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Investigation of the Antibacterial Activity of Khat )Catha edulis) and Its Endophytes
Naael Asad1; naaelasd@gmail.com
Dr. Yoram Shotland1, Prof. Efraim Lewinsohn2
1Sami Shamoon College of Engineering, Be'er-Sheva, Israel.
2Newe Ya’ar Research Center, Agricultural Research Organization, Volcani Center, Ramat Yishay, Israel.
Khat, originating from the Horn of Africa and the southern Arabian Peninsula, is renowned for its stimulating and euphoric and effects when chewed. Integral to the culture of these regions, it serves ritualistic and medicinal purposes, particularly for treating intestinal and respiratory inflammations. Recent research has delved into its antibacterial properties to identify those compounds responsible for its medicinal efficacy and to explore its potential antimicrobial resistance mechanisms in various agricultural settings.
This research study pursued three objectives: to identify the antibacterial compounds of Khat; to investigate the role of endophytic bacteria associated with Khat; and to assess the efficacy of Khat extracts for protecting other agricultural crops. In part 1, methanol extraction from Khat proved most effective for yielding antibacterial effects. Fractionation revealed a diverse array of hydrophilic compounds with acidic or basic characteristics that inhibit bacterial growth, likely enhancing other crops’] resistance against infection. We found that extracts from Khat leaves injured 48 hours before harvesting produced a significantly greater inhibition zone against a variety of bacteria, compared to extracts from regular leaves. We suggest that physical damage to leaves causes increased production of antibacterial compounds.
During the second part, the testing of bacterial growth inhibition, clear zones formed around the discs, which we examined. In these clear zones, bacteria, other than the bacteria being tested, were found scattered around the disc. We isolated and identified these incidental bacteria using 16SrRNA gene sequencing, revealing them to be endophytes of Khat growing around the discs. These endophytes, resistant to Khat’s active compounds, also showed antibacterial activity against other bacteria. Seven different bacteria were identified, mainly originating from hexane and water extracts. We propose that these incidental bacteria live with Khat and contribute to its growth and defense. We demonstrated that it is possible to obtain antibacterial compounds, released into the growth medium, by growing the endophytes in the presence of an additional bacterium, Enteropathogenic Escherichia coli )EPEC(, which likely induces antibacterial secretion mechanisms.In the third part, we evaluated the protective effects of endophytes and Khat extracts against bacterial pathogens on melon and tomato seedlings. Specifically, we focused on two infections—bacterial fruit blotch in melons )caused by Acidovorax citrulli( and bacterial leaf spot in tomatoes )caused by Xanthomonas euvesicatoria(. Seedlings were inoculated with the pathogens and then treated with either a mixture of the seven endophytes or Khat extract. In each experiment, five seedlings were sprayed with buffer as a control. While the effect of the treatment with the seven endophytes was ambiguous, the treatment with Khat extract showed significant protection of the seedlings from attacking pathogens. The application of Khat extract immediately after pathogen
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