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The cucumber inbred line Xintai mici was used for all experiments. The PM pathogen (Podosphaera xanthii) used in this test was isolated from diseased cucumber leaves from the greenhouse at Zhejiang University of Agriculture and Forestry (Zhejiang, China) and maintained by infection of susceptible cucumber cultivar plants.
Orthogonal test for indoor seedling stage resistance identification method
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Cucumber plants were grown in soil (nursery substrate : garden soil = 2:1, V:V) under a long-day photoperiod (16-h light/8-h dark at 26 °C, 20,000 lx) up to one-true-leaf stage~three-true-leaf stage[11]. For the disease resistance test, a four-factor three-level (L9(3)4) orthogonal experimental design was employed, with factors including seedling age (a), spore suspension concentration (b), inoculation method (c), and moisturizing time (d). The detailed factors and levels are shown in Tables 1 & 2. Severely diseased cucumber plants from the greenhouse were selected, and fresh powdery mildew spores were collected and re-suspended in a water solution containing 0.01% Tween-20. The spore suspension was adjusted to the desired concentration (104, 105, or 106) spores per mL, and the spores were inoculated onto leaves at different seedling ages (one-true-leaf stage, two-true-leaf stage, or three-true-leaf stage) using different inoculation methods (spraying: spray a certain concentration of spore suspension onto flattened cucumber leaves with a spray bottle; rubbing: mix a specific concentration of spore suspension with quartz sand, and use gauze to evenly spread the mixture onto cucumber leaves; leaf brushing: use a specially made soft-bristled brush to evenly apply a certain concentration of spore suspension onto the leaves of cucumber plants). The inoculated plants were then placed in a growth chamber under controlled lighting conditions and subjected to 12, 24, and 36 h of moisturizing treatment. Furthermore, infection of leaf was recorded every 3 d after inoculation, and the incidence and disease index were investigated 15 d later. The experiment was set up with two biological replicates, each replicate experiment contains 90 samples (for each orthogonal scheme, 10 samples were prepared).
Table 1. Factors and levels of cucumber powdery mildew seedling resistance identification test.
Factor Level 1 Level 2 Level 3 a Seedling age one-true-leaf two-true-leaf three-true-leaf b Spore suspension concentration: spores/mL 104 105 106 c Inoculation method spraying rubbing leaf brushing d Moisturizing time 12 h 24 h 36 h Table 2. Cucumber powdery mildew seedling resistance identification implementation programme.
Test
no.Factor Scheme of orthogonal design a b c d T1 1 1 2 2 a1b1c2d2 one-true-leaf 104 rubbing 24 h T2 2 1 3 3 a2b2c3d3 two-true-leaf 104 leaf brushing 36 h T3 3 3 3 1 a3b3c1d1 three-true-leaf 106 leaf brushing 12 h T4 1 1 1 1 a1b1c1d3 one-true-leaf 104 spraying 12 h T5 2 2 2 1 a2b2c2d1 two-true-leaf 105 rubbing 12 h T6 3 2 1 3 a3b3c3d2 three-true-leaf 105 spraying 36 h T7 1 2 3 2 a1b1c3d1 one-true-leaf 105 leaf brushing 24 h T8 2 3 1 2 a2b2c1d2 two-true-leaf 106 spraying 24 h T9 3 3 2 3 a3b3c2d3 three-true-leaf 106 rubbing 36 h The calculation of disease index (DI) followed the method described by Nie et al.[12]. For details, refer to Table 3. The formula for calculating the disease index (DI) is:
$ \text{DI =}\ \dfrac{\sum_{ }^{ }\left({S}_{{i}}\cdot {n}_{{i}}\right)}{{4}{N}}\times100 $ (1) Where Si represents the disease grade, ni represents the number of plants in the corresponding disease grade, i represents each level of disease severity, and N represents the total number of plants investigated.
Table 3. Disease grading criteria for indoor seedling resistance identification.
Grade Symptom 0 no symptom 1 < 5% of the surface area of the leaf 2 6%−25% of the leaf infected 3 26%−50% of the leaf infected 4 > 50% of the leaf infected Identification of disease resistance of cucumber germplasm resources during the indoor seedling stage
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In this study, four cucumber varieties with distinct resistance profiles, as determined through preliminary experiments, were chosen as test subjects. Among them, R1641 was designated as a highly resistant variety, BK2 as disease-resistant, 9930 as moderately resistant, and H136 as susceptible. At the stage when the cucumber plants developed three true leaves, the leaves were cut off and laid flat in sterilized plastic Petri dishes (with a moist filter paper placed at the bottom of the dish). The optimal indoor seedling disease resistance identification method, which had been screened, was utilized to inoculate powdery mildew on the leaves of the four germplasm resources. Details regarding the investigation of the incidence rate refer to Table 3. Furthermore, the four cucumber germplasms were graded based on their resistance levels: 0 ≤ DI < 15 was classified as highly resistant, 15 ≤ DI < 35 as resistant, 35 ≤ DI < 55 as moderately resistant, 55 ≤ DI < 75 as susceptible, and DI ≥ 75 as highly susceptible. Three plants were cultivated for each material, and three leaves were cut from each plant.
Observation of the germination process of powdery mildew conidia on cucumber leaves under different treatments
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At 72 h after inoculation with powdery mildew, samples were taken from the leaves of the nine schemes of orthogonal design using a puncher, and the leaf discs were placed in 10 mL centrifuge tubes. Three plants were randomly selected for each treatment, and one leaf was taken from each plant. Three leaf discs were taken from each leaf, resulting in a total of nine replicates. Before observing the conidia, the leaf discs were placed in a decolorizing solution (acetic acid : ethanol = 1:3) for 3 h. They were then rinsed with deionized water two to three times and stained with cotton blue in lactophenol for 1−2 min. Subsequently, the leaf discs were clamped onto a glass slide with forceps, covered with a cover glass, and observed under a 10 × 40 magnification microscope to observe the germination status of powdery mildew conidia on cucumber leaves under different treatment combinations. Twenty conidia were observed per leaf disc.
Measurement of chlorophyll (Chl) contents
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Twenty days after inoculation with powdery mildew, the chlorophyll content of cucumber in each orthogonal design scheme was measured. The third leaf from the top of the cucumber plants was selected for measurement. Chl contents of cucumber leaves were determined using a SPAD-502 PLUS chlorophyll meter. The experiment was set up with two biological replicates, and in each replicate, five strains were randomly selected for measurement.
Measurement of photosynthetic rate
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Twenty days after inoculation with powdery mildew, the Pn, Ci and Gs of the third leaf from the top of the cucumber in each orthogonal design scheme was measured by a portable photosynthetic instrument LI-6800 (LI-COR, Lincoln, USA). The experiment was set up with two biological replicates, and in each replicate, five strains were randomly selected for measurement.
Biomass measurement
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Twenty-one days after inoculation with powdery mildew, the fresh weight and dry weight of cucumber plants were measured for each orthogonal design scheme as well as the control group (sprayed with water). After being killed at 105 °C for 15 min, the samples were dried at 80 °C until reaching a constant weight, and the dry weight of all cucumber plants in each treatment combination was determined. The experiment was set up with two biological replicates, and in each replicate, five strains were randomly selected for measurement.
Statistical analysis
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All data were expressed as the mean ± standard error of the mean (SEM). Software of statistical product and service solutions (SPSS) 18.0 was used for the evaluation of the statistical experimental design.
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At 15 d post-inoculation, the cucumber Xintai mici demonstrated full disease onset. Initially, an individual factor analysis was conducted. As depicted in Fig. 1a, with the increase in cucumber seedling age, both the disease incidence and disease index exhibited a gradual ascension, with no significant differences in disease incidence among different treatments. The disease index for inoculation at the three-true-leaf stage was the highest at 82.40, significantly higher than at the one-true-leaf stage, but bore no significant difference with the two-true-leaf stage inoculation. As illustrated in Fig. 1b, an upward trend in both disease incidence and index is observed by an increase in the concentration of inoculating spore suspension. At an inoculation concentration of 106 per mL, the disease index stands at 86.78, remarkably outstripping those of the two treatments with inoculation concentrations of 105 per mL (65.70) and 104 per mL (58.83). Conversely, there are no notable disparities in either the disease index or the incidence among treatments differing in inoculation method and post-inoculation hydration time (Fig. 1c & d).
Figure 1.
Resistance identification results of combinations of indoor seedling resistance identification methods (15 d). Effects of (a) different seedling ages, (b) spore suspension concentrations, (c) inoculation methods, and (d) moisturizing time on the incidence rate and disease index of cucumber seedlings. (e) The influence of different inoculation schemes on the incidence rate and disease index of cucumber seedlings. Values are means of minimum two replicates, with each containing 9 to 10 seedlings. Data are expressed as mean ± Standard error of mean, and significances were examined by one-way ANOVA, followed by Duncan's multiple comparisons test and different letters represent significant differences (p < 0.05).
Upon analyzing the disease incidences and indices of nine treatment combinations, it is discerned that there are no significant differences in disease incidence among the treatments, all being above 96.67%. The disease progression of treatment T3 is the swiftest, with a disease incidence of 100%, and the symptoms are distinct. Fifteen days after inoculation with powdery mildew, the disease index of T3 reaches 89.03, significantly surpassing most treatments. Consequently, this treatment is regarded as the optimal method for early-stage powdery mildew resistance determination in cucumber seedlings, namely, inoculating at the three-true-leaf stage, with a spore suspension concentration of 106 per mL, using the leaf-brushing method, and maintaining hydration for 12 h post-inoculation (Fig. 1e).
Disease resistance identification of four cucumber germplasm resources
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The indoor seedling disease resistance identification method was adopted to evaluate the powdery mildew resistance of four cucumber germplasm resources. The results indicated that R1461 exhibited high resistance, BK2 demonstrated resistance, while both 9930 and H136 showed susceptibility to the disease (Fig. 2). Except for 9930, the resistance types of the other three materials were consistent with those determined in previous experiments. These findings suggest that the indoor seedling disease resistance identification method screened in this study is reliable.
Figure 2.
Identification of disease resistance in different cucumber germplasm. The onset of disease in (a) R1461, (b) BK2, (c) 9930, and (d) H136 after 15 d of inoculation with powdery mildew.
Growth of powdery mildew spores under different treatment combinations
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At 72 h after inoculation of powdery mildew on cucumber leaves, the spores in all treatment combinations began to germinate (Fig. 3). The spore growth on treatment combinations T1, T4, T5, and T7 were slow, with few germ tube branches, and some germ tubes had not yet begun to elongate. In contrast, the germ tubes of the spores on treatment combinations T2 and T6 had extended, branched, and started to spread on the leaf surface, forming hyphae. Meanwhile, the spores on treatment combinations T3, T8, and T9 also began to form hyphae, but the growth process was slightly faster than treatments T2 and T6.
Figure 3.
Growth and development of powdery mildew fungus spores under different treatment combinations. (a)−(i) Correspond to the spore germination of treatment combinations T1−T9 after 72 h of inoculation.
Effect of powdery mildew on the growth of cucumber seedlings
The impact of powdery mildew on cucumber biomass accumulation
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As shown in Fig. 4, compared to the control group, the fresh weight and dry weight of cucumber plants inoculated with powdery mildew fungi decreased significantly. In addition, it is observable that the impact of powdery mildew on the dry weight and fresh weight of plants diminishes as the disease index decreases. Compared to other treatments inoculated with powdery mildew at the one-true-leaf stage, the fresh and dry weights of cucumber seedlings in treatment T4 were the highest, but there was no significant difference between treatments T1 and T7. Among the treatments inoculated with powdery mildew at the two-true-leaf stage, the fresh weight under treatment T8 significantly decreased by 14.05% and 13.74% compared to T2 and T5, respectively, and the dry weight significantly decreased by 38.89% and 37.74%. When inoculated with powdery mildew at the three-true-leaf stage, compared to treatments T6 and T9, the fresh weight of cucumber plants under treatment T3 decreased by 34.01% and 13.83% respectively, and the dry weight decreased by 31.28% and 3.12%. It can be seen that the infection of powdery mildew inhibits the accumulation of dry matter in cucumber seedlings, and this inhibitory effect gradually intensifies with the worsening of the disease.
Figure 4.
Effect of powdery mildew on dry and fresh weight of plants at different seedling ages. (a) The impact of inoculating powdery mildew at the one-true-leaf stage on the growth of cucumber seedlings. (b) The impact of inoculating powdery mildew at the two-true-leaf stage on the growth of cucumber seedlings. (c) The impact of inoculating powdery mildew at the three-true-leaf stage on the growth of cucumber seedlings. Values are means of minimum two replicates, with each containing five seedlings. Data are expressed as mean ± Standard error of mean, and significances were examined by one-way ANOVA, followed by Duncan's multiple comparisons test and different letters represent significant differences (p < 0.05).
The impact of powdery mildew on photosynthesis of cucumber seedlings
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As shown in Table 4, after inoculation with powdery mildew, cucumber leaves with different degrees of disease showed significant differences in Pn values. Compared with other treatments, T3, T8, and T9 had the greatest decline in Pn, which were only 2.42, 2.98, and 1.92, respectively. Similarly to Pn, the transpiration rate and stomatal conductance under treatments T3, T8, and T9 also significantly decreased compared to other treatments, with a decrease of more than 40%. Among the nine treatments, there was no significant difference in Ci values of most of the leaf treatments, but the Ci values of treatments T3, T8, and T9 were significantly lower than other treatments, indicating that powdery mildew inhibits the photosynthetic rate of cucumber leaves. When the disease index is low, the reduction in Pn is mainly due to non-stomatal limitations, and when the disease is serious, the reduction in Pn is mainly due to stomatal limitations.
Table 4. Comparison of photosynthetic parameters of diseased plants in different treatments.
Treatments Pn
(μmol/m2/s)Ci
(μmol/mol)E
(mmol/m2/s)Gs
(mol/m2/s)T1 3.29 bc 341.17 a 1.78 ab 0.13 ab T2 3.08 bc 325.84 a 1.41 b 0.09 b T3 2.42 de 229.12 b 0.49 cd 0.03 c T4 3.91 a 321.45 a 1.56 b 0.10 b T5 3.84 ab 343.73 a 2.18 a 0.15 a T6 3.71 ab 332.47 a 1.83 ab 0.12 ab T7 4.01 a 335.53 a 1.98 a 0.14 a T8 2.98 cd 220.68 b 0.81 c 0.05 c T9 1.92 e 208.43 b 0.29 d 0.02 c Different lowercase letters indicate significant differences between treatments (p < 0.05). The impact of powdery mildew on chlorophyll content in cucumber seedling leaves
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Analysis of Fig. 5 shows that among the treatments of powdery mildew inoculation at the one-true-leaf stage, the SPAD value of the functional leaves in treatment T4 was the largest, but there was no significant difference with T1 and T7. At the two-true-leaf stage, compared to treatments T5 and T8, the SPAD value under treatment T2 significantly increased by 14.25% and 29.95%, respectively. Among the treatments of powdery mildew inoculation at the three-true-leaf stage, the SPAD value of treatment T6 was the largest, significantly higher than T3 and T9 by 16.37% and 13.86%. It can be seen that the infection of powdery mildew will reduce the chlorophyll content in cucumber leaves, thereby affecting the photosynthesis of cucumbers.
Figure 5.
Statistics of SPAD values of plants at different seedling ages under powdery mildew stress. (a) The effect of treatments T1, T4, and T7 on chlorophyll content in cucumber. (b) The effect of treatments T2, T5, and T8 on chlorophyll content in cucumber. (c) The effect of treatments T2, T5, and T8 on chlorophyll content in cucumber. Values are means of minimum two replicates, with each containing five seedlings. Data are expressed as mean ± Standard error of mean, and significances were examined by one-way ANOVA, followed by Duncan's multiple comparisons test and different letters represent significant differences (p < 0.05).
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All data generated or analyzed during this study are included in this published article.
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About this article
Cite this article
Tan J, Zhong L, Fan S, Cheng S, Gao Y, et al. 2024. Methods for seedling identification of cucumber resistance to powdery mildew and its effect on the growth of cucumber seedlings. Vegetable Research 4: e030 doi: 10.48130/vegres-0024-0028
Methods for seedling identification of cucumber resistance to powdery mildew and its effect on the growth of cucumber seedlings
- Received: 24 March 2024
- Revised: 01 June 2024
- Accepted: 24 June 2024
- Published online: 08 October 2024
Abstract: The severity of powdery mildew in cucumbers gravely impinges upon both yield and quality. In order to probe efficacious methods for early-stage resistance determination against powdery mildew in cucumbers, we utilized Xintai mici as an experimental subject and adopted a four-factor, three-level (L9(3)4) orthogonal experimental design. This design encompassed four factors: inoculation seedling age, inoculation concentration, inoculation method, and different hydration periods post-inoculation. Through this, we sifted and discerned the most optimal indoor method for early-stage resistance determination against powdery mildew in cucumbers. Concurrently, we studied the influence of different determination methods on the accumulation of cucumber plant biomass, chlorophyll content, and leaf photosynthetic parameters to delineate the impact patterns of powdery mildew on cucumber leaf photosynthetic traits. The results underscored that disease incidence amongst different treatments were minute, all exceeding 96.67%. In contrast to other treatments, T3 (inoculating a spore suspension of powdery mildew at a concentration of 106 per mL on one-true-leaf stage using the leaf-brushing method and maintaining hydration for 12 h post-inoculation) exhibited a rapid onset of disease, with a disease index reaching 89.03, thereby proving to be the most superior method for early-stage powdery mildew determination. Various determination methods wielded a significant influence on dry and fresh weight, chlorophyll content, and leaf photosynthetic activity of cucumber seedlings. Relative to other treatments, the net photosynthetic rate (Pn) of T3, T8, and T9 was significantly suppressed post-inoculation with powdery mildew, all lingering below 3 μmol/m2/s, while the transpiration rate (E), intercellular CO2 concentration (Ci), as well as the stomatal conductance (Gs), drastically declined, the decrease fell between 25%−80%. The relative chlorophyll content maintained at a lower level, ultimately leading to a significant reduction in the accumulation of dry matter. This demonstrates that powdery mildew can significantly stifle the growth of cucumber seedlings.
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Key words:
- Cucumbers /
- Methods /
- Seedlings /
- Identification /
- Resistance /
- Powdery mildew