Inhibitory effects of the natural product esculetin on Phytophthora capsici and its possible mechanism

Esculetin is an important plant-derived natural product that has multiple bioactivities and applications. Phytophthora capsici is a notorious plant pathogen capable of infecting a broad range of hosts. In this study, we evaluated the antifungal activity of esculetin against P. capsici. The baseline sensitivity of P. capsici to esculetin was established using 108 isolates collected from various geographical regions in the Jiangsu and Shandong Provinces of China. The median effective concentration (EC50) values for esculetin ranged from 2.08 to 16.46 μg/mL, with a mean of 6.87 ± 2.70 μg/mL, and were normally distributed. Furthermore, both zoospore production and germination were also strongly inhibited by esculetin. Importantly, esculetin exhibited protective as well as curative activities against P. capsici on tomato, and was capable of restricting the early infection of P. capsici on Nicotiana benthamiana. We found that the esculetin treatment led to cell membrane damage of P. capsici, as revealed by morphological observations and measurements of relative conductivity and malondialdehyde (MDA). Finally, our results also suggested esculetin may adversely affect P. capsici by inhibiting its DNA and protein synthesis. These findings will contribute to a broader evaluation of using esculetin to control diseases caused by P. capsici and towards a better understanding of its mode of action as a potential fungicide.

Effect of photosensitisers on growth and morphology of Phytophthora citrophthora coupled with leaf bioassays in pear seedlings

The phytopathogenic oomycetes of the genus Phytophthora cause devastating economic losses worldwide.<br />Naphthodianthrone compounds, present in plant extracts of buckwheat and Saint John’s wort act as photosensitiser<br />agents and exhibit antimicrobial activity against a number of pathogens. In this study, we investigated the potential<br />inhibitory effects of fagopyrin and hypericin on Phytophthora citrophthora (R.E. Sm. &amp; E.H. Sm.) Leonian 1906, the<br />main causal agent of rot diseases in deciduous trees. Fagopyrin had the highest inhibitory effect in the colony growth<br />at a concentration of 2% of a stock solution (3 mg/mL), inducing clubbed hyphae with round tips. Notably, hypericin<br />also inhibited the radial colony growth and increased the hyphal branching at the subapical region, while also promoting<br />the formation of enlarged cells with irregular shapes growing collectively as biofilm-like structures. In terms of the<br />mycelial dry weight, although both photosensitisers had considerable inhibitory effects, the fagopyrin treatment was<br />most effective. Leaf bioassays showed that under dark conditions the photosensitiser pre-treated zoospores formed a<br />dense, but aberrant, mycelial growth with penetration defects. In contrast, when the zoospore production was performed<br />under light conditions, the zoospores failed to cause necrotic lesions and penetration events implying that their<br />virulence was impaired. These findings shed light on the biological effects of fagopyrin and hypericin in the regulation<br />of the mycelial growth, morphology and pathogenicity of P. citrophthora.

Globally invasive genotypes of the amphibian chytrid outcompete an enzootic lineage in coinfections

Competition between genotypes is likely to be a key driver of pathogen evolution, particularly following a geographical invasion by distant strains. Theory predicts that competition between disease strains will result in the most virulent strain persisting. Despite its evolutionary implications, the role of strain competition in shaping populations remains untested for most pathogens. We experimentally investigated the in vivo competitive differences between two divergent lineages of the amphibian-killing chytrid fungus ( Batrachochytrium dendrobatidis , Bd ). These Bd lineages are hypothesized to have diverged in allopatry but been recently brought back into secondary contact by human introduction. Prior studies indicate that a panzootically-distributed, global lineage of Bd was recently introduced into southern Brazil, and is competitively excluding enzootic lineages in the southern Atlantic Forest. To test for differences in competitive ability between invasive and enzootic Brazilian Bd isolates, we coinfected a model host frog system which we developed for this study ( Hymenochirus curtipes ). We tracked isolate-specific zoospore production over the course of the coinfection experiment with chip-based digital PCR (dPCR). The globally invasive panzootic lineage had a competitive advantage in spore production especially during the first one to four weeks of infection, and on frogs that eventually succumbed to Bd infection. Our study provides new evidence that competitive pressure resulting from the human movement of pathogen strains can rapidly alter the genetics, community dynamics and spatial epidemiology of pathogens in the wild.

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.

Chlorophyta, Xanthophyceae and Cyanobacteria in Wright Valley, Antarctica

Wright Valley, Victoria Land contains numerous aquatic habitats suitable for the growth of algae in summer. Excepting diatoms and lichen phycobionts, algal diversity and distribution in the valley was documented. Using cultures and environmental cloning eight cyanobacterial and 14 eukaryotic species were revealed. The cyanobacterium Microcoleus vaginatus and the chlorophycean Chlorococcum sp. 1 were the most common, both occurring in more than one habitat (ponds, soils or streams). Ponds harboured the most diverse communities. Habitat specialization was rare. Chlamydomonads were not found outside ponds, but species capable of zoospore production were able to colonize ponds and soils. Nostocalean cyanobacteria were not detected. Results suggest dispersal within and between valleys, with little evidence of Antarctic endemism. All but one cyanobacterium with similar internally transcribed spacer (ITS) length to clones from Miers Valley proved to be different species when 16S rRNA gene sequences were also considered; thus, ITS length is unreliable for assessing identity and biogeography of these cyanobacteria. Comparison with a 454 16S rRNA gene soil dataset from Wright Valley indicated the occurrence of only one of the cyanobacterial species, the distribution of which may be limited by salinity.

Phytophthora sojae TatD Nuclease Positively Regulates Sporulation and Negatively Regulates Pathogenesis

During pathogenic interactions, both the host and pathogen are exposed to conditions that induce programmed cell death (PCD). Certain aspects of PCD have been recently examined in eukaryotic microbes but not in oomycetes. Here, we identified conserved TatD proteins in Phytophthora sojae; the proteins are key components of DNA degradation in apoptosis. We selected PsTatD4 for further investigation because the enzyme is unique to the oomycete branch of the phylogenetic tree. The purified protein exhibited DNase activity in vitro. Its expression was upregulated in sporangia and later infective stages but downregulated in cysts and during early infection. Functional analysis revealed that the gene was required for sporulation and zoospore production, and the expression levels were associated with the numbers of hydrogen-peroxide-induced terminal dUTP nick end-labeling-positive cells. Furthermore, overexpression of PsTatD4 gene reduced the virulence in a susceptible soybean cultivar. Together, these data suggest that apoptosis may play different roles in the early and late infective stages of P. sojae, and that PsTatD4 is a key regulator of infection. The association of PsTatD4 and apoptosis will lay a foundation to understanding the basic biology of apoptosis and its roles in P. sojae disease cycle.