by examining egg production at 42 DPI. Results showed that M. enterolobii developed and reproduced only in 'Beauregard' and 'LA18-100'. In resistant genotypes such as 'Jewel', 'LA14-31', and 'LA19-65', M. enterolobii remained at the pre-parasitic J2-stage, with halted development linked to localized cell death in response to M. enterolobii penetration. For M. incognita, the defense response was most notable in 'LA18-100', where infective juveniles either died, matured as males, or experienced delayed development into adult females, with a marked reduction in M. incognita reproduction. These findings suggest that resistance to M. enterolobii likely involves a hypersensitive-like response that prevents feeding site establishment, whereas resistance to M. incognita appears quantitative, as evidenced by delayed nematode development and reduced reproduction in resistant genotypes.

Transglutaminases (TGases) are enzymes highly conserved among prokaryotic and eukaryotic organisms, where their role is to catalyze protein cross-linking. One of the putative TGases of Phytophthora infestans has previously been shown to be localized to the cell wall. Based on sequence similarity we were able to identify six more genes annotated as putative TGases and show that these seven genes group together in phylogenetic analysis. These seven proteins are predicted to contain both a TGase domain and a MANSC domain, the latter of which was previously shown to play a role in protein stability. Chemical inhibition of transglutaminase activity and silencing of the entire family of the putative cell wall TGases are both lethal to P. infestans indicating the importance of these proteins in cell wall formation and stability. The intermediate phenotype obtained with lower drug concentrations and less efficient silencing displays a number of deformations to germ tubes and appressoria. Both chemically treated and silenced lines show lower virulence than the wild type in leaf infection assays. Finally, we show that appressoria of P. infestans possess the ability to build up turgor pressure and that this ability is decreased by chemical inhibition of TGases.

Brown rot is a disease that affects stone and pome fruit crops worldwide. It is caused by fungal members of the genus Monilinia, mainly M. fructicola, M. laxa and M. fructigena. This study presents evidence that, despite having a very similar battery of Cell Wall Degrading Enzymes (CWDEs), the three species behave differently during the early stages of infection, suggesting differences at the regulatory level, which could also explain the differences in host preference among the three species. We have shown that M. fructicola infection is accelerated by red light, and the first symptoms appear much earlier than in darkness or in the other two species. The overexpression of genes encoding for CAZymes such as pme3, pme2, pg1, cel1, pnl1 and pnl2, as well as the necrosis factor nep2, can be associated with the etiology of Monilinia spp. In addition, we found that nep2 in M. fructigena lacks binding sites in its promoter sequence for the White-Collar Complex (WCC), which is the major transcription factor responsible for regulating photo-reception processes in fungi. Finally, we found that AlphaFold models of the NEP1-like proteins (NLPs) present on the three Monilinia species predict proteins with a very high degree of similarity.