Inhibitory Effect of Endophytic Bacteria with Auxin Production Ability on Diplodia bulgarica, the Causative Agent of Apple Canker in East Azerbaijan Province

پژوهش های حفاظت گیاهان ایران

Article 4, Volume 38, Issue 2 - Serial Number 64, June 2024, Pages 147-157   

Document Type: Research Article

DOI: 10.22067/jpp.2024.82876.1148

Authors

Samaneh Hagverdi¹; Reza Khakvar^{* 1}; Mehdi Arzanlou²; Nasser Aliasgharzad²

¹Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

²Department of Soil Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

Abstract

Introduction
 The apple (Malus domestica Borkh.), a cornerstone of Iran's agricultural exports, plays an important role in the nation's economy. The cultivation of this fruit, however, is frequently exposed to a multitude of pests and diseases, especially fungi from the Botryosphaeriaceae family emerging as a significant threat. Among these, Diplodia bulgarica is a particularly harmful fungus that causes a lot of damage to apple orchards. Traditional management strategies have predominantly relied on chemical fungicides; however, these methods have several problems, including environmental contamination, food safety concerns, and the potential for pathogen resistance. These issues highlight the critical need for alternative solutions. To deal with these problems, experts in plant diseases are looking more at using biological control agents as a viable and environmentally friendly option. Endophytic bacteria, a group of non-pathogenic microorganisms that inhabit plant tissues, are leading this change. By producing essential growth factors, these bacteria not only enhance the metabolic processes of their host plants but also protect them from many diseases and environmental stress. Their mode of action is not limited to direct antagonistic actions like nutrient competition and antibiotic production; they also take in a range of indirect strategies that effectively inhibit plant diseases. Using endophytic bacteria to control plant diseases is a new, exciting and promising direction toward sustainable agriculture. Their dual role as biocontrol agents and promoters of plant health offers a comprehensive strategy for enhancing crop resilience. Ongoing research is revealing how endophytic bacteria could transform the way we protect plants and increase farm yields, marking a new era in the management of apple and other fruit tree diseases.
Materials and Methods
 This study aimed to isolate auxin-producing endophytic bacteria from apple trees in the orchards of East Azerbaijan province and assess their capacity to inhibit the pathogenic fungus Diplodia bulgarica the major causal agent of apple tree decline in Northwest Iran. To do this, healthy branches from apple trees in East Azerbaijan province in northwest Iran were sampled, resulting in 110 isolates of endophytic bacteria being isolated and subsequently analyzed. Employing the dual-culture method, researchers identified the most effective isolate exhibiting the strongest antagonistic effect. Subsequently, fifty isolates out of the initial 110 were chosen for further investigation based on their promising antifungal properties. To statistically analyze the auxin production data, researchers conducted a variance analysis (ANOVA). Finally, Duncan's multiple range test (DMRT) at a 1% significance level was used to compare the mean auxin production values among these selected isolates. Identification was carried out based on polymerase chain reaction (PCR) and sequencing of the 16S rDNA region, along with biochemical assays including gram staining, catalase, oxidase, arginine hydrolase, and others.
 Results and Discussion
 The dual-culture assay results led to the selection and preservation of 50 isolates demonstrating varying degrees of antagonistic potential. Of these, 37 isolates (74%) were identified as gram-positive, while the remaining 13 isolates were gram-negative. The inhibition percentage of these isolates ranged from 10% to 79%. Notably, five endophytes exhibited high inhibition rates (74-79%), were also effective in auxin production. The auxin production assay revealed that, on average, bacterial isolates produced 5.5 mg/ml of auxin in the absence of tryptophan and 8.58 mg/ml in its presence. Based on biochemical and molecular identification, all five isolates belonged to the Bacillus genus, with more than 99% similarity. These included Bacillus xiamenensis, Bacillus sonorensis, Bacillus tequilensis, Bacillus mojavensis, and Bacillus subtilis. Bacillus mojavensis demonstrated the highest auxin production, yielding up to tenfold more auxin without tryptophan and fivefold more with tryptophan compared to other isolates. The study also found that adding L-tryptophan to the bacterial growth medium generally increased auxin production. In the qualitative evaluation, a pink color change was observed exclusively in the B. mojavensis isolate.
 Conclusion
 While fungicides are highly effective, their escalating use has led to serious repercussions, including environmental pollution, toxic residues in food, pathogen resistance, and socio-economic issues. The substantial costs and consumer demand for chemical-free produce have spurred researchers to seek viable alternatives to these agents. In this study, five Bacillus bacterial isolates were identified from apple trees in Iran's northwestern regions. These isolates not only exhibit strong inhibitory effects against the fungus Diplodia bulgarica but also possess the capability to produce significant amounts of the phytohormone auxin (IAA). Consequently, they can be recommended to farmers as both an alternative to fungicides and as biofertilizers. The deployment of these bacteria promises not only the effective and safe mitigation of pathogens but also supports plant growth and development through hormone production.

Keywords

Bacillus; Biocontrol; Pphytohormone; Trunk canker

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