EVALUATION OF THE INFLUENCE OF Azotobacter chroococcum K2020 ON CHLOROPHYLL BIOSYNTHESIS IN COTTON CULTIVARS INFECTED WITH Fusarium oxysporum f. sp. vasinfectum

ABSTRACT

This study aimed to analyze chlorophyll synthesis in different cotton (Gossypium hirsutum L.) cultivars under the influence of Azotobacter chroococcum K2020 and Fusarium oxysporum f. sp. vasinfectum. Six cultivars — “Gulbahor-2”, “Bukhara-102”, “UzRFA-709”, “UzRFA-710”, “Afsona” and “Sadaf” — were inoculated under four conditions: control (distilled water), A. chroococcum K2020, Fusarium oxysporum f. sp. vasinfectum and a mixed treatment of both. The chlorophyll content was measured at the true leaf stage and expressed as milligrams per gram of fresh weight (mg/g FW). The results demonstrated that A. chroococcum K2020 treatment consistently led to higher chlorophyll content compared to the control across all cultivars. For example, in “Gulbahor-2”, total chlorophyll content increased from 1,47 mg/g (control) to 2,31 mg/g with A. chroococcum K2020, while it decreased to 1,36 mg/g with F.oxysporum f.sp. vasinfectum and reached  2,11 mg/g  in the mixed treatment. Consistent trends were observed across other cultivars:

“Bukhara-102”: Control –1,45; A. chroococcum K2020– 2,21; F.oxysporum f.sp. vasinfectum – 1,66; Mixed – 2,06.

“UzRFA-710”: Control – 1,39; A. chroococcum K2020 – 2,23; F.oxysporum f.sp. vasinfectum – 1,54; Mixed – 2,09.

“UzRFA-709”: Control – 1,99; A. chroococcum K2020 – 2,19; F.oxysporum f.sp. vasinfectum – 1,75; Mixed – 2,11.

“Afsona”: Control – 2,28; A. chroococcum K2020 – 2,51; F.oxysporum f.sp. vasinfectum – 1,91; Mixed – 2,35.

“Sadaf”: Control – 1,90; A. chroococcum K2020 – 2,27; F.oxysporum f.sp. vasinfectum – 1,87; Mixed – 2,17.

The obtained results demonstrate that Azotobacter chroococcum K2020 plays a significant regulatory role in enhancing early-stage chlorophyll biosynthesis and alleviating the physiological disruptions caused by Fusarium oxysporum f. sp. vasinfectum. The genotype-dependent variation in response intensity underscores the importance of physiological plasticity and provides a basis for targeted selection of resilient cotton cultivars and the rational application of microbial biostimulants under biotic stress conditions.

АННОТАЦИЯ

В данном исследовании проанализирован синтез хлорофилла у различных сортов хлопчатника (Gossypium hirsutum L.) под воздействием бактерии Azotobacter chroococcum K2020 и фитопатогенного гриба Fusarium oxysporum f. sp. vasinfectum. Были использованы шесть сортов: «Гулбахор-2», «Бухара-102», «УзРФА-709», «УзРФА-710», «Афсона» и «Садаф». Семена подвергались обработке по четырём вариантам: контроль (дистиллированная вода), инокуляция A. chroococcum K2020, инокуляция F. oxysporum f. sp. vasinfectum, а также смешанная инокуляция обоими микроорганизмами. Содержание хлорофилла измеряли на стадии настоящих листьев и выражали в миллиграммах на грамм сырой массы (мг/г  СМ). Результаты показали, что обработка A. chroococcum K2020 стабильно приводила к повышению содержания хлорофилла по сравнению с контролем во всех сортах. Например, у сорта «Гулбахор-2» общее содержание хлорофилла увеличилось с 1,47 мг/г (контроль) до 2,31 мг/г при обработке A. chroococcum KO2020, тогда как при воздействии F. oxysporum f.sp. vasinfectum оно снизилось до 1,36 мг/г, а при смешанной обработке составило 2,11 мг/г. Похожие тенденции наблюдались и у других сортов:

«Бухара-102»: Контроль – 1,45; A. chroococcum K2020 – 2,21; F. oxysporum f.sp. vasinfectum – 1,66; Смешанный – 2,06.

«УзРФА-709»: Контроль – 1,99; A. chroococcum K2020 – 2,19; F. oxysporum f.sp. vasinfectum – 1,75; Смешанный – 2,11.

«УзРФА-710»: Контроль – 1,39; A. chroococcum K2020 – 2,23; F.oxysporum f.sp. vasinfectum – 1,54; Смешанный – 2,09.

«Афсона»: Контроль – 2,28; A. chroococcum K2020 – 2,51; F.oxysporum f.sp. vasinfectum – 1,91; Смешанный – 2,35.

«Садаф»: Контроль – 1,90; A. chroococcum K2020 – 2,27; F.oxysporum f.sp. vasinfectum – 1,87; Смешанный – 2,17.

Полученные результаты демонстрируют, что Azotobacter chroococcum K2020 играет важную регуляторную роль в усилении биосинтеза хлорофилла на ранних стадиях развития и в смягчении физиологических нарушений, вызванных Fusarium oxysporum f.sp.vasinfectum. Генотип-зависимая вариабельность интенсивности ответа подчёркивает значение физиологической пластичности и служит основой для целенаправленного отбора устойчивых сортов хлопчатника, а также рационального применения микробных биостимуляторов в условиях биотического стресса.

Keywords: Cotton cultivars, Azotobacter chroococcum K2020, Fusarium oxysporum f. sp. vasinfectum, chlorophyll synthesis, biotic stress, seed inoculation, microbial interaction, plant physiology.

Ключевые слова: Сорта хлопчатника, Azotobacter chroococcum K2020, Fusarium oxysporum f. sp. vasinfectum, синтез хлорофилла, биотический стресс, инокуляция семян, микробные взаимодействия, физиология растений.

Introduction

Cotton (Gossypium hirsutum L.) is one of the most important industrial crops cultivated worldwide, not only for fiber production but also for its valuable seeds and oil content. However, biotic stress factors, including soil-borne pathogens such as Fusarium oxysporum f. sp. vasinfectum, pose a significant threat to cotton production by negatively affecting seedling development and physiological processes such as photosynthesis. In recent years, the use of beneficial microorganisms, especially nitrogen-fixing bacteria like Azotobacter chroococcum, has attracted attention as a sustainable strategy to enhance plant tolerance against stress factors and improve growth.

Chlorophyll content is a crucial physiological indicator of plant health and photosynthetic efficiency. Alterations in chlorophyll synthesis under biotic stress conditions can serve as an early marker of plant stress responses. The present study focuses on evaluating the impact of Azotobacter chroococcum K2020 strain, alone and in combination with Fusarium oxysporum f. sp. vasinfectum, on the chlorophyll content in cotton seedlings. The results of this study may contribute to understanding microbial interactions that influence chlorophyll biosynthesis under stress, and to identifying eco-friendly approaches for improving cotton seedling vigor.

Several studies have demonstrated the positive effects of beneficial soil bacteria on plant physiological traits under both normal and stress conditions. According to Bashan and de-Bashan [1], Azotobacter species are capable of producing phytohormones such as auxins, cytokinins and gibberellins, which enhance seed germination and chlorophyll accumulation. Similar findings were reported by Narula et al. [2], who observed improved photosynthetic pigment content in wheat inoculated with Azotobacter chroococcum.

Conversely, pathogenic fungi like Fusarium oxysporum are known to disrupt chloroplast integrity and reduce chlorophyll synthesis through the production of mycotoxins and reactive oxygen species (ROS), as noted by Di Pietro et al. [3]. In response to such stress, plants may attempt to compensate by activating antioxidant systems, yet this may not always prevent damage to the photosynthetic apparatus.

Moreover, studies exploring the dual application of beneficial and pathogenic microorganisms are limited. However, preliminary evidence suggests that certain bioinoculants can mitigate pathogen-induced chlorophyll degradation by enhancing systemic resistance and stabilizing physiological functions [4]. In particular, free-living nitrogen-fixing bacteria such as Azotobacter chroococcum not only positively influence plant growth, but also stimulate the synthesis of phytohormones, improve the root system, and increase stress tolerance. Their application in the biological control of pathogens is of great importance in the development of environmentally friendly and sustainable agro-technologies today.

This research aims to comprehensively assess the influence of the beneficial bacterium Azotobacter chroococcum K2020 on chlorophyll biosynthesis in cotton (Gossypium hirsutum L.) cultivars, both in the presence and absence of the pathogenic fungus Fusarium oxysporum f. sp. vasinfectum. The study emphasizes the importance of microbial interactions under biotic stress conditions and seeks to determine the potential of A. chroococcum K2020 as a bioinoculant for enhancing plant resilience and maintaining photosynthetic efficiency during early seedling development.

Materials and methods

Plant material and experimental design

Six cotton (Gossypium hirsutum L.) cultivars —“Gulbahor-2”, “Bukhara-102”, “UzRFA-709”, “UzRFA-710”, “Afsona” and “Sadaf” — were selected for this study. The seeds were surface-sterilized using 96% ethanol for 1 minute, then thoroughly rinsed with distilled water for 3 minutes to remove any residual sterilizing agent.

The seeds were then placed in sterile Petri dishes lined with filter paper. Each Petri dish contained 10 seeds, and the experiment was laid out in a completely randomized design with 5 replicates per treatment.

Treatment conditions

Four treatment conditions were applied:

1. Control – seeds soaked in distilled water

2. Azotobacter treatment (Treatment 1) – seeds inoculated with Azotobacter chroococcum K2020

3. Fusarium treatment (Treatment 2) – seeds inoculated with Fusarium oxysporum f. sp. vasinfectum

4. Mixed treatment (Treatment 3) – seeds inoculated with both 

A. chroococcum K2020 and F. oxysporum f. sp. vasinfectum.

The inoculants were prepared at concentrations of 10^х7 CFU/Ml (A. chroococcum K2020) and 10^х5 spores/mL ( F. oxysporum f. sp. vasinfectum). The seeds were inoculated for 18 hours, after which they were incubated in Petri dishes under optimal conditions: 25–28°C, adequate humidity and light, to promote germination and seedling growth.

Typically, seeds germinated within 2–3 days, cotyledons fully opened within 5–7 days, and true leaves appeared by 7–10 days. Slight variations in development time were observed depending on the cultivar, environmental factors and microbial treatment.

Chlorophyll extraction and measurement

At the true leaf stage, seedlings were harvested for chlorophyll analysis. The leaves were not frozen but slightly chilled in a refrigerator to maintain freshness prior to extraction.

For each experiment, 50 mg of fresh leaf tissue was taken and extracted with 5 mL of 96% ethanol that had been pre-cooled. The slight pre-cooling of the ethanol improved the efficiency of compound extraction. The resulting homogenate was filtered and centrifuged at 5000 rpm for 5 minutes to separate the supernatant.

It is important to note that the optimal centrifugation speed for chlorophyll extraction from leaf tissue is generally within the range of 4000 to 6000 rpm. In this study, centrifugation at 5000 rpm for 5 minutes was found to be sufficient for obtaining a clear supernatant. Higher centrifugation speeds may lead to chlorophyll degradation or loss with sediment. Figure 1 shows the image of extracts prepared for chlorophyll content determination in cotton leaves.

Figure 1. The process of chlorophyll content determination

Spectrophotometric measurements were taken at three wavelengths:

664 nm – Chlorophyll “a”; 649 nm – Chlorophyll “b”; 470 nm – Carotenoids.

Chlorophyll concentrations were calculated using Lichtenthaler & Wellburn’s equations:

For 96% Ethanol (Lichtenthaler & Wellburn, 1983):

Chlorophyll “a” (mg/L):  Chl a = 13.36 *A ₆₆₄ - 5.19 *A₆₄₉

Chlorophyll “b” (mg/L):  Chl b = 27.43 * A₆₄₉- 8.12 *A₆₆₄

Total chlorophyll (mg/L):  Total Chl = 17.76 *A₆₄₉ + 7.34*A₆₆₄

Carotenoids (mg/L):

C _x+c = {(1000 * A₄₇₀) - (1,82 * Chl a) - (85,02 * Chl b)} / 198

Note: Here:  Absorbance value measured at 470 nm. Total carotenoids (carotene + xanthophyll), mg/L

Chl a  and Chl b — previously calculated chlorophyll contents (mg/L).

The results from the above formulas are expressed in mg/L. However, to express chlorophyll content in mg/g of fresh leaf weight, the amount of leaf material used and the volume of ethanol for extraction must be taken into account.

Specifically, since 50 mg = 0.05 g of leaf tissue was extracted with 5 mL = 0.005 L of 95% ethanol, the calculated concentrations (in mg/L) should be divided by 10 to convert the values to mg/g of fresh weight.

Chl (mg/g) = {Chl (mg/L) * V (L)} / Fresh weight (g)

Results

The analysis of photosynthetic pigments in cotton seedlings revealed significant differences in chlorophyll “a”, chlorophyll “b”, total chlorophyll and carotenoids content across the different treatment groups. Seedlings treated with Azotobacter chroococcum K2020 consistently demonstrated elevated pigment concentrations compared to the control. This enhancement suggests a stimulatory effect of Azotobacter on chloroplast development and pigment biosynthesis, potentially linked to the bacterium’s ability to produce plant growth-promoting substances such as auxins and cytokinins (Table 1).

Table 1.

Determination of chlorophyll content

 (According to Table 1, the highest chlorophyll concentrations were observed in seedlings treated with Azotobacter chroococcum K2020, while Fusarium treatment led to a reduction in pigment levels).

Quantitative analysis of chlorophyll and carotenoid contents revealed marked treatment-dependent variations across all tested cotton varieties.

In all genotypes, Azotobacter inoculation (Treatment 1) led to a notable increase in pigment levels compared to the control. For instance, in “Gulbahor-2”, total chlorophyll content increased from 1.62 mg/g FW (control) to 2.08 mg/g FW, representing an approximate 28% rise. Similarly, “Bukhara-102” exhibited a 52% enhancement, and “Afsona” showed a 12% increase in total chlorophyll under this treatment. Carotenoid content also rose by 15–22% relative to the control, confirming the stimulatory role of Azotobacter chroococcum K2020 in promoting photosynthetic pigment biosynthesis.

In contrast, Fusarium oxysporum f.sp. vasinfectum infection (Treatment 2) caused a drastic reduction in pigment accumulation. Compared to the control, total chlorophyll declined by 27–38%, with chlorophyll a showing a more pronounced decrease (up to 40% in “Bukhara-102” and “UzRFA-710”). Carotenoids dropped by 18–25%, indicating severe impairment of the photosynthetic apparatus due to fungal-induced oxidative stress and chloroplast disruption.

Interestingly, in the combined treatment (Treatment 3: Azotobacter + Fusarium), pigment levels were intermediate between those of the Fusarium-infected and Azotobacter-only groups. For example, total chlorophyll in “Gulbahor-2” was 2.11 mg/g FW,  about 5% lower than Treatment 1, but 25% higher than Treatment 2. Similar partial restoration was observed in carotenoid levels (increase by ~20% relative to Fusarium-only). This suggests that Azotobacter provided partial protection by mitigating pathogen-induced damage.

Across all varieties, carotenoid concentration followed the same trend — the highest values were found in the Azotobacter-treated plants (0.57–0.71 mg/g FW), whereas the lowest occurred under Fusarium stress (0.41–0.56 mg/g FW). Carotenoids are essential for photoprotection and antioxidative defense; hence, their higher accumulation under bacterial inoculation reflects improved stress tolerance.

Overall, the data demonstrate that Azotobacter treatment enhances photosynthetic pigment synthesis, while Fusarium infection suppresses it. The combined treatment partially restores pigment levels, emphasizing the bioprotective potential of Azotobacter in pathogen-challenged cotton plants.

Discussion

The results of this study provide strong evidence for the beneficial role of Azotobacter chroococcum K2020 in enhancing chlorophyll and carotenoid biosynthesis in cotton seedlings. In particular, seedlings treated with A. chroococcum K2020 demonstrated significantly higher levels of chlorophyll “a”, chlorophyll “b”, total chlorophyll, and carotenoids compared to the control and pathogen-inoculated groups. These findings are consistent with previous reports suggesting that plant growth-promoting rhizobacteria (PGPR) can stimulate pigment production by improving nutrient availability, producing phytohormones, and enhancing photosynthetic capacity [1, 2, 8].

The observed increase in pigment concentration may be attributed to the production of auxins, cytokinins, and other bioactive compounds by Azotobacter, which facilitate chloroplast development and delay senescence. As noted by Ahmad et al. [2] and Khan et al. [8], Azotobacter species enhance photosynthetic performance not only by stimulating chlorophyll synthesis but also by improving plant nitrogen status and overall vigor.

Conversely, treatment with Fusarium oxysporum f.sp. vasinfectum led to a 27–38% reduction in total chlorophyll and an 18–25% decrease in carotenoids compared to the control. This decline is likely associated with fungal-induced oxidative stress and chloroplast damage, as evidenced by visible leaf yellowing in infected plants. Similar findings were reported by Amini & Sidovich [3] and Guo et al. [9], who demonstrated that pathogenic fungi produce mycotoxins and reactive oxygen species (ROS) that disrupt chloroplast integrity and inhibit pigment biosynthesis. Our results therefore confirm that Fusarium oxysporum f.sp. vasinfectum infection suppresses photosynthetic pigment formation, leading to reduced photosynthetic efficiency and overall plant weakening.

Interestingly, In the combined treatment (A. chroococcum + Fusarium), total chlorophyll content was 20–25% higher than in the Fusarium-only group, but 5–10% lower than in the Azotobacter-only variant. Carotenoid levels showed a similar pattern, being ~20% higher than under Fusarium infection yet 10–15% lower than with Azotobacter alone.

This intermediate response confirms the partial protective effect of A. chroococcum, likely mediated through induced systemic resistance (ISR), microbial competition, and modulation of stress-related hormones [4, 6].

These findings align with recent studies indicating that beneficial microbes can mitigate the effects of biotic stress through both direct and indirect interactions with pathogens and host plants [6, 7]. For instance, Raza et al. [4] reported that cotton plants treated with Azotobacter under Fusarium wilt stress exhibited improved physiological traits and reduced disease symptoms.

Taken together, the data from this study reinforce the hypothesis that Azotobacter chroococcum K2020 can act as an effective bioinoculant under biotic stress conditions. Its ability to stimulate pigment biosynthesis and partially counteract pathogen damage makes it a promising component of sustainable, eco-friendly agricultural technologies aimed at enhancing plant health and resilience in cotton cultivation systems.

Conclusion

This study comprehensively evaluated the influence of Azotobacter chroococcum  K2020 and Fusarium oxysporum f.sp. vasinfectum on the biosynthesis of photosynthetic pigments in cotton (Gossypium hirsutum L.) seeds and seedlings. Quantitative analysis revealed significant treatment-dependent variations in chlorophyll and carotenoid contents across all tested varieties.

Application of A. chroococcum led to a pronounced enhancement in pigment accumulation, with total chlorophyll increasing by 25–50% and carotenoid levels by 15–22% relative to the untreated control. The most notable responses were observed in the varieties “Gulbahor-2” and “Afsona”, which displayed the highest pigment concentrations, indicating strong photosynthetic activity and improved physiological status.

In contrast, infection with Fusarium oxysporum f.sp. vasinfectum resulted in a substantial pigment decline, characterized by a 27–38% reduction in total chlorophyll and an 18–25% decrease in carotenoids. This suppression is likely linked to oxidative stress, chloroplast membrane disruption, and inhibition of pigment biosynthetic enzymes under fungal attack.

Interestingly, under the combined treatment with both A. chroococcum and Fusarium, pigment values were 20–25% higher than in the Fusarium-only variant, though 5–10% lower than in the Azotobacter-only treatment. These intermediate results demonstrate a partial bioprotective effect of A. chroococcum K2020, which likely mitigates pathogen-induced oxidative stress through mechanisms such as induced systemic resistance (ISR), antioxidant activation, and microbial competition in the rhizosphere.

Overall, the findings confirm that Azotobacter chroococcum K2020 acts as an effective plant growth-promoting rhizobacterium (PGPR), capable of enhancing pigment synthesis, sustaining photosynthetic efficiency, and strengthening the biotic stress tolerance of cotton plants. These results highlight the potential of A. chroococcum as a biological inoculant for sustainable cotton cultivation and integrated disease management strategies.

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