Endophytes and Plant Extracts as Potential Antimicrobial Agents against Candidatus Liberibacter Asiaticus, Causal Agent of Huanglongbing

Jessica Dominguez; Krishnaswamy Jayachandran; Ed Stover; Joseph Krystel; Kateel G Shetty

doi:10.3390/microorganisms11061529

Endophytes and Plant Extracts as Potential Antimicrobial Agents against Candidatus Liberibacter Asiaticus, Causal Agent of Huanglongbing

Microorganisms. 2023 Jun 8;11(6):1529. doi: 10.3390/microorganisms11061529.

Authors

Jessica Dominguez¹, Krishnaswamy Jayachandran¹, Ed Stover², Joseph Krystel², Kateel G Shetty¹

Affiliations

¹ Department of Earth and Environment, Florida International University, Miami, FL 33199, USA.
² United States Department of Agriculture/Agricultural Research Service, Ft. Pierce, FL 34945, USA.

Abstract

Huanglongbing (HLB), also known as citrus greening, is an insidious disease in citrus and has become a threat to the sustainability of the citrus industry worldwide. In the U.S., Candidatus Liberibacter asiaticus (CLas) is the pathogen that is associated with HLB, an unculturable, phloem-limited bacteria, vectored by the Asian Citrus Psyllid (ACP, Diaphorina citri). There is no known cure nor treatment to effectively control HLB, and current control methods are primarily based on the use of insecticides and antibiotics, where effectiveness is limited and may have negative impacts on beneficial and non-target organisms. Thus, there is an urgent need for the development of effective and sustainable treatment options to reduce or eliminate CLas from infected trees. In the present study, we screened citrus-derived endophytes, their cell-free culture supernatants (CFCS), and crude plant extracts for antimicrobial activity against two culturable surrogates of CLas, Sinorhizobium meliloti and Liberibacter crescens. Candidates considered high-potential antimicrobial agents were assessed directly against CLas in vitro, using a propidium monoazide-based assay. As compared to the negative controls, statistically significant reductions of viable CLas cells were observed for each of the five bacterial CFCS. Subsequent 16S rRNA gene sequencing revealed that each of the five bacterial isolates were most closely related to Bacillus amyloliquefaciens, a species dominating the market of biological control products. As such, the aboveground endosphere of asymptomatic survivor citrus trees, grown in an organic orchard, were found to host bacterial endophytes capable of effectively disrupting CLas cell membranes. These results concur with the theory that native members of the citrus microbiome play a role in the development of HLB. Here, we identify five strains of Bacillus amyloliquefaciens demonstrating notable potential to be used as sources of novel antimicrobials for the sustainable management of HLB.

Keywords: Candidatus Liberibacter asiaticus; antimicrobial activity; citrus greening; endophytes; medicinal plants; psyllid homogenate assay.

Grants and funding

This research was made possible through an educational grant to the first author, Jessica Dominguez, under the USDA National Institute of Food and Agriculture’s Hispanic-Serving Institutions Higher Education Grants Program, number 2016-38422-25549 and also USDA-NIFA Emergency Citrus Disease Research & Extension (ECDRE) program, grant number 2022-70029-38526. We also acknowledge the financial support of the Doctoral Evidence Acquisition Fellowship, granted by the University Graduate School of Florida International University, as well as financial support through teaching assistantships from the Department of Earth and Environment at Florida International University.