Fig 1.
Phytophthora nicotianae pathogenicity assays on Capsicum annuum plants.
(A) Capsicum annuum plants were soil inoculated with 375,000 P. nicotianae (isolate NMT1: Genbank: HQ711620) zoospores per plant or 10,000 P. capsici (strain PWB24: ATCC MYA-2289) zoospores per plant and watered daily for two weeks. (B) Capsicum annuum plants were foliar inoculated with 100,000 P. nicotianae zoospores or mock inoculated with water and incubated in a humidity chamber for 5 days at 28°C. No symptom development was observed on the crown, roots, or foliage of P. nicotianae inoculated plants.
Fig 2.
Treatment of Capsicum annuum with non-host Phytophthora nicotianae or BABA elicits localized autofluorescent compounds and reduces photosynthetic rate in C. annuum.
(A) C. annuum leaves were inoculated with 2,000 P. nicotianae zoospores, 2.5 mM BABA or mock inoculated with water and incubated in a humidity chamber. After 48 hours, inoculated leaf tissue was excised and imaged using a stereofluorescent microscope. (B) C. annuum plants were soil drenched with P. nicotianae or P. capsici zoospore solutions (100,000 zoospores per plant), 2.5 mM BABA or water and photosynthetic rate was measured using a Licor 6400 portable photosynthesis system 3 days post inoculation. Standard deviation bars are shown.
Fig 3.
Differential response to P. capsici foliar inoculation with BABA or Phytophthora nicotianae treated Capsicum annuum.
Plants were soil drenched with 100,000 P. nicotianae zoospores, 2.5 mM BABA, or water and foliar inoculated with 2,000 P. capsici zoospores per leaf, and plants were incubated in a humidity chamber at 28°C. for 48 h Plants were removed from humidity chamber and watered normally and grown under fluorescent light for an additional two weeks. (A) P. capsici caused systemic foliar blight and death in all untreated plants, whereas 50%- 83% of P. nicotianae or BABA treated plants did not develop disease symptoms outside of the inoculated leaves. (B) Histochemical x-gluc staining of GUS expressing P. capsici was utilized to visualize pathogen structures on inoculated C. annuum leaves. At 72 h post inoculation, abundant P. capsici hyphae and sporangia were visible on untreated plants, while BABA and P. nicotianae treated plants displayed confined areas of x-gluc staining with no identifiable P. capsici structures. Scale bar 30 μm for all images.
Fig 4.
Enhanced production of hydrogen peroxide and development of hypersensitive response in BABA and P. nicotianae induced C. annuum plants during early P. capsici infection.
The hydrogen peroxide indicator stain 3-3- diaminobenzidine (DAB) was used to visualize reactive oxygen species production at 12, 24, and 48 h post P. capsici inoculation. The presence of hydrogen peroxide is indicated by the formation of a dark precipitate. P. capsici hyphae and germtubes are indicated with arrows. Tissue was imaged using a compound microscope with a digital camera mounted using an eyepiece adapter (Nikon, Melville, NY). Scale bar is 10 μm for all images.
Fig 5.
Principle components analysis (PCA) of relative concentrations for 62 metabolites identified in Capsicum annuum through GCMS analysis.
Capsicum annuum seedlings were treated with BABA, P. nicotianae (P.nic), or water (H2O) and challenged with P. capsici (+) or mock inoculated with water (-) for a total of 6 treatments, with each treatment replicated on 4 blocks with 15 seedlings each in each block. Normalized peak area for each metabolite was log transformed and autoscaled to focus on relative changes across treatments. Substantial shifts in metabolite concentrations were identified for BABA treated plants, with no clear distinction between P. capsici (+) or mock challenged (-) groups. P. nicotianae (P.nic) induced plants that were challenged with P. capsici were distinguishable from non-induced (H2O) plants at the 95% confidence interval.
Fig 6.
Heat map of carbohydrate metabolites that significantly changed in concentration for BABA and P. nicotianae induced plants.
Capsicum annuum seedlings were treated with BABA, P. nicotianae (P.nic), or water (H2O) and challenged with P. capsici (+) or mock inoculated with water (-) for a total of 6 treatments, with each treatment replicated on 4 blocks with 15 seedlings each in each block. Metabolites in red indicate higher relative concentrations while those in blue indicate lower relative concentrations.
Fig 7.
Heat map and dendrogram of non-carbohydrate metabolites that significantly changed in Phytophthora nicotianae induced Capsicum annuum plants.
Capsicum annuum seedlings were treated with BABA, P. nicotianae (P.nic), or water (H2O) and challenged with P. capsici (+) or mock inoculated with water (-) for a total of 6 treatments, with each treatment replicated on 4 blocks with 15 seedlings each in each block. Dendrogram was constructed using the Spearman distance measure and Ward clustering algorithm. Metabolites in red indicate higher relative concentrations while those in blue indicate lower relative concentrations.
Fig 8.
Box and whisker plots for metabolites that were statistically distinct between Phytophthora nicotianae induced plants that were challenged with P. capsici [P.nic (+)] and mock inoculated plants [P.nic (-)].
Data were log transformed and autoscaled (mean centered and divided by the standard deviation of each variable) to focus on relative changes in metabolite profiles across treatments.
Table 1.
Metabolites with fold changes greater than 2 between BABA and water treated C. annuum plants.
Fig 9.
Impact of selected carbohydrates on Phytophthora capsici growth rate.
P. capsici colonies were grown on water agar supplemented with 1% (w / v) of listed sugars at 28°C for five days. Average growth (mm / day) rate is listed above each label. * Indicates significant differences in growth rate compared to water agar, α equal to 0.05.
Fig 10.
Overview of selected carbohydrate pathways.
Metabolites boxed in red were significantly increased in concentration in induced plants while metabolites boxed in blue were significantly decreased. Abbreviations: oxidative pentose phosphate pathway (OPPP), non-oxidative pentose phosphate pathway (NPPP), sucrose synthetase (SuSY), UTP glucose-1-phosphate uridylyltransferase (UGP2), phosphoglucomutase (PGM), trehalose 6-phosphate synthase (T6PS), Trehalose 6- phosphate phosphatase (T6PP), sucrose phosphate synthase (SPS), sucrose phosphate phosphatase (SPP), invertase (INV), glucose 6-phosphate isomerase (G6P-I), hexose kinase (HXK), fructose kinase (FK), fructose 1-phosphate aldolase (F1P-A), glyceraldehyde dehydrogenase (Gdh), fructose 6-phosphate kinase (F6P- K), fructose 1,6 bisphosphatase (F1,6bp), fructose 1,6 bisphosphate aldolase (F1,6- A), triose phosphate isomerase (TPI), glycerol 3-phosphate dehydrogenase (G3Pdh), glycerol kinase (GK), glucose 6-phosphate dehydrogenase (G6Pdh), 6- phosphogluconate dehydrogenase (6PGdh).
Fig 11.
Overview of TCA cycle and associated biochemical pathways.
Metabolites boxed in red were significantly increased in concentration in induced plants while metabolites boxed in blue were significantly decreased.