Chilling Stress—The Key Predisposing Factor for Causing Alternaria alternata Infection and Leading to Cotton (Gossypium hirsutum L.) Leaf Senescence

Leaf senescence plays a vital role in nutrient recycling and overall capacity to assimilate carbon dioxide. Cotton premature leaf senescence, often accompanied with unexpected short-term low temperature, has been occurring with an increasing frequency in many cotton-growing areas and causes serious reduction in yield and quality of cotton. The key factors for causing and promoting cotton premature leaf senescence are still unclear. In this case, the relationship between the pre-chilling stress and Alternaria alternata infection for causing cotton leaf senescence was investigated under precisely controlled laboratory conditions with four to five leaves stage cotton plants. The results showed short-term chilling stress could cause a certain degree of physiological impairment to cotton leaves, which could be recovered to normal levels in 2–4 days when the chilling stresses were removed. When these chilling stress injured leaves were further inoculated with A. alternata, the pronounced appearance and development of leaf spot disease, and eventually the pronounced symptoms of leaf senescence, occurred on these cotton leaves. The onset of cotton leaf senescence at this condition was also reflected in various physiological indexes such as irreversible increase in malondialdehyde (MDA) content and electrolyte leakage, irreversible decrease in soluble protein content and chlorophyll content, and irreversible damage in leaves' photosynthesis ability. The presented results demonstrated that chilling stress acted as the key predisposing factor for causing A. alternata infection and leading to cotton leaf senescence. It could be expected that the understanding of the key factors causing and promoting cotton leaf senescence would be helpful for taking appropriate management steps to prevent cotton premature leaf senescence

Effects of evodiamine (EVO) on the protein expression of Cyt C, caspase-12, -8, -9 and -3, Fas and Trail in the H446 and H1688 SCLC cells.

<p>Cell lysates were analyzed by Western blot. Each experiment was repeated 3 times. Data presented as mean ± standard deviation (n = 3). Untreated H446 or H1688 cells were used as a negative control group. *<i>P</i><0.05 as compared to corresponding control group. Fas: factor associated suicide; Trail: tumor necrosis factor-related apoptosis inducing ligand; Cyt C: cytochrome C.</p

Effects of evodiamine (EVO) on the mRNA expression of Bax and Bcl-2 in H446 and H1688 cells.

<p>Cell lysates were analyzed by RT-PCR. Each experiment was repeated 3 times. Data presented as mean ± standard deviation (n = 3). Untreated H446 or H1688 cells were used as a negative control group. *<i>P</i><0.05 as compared to the control group. ^#<i>P</i><0.05 as compared to corresponding EVO treated group at 24 h. <i>P</i><0.05 as compared to corresponding EVO treated group at 48 h.</p

Changes in chlorophyll contents during Alternaria disease development promoted by chilling stress pre-treatment.

<p>a: the control cotton plants sustained growing at optimal temperature of 28/20°C. b, c, d: Cotton plants performed with chilling stress pre-treatments at 20/16°C, 16/12°C, 12/8°C for 3 days respectively, then inoculated with 1.2×10⁴ conidial/mL inoculum suspension of <i>A. alternata</i> isolate A1, and returned to grow at 28/20°C. All collected data (mean ± standard deviation SD with 6 replicates) were presented as relative values to the chlorophyll contents (SPAD unit) at −3 d (100% chlorophyll content = 41.5 for XLZ13 and 44.0 for XLZ33 leaves, respectively). The chilling stress pre-treatment period was indicated by time points from −3 to 0, and the period after inoculation was indicated by time points from 0 to 15.</p

Appearance of Alternaria disease on cotton leaves pre-treated by various durations of chilling stress at 16/12°C.

<p>Various durations of chilling stress pre-treatments were performed at 16/12°C. Then the pre-treated cotton leaves were inoculated with 1.2×10⁴ conidial/mL inoculum suspension of <i>A. alternata</i> isolate A1 by slightly brushing method and returned to grow at optimal temperature of 28/20°C. The mock inoculations were performed with sterilized water. Presented pictures photographed at 15 days after inoculation.</p

Effects of evodiamine (EVO) on the activities of caspase-8 (A), -9 (B) and -3 (C) in H446 cells.

<p>Cell lysates were analyzed by a colorimetric assay of Ac-DEVD-pNA. Each experiment was repeated 3 times. Data presented as mean ± standard deviation (n = 3). Caspase activities were given as arbitrary units (AU) per milligram of protein. Untreated H446 cells were used as a negative control group. *<i>P</i><0.05 as compared to the corresponding control group. ^#<i>P</i><0.05 as compared to corresponding EVO treated group at 24 h. <i>P</i><0.05 as compared to corresponding EVO treated group at 48 h.</p

Changes in MDA content during Alternaria disease development promoted by chilling stress pre-treatment.

<p>a: the control cotton plants sustained growing at optimal temperature of 28/20°C. b, c, d: Cotton plants performed with chilling stress pre-treatments at 20/16°C, 16/12°C, 12/8°C for 3 days respectively, then inoculated with 1.2×10⁴ conidial/mL inoculum suspension of <i>A. alternata</i> isolate A1, and returned to grow at 28/20°C. All collected data (mean ± standard deviation SD with 6 replicates) were presented as relative values to the malondialdehyde contents at −3 d (100% MDA content = 22.4 nmol/gFW for XLZ13 and 21.8 nmol/gFW for XLZ33 leaves, respectively). The chilling stress pre-treatment period was indicated by time points from −3 to 0, and the period after inoculation was indicated by time points from 0 to 15.</p

Evodiamine (EVO) induces apoptosis through two intrinsic caspase-dependent pathways, but not through an extrinsic caspase-dependent pathway.

<p>Evodiamine (EVO) induces apoptosis through two intrinsic caspase-dependent pathways, but not through an extrinsic caspase-dependent pathway.</p

Influences of chilling stress pre-treatments performed with various low temperatures on occurrence and development of Alternaria disease.

<p>Cotton seedlings were pre-treated with chilling stress for 3 days, then inoculated with <i>A. alternata</i> isolate A1, and removed to grow at optimal temperature of 28/20°C; Different letters behind the disease index indicate significant difference with the <i>P</i> value of 0.05. The first letters indicate the comparison among different temperatures of chilling stress pre-treatment and the second letters indicate the comparison between XLZ13 and XLZ33.</p

Changes in Fv/Fm ratio during Alternaria disease development promoted by chilling stress pre-treatment.

<p>a: the control cotton plants sustained growing at optimal temperature of 28/20°C. b, c, d: Cotton plants performed with chilling stress pre-treatments at 20/16°C, 16/12°C, 12/8°C for 3 days respectively, then inoculated with 1.2×10⁴ conidial/mL inoculum suspension of <i>A. alternata</i> isolate A1, and returned to grow at 28/20°C. All collected data (mean ± standard deviation SD with 6 replicates) were presented as relative values to the maximal quantum yield of photosystem II photochemistry (Fv/Fm ratio) at −3 d (100% Fv/Fm ratio = 0.805 for XLZ13 and 0.812 for XLZ33 leaves). The chilling stress pre-treatment period was indicated by time points from −3 to 0, and the period after inoculation was indicated by time points from 0 to 15.</p