Effects on the yield and fiber quality components of Bt cotton inoculated with Azotobacter chroococcum under elevated CO₂

CO₂ levels

This experiment was conducted in CO₂-controlled growth cabinets (i.e., CCGC; GXM-508C-4-CO₂; Ningbo Jiangnan Instrument CO., Ningbo, China) at the Experiment Station of Cotton Research Center, Shandong Academy of Agricultural Sciences (SAAS) located in Linqing County, Shandong Province of China (36.51°N, 115.42°E) on 24 April of 2018 and 2019. Two CO₂ levels, ambient (400 μl/L, abbreviated as aCO₂) and elevated (800 μl/L or double ambient, abbreviated as eCO₂), were applied continuously during the experiment in both years. The CCGCs of elevated CO₂ treatments were inflated with canned 99.95% CO₂ gas and automatically controlled by the infrared CO₂ analyzer (Range: 0–2,000 ppm; Ventostat 8102; Telaire Co., St. Marys, PA, USA) (Guo et al., 2012).

Cotton cultivar

The transgenic Bt cotton (Line SCRC 37 with Cry1Ab/Cry1Ac, abbreviated as Bt) was obtained from the Cotton Research Center, Shandong Academy of Agricultural Sciences. The cotton cultivar line had middle-mature duration (approximately 129 d) and was suited to grow in Northern China (Zhang et al., 2009). These cottons were grown in plastic cases (38 cm in diameter and 35 cm in height) laden with 22 kg sterilized soil and 12 g NPK compound fertilizer (N: P: K = 4: 1: 2) on 24 April of 2018 and 2019. Soil available nutrients of N, P and K were quantified as 0.19%, 16.74 mg/kg and 317.62 mg/kg, respectively.

Inoculation of cotton seeds with Azotobacter chroococcum

The glass tubes (diameter × height = 1.5 cm × 10 cm) with lyophilized powder of Azotobacter chroococcum (No. 10006, abbreviated as AC) were provided by the Agriculture Culture Collection of China (ACCC), Institute of Agriculture Resources and Regional Planning. A. chroococcum were cultivated by the specific culture medium in constant temperature shaker (28 °C, 200 rpm) until the absorbance of AC (1.001) reached to 1.000 at 600 nm. The culture medium for Azotobacter chroococcum with the ph of 7.0–7.2 contains 10.0 g Sucrose or Mannitol, 1.0 g CaCO₃, 0.5 g K₂HPO₄·3 H₂O, 0.2 g MgSO₄·7H₂O, 0.2 g NaCl, 15–20 g Agar, 1,000.0 ml distilled water. Before the inoculation of azotobacter, the supernatant of culture medium was discarded after centrifugation and then re-suspended the liquid medium until the azotobacter density was 10⁸ copies per milliliter. The cotton seeds were immersed by two different inoculations liquid mediums, the one is used with A. chroococcum (abbreviated as AC), the other is used without A. chroococcum (abbreviated as CK). Then those cotton seeds with azotobacter inoculation was dried for 4 h in the sterile draught cupboard at 28 °C.

Pot-culture experiment setup

There were two CO₂ levels (aCO₂ vs eCO₂) and two azotobacter inoculations (AC vs CK) in this experiment, with 20 replicates for a total of 80 pots. Each case with four cotton seeds at 1.5 cm depth were randomly distributed in the field; the soil was sterilized with a high temperature sterilizer (121 °C) and brought to ambient temperature prior to sowing of cotton seeds. These pots were maintained in CCGCs of aCO₂ and eCO₂ under controlled conditions (T = 26 ± 1 °C, RH = 65 ± 5%, L: D = 14 h: 10 h) without applications of pesticide and herbicide during the experiment. The rhizosphere soil samples were randomly sampled from each pot at three stages: pre-plant stage (1 day before sowing), seedling stage (14 days after sowing) and maturity stage (1 day before harvest). Then, those samples were used for analyzing the relative rhizobacteria densities by qRT-PCR with the specific primer (Table 1). One-day prior to sowing measure of A. chroococcum density was 5.31 ± 0.04 10⁵ in 2018 5.27 ± 0.07 10⁵ in 2019, respectively (Table 2).

Nitrogen metabolism-related substances and enzyme activities of Bt cotton

Five fully-expanded leaf samples measured to 1.00 g were randomly selected from Bt cotton inoculated with azotobacter treatments (AC vs CK) at squaring stage. These frozen samples were thoroughly homogenized into the extraction buffer by fitted pestles, and then the supernatants were respectively collected into a 1.5 ml microcentrifuge tube after centrifugation. The content of free amino acids in samples were respectively quantified spectrophotometrically in contrast to a standard curve by ninhydrin colorimetry method (Jeong et al., 2023; Zhang, 1992). Then the soluble protein content and nitrogen metabolism-related enzyme activities of Bt cotton leaves were respectively determined by Coomassie Brillant Blue G-250 Dye Binding (Qiang et al., 2011; Ribeiro et al., 2013; Tosi & Ré, 2013) and Lal’s colorimetric method (Chen et al., 2014; Zhu et al., 2021).

Yield and fiber quality components of Bt cotton

At maturity, ten plants were randomly selected from each treatment to record the yield characters viz., cotton weight per 20 bolls (g), lint yield per 20 bolls (g), boll number per plant and ginning outturn (%, abbreviated as GOT). Kapas were taken from the first picking to subject the fiber quality testing (i.e., length, strength, micronaire value, uniformity index and elongation) by using high volume instrument (HVI-900-A) at Institute of Cotton Research of Chinese Academy of Agricultural Sciences (CAAS), Anyang, Henan province of China (Nie et al., 2019).

Data analysis

The data disposing and analyzing were used by SPSS Statistics 26.0 (IBM, Armonk, NY, USA, 2022).

Two-factor and Three-factor analysis of variances (ANOVAs) were used to test the influences of CO₂ levels (aCO₂ vs eCO₂), azotobacter inoculation (AC vs CK), sampling years (2018 & 2019) and their interactions on the measured indexes of nitrogen metabolism-related substances (i.e., soluble protein and free amino acids content), enzyme activities (i.e., GPT and protease activity), yield (i.e., cotton weight per 20 bolls, lint yield per 20 bolls, boll number per plant and GOT) and fiber quality (length, strength, micronaire, uniform and elongation). The differences in average values of rhizobacteria densities, yield and fiber quality between cotton plants inoculated by AC and CK, between aCO₂ and eCO₂ were analyzed by t test at P < 0.05. The dissimilarity of those measured indexes between/among treatments was analyzed by the Duncan-test at P < 0.05.

Incremental densities of A. chroococcum in cotton rhizosphere soil under elevated CO₂ during different sampling stage

Inoculation with A. chroococcum (abbreviated as AC) have significant effects on the azotobacter density (P < 0.001), but non-significant influence (P ≥ 0.1, Table 3) of sampling year or any two- and three-interactions was measured on AC abundances at the seeding and maturity stages in each year (Tables 2 and 3). Compared to the ambient CO₂, elevated CO₂ raised obviously the AC density of the rhizosphere soil at cotton maturity stage; compared with culture medium without A. chroococcum (CK), we observed significant incremental trends of azotobacter density in the cotton root soil inoculated with AC during the seedling and maturity stages in 2018 and 2019 (P < 0.001; Tables 2 and 3). The interaction between CO₂ and azotobacter inoculation was significant influence (P < 0.001) of the AC abundance in the soil samples at the maturity stage (Table 3).

Nitrogen metabolism-related substances and enzyme activities of Bt cotton inoculated with A. chroococcum under elevated CO₂

Significant effects of azotobacter inoculation (P < 0.01 or P < 0.001) were observed on the soluble protein content, free amino acids content, GPT and protease activity in both years (Table 4). But non-significant effect (P > 0.05) of sampling year and CO₂ treatment on these parameters except for GPT activity under CO₂ treatment (P < 0.001, Table 4). Compared with culture medium without azotobacter, AC inoculation significantly increased the soluble protein content (+21.79%) and GPT activity (+63.27%) except for the free amino acids content (−1.86%) and protease activity (−2.22%) of Bt cotton, respectively (P < 0.01, Table 5).

In addition to the significant main effects of azotobacter inoculation and CO₂ treatment, there were significant interactions between two main impact factors on the soluble protein content, GPT and protease activity (P < 0.01 or P < 0.001) of Bt cotton except for the free amino acids content (P = 0.27 > 0.05) in both study years (Table 4). Compared with culture medium without azotobacter (CK), AC inoculation increased the soluble protein content (aCO₂: +17.93%; eCO₂: +25.69%) and GPT activity (aCO₂: +43.86%; eCO₂: +84.15%) except for the free amino acids content (eCO₂: −2.48%) and protease activity (aCO₂: −1.22%; eCO₂: −3.31%) of Bt cotton (P < 0.05 or P < 0.001, Fig. 1); On the other hand, compared with ambient CO₂, elevated CO₂ significantly increased the soluble protein content (AC: +5.02%) of Bt cotton (P < 0.01, Fig. 1); Moreover, compared with ambient CO₂, inverse trend was observed between two azotobacter inoculations in GPT activity (AC: +19.51%; CK: −7.11%) and protease activity (AC: −0.95%; CK: +1.10%) of Bt cotton growing under elevated CO₂ (P < 0.05 or P < 0.001, Fig. 1).

Yield of Bt cotton inoculated with A. chroococcum under elevated CO₂

The yield characters of Bt cotton, including cotton weight per 20 bolls, lint yield per 20 bolls, boll number per plant and GOT, were significantly affected by azotobacter inoculation (P < 0.001, Table 6). Elevated CO₂ treatment significantly influenced the measured indexes (P < 0.05 or P < 0.001) except for GOT (P = 0.88 > 0.05, Table 6). Compared with culture medium without azotobacter, AC inoculation significantly raised cotton weight per 20 bolls (+1.98%), lint yield per 20 bolls (+3.52%), boll number per plant (+26.55%) and GOT (+1.33%) of Bt cotton respectively (P < 0.001, Table 7). Moreover, elevated CO₂ treatment increased cotton weight per 20 bolls (+1.35%) of Bt cotton compared with ambient CO₂ (P = 0.02 < 0.05, Table 7).

In addition to the obvious influences of azotobacter inoculation and CO₂ treatment, there were significant interactions between two main impact factors on cotton weight per 20 bolls, lint yield per 20 bolls and GOT of Bt cotton in both study years (P < 0.05 or P < 0.001, Table 6). Compared with culture medium without azotobacter (CK), AC inoculation increased the cotton weight per 20 bolls (aCO₂: +1.46%; eCO₂: +2.82%), lint yield per 20 bolls (aCO₂: +2.58%; eCO₂: +4.45%), boll number per plant (aCO₂: +30.16%; eCO₂: +23.19%) and GOT (aCO₂: +1.10%; eCO₂: +1.59%) of Bt cotton (P < 0.05 or P < 0.001, Fig. 2). On the other hand, compared with ambient CO₂, elevated CO₂ significantly increased the cotton weight per 20 bolls (AC: +1.84%; CK: +0.49%), lint yield per 20 bolls (AC: +2.11%), and boll number per plant (CK: +9.52%) of Bt cotton (P < 0.05, P ≤ 0.01 or P < 0.001, Fig. 2).

Fiber quality components of Bt cotton inoculated with A. chroococcum under elevated CO₂

There was significant influence of azotobacter inoculation (P < 0.001) on the fiber quality components of Bt cotton in both years (Table 8), but no significant influence (P > 0.1) of sampling year and CO₂ treatment on the fiber quality parameters except for the uniform under CO₂ treatment (P ≤ 0.04, Table 8). Compared with culture medium without azotobacter, AC inoculation significantly increased length (+7.64%), uniform (+1.70%), strength (+5.67%) and elongation (+2.26%) except for the micronaire (−5.54%) of Bt cotton, respectively (P < 0.001, Table 9).

In addition to the positive influences of azotobacter inoculation and CO₂ treatment, there had significant interactions between two main impact factors on length, uniform, strength, micronaire and elongation of Bt cotton in both study years (P < 0.05, P < 0.01 or P ≤ 0.001, Table 8). Compared with culture medium without azotobacter (CK), AC inoculation increased length (aCO₂: +3.86%; eCO₂: +11.60%), uniform (aCO₂: +1.23%; eCO₂: +2.19%), strength (aCO₂: +3.89%; eCO₂: +7.44%) and elongation (aCO₂: +1.15%; eCO₂: +3.07%) of Bt cotton (P < 0.01 or P ≤ 0.001), except for micronaire (aCO₂: −3.59%; eCO₂: −7.29%) (P < 0.05, Fig. 3). On the other hand, compared with ambient CO₂, elevated CO₂ significantly increased the uniform (AC: +0.76%) and micronaire (CK: +1.92%), respectively (P = 0.03 or P < 0.05); Moreover, the inverse trend from ambient CO₂ to elevated CO₂ levels was observed between two azotobacter inoculations in length (AC: +4.42%; CK: −2.91%), strength (AC: +1.57%; CK: −1.82%) and elongation (AC: +0.86%; CK: −1.03%) of Bt cotton growing under elevated CO₂ (P < 0.05 or P < 0.001, Fig. 3).