Impact of temperature on the growth of Klebsiella aerogenes, which causes pearl millet stem rot

Introduction

One of the most important and ancient staple millets for millions of poor people living in tropical dry and semi-arid areas is pearl millet [Pennisetum glaucum (L.) R. Br.], a plant belonging to the Poaceae family. Pearl millet, the world's fifth-most significant cereal crop after rice, wheat, maize, and sorghum, is essential to maintaining food and energy security, especially in locations that receive rain. The All India Coordinated Research Project on Pearl Millet (2022) reports that this crop is cultivated on more than 30 million hectares globally, with Africa accounting for the majority of its cultivation (>18 million ha) and Asia for the majority (>10 million ha). According to Yadav et al. (2012), 90% of the world's pearl millet land is found in Africa and India. Interestingly, since 1980, millet production has increased by 130% in central and west Africa, accounting for 50% of the world's total production. Rajasthan is the state that produces the most, and India is the leader in production. Over 68.40 million hectares, pearl millet was grown in India in 2021-2022, with a total production of 97.80 million tonnes and a productivity rate of 1,430 kg/ha. With a productivity rate of 2,318 kg/ha, the 4.83 lakh hectares of cultivable land in Haryana produced 11.19 lakh tonnes of output. Districts Bhiwani, Mahendragarh, Rewari, Hisar, Charkhi Dadri, Jhajjar, Rohtak and Palwal are the main ones in Haryana that grow pearl millet. The diseases that can affect pearl millet in India include downy mildew (also known as green ear disease), which is caused by Sclerospora graminicola; rust, which is caused by Puccinia substriata var. indica; smut, which is caused by Moesziomyces bullatus; sugary disease (also known as ergot) caused by Claviceps fusiformis; pyricularia leaf spot (blast) caused by Magnaporthe grisea; stem rot caused by Klebsiella aerogenes; bacterial leaf blight, which is caused by Pantoea stewartii subspecies indologenes; and stunting, little leaf and phyllody disease, which is caused by Candidatus. In particular, stem rot, bacterial leaf blight, stunting, small leaf, and phyllody disease were all observed to be prevalent in India in 2021-2022 (Hemalatha et al., 2022; Malik et al., 2022; Mushineni et al., 2021) [6,8,10] . The genus Klebsiella, which has historically been linked to diseases in humans and animals, has recently become an important plant pathogen. It has been linked to a number of crop diseases, including wilting in many plantations (Ajayasree and Borkar, 2018) [3] , stem rot in pearl millet (Malik et al., 2022) [8] and top rot in maize (Huang et al., 2016) [7] .

Klebsiella aerogenes is the causative agent of pearl millet stem rot. The Enterobacteriaceae family of bacteria is distinguished by a wide range of biochemical, morphological, and cultural characteristics. According to genome-based comparative bacterial phylogenetics, Enterobacter aerogenes was reclassified as Klebsiella aerogenes in 2017 (Tindall et al., 2017; Wesevich et al., 2019) [11,12] . On nutrient agar, rod-shaped, gram-negative, catalase-positive, citrate-positive, indole-negative, facultative anaerobe Klebsiella aerogenes forms whitishcream colonies. Some strains of these organisms are naturally occurring parts of the flora of the human gastrointestinal tract, and they can be found on plants, in soil, and in water. Klebsiella affects people and animals in addition to plants (Huang et al., 2016) [7] . Disease incidence, overall severity of the disease, and the source of the inoculum all affect crop output. In order to identify the ideal circumstances for the pathogen's proliferation, experiments were carried out to assess its growth pattern across a range of temperature regimes.

Materials and Methods

7 different isolates namely KA1, KA2, KA3, KA4, KA5, KA6 and KA7 of Klebsiella aerogenes causing stem rot of pearl millet were used during study. Their broths were prepared and from them serial dilutions were performed. Procedure 1. Arrange test tubes and fill them with 9 ml double distilled water and label them. 2. Add 1ml bacterial suspension from the broth to first test tube. 3. Make up further dilution upto 10 -5 . 4. Perform plating from the last dilution by pour plate method for obtaining single colonies. 5. Add 1ml of diluted sample to appropriately labelled petri plates after dilution. 6. Incubate the plates as per the temperature range for recording observations. 7. Repeat the same process for all 7 isolates Observations Pathogen colonies were counted after 48 hours of incubation using following formula:

(No. of colonies x Total Dilution factor) CFU /ml = Volume of culture plated in ml Bacterial isolates were grown on nutrient agar (Table 1 denotes its composition) plates and their growth were recorded at different temperature regimes (20±2 °C, 25±2 °C, 30±2 °C, 35±2 °C and 40±2 °C).

Statistical Analysis

Analysis was performed by using opstat software.

Results and Discussion

7 isolates i.e., KA1, KA2, KA3, KA4, KA5, KA6 and KA7 of Klebsiella aerogenes causing stem rot of pearl millet were undertaken in the study and the CFU x 10 5 /ml were calculated at 5 different temperature ranges varying from 20±2 °C to 40±2 °C. It was observed that 25±2 °C was found to be the most optimum temperature for all isolates of Klebsiella aerogenes as maximum number of colonies were observed at this temperature (Table 2). The least number of colonies were observed at 40±2 °C in all seven isolates of the stem rot causing pathogen. After 25±2 °C, 30±2 °C was second best temperature for Klebsiella aerogenes isolates growth followed by 20±2 °C. The findings indicate that Klebsiella aerogenes thrives best at 25±2 °C (Figure 1). This temperature likely provides the ideal conditions for the metabolic activities of the bacterium, including nutrient uptake, enzyme activity and overall cellular function. At 20 °C, the bacterial growth was significantly lower, suggesting that the cooler temperature slows down metabolic processes, leading to reduced proliferation. At 30 °C and 35 °C, although the bacteria still grew, the conditions were less favourable compared to 25 °C. This may be due to the increased metabolic strain or the denaturation of proteins at higher temperatures. Notably, at 40 °C, the growth was minimal, likely because the higher temperature approaches the upper limit of the bacterium's thermal tolerance, leading to thermal stress and potential denaturation of critical cellular components. Figure 2 shows individual isolate growth at different temperature range.

Understanding the temperature preferences of Klebsiella aerogenes is crucial for various applications, including controlling its growth in field areas, lab conditions and optimizing conditions for industrial processes where this bacterium might cause loss. Additionally, these findings underscore the importance of temperature control in preventing the spread of bacterial infections, as certain temperatures can either inhibit or promote bacterial growth.

Further research into the molecular mechanisms governing temperature-dependent growth could provide deeper insights into the adaptability and resilience of Klebsiella aerogenes. This knowledge can inform strategies for managing bacterial populations in both environmental and medical contexts.

Conclusion

In conclusion, this study investigated the temperature preferences of seven isolates of Klebsiella aerogenes responsible for stem rot in pearl millet. Our findings indicate that 25±2 °C is optimal for the growth of these isolates, with maximum colony counts observed at this temperature. Lower temperatures, such as 20±2 °C, slowed bacterial growth significantly, while higher temperatures up to 40±2 °C showed progressively less favorable conditions, likely due to metabolic strain and thermal stress. These results align with previous studies on related bacteria, highlighting the importance of temperature control in managing Klebsiella aerogenes populations. Further research into the underlying molecular mechanisms could enhance our understanding and aid in developing effective strategies for bacterial control in diverse environments and industries.

Conflict of Interest

Author confirms no conflict of Interest.

Acknowledgement

AcknowledgementWe highly acknowledge HOD, Department of Plant Pathology, CCSHAU, Hisar (Haryana) for providing all the necessary requirements on time.

References

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