Stem canker pathogen Botryosphaeria dothidea inhibits poplar leaf photosynthesis in the early stage of inoculation

Abstract

Fungal pathogens can induce canker lesions, wilting, and even dieback in many species. Trees can suffer serious physiological effects from stem cankers. In this study, we investigated the effects of Botryosphaeria dothidea (B. dothidea) on Populus bolleana (P. bolleana) leaves photosynthesis and stomatal responses, when stems were inoculated with the pathogen. To provide experimental and theoretical basis for preventing poplar canker early. One-year-old poplar stems were inoculated with B. dothidea using an epidermal scraping method. In the early stage of B. dothidea inoculation (2-14 days post inoculation, dpi), the gas exchange, stomatal dynamics, hormone content, photosynthetic pigments content, chlorophyll fluorescence parameters, and non-structural carbohydrate (NSC) were evaluated to elucidate the pathophysiological mechanism of B. dothidea inhibiting photosynthesis. Compared with the control groups, B. dothidea noteworthily inhibited the net photosynthetic rate (P _n), stomatal conductance (G _s), intercellular CO₂ concentration (C _i), transpiration rate (T _r), and other photosynthetic parameters of poplar leaves, but stomatal limit value (L _s) increased. Consistent with the above results, B. dothidea also reduced stomatal aperture and stomatal opening rate. In addition, B. dothidea not only remarkably reduced the content of photosynthetic pigments, but also decreased the maximum photochemical efficiency (F _v/F _m), actual photochemical efficiency (Φ _PSII), electron transfer efficiency (ETR), and photochemical quenching coefficient (q _P). Furthermore, both chlorophyll and Φ _PSII were positively correlated with P _n. In summary, the main reason for the abated P _n under stem canker pathogen was that B. dothidea not merely inhibited the stomatal opening, but hindered the conversion of light energy, electron transfer and light energy utilization of poplar leaves. In general, the lessened CO₂ and P _n would reduce the synthesis of photosynthetic products. Whereas, sucrose and starch accumulated in poplar leaves, which may be due to the local damage caused by B. dothidea inoculation in phloem, hindering downward transport of these products.

Keywords: Botryosphaeria dothidea; chlorophyll fluorescence; fungal pathogens; photosynthesis; poplar stem canker; stomatal closure.

FIGURE 1

B. dothidea inoculation on photosynthetic parameters in P. bolleana leaves. One-year old poplar stems were inoculated, respectively, by B. dothidea (Bd) or PDA (CTR). (A) Net photosynthetic rate (P_n), (B) stomatal conductance (G_s), (C) intercellular CO₂ concentration (C_i), and (D) limiting value of stomata (L_s) were measured at 0, 2, 4, 6, 8, 10, and 14 dpi. Data are presented as the mean of seven replicates. Error bars represent the standard error of the mean. Asterisks denote significant difference at P < 0.05 between treatments.

FIGURE 2

B. dothidea inoculation on water transportation in P. bolleana leaves. One-year old poplar stems were inoculated, respectively, by B. dothidea (Bd) or PDA (CTR). (A) Transpiration rate (T_r), (B) vapor pressure deficit (VPD), and (C) water use efficiency (WUE) were measured at 0, 2, 4, 6, 8, 10, and 14 dpi. Data are presented as the mean of seven replicates. Error bars represent the standard error of the mean. Asterisks denote significant difference at P < 0.05 between treatments.

FIGURE 3

B. dothidea inoculation on stomatal movement in P. bolleana leaves. One-year old poplar stems were inoculated, respectively, by B. dothidea (Bd) or PDA (CTR). Sample collection and stomatal movement parameters measurement were performed at 6 and 14 dpi. (A) Stomatal density, (B) stomatal aperture, (C) stomatal opening rate. Each column is the mean of seven replicates. Error bars represent the standard error of the mean. Columns labeled with different letters (a–c) denote a significant difference (P < 0.05) between treatments.

FIGURE 4

B. dothidea inoculation on stomatal characteristics in P. bolleana leaves. Representative images of stomatal characteristics under poplar stems inoculated by B. dothidea (Bd) or PDA (CTR) at 6 and 14 dpi (A–H). (B,D,F,H) Showed the enlarged images of stomatal characteristics. Yellow scale bars (A,C,E,G) = 50 μm. Green scale bars (B,D,F,H) = 5 μm.

FIGURE 5

B. dothidea inoculation on phytohormone content in P. bolleana leaves. One-year old poplar stems were inoculated, respectively, by B. dothidea (Bd) or PDA (CTR). Sample collection and phytohormone content measurement were performed at 6 and 14 dpi. (A) ABA content, (B) JA-me content, (C) IAA content, (D) ZR content. Each column is the mean of seven replicates. Error bars represent the standard error of the mean. Columns labeled with different letters (a–c) denote a significant difference (P < 0.05) between treatments.

FIGURE 6

B. dothidea inoculation on photosynthetic pigments in P. bolleana leaves. One-year old poplar stems were inoculated, respectively, by B. dothidea (Bd) or PDA (CTR). Sample collection and photosynthetic pigments measurement were performed at 6 and 14 dpi. (A) Chlorophyll a content (Chl a), (B) Chlorophyll b content (Chl b), (C) total Chlorophyll content (Chl), (D) Carotenoids content (Car). (E) Chlorophyll a/b ratio, (Chl a/b), (F) Chlorophyll/Carotenoids ratio, (Chl/Car). Each column is the mean of seven replicates. Error bars represent the standard error of the mean. Columns labeled with different letters (a–c) denote a significant difference (P < 0.05) between treatments.

FIGURE 7

B. dothidea inoculation on chlorophyll fluorescence parameters in P. bolleana leaves. One-year old poplar stems were inoculated, respectively, by B. dothidea (Bd) or PDA (CTR). Chlorophyll fluorescence parameters were measured at 0, 2, 4, 6, 8, 10, and 14 dpi. (A) Ratio of variable to maximal chlorophyll fluorescence (F_v/F_m), (B) Actual photochemical efficiency of PSII (Φ_PSII), (C) Electron transfer rate (ETR), (D) Photochemical quenching coefficient (q_P). (E) Non-photochemical quenching coefficient (NPQ). Data are presented as the mean of seven replicates. Error bars represent the standard error of the mean. Asterisks denote significant difference at P < 0.05 between treatments.

FIGURE 8

Correlation analysis of parameters. (A) Chlorophyll content (Chl) Vs. net photosynthetic rate (P_n), (B) actual photochemical efficiency (Φ_PSII) Vs. net photosynthetic rate (P_n).

FIGURE 9

B. dothidea inoculation on sucrose and starch content in P. bolleana leaves. One-year old poplar stems were inoculated, respectively, by B. dothidea (Bd) or PDA (CTR). Sample collection, sucrose and starch content measurements were performed at 6 and 14 dpi. (A) Sucrose content, (B) starch content. Each column is the mean of seven replicates. Error bars represent the standard error of the mean. Columns labeled with different letters (a–c) denote a significant difference (P < 0.05) between treatments.